In O
spite O
of O
medical O
help O
: O
the O
puzzle O
of O
an O
eighteenth O
- O
century O
Prime O
Minister O
' O
s O
illness O
. O

Abstract O

Medical O
History O
, O
1990 O
, O
34 O
: O
178 O
- O
184 O
. O

IN O
SPITE O
OF O
MEDICAL O
HELP O
: O
THE O
PUZZLE O
OF O
AN O

EIGHTEENTH O
- O
CENTURY O
PRIME O
MINISTER O
' O
S O

ILLNESS O

by O

MARJORIE O
BLOY O
* O

Charles O
Watson O
Wentworth O
, O
second O
Marquis O
of O
Rockingham O
, O
died O
suddenly O
and O
unexpectedly O
on O
1 O
July O
1782 O
, O
when O
he O
was O
only O
52 O
years O
old O
. O

He O
had O
suffered O
- O
or O
enjoyed O
- O
ill O
health O
all O
his O
life O
but O
in O
1782 O
appeared O
to O
be O
no O
worse O
than O
he O
had O
ever O
been O
. O

His O
death O
in O
London O
terminated O
his O
second O
period O
of O
office O
as O
Prime O
Minister O
, O
to O
which O
he O
had O
been O
appointed O
only O
14 O
weeks O
earlier O
. O

In O
May O
he O
had O
reported O
to O
the O
Duke O
of O
Portland O
that O
he O
had O
" O
for O
some O
weeks O
past O
undergone O
much O
Pain O
and O
much O
inconvenience O
from O
something O
similar O
to O
my O
old O
Complaint O
in O
my O
Side O
and O
Stomach O
" O
but O
that O
he O
felt O
much O
better O
than O
he O
had O
. O
' O
By O
17 O
June O
he O
was O
recovering O
from O
both O
influenza O
and O
his O
" O
old O
complaint O
" O
. O
2 O
On O
1 O
July O
he O
died O
and O
on O
the O
20th O
he O
was O
interred O
in O
York O
Minster O
. O

The O
first O
recorded O
bout O
of O
illness O
suffered O
by O
the O
marquis O
, O
then O
Lord O
Higham O
, O
was O
in O
July O
1741 O
when O
the O
11 O
- O
year O
- O
old O
was O
" O
a O
little O
indisposed O
, O
something O
Feaverish O
I O
guess O
it O
proceeds O
from O
Worms O
and O
will O
Soon O
be O
removed O
" O
. O
3 O
He O
also O
had O
a O
rash O
and O
it O
was O
thought O
that O
the O
cause O
of O
the O
problem O
was O
that O
the O
boy B
had O
overheated O
himself O
. O
4 O
He O
was O
still O
ill O
at O
the O
beginning O
of O
August O
: O
he O
had O
been O
" O
much O
out O
of O
order O
" O
for O
a O
long O
time O
but O
had O
been O
recommended O
to O
take O
warm O
baths O
by O
Dr O
Wilmot O
and O
Mr O
Ranby O
when O

they O
were O
consulted O
in O
London O
. O
5 O
Charles O
' O
s O
aunt O
, O
Lady O
Isabella O
Finch O
, O
was O
sure O
that O
the O
baths O
" O
and O
other O
Things O
They O
' O
ll O
prescribe O
will O
in O
a O
short O
Time O
entirely O
Cure O
his O
Complaints O
w O
[ O
hic O
] O
h O
neither O
of O
Them O
thought O
proceed O
from O
any O
dangerous O
Causes O
" O
. O
6 O
In O
spite O
of O
Lady O
Isabella O
' O
s O
hopes O
, O
Charles O
did O
not O
greatly O
improve O
, O
even O
though O
his O
mother O
believed O
that O
he O
continued O
mending O
every O
day O
. O

The O
main O
reason O
that O
Higham O
and O
his O
mother O
had O
gone O
to O
London O
to O
consult O
Dr O
Wilmot O
and O
Mr O

* O
Marjorie O
Bloy O
, O
Ph O
. O
D O
. O
, O
18 O
Farm O
View O
Road O
, O
Kimberworth O
, O
Rotherham O
, O
S O
. O

Yorks O
. O

S61 O
2BA O
. O

The O
Rockingham O
Papers O
are O
in O
the O
holdings O
of O
the O
Wentworth O
Woodhouse O
Muniments O
at O
Sheffield O
City O
Archives O
Department O
, O
Sheffield O
City O
Library O
. O

I O
am O
grateful O
to O
Dr O
R O
. O

S B
. I

Morton O
for O
his O
advice O
and O
help O
with O
the O
diagnostic O
sections O
of O
this O
essay O
. O

' O
WWM O
, O
RI O
- O
2094 O
. O

Rockingham O
to O
Portland O
, O
25 O
May O
1782 O
. O

2WWM O
, O
RI O
- O
2094 O
. O

Rockingham O
to O
Charlemont O
, O
17 O
June O
1782 O
. O

3 O
WWM O
, O
M8 O
- O
25 O
. O

Malton O
to O
Nottingham O
, O
after O
16 O
June O
1741 O
. O

4 O
WWM O
, O
M8 O
- O
26 O
. O

Lady O
Finch O
to O
Lady O
Malton O
, O
30 O
July O
1741 O
. O

5 O
WWM O
, O
M8 O
- O
28 O
. O

Winchelsea O
to O
Malton O
, O
7 O
August O
1741 O
. O

6 O
WWM O
, O
M8 O
- O
29 O
. O

Lady O
Finch O
to O
Malton O
, O
7 O
August O
1741 O
. O

178 O

An O
eighteenth O
- O
century O
Prime O
Minister O
' O
s O
illness O

Ranby O
was O
his O
mother O
' O
s O
concern O
about O
a O
" O
swelling O
in O
a O
certain O
part O
which O
was O
larger O
than O
when O
we O
left O
Wentworth O
" O
. O

The O
doctors O
hoped O
that O
it O
would O
burst O
outwards O
" O
which O
they O
assure O
me O
will O
be O
the O
safest O
way O
and O
give O
the O
poor O
Monkey B
but O
very O
little O
pain O
' O
7 O

On O
20 O
August O
Lord O
Winchelsea O
, O
Higham O
' O
s O
uncle O
, O
surprised O
to O
see O
the O
boy O
so O
well O
and O
brisk O
, O
hoped O
that O
Charles O
was O
" O
now O
safe O
from O
this O
complaint O
" O
- O
the O
same O
one O
from O
which O
he O
had O
suffered O
in O
1738 O
- O
39 O
- O
but O
thought O
that O
he O
would O
never O
be O
safe O
" O
if O
he O
continues O
the O
practice O
of O
overheating O
himself O
and O
then O
drinking O
Cold O
Water O
" O
. O

He O
said O
that O
Charles O
was O
of O
a O
" O
pretty O
healthy O
strong O
Constitution O
" O
; O
8 O
Lady O
Malton O
was O
not O
so O
sure O
. O

The O
same O
day O
she O
wrote O
a O
progress O
report O
to O
her O
husband O
saying O
that O
Charles O
' O
s O
swelling O
continued O
to O
grow O
, O
as O
did O
the O
pain O
" O
in O
that O
part O
( O
but O
not O
the O
lease O
[ O
sic O
] O
trouble O
in O
making O
Water O
or O
going O
to O
Stool O
) O
& O
less O
Fever O
than O
c O
[ O
oulJd O
be O
imagined O
where O
Matter O
is O
as O
they O
now O
imagine O
certainly O
gathering O
and O
must O
end O
in O
an O
operation O
" O
. O

In O
spite O
of O
it O
all O
, O
Charles O
was O
in O
fine O
spirits O
. O
9 O
Lady O
Malton O
dosed O
the O
boy O
with O
cinchona O
bark O
, O
which O
removed O
the O
pains O
in O
his O
legs O
and O
reduced O
his O
fever O
, O
and O
she O
was O
convinced O
that O
they O
would O
soon O
have O
" O
a O
clear O
Stage O
to O
act O
in O
a O
proper O
manner O
a O
[ O
bou O
] O
t O
his O
other O
Complaints O
w O
[ O
hic O
] O
h O
the O
Learned O
assure O
me O
are O
to O
be O
conquered O
also O
" O
. O

10 O
Charles O
was O
soon O
allowed O
to O
eat O
meat O
and O
Mr O
Ranby O
still O
assured O
her O
that O
the O
swelling O
would O
break O
outwards O
. O

1 O
" O
Three O
days O
later O
he O
decided O
to O
lance O
it O
, O
even O
though O
Dr O
Bourne O
disagreed O
. O

The O
boy B
' O
s O
mother O
was O
puzzled O
because O
the O
swelling O
" O
sometimes O
pushes O
forward O
very O
fast O
then O
retires O
a O
little O
" O
but O
the O
doctor O
and O
Ranby O
seemed O
happy O
with O
his O
condition O
. O
' O
2 O
At O
this O
point O
the O
letters O
cease O
, O
presumably O
because O
Malton O
arrived O
in O
London O
with O
his O
daughters O
, O
to O
have O
them O
inoculated O
against O
smallpox O
, O
but O
a O
later O
letter O
states O
that O
surgery O
to O
open O
the O
swelling O
was O
not O
undertaken O
. O

13 O

By O
the O
end O
of O
October O
the O
correspondence O
had O
recommenced O
. O

Charles O
was O
ill O
again O
. O

He O
was O
just O
the O
same O
as O
when O
he O
left O
Kensington O
, O
so O
John O
Bourne O
had O
bled O
him O
and O
the O
child B
had O
started O
on O
Sir O
Edward O
Hulse O
' O
s O
prescription O
, O
unfortunately O
not O
defined O
in O
the O
letter O
, O
but O
which O
was O
apparently O
as O
bad O
as O
the O
last O
one O
, O
if O
not O
worse O
. O

Lady O
Malton O
thought O
that O
" O
with O
such O
a O
State O
of O
Blood O
the O
Continuation O
of O
Health O
cannot O
be O
expected O
" O
but O
was O
hopeful O
that O
the O
" O
Cinnabar O
may O
prove O
a O
more O
Efficacious O
remedie O
than O
any O
than O
has O
been O
tryed O
yet O
" O
" O
. O
" O
4 O
That O
night O
she O
applied O
" O
a O
Blister O
. O
. O
. O
without O
the O
least O
Symptom O
or O
tendency O
to O
anything O
like O
Strangury O
" O
. O

He O
bore O
the O
treatment O
well O
, O
as O
he O
had O
done O
three O
years O
previously O
, O
and O
it O
seemed O
so O
successful O
that O
Lady O
Malton O
was O
" O
determined O
to O
keep O
it O
running O
full O
as O
long O
as O
I O
did O
last O
time O
by O
the O
help O
of O
John O
Borne O
[ O
sic O
] O
with O
much O
ease O
to O
the O
Dear O
Child B
" O
. O
' O
5 O
She O

7 O
WWM O
, O
M7 O
- O
51 O
. O

Lady O
to O
Lord O
Malton O
, O
18 O
August O
1741 O
. O

8 O
WWM O
, O
M2 O
- O
84 O
. O

Winchelsea O
to O
Malton O
, O
20 O
August O
1741 O
. O

9 O
WWM O
, O
M7 O
- O
52 O
. O

Lady O
to O
Lord O
Malton O
, O
20 O
August O
1741 O
. O

' O
0 O
WWM O
, O
M7 O
- O
53 O
. O

Lady O
to O
Lord O
Malton O
, O
25 O
August O
1741 O
. O

WWM O
, O
M7 O
- O
54 O
. O

Lady O
to O
Lord O
Malton O
, O
29 O
August O
1741 O
. O

WWM O
, O
M7 O
- O
55 O
. O

Lady O
to O
Lord O
Malton O
, O
1 O
September O
1741 O
. O

3 O
WWM O
, O
R170 O
- O
20 O
. O

Nicol6 O
Scanagati O
of O
Padua O
, O
20 O
July O
1750 O
. O

I O
am O
grateful O
to O
Fr O
John O
McMahon O
and O
Dr O
Stephen O
Bemrose O
for O
their O
translations O
of O
this O
letter O
. O

4 O
WWM O
, O
M7 O
- O
14 O
. O

Lady O
to O
Lord O
Malton O
, O
31 O
October O
1741 O
. O

5 O
WWM O
, O
M7 O
- O
19 O
. O

Lady O
Malton O
to O
Lady O
Finch O
, O
2 O
November O
1741 O
. O

179 O

Marjorie O
Bloy O

continued O
with O
the O
blister O
and O
applied O
" O
ointment O
with O
flyes O
" O
, O
apparently O
some O
sort O
of O
irritant O
potion O
, O
with O
no O
sign O
of O
strangury O
. O

Charles O
found O
her O
treatment O
" O
not O
near O
the O
pain O
he O
expected O
" O
and O
she O
was O
" O
full O
of O
hopes O
that O
he O
will O
rec O
[ O
eiv O
] O
e O
great O
benefit O
from O
it O
" O
. O
1 O

Apart O
from O
his O
other O
troubles O
, O
one O
of O
Charles O
' O
s O
knees O
had O
swollen O
but O
this O
had O
much O
abated O
since O
the O
application O
of O
the O
blisters O
which O
Lady O
Malton O
believed O
" O
must O
be O
acting O
upon O
the O
whole O
Mass O
of O
Blood O
" O
since O
it O
had O
" O
reached O
the O
remote O
part O
" O
. O

She O
thought O
that O
Sir O
Edward O
Hulse O
' O
s O
powders O
were O
too O
slow O
in O
taking O
effect O
although O
the O
boy B
took O
them O
very O
quietly O
. O
' O
7 O
Sir O
Edward O
did O
not O
" O
apprehend O
any O
great O
danger O
from O
the O
Siziness O
[ O
thickness O
] O
of O
Charles O
' O
blood O
" O
; O
Lady O
Malton O
thought O
that O
the O
condition O
was O
the O
cause O
of O
all O
the O
child B
' O
s O
problems O
, O
which O
would O
not O
end O
until O
it O
was O
set O
to O
rights O
. O

At O
any O
rate O
, O
he O
was O
fit O
enough O
to O
go O
hunting O
. O
' O
8 O
Charles O
continued O
in O
the O
same O
state O
of O
health O
. O

He O
slept O
well O
at O
night O
, O
ate O
more O
than O
his O
mother O
thought O
was O
good O
for O
him O
, O
and O
was O
able O
to O
exercise O
strenuously O
without O
tiring O
. O

He O
put O
on O
no O
weight O
though O
, O
and O
" O
as O
for O
them O
swellings O
at O
his O
throat O
, O
they O
are O
almost O
gone O
one O
day O
and O
rise O
the O
next O
" O
. O

His O
mother O
did O
not O
expect O
a O
speedy O
recovery O
and O
" O
if O
the O
D O
[ O
octo O
] O
rs O
think O
him O
in O
a O
good O
state O
of O
health O
now O
, O
I O
s O
[ O
houl O
] O
d O
be O
glad O
to O
see O
him O
in O
a O
better O
" O
. O
' O
9 O
He O
began O
to O
improve O
and O
by O
the O
end O
of O
November O
even O
she O
thought O
he O
was O
on O
the O
mend O
and O
gaining O
weight O
. O
20 O
Unfortunately O
, O
Lady O
Malton O
again O
had O
cause O
for O
concern O
over O
his O
health O
in O
January O
1742 O
when O
he O
began O
to O
suffer O
from O
an O
intermittent O
hoarseness O
. O
2 O
' O
Otherwise O
he O
was O
as O
well O
as O
one O
could O
expect O
, O
with O
no O
other O
complaints O
. O
22 O
It O
was O
not O
to O
last O
. O

In O
May O
1742 O
Charles O
and O
his O
mother O
were O
again O
in O
Bristol O
, O
taking O
the O
waters O
because O
he O
had O
been O
indisposed O
. O

Lady O
Malton O
thought O
the O
waters O
were O
doing O
them O
good O
because O
they O
were O
both O
being O
violently O
sick O
. O
23 O
However O
, O
Charles O
had O
had O
no O
dinner O
on O
25 O
or O
26 O
May O
and O
was O
hot O
, O
lazy O
, O
and O
inclined O
to O
stir O
, O
" O
from O
which O
I O
conclude O
he O
is O
not O
well O
, O
. O

. O
. O
and O
therefore O
Intend O
to O
give O
him O
a O
gentle O
Vomit O
. O
. O
. O
and O
to O
let O
him O
take O
his O
old O
Remedie O
the O
Salt O
Draughts O
for O
a O
few O
Daies O
which O
I O
dare O
say O
will O
set O
him O
quite O
to O
rights O
" O
. O
24 O
By O
29 O
May O
Dr O
Boume O
had O
bled O
the O
boy B
" O
which O
succeeded O
very O
well O
but O
. O
. O
. O
found O
it O
[ O
his O
blood O
] O
as O
bad O
as O
ever O
" O
. O

The O
waters O
were O
not O
working O
" O
but O
there O
is O
a O
great O
deal O
for O
them O
to O
do O
which O
grant O
God O
they O
may O
effect O
" O
. O

The O
weather O
had O
turned O
warm O
so O
Lady O
Malton O
had O
" O
shorn O
him O
. O
. O
. O
which O
has O
display O
' O
d O
a O
most O
scabby O
head O
and O
indeed O
several O
other O
untoward O
Blotches O
he O
has O
out O
upon O
other O
parts O
of O
his O
Body O
" O
, O
which O
made O
her O
uneasy O
. O
25 O
The O
blotches O
on O
his O
head O
were O
not O
numerous O
" O
yet O
they O
made O
up O
in O
quality O
for O
so O
virulent O
a O
Corrosive O
Humour O
is O
not O
easily O
conceived O
without O
seeing O
it O
" O
. O

The O
pustules O
on O
his O
body O
were O
of O
the O
same O
sort O

16 O
WWM O
, O
M7 O
- O
17 O
. O

Lady O
to O
Lord O
Malton O
, O
4 O
November O
1741 O
. O

7 O
WWM O
, O
M7 O
- O
18 O
. O

Lady O
to O
Lord O
Malton O
, O
4 O
November O
1741 O
. O

18 O
WWM O
, O
M7 O
- O
16 O
. O

Lady O
to O
Lord O
Malton O
, O
7 O
November O
1741 O
. O

' O
9WWM O
, O
M7 O
- O
15 O
. O

Lady O
to O
Lord O
Malton O
, O
9 O
November O
1741 O
. O

20 O
WWM O
, O
M7 O
- O
22 O
. O

Lady O
to O
Lord O
Malton O
, O
25 O
November O
1741 O
. O

21 O
WWM O
, O
M7 O
- O
1 O
. O

Lady O
to O
Lord O
Malton O
, O
25 O
January O
1742 O
. O

22 O
WWM O
, O
M7 O
- O
4 O
. O

Lady O
to O
Lord O
Malton O
, O
8 O
February O
1742 O
and O
WWM O
, O
M7 O
- O
9 O
. O

Lady O
to O
Lord O
Malton O
, O
22 O
February O
1742 O
. O

23 O
WWM O
, O
M7 O
- O
29 O
. O

Lady O
to O
Lord O
Malton O
, O
12 O
May O
1742 O
. O

24 O
WWM O
, O
M7 O
- O
35 O
. O

Lady O
to O
Lord O
Malton O
, O
26 O
May O
1742 O
. O

25 O
WWM O
, O
M7 O
- O
36 O
. O

Lady O
to O
Lord O
Malton O
, O
29 O
May O
1742 O
. O

180 O

An O
eighteenth O
- O
century O
Prime O
Minister O
' O
s O
illness O

and O
his O
mother O
intended O
to O
put O
plasters O
on O
them O
to O
prevent O
them O
from O
spreading O
. O

Charles O
was O
also O
feverish O
; O
his O
glands O
were O
swollen O
and O
his O
pulse O
was O
erratic O
" O
but O
out O
of O
compassion O
to O
you O
I O
must O
tell O
you O
that O
he O
is O
with O
me O
as O
Brisk O
and O
lively O
as O
you O
ever O
Saw O
him O
" O
. O

Lady O
Malton O
had O
called O
in O
two O
eminent O
Bristol O
men B
, O
Dr O
Logan O
and O
Mr O
Pye O
, O
to O
treat O
the O
boy O
; O
Mr O
Pye O
prescribed O
" O
the O
Precipitate O
Per O
se O
" O
as O
the O
cure O
for O
the O
" O
hectic O
" O
. O

Pye O
made O
it O
himself O
and O
said O
that O
it O
was O
the O
only O
remedy O
that O
would O
work O
. O

Clearly O
Charles O
was O
impatient O
to O
be O
cured O
because O
he O
told O
his O
mother O
to O
give O
him O
the O
medicine O
" O
to O
cure O
me O
which O
I O
am O
sure O
it O
will O
do O
or O
shoot O
me O
through O
the O
head O
at O
once O
" O
. O

She O
thought O
that O
this O
attitude O
was O
" O
odd O
from O
one O
of O
his O
Age O
and O
[ O
it O
] O
does O
not O
a O
little O
disturb O
" O
. O
26 O
The O
blotches O
began O
to O
burst O
and O
indent O
but O
the O
doctor O
thought O
that O
all O
would O
be O
well O
in O
the O
end O
. O
27 O
Meanwhile O
, O
Charles O
was O
still O
losing O
weight O
even O
though O
" O
he O
had O
none O
to O
spare O
before O
" O
and O
he O
was O
inclined O
to O
be O
lazy O
which O
was O
not O
his O
natural O
turn O
. O

His O
father O
recommended O
some O
unknown O
cure O
which O
he O
called O
Gascoin O
' O
s O
Powder O
- O
a O
dose O
of O
five O
grains O
made O
up O
with O
syrup O
into O
a O
pill O
- O
every O
night O
. O

To O
make O
matters O
worse O
, O
the O
doctors O
disagreed O
about O
the O
treatment O
. O

" O
Dr O
Pye O
is O
Vehemently O
for O
the O
P O
. O

Per O
se O
, O
Dr O
Logan O
saies O
that O
it O
is O
a O
Medicine O
that O
may O
prove O
too O
rough O
in O
its O
operation O
for O
his O
Constitution O
& O
therefore O
begs O
a O
tryal O
of O
Beazor O
mineral O
[ O
gall O
stones O
from O
a O
goat B
] O
and O
Viper O
Broth O
" O
. O

The O
Bristol O
water O
had O
not O
yet O
acted O
" O
because O
his O
case O
is O
of O
too O
obstinate O
a O
Nature O
" O
and O
Lady O
Malton O
herself O
was O
satisfied O
that O
since O
nothing O
else O
had O
worked O
to O
cure O
the O
boy B
, O
the O
time O
had O
come O
to O
try O
mercurials O
, O
even O
though O
she O
knew O
that O
they O
were O
" O
powerful O
and O
perhaps O
in O
some O
cases O
hazardous O
medicines O
" O
. O
28 O
She O
wanted O
to O
see O
some O
remedy O
succeed O
but O
was O
" O
afraid O
of O
violent O
ones O
and O
at O
the O
same O
time O
vastly O
distrustfull O
[ O
sic O
] O
of O
mild O
ones O
" O
. O

It O
would O
appear O
that O
the O
" O
precipitate O
Per O
se O
" O
, O
probably O
mercury O
- O
based O
, O
could O
be O
a O
kill O
- O
or O
- O
cure O
remedy O
. O

Her O
" O
terrors O
" O
did O
not O
arise O
from O
any O
immediate O
danger O
to O
her O
son O
, O
and O
her O
" O
perfect O
Knowledge O
" O
of O
his O
disorder O
convinced O
her O
that O
whatever O
remedies O
he O
took O
, O
the O
cure O
was O
in O
the O
hands O
of O
God O
. O
29 O

To O
add O
to O
Charles O
' O
disorders O
, O
on O
12 O
June O
he O
developed O
a O
" O
very O
inflamed O
bad O
Eye O
. O
. O
. O
the O
same O
Eye O
that O
. O
. O
. O

he O
did O
not O
see O
so O
well O
of O
[ O
as O
] O
the O
other O
. O
. O
. O

He O
sais O
[ O
sic O
] O
that O
from O
that O
eye O
Alone O
he O
can O
Scarcely O
distinguish O
anything O
" O
. O

The O
doctors O
suggested O
bathing O
the O
eye O
: O
Lady O
Malton O
knew O
that O
the O
" O
frightful O
symptoms O
" O
which O
were O
" O
shocking O
to O
behold O
" O
were O
a O
result O
of O
" O
the O
Same O
as O
produces O
all O
the O
rest O
of O
his O
complaints O
in O
whichever O
Shape O
they O
appear O
" O
. O

30 O
The O
eye O
was O
very O
bloodshot O
and O
inflamed O
; O
the O
eyelid O
was O
swollen O
so O
he O
could O
hardly O
open O
it O
. O

The O
other O
eye O
was O
dull O
and O
" O
he O
had O
very O
little O
sight O
of O
it O
" O
. O
31 O
By O
14 O
June O
the O
eye O
problem O
had O
eased O
somewhat O
but O
Lady O
Malton O
could O
find O
no O
cause O
to O
attribute O
the O
improvement O
to O
any O
of O
the O
" O
cures O
" O
. O

Charles O
was O
still O
being O
subjected O
to O
Bristol O
water O
, O
Beazor O
mineral O
, O
Viper O
broth O
, O
cinnabar O
and O
the O
precipitate O
per O
se O
. O
32 O
She O
decided O
to O
take O
the O
boy B
home O
to O
Wentworth O
because O
he O
was O

26 O
WWM O
, O
M7 O
- O
38 O
. O

Lady O
to O
Lord O
Malton O
, O
1 O
June O
1742 O
. O

27 O
WWM O
, O
M7 O
- O
39 O
. O

Lady O
to O
Lord O
Malton O
, O
2 O
June O
1742 O
. O

28 O
WWM O
, O
M7 O
- O
41 O
. O

Lady O
to O
Lord O
Malton O
, O
5 O
June O
1742 O
. O

29 O
WWM O
, O
M7 O
- O
43 O
. O

Lady O
to O
Lord O
Malton O
, O
7 O
June O
1742 O
. O

30 O
WWM O
, O
M7 O
- O
45 O
. O

Lady O
to O
Lord O
Malton O
, O
12 O
June O
1742 O
. O

31 O
WWM O
, O
M7 O
- O
56 O
. O

Lady O
to O
Lord O
Malton O
, O
undated O
: O
12 O
June O
? O

1742 O
. O

32 O
WWM O
, O
M7 O
- O
46 O
. O

Lady O
to O
Lord O
Malton O
, O
14 O
June O
? O

1742 O
. O

181 O

Marjorie O
Bloy O

more O
likely O
to O
recover O
there O
than O
anywhere O
else O
. O
33 O
He O
still O
ate O
and O
slept O
well O
and O
was O
" O
pretty O
cheerful O
but O
his O
looks O
are O
bitter O
bad O
still O
. O

The O
flesh O
he O
lost O
in O
the O
Accidental O
Feavour O
he O
has O
not O
Recover O
' O
d O
and O
his O
complexion O
is O
of O
the O
most O
sickly O
sort O
his O
hands O
of O
the O
same O
Hue O
his O
legs O
are O
tollerable O
[ O
sic O
] O
well O
" O
. O
34 O

They O
returned O
to O
Wentworth O
in O
short O
stages O
and O
by O
6 O
September O
Higham O
was O
" O
perfectly O
recovered O
. O

. O
. O
after O
the O
long O
and O
successful O
Care O
that O
Lady O
Malton O
has O
taken O
" O
of O
him O
. O
35 O
In O
May O
1743 O
he O
was O
inoculated O
against O
smallpox O
and O
made O
a O
perfect O
recovery O
after O
which O
he O
caught O
cold O
" O
by O
stripping O
when O
He O
was O
hot O
" O
. O
36 O
Lord O
Higham O
does O
not O
seem O
to O
have O
been O
seriously O
ill O
after O
that O
until O
, O
at O
the O
age O
of O
19 O
, O
he O
undertook O
his O
Grand O
Tour O
in O
1749 O
. O

In O
July O
1750 O
, O
by O
then O
Lord O
Malton O
, O
he O
had O
cause O
to O
consult O
Nicolo O
Scanagati O
in O
Padua O
for O
the O
treatment O
of O
gonorrhoea O
. O

Scanagati O
produced O
a O
lengthy O
medical O
report O
of O
Malton O
' O
s O
treatment O
, O
presumably O
for O
his O
English O
doctor O
' O
s O
enlightenment O
. O
37 O
The O
initial O
treatment O
was O
an O
" O
electuary O
, O
consisting O
of O
three O
ounces O
of O
emollient O
, O
three O
drams O
of O
powdered O
jalap O
, O
a O
half O
[ O
dram O
] O
of O
purified O
nitre O
, O
bound O
together O
with O
lemon O
juice O
taken O
twice O
a O
day O
" O
. O

The O
result O
was O
satisfactory O
: O
" O
The O
dark O
greenish O
poison O
was O
oozing O
slowly O
from O
his O
penis O
, O
which O
was O
all O
contracted O
and O
the O
sharp O
and O
constant O
pain O
extended O
from O
the O
perineum O
up O
to O
the O
urinary O
bladder O
, O
producing O
small O
swellings O
now O
in O
this O
place O
, O
now O
in O
that O
. O
" O
There O
was O
a O
fierce O
burning O
sensation O
in O
the O
glands O
, O
which O
prevented O
him O
from O
sleeping O
. O

Because O
of O
this O
, O
it O
seemed O
reasonable O
to O
bathe O
that O
part O
in O
tepid O
water O
and O
milk O
, O
and O
to O
apply O
poultices O
to O
the O
areas O
affected O
by O
swelling O
and O
contractions O
, O
together O
with O
cold O
drinks O
and O
a O
few O
grains O
of O
laudanum O
at O
night O
. O

Malton O
was O
blooded O
regularly O
besides O
being O
given O
purgatives O
; O
the O
treatment O
then O
moved O
on O
to O
the O
administration O
of O
mercury O
, O
both O
internal O
and O
on O
the O
gums O
, O
since O
it O
was O
widely O
believed O
at O
the O
time O
that O
gonorrhoea O
and O
syphilis O
were O
steps O
of O
the O
same O
disease O
, O
" O
the O
Venereal O
" O
. O

Scanagati O
at O
this O
point O
ruled O
out O
the O
suggestion O
of O
syphilitic O
chancre O
because O
Malton O
' O
s O
urine O
was O
fine O
and O
light O
with O
a O
pungent O
odour O
. O

Scanagati O
did O
ask O
if O
Malton O
had O
previously O
ever O
had O
a O
similar O
peculiarity O
of O
his O
urine O
. O

Malton O
replied O
that O
when O
he O
was O
very O
young O
and O
still O
inexperienced O
sexually O
, O
for O
some O
time O
following O
a O
fever O
he O
had O
had O
the O
same O
unusual O
urine O
, O
and O
indeed O
that O
on O
one O
occasion O
this O
symptom O
coincided O
with O
certain O
tumours O
on O
the O
testicles O
. O

It O
was O
only O
by O
chance O
that O
he O
had O
not O
had O
recourse O
to O
surgery O
, O
the O
reason O
being O
that O
he O
was O
also O
afflicted O
with O
a O
throat O
infection O
- O
to O
which O
he O
was O
prone O
- O
and O
therefore O
had O
his O
vein O
opened O
four O
times O
. O

Thereupon O
the O
inflammation O
subsided O
, O
and O
equally O
the O
tumours O
and O
sediment O
disappeared O
. O

He O
told O
me O
that O
as O
a O
youth O
he O
had O
sometimes O
experienced O
some O
difficulty O
and O
a O
burning O
sensation O
when O
urinating O
, O
which O
subsided O
when O
his O
blood O
was O
let O
and O
with O
the O
application O
of O
poultices O
. O

I O
observed O
that O
from O
time O
to O
time O
his O
face O
and O
body O
were O
covered O
with O
purplish O
spots O
, O
which O
, O
having O
produced O
a O
little O
fluid O
, O
would O
disappearas O
indeed O
happened O
in O
the O
course O
of O
the O
cure O
, O
at O
the O
end O
of O
which O
his O
face O
was O
entirely O

33 O
WWM O
, O
M7 O
- O
47 O
. O

Lady O
to O
Lord O
Malton O
, O
15 O
June O
1742 O
. O

34 O
WWM O
, O
M7 O
- O
48 O
. O

Lady O
to O
Lord O
Malton O
, O
16 O
June O
1742 O
. O

35 O
WWM O
, O
M2 O
- O
104 O
/ O
5 O
. O

Lady O
Finch O
to O
Lady O
Malton O
, O
6 O
September O
1742 O
. O

36 O
WWM O
, O
M2 O
- O
135 O
. O

Lady O
Finch O
to O
Malton O
, O
18 O
June O
1742 O
. O

37 O
WWM O
, O
R170 O
- O
20 O
. O

Nicol6 O
Scanagati O
' O
s O
report O
. O

182 O

An O
eighteenth O
- O
century O
Prime O
Minister O
' O
s O
illness O

free O
from O
these O
spots O
. O

From O
this O
observation O
, O
it O
seemed O
to O
me O
simple O
to O
deduce O
both O
the O
original O
cause O
and O
the O
more O
immediate O
cause O
of O
the O
said O
sediment O
: O
namely O
a O
natural O
complexion O
of O
humours O
which O
are O
exacerbated O
by O
muriatic O
[ O
i O
. O
e O
. O
, O
acidic O
] O
sourness O
, O
together O
with O
the O
marked O
inflammation O
of O
the O
blood O
and O
the O
motion O
of O
the O
contracted O
poison O
. O

Scanagati O
then O
recommended O
the O
continuation O
of O
the O
electuary O
made O
of O
emollient O
, O
guaiacum O
resin O
, O
balsam O
, O
rhubarb O
, O
and O
nitre O
. O

What O
does O
all O
this O
add O
up O
to O
in O
terms O
of O
diagnosis O
? O

The O
early O
illness O
is O
a O
mystery O
. O

Did O
he O
have O
an O
inguinal O
hernia O
; O
or O
perhaps O
mumps O
or O
epididymitis O
? O

His O
was O
a O
childless O
marriage O
. O

He O
seems O
to O
have O
been O
too O
fit O
for O
the O
illness O
to O
have O
been O
rheumatic O
fever O
. O

Cystitis O
was O
not O
uncommon O
and O
this O
could O
certainly O
lead O
to O
" O
strangury O
" O
. O

Perhaps O
Rockingham O
suffered O
from O
a O
congenital O
defect O
of O
his O
urinogenitary O
system O
which O
would O
result O
in O
recurrent O
attacks O
of O
cystitis O
and O
might O
cause O
long O
- O
term O
damage O
to O
the O
urinary O
tract O
and O
eventual O
destruction O
of O
the O
kidneys O
, O
precipitating O
sudden O
and O
unexpected O
death O
. O

Another O
possibility O
might O
be O
diabetes O
. O

Rockingham O
had O
a O
urinary O
infection O
and O
certainly O
suffered O
from O
recurrent O
skin O
infections O
, O
although O
it O
appears O
from O
Scanagati O
' O
s O
report O
that O
these O
cleared O
up O
when O
mercury O
was O
administered O
. O

Certainly O
the O
marquis O
complained O
often O
about O
pains O
in O
his O
side O
and O
stomach O
and O
made O
no O
secret O
of O
his O
" O
old O
complaint O
" O
. O

He O
was O
noticeably O
less O
physically O
active O
as O
he O
moved O
into O
his O
thirties O
and O
only O
occasionally O
exerted O
himself O
by O
riding O
any O
distance O
, O
even O
though O
he O
had O
enjoyed O
hunting O
when O
he O
was O
younger O
. O

He O
found O
the O
pains O
caused O
by O
his O
" O
old O
complaint O
" O
made O
him O
feel O
so O
ill O
that O
he O
was O
unable O
to O
concentrate O
on O
" O
any O
Manner O
of O
Business O
" O
and O
on O
occasion O
it O
seemed O
likely O
to O
prevent O
him O
from O
attending O
Parliament O
. O
38 O
He O
may O
well O
have O
suffered O
from O
a O
problem O
with O
gallstones O
from O
an O
early O
age O
. O
39 O
He O
appears O
to O
have O
suffered O
from O
a O
nervous O
disorder O
which O
manifested O
itself O
in O
severe O
palpitations O
, O
trembling O
, O
and O
other O
types O
of O
physical O
discomforts O
such O
as O
boils O
and O
headaches O
with O
which O
he O
was O
frequently O
afflicted O
. O
40 O
He O
probably O
had O
constipation O
too O
, O
since O
he O
was O
always O
dosing O
himself O

with O
purgatives O
. O

In O
fact O
in O
April O
1772 O
the O
Duke O
of O
Richmond O
decided O
that O
Rockingham O
' O
s O
real O
problem O
was O
a O
" O
surfeit O
of O
physick O
" O
41 O
although O
Edmund O
Burke O
noted O
in O
June O
1772 O
that O
the O
marquis O
had O
had O
a O
long O
and O
severe O
illness O
. O
42 O
Whatever O
his O
many O
and O
varied O
ailments O
, O
Rockingham O
survived O
until O
he O
was O
52 O
in O
spite O
of O
the O
attentions O
of O
both O
doctors O
and O
quacks O
and O
that O
, O
for O
the O
mid O
- O
eighteenth O
century O
, O
was O
a O
good O
age O
. O

The O
mystery O
still O
remains O
, O
however O
. O

Contemporary O
opinion O
had O
it O
that O
he O
died O
of O
pneumonia O
but O
it O
must O
have O
struck O

38 O
WWM O
, O
R153 O
- O
1 O
. O

Rockingham O
to O
Burke O
, O
31 O
October O
1767 O
; O
WWM O
, O
RI O
- O
1238 O
. O

Rockingham O
to O
Dowdeswell O
, O
20 O
October O
1769 O
; O
WWM O
, O
RI O
- O
1928 O
. O

Rockingham O
to O
Savile O
, O
September O
1780 O
. O

39 O
Ross O
J O
. O

S B
. I

Hoffman O
, O
The O
Marquis O
. O

A O
study O
of O
Lord O
Rockingham O
, O
1730 O
- O
1782 O
, O
New O
York O
, O
Fordham O
University O
Press O
, O
1973 O
, O
p O
. O

35 O
. O

This O
is O
the O
only O
recent O
biography O
of O
the O
second O
Marquis O
of O
Rockingham O
, O
and O
does O
not O
deal O
with O
his O
illnesses O
. O

Although O
he O
does O
not O
give O
the O
source O
of O
his O
information O
, O
Hoffman O
asserts O
that O
Rockingham O
was O
in O
Bath O
between O
March O
and O
August O
1761 O
suffering O
from O
gallstones O
. O

The O
marquis O
would O
then O
have O
been O
31 O
years O
old O
. O

40 O
Historical O
Manuscripts O
Commission O
, O
Lindley O
Wood O
, O
p O
. O

184 O
. O

41 O
WWM O
, O
RI O
- O
1403 O
. O

Richmond O
to O
Rockingham O
, O
26 O
April O
1772 O
. O

42 O
Burke O
to O
James O
de O
Lancey O
, O
30 O
June O
1772 O
. O

The O
correspondence O
of O
Edmund O
Burke O
, O
vol O
. O

2 O
, O
ed O
. O

T O
. O

W O
. O

Copeland O
and O
others O
, O
Cambridge O
University O
Press O
, O
1958 O
- O
1978 O
, O
p O
. O

311 O
. O

183 O

Marjorie O
Bloy O

suddenly O
: O
it O
was O
only O
two O
weeks O
from O
him O
" O
recovering O
" O
to O
dying O
. O

Another O
almost O
contemporary O
account O
of O
the O
marquis O
' O
s O
death O
came O
from O
the O
Earl O
of O
Albemarle O
. O

He O
noted O
that O
Rockingham O
had O
" O
for O
some O
time O
past O
been O
afflicted O
with O
water O
on O
the O
chest O
: O
and O
to O
this O
well O
- O
known O
malady O
was O
superadded O
the O
then O
novel O
disease O
of O
influenza O
" O
. O
43 O
This O
conceivably O
could O
be O
an O
uninformed O
account O
, O
handed O
down O
orally O
by O
surviving O
members O
of O
the O
marquis O
' O
s O
family O
, O
of O
emphysema O
. O

More O
fascinating O
than O
the O
diagnosis O
of O
the O
cause O
of O
death O
, O
however O
, O
is O
- O
what O
was O
wrong O
with O
him O
during O
his O
lifetime O
? O

Or O
is O
it O
yet O
another O
example O
of O
the O
" O
English O
disease O
" O
: O
hypochondria O
? O

43Albemarle O
, O
George O
Thomas O
, O
Earl O
of O
, O
Memoirs O
of O
the O
Marquis O
of O
Rockingham O
and O
his O
contemporaries O
, O
London O
, O
Richard O
Bentley O
, O
2 O
vols O
. O
, O
1852 O
, O
vol O
. O

2 O
, O
p O
. O

483 O
. O

This O
is O
the O
only O
contemporary O
work O
concerning O
Rockingham O
, O
but O
does O
not O
mention O
his O
early O
life O
and O
illnesses O
. O

184 O

Tissue O
- O
dependent O
isoforms O
of O
mammalian O
Fox O
- O
1 O
homologs O
are O
associated O
with O
tissue O
- O
specific O
splicing O
activities O

Abstract O

An O
intronic O
hexanucleotide O
UGCAUG O
has O
been O
shown O
to O
play O
a O
critical O
role O
in O
the O
regulation O
of O
tissue O
- O
specific O
alternative O
splicing O
of O
pre O
- O
mRNAs O
in O
a O
wide O
range O
of O
tissues O
. O

Vertebrate O
Fox O
- O
1 O
has O
been O
shown O
to O
bind O
to O
this O
element O
, O
in O
a O
highly O
sequence O
- O
specific O
manner O
, O
through O
its O
RNA O
recognition O
motif O
( O
RRM O
) O
. O

In O
mammals O
, O
there O
are O
at O
least O
two O
Fox O
- O
1 O
- O
related O
genes O
, O
ataxin O
- O
2 O
binding O
protein O
1 O
( O
A2BP1 O
) O
/ O
Fox O
- O
1 O
and O
Fxh O
/ O
Rbm9 O
, O
which O
encode O
an O
identical O
RRM O
. O

Here O
, O
we O
demonstrate O
that O
both O
mouse B
Fxh O
and O
A2BP1 O
transcripts O
undergo O
tissue O
- O
specific O
alternative O
splicing O
, O
generating O
protein O
isoforms O
specific O
to O
brain O
and O
muscle O
. O

These O
tissue O
- O
specific O
isoforms O
are O
characterized O
for O
their O
abilities O
to O
regulate O
neural O
cell O
- O
specific O
alternative O
splicing O
of O
a O
cassette O
exon O
, O
N30 O
, O
in O
the O
non O
- O
muscle O
myosin O
heavy O
chain O
II O
- O
B O
pre O
- O
mRNA O
, O
previously O
shown O
to O
be O
regulated O
through O
an O
intronic O
distal O
downstream O
enhancer O
( O
IDDE O
) O
. O

All O
Fxh O
and O
A2BP1 O
isoforms O
with O
the O
RRM O
are O
capable O
of O
binding O
to O
the O
IDDE O
in O
vitro O
through O
the O
UGCAUG O
elements O
. O

Each O
isoform O
, O
however O
, O
shows O
quantitative O
differences O
in O
splicing O
activity O
and O
nuclear O
distribution O
in O
transfected O
cells O
. O

All O
Fxh O
isoforms O
and O
a O
brain O
isoform O
of O
A2BP1 O
show O
a O
predominant O
nuclear O
localization O
. O

Brain O
isoforms O
of O
both O
Fxh O
and O
A2BP1 O
promote O
N30 O
splicing O
much O
more O
efficiently O
than O
do O
the O
muscle O
- O
specific O
isoforms O
. O

Skeletal O
muscles O
express O
additional O
isoforms O
that O
lack O
a O
part O
of O
the O
RRM O
. O

These O
isoforms O
are O
incapable O
of O
activating O
neural O
cell O
- O
specific O
splicing O
and O
, O
moreover O
, O
can O
inhibit O
UGCAUG O
- O
dependent O
N30 O
splicing O
. O

These O
findings O
suggest O
that O
tissue O
- O
specific O
isoforms O
of O
Fxh O
and O
A2BP1 O
play O
an O
important O
role O
in O
determining O
tissue O
specificity O
of O
UGCAUG O
- O
mediated O
alternative O
splicing O
. O

INTRODUCTION O

Alternative O
splicing O
of O
pre O
- O
mRNA O
is O
one O
of O
the O
fundamental O
mechanisms O
for O
the O
regulation O
of O
gene O
expression O
in O
higher O
eukaryotes O
( O
1 O
, O
2 O
) O
. O

Developmentally O
regulated O
, O
cell O
type O
- O
or O
tissue O
- O
specific O
, O
and O
signal O
- O
induced O
alternative O
splicing O
of O
pre O
- O
mRNAs O
takes O
place O
in O
multicellular O
organisms O
throughout O
their O
lifetimes O
. O

Misregulation O
or O
abnormalities O
in O
pre O
- O
mRNA O
splicing O
, O
in O
some O
instances O
, O
leads O
to O
cellular O
dysfunctions O
found O
in O
human B
and O
animal O
diseases O
( O
3 O
- O
5 O
) O
. O

Using O
various O
model O
systems O
of O
regulated O
alternative O
splicing O
, O
a O
number O
of O
pre O
- O
mRNA O
features O
that O
influence O
alternative O
splice O
site O
selection O
have O
been O
defined O
( O
1 O
, O
2 O
) O
. O

These O
include O
enhancer O
and O
repressor O
RNA O
sequences O
located O
in O
exons O
and O
introns O
. O

Identification O
of O
RNA O
- O
binding O
proteins O
targeting O
these O
cis O
- O
regulatory O
elements O
is O
currently O
in O
progress O
. O

In O
vertebrates O
, O
participation O
of O
the O
SR O
family O
proteins O
and O
hnRNP O
proteins O
, O
such O
as O
PTB O
and O
hnRNPA1 O
, O
in O
alternative O
splicing O
regulation O
via O
binding O
to O
the O
cis O
- O
regulatory O
elements O
have O
been O
shown O
in O
many O
tissue O
- O
specific O
splicing O
models O
( O
6 O
, O
7 O
) O
. O

Although O
these O
RNA O
- O
binding O
proteins O
are O
ubiquitously O
expressed O
, O
their O
different O
abundance O
in O
different O
cells O
, O
differences O
in O
their O
post O
- O
translational O
modifications O
in O
different O
cellular O
contexts O
and O
their O
different O
abilities O
to O
assemble O
multiprotein O
complexes O
in O
different O
pre O
- O
mRNA O
contexts O
are O
thought O
to O
contribute O
to O
the O
determination O
of O
cell O
type O
- O
specific O
patterns O
of O
alternative O
splicing O
. O

For O
the O
last O
few O
years O
, O
tissue O
- O
specific O
and O
tissue O
- O
enriched O
RNA O
- O
binding O
proteins O
have O
begun O
to O
be O
identified O
as O
splicing O
regulators O
. O

These O
include O
brain O
- O
specific O
( O
or O
enriched O
) O
Nova O
- O
1 O
, O
nPTB O
( O
brPTB O
) O
and O
some O
of O
the O
CELF O
family O
proteins O
( O
8 O
- O
12 O
) O
. O

Discovery O
of O
these O
proteins O
has O
had O
a O
great O
impact O
on O
studies O
aimed O
at O
understanding O
the O
molecular O
mechanisms O
of O
alternative O
splicing O
regulation O
. O

One O
of O
the O
intronic O
cis O
- O
elements O
, O
which O
are O
involved O
in O
tissue O
- O
specific O
or O
differentiation O
stage O
- O
dependent O
regulation O
of O
alternative O
splicing O
, O
is O
the O
hexanucleotide O
UGCAUG O
. O

The O
importance O
of O
this O
element O
was O
originally O
recognized O
in O
fibronectin O
pre O
- O
mRNA O
by O
Huh O
and O
Hynes O
( O
13 O
) O
. O

Since O
then O
, O
alternative O
splicing O
specific O
to O
a O
variety O
of O
tissues O
or O
cell O
types O
, O
including O
neural O
cells O
, O
muscles O
, O
epithelial O
cells O
and O
erythrocytes O
, O
has O
been O
shown O
to O
be O
modulated O
via O
this O
element O
( O
14 O
- O
21 O
) O
. O

Recently O
, O
Jin O
et O
al O
. O

( O
21 O
) O
discovered O
that O
a O
zebrafish B
homolog O
of O
Caenorhabditis B
elegans I
Fox O
- O
1 O
( O
22 O
) O
could O
bind O
specifically O
to O
the O
pentanucleotide O
GCAUG O
by O
in O
vitro O
selection O
from O
randomized O
RNA O
sequences O
. O

This O
pentanucleotide O
is O
almost O
identical O
to O
the O
hexanucleotide O
UGCAUG O
except O
for O
the O
first O
U O
. O

Moreover O
, O
the O
zebrafish B
Fox O
- O
1 O
homolog O
, O
as O
well O
as O
the O
mouse B
Fox O
- O
1 O
homolog O
, O
are O
capable O
of O
repressing O
the O
inclusion O
of O
an O
alternative O
cassette O
exon O
of O
the O
ATP O
synthase O
F1 O
gamma O
pre O
- O
mRNA O
via O
binding O
to O
GCAUG O
, O
which O
mimics O
muscle O
- O
specific O
exclusion O
of O
this O
exon O
. O

This O
mouse B
homolog O
is O
identical O
to O
the O
ataxin O
- O
2 O
binding O
protein O
1 O
( O
A2BP1 O
) O
, O
which O
has O
been O
previously O
cloned O
in O
humans B
and O
mice B
as O
the O
cDNA O
encoding O
a O
protein O
, O
which O
interacts O
with O
ataxin O
- O
2 O
, O
the O
product O
of O
the O
causative O
gene O
for O
spinocerebellar O
ataxia O
type O
2 O
( O
23 O
, O
24 O
) O
. O

In O
addition O
to O
A2BP1 O
/ O
Fox O
- O
1 O
, O
another O
mouse B
homolog O
of O
C B
. I
elegans I
Fox O
- O
1 O
, O
Fxh O
, O
has O
been O
independently O
cloned O
as O
a O
cDNA O
, O
which O
is O
induced O
by O
androgen O
in O
motor O
neurons O
( O
25 O
) O
. O

Of O
note O
is O
that O
A2BP1 O
/ O
Fox O
- O
1 O
and O
Fxh O
share O
an O
identical O
RNA O
recognition O
motif O
( O
RRM O
) O
at O
the O
amino O
acid O
level O
. O

Therefore O
, O
two O
genes O
in O
the O
mouse B
genome O
encode O
homologs O
of O
nematode O
Fox O
- O
1 O
. O

According O
to O
the O
names O
given O
by O
the O
first O
cDNA O
cloning O
, O
we O
used O
the O
nomenclature O
of O
A2BP1 O
and O
Fxh O
in O
this O
report O
. O

A2BP1 O
and O
Fxh O
have O
been O
named O
A2BP1 O
and O
Rbm9 O
, O
respectively O
, O
in O
the O
human B
and O
mouse B
genomes O
. O

We O
have O
been O
studying O
regulatory O
mechanisms O
of O
neural O
cell O
- O
specific O
alternative O
splicing O
using O
the O
non O
- O
muscle O
myosin O
heavy O
chain O
II O
- O
B O
( O
NMHC O
- O
B O
) O
gene O
as O
a O
model O
system O
( O
14 O
, O
26 O
) O
. O

NMHC O
- O
B O
mRNA O
is O
expressed O
ubiquitously O
. O

However O
, O
an O
alternative O
exon O
, O
N30 O
, O
which O
encodes O
a O
30 O
nt O
coding O
sequence O
, O
is O
included O
in O
the O
mRNAs O
from O
some O
neural O
cells O
, O
but O
is O
skipped O
in O
those O
from O
all O
other O
cells O
in O
mammals O
and O
birds B
( O
27 O
, O
28 O
) O
. O

In O
cultured O
cells O
, O
a O
switch O
in O
N30 O
splicing O
from O
exclusion O
to O
inclusion O
can O
be O
seen O
in O
neural O
retinoblastoma O
Y79 O
cells O
during O
the O
post O
- O
mitotic O
and O
differentiated O
stages O
triggered O
by O
butyrate O
treatment O
. O

We O
have O
previously O
defined O
an O
intronic O
distal O
downstream O
enhancer O
( O
IDDE O
) O
, O
which O
confers O
neural O
cell O
specificity O
on O
N30 O
inclusion O
, O
using O
this O
cell O
line O
( O
14 O
) O
. O

The O
IDDE O
includes O
two O
copies O
of O
UGCAUG O
. O

Mutation O
of O
these O
hexanucleotides O
results O
in O
N30 O
skipping O
in O
post O
- O
mitotic O
differentiated O
Y79 O
cells O
. O

In O
this O
study O
, O
we O
investigated O
the O
possible O
involvement O
of O
A2BP1 O
and O
Fxh O
in O
the O
regulation O
of O
N30 O
splicing O
. O

To O
this O
end O
, O
we O
have O
isolated O
cDNA O
clones O
for O
A2BP1 O
and O
Fxh O
from O
brain O
and O
muscles O
. O

cDNA O
cloning O
revealed O
the O
existence O
of O
tissue O
- O
specific O
( O
enriched O
) O
isoforms O
of O
both O
A2BP1 O
and O
Fxh O
. O

Of O
importance O
, O
different O
isoforms O
of O
A2BP1 O
and O
Fxh O
show O
different O
activities O
with O
respect O
to O
N30 O
splicing O
as O
well O
as O
different O
subcellular O
localizations O
. O

MATERIALS O
AND O
METHODS O

Database O
disposition O

The O
sequences O
reported O
in O
this O
paper O
have O
been O
deposited O
in O
the O
GenBank O
database O
with O
accession O
numbers O
AY659951 O
( O
F011 O
) O
, O
AY659952 O
( O
F411 O
) O
, O
AY659953 O
( O
F402 O
) O
, O
AY659954 O
( O
A016 O
) O
, O
AY659955 O
( O
A030 O
) O
, O
AY659956 O
( O
A713 O
) O
, O
AY659957 O
( O
A715 O
) O
and O
AY659958 O
( O
A704 O
) O
. O

RNA O
preparation O
and O
RT O
- O
PCR O

Total O
RNAs O
were O
isolated O
from O
mouse B
tissues O
and O
cultured O
cells O
using O
an O
RNA O
isolation O
kit O
( O
Stratagene O
) O
or O
an O
RNeasy O
mini O
kit O
( O
Qiagen O
) O
. O

To O
obtain O
the O
full O
- O
length O
coding O
regions O
of O
cDNAs O
for O
Fxh O
and O
A2BP1 O
, O
RT O
- O
PCRs O
were O
performed O
using O
Superscript O
II O
RNase O
H O
- O
reverse O
transcriptase O
( O
Invitrogen O
) O
and O
Pfu O
Turbo O
DNA O
polymerase O
( O
Stratagene O
) O
. O

The O
PCR O
primers O
used O
to O
obtain O
all O
Fxh O
cDNAs O
were O
5 O
' O
- O
ctcaggcctccactagttAT O
- O
3 O
' O
and O
5 O
' O
- O
ctcaggcctcctctagaaGT O
- O
3 O
' O
. O

The O
upstream O
primers O
for O
the O
brain O
( O
A016 O
and O
A030 O
) O
and O
muscle O
( O
A713 O
, O
A715 O
and O
A704 O
) O
A2BP1 O
cDNAs O
were O
5 O
' O
- O
ctcaggcctccactagtgAT O
- O
3 O
' O
and O
5 O
' O
- O
ctcaggcctccactagtcAT O
- O
3 O
' O
, O
respectively O
, O
and O
the O
downstream O
primer O
for O
all O
A2BP1 O
cDNAs O
was O
5 O
' O
- O
ctcaggcctcctctagagAT O
- O
3 O
' O
. O

Lower O
case O
letters O
represent O
adapter O
sequences O
including O
restriction O
enzyme O
sites O
. O

5 O
' O
Rapid O
amplification O
of O
cDNA O
ends O
( O
RACE O
) O
was O
performed O
using O
Marathon O
- O
Ready O
cDNA O
( O
BD O
Biosciences O
Clontech O
) O
. O

For O
the O
analysis O
of O
minigene O
and O
NMHC O
- O
B O
mRNAs O
, O
RT O
- O
PCRs O
were O
performed O
as O
described O
previously O
( O
14 O
, O
26 O
) O
. O

Sequences O
of O
primers O
P1 O
- O
P9 O
shown O
in O
Figures O
1 O
, O
4 O
and O
6 O
are O
as O
follows O
: O
P1 O
, O
5 O
' O
- O
AATTCACCCAGCAACCAGAA O
- O
3 O
' O
; O
P2 O
, O
5 O
' O
- O
TAGAGGGATGTAAGTGTTGA O
- O
3 O
' O
; O
P3 O
, O
5 O
' O
- O
CAGAGGGCGGACAGTGTATG O
- O
3 O
' O
; O
P4 O
, O
5 O
' O
- O
GGCGGCAGGGGCGAGGGCAT O
- O
3 O
' O
; O
P5 O
, O
5 O
' O
- O

CCGTGGTCGCACCGTGTACA O
- O
3 O
' O
; O
P6 O
, O
5 O
' O
- O
CAGCGGCAGTGGCAGGGGTG O
- O
3 O
' O
; O
P7 O
, O
5 O
' O
- O
AGGAAGAAAGGACCATAATA O
- O
3 O
' O
; O
P8 O
, O
5 O
' O
- O
CCTCCACCCAGCTCCAGTTG O
- O
3 O
' O
; O
and O
P9 O
, O
5 O
' O
- O
CCTGTAGTTATTAAATCCTT O
- O
3 O
' O
. O

Preparation O
of O
expression O
constructs O
and O
minigenes O

The O
cDNAs O
of O
Fxh O
and O
A2BP1 O
were O
introduced O
into O
a O
plasmid O
pCS3 O
+ O
MT O
, O
which O
contains O
a O
myc O
- O
epitope O
, O
and O
its O
modified O
version O
pCS3 O
+ O
MT O
+ O
NLS O
, O
which O
in O
addition O
contains O
the O
nuclear O
localization O
signal O
( O
NLS O
) O
of O
the O
SV40 O
large O
T O
antigen O
( O
26 O
) O
. O

Minigenes O
G O
, O
J O
without O
the O
IDDE O
, O
and O
H O
with O
the O
wild O
- O
type O
IDDE O
are O
the O
same O
as O
minigenes O
W O
, O
D4 O
and O
Cm0 O
, O
respectively O
in O
ref O
. O

( O
14 O
) O
. O

The O
201 O
nt O
IDDEs O
with O
mutations O
were O
generated O
by O
recombinant O
PCR O
using O
the O
appropriate O
primers O
, O
which O
included O
mutated O
sequences O
. O

The O
hexanucleotide O
TGCATG O
sequences O
at O
the O
5 O
' O
and O
3 O
' O
sides O
were O
changed O
to O
GTTACT O
and O
ACCTAC O
, O
respectively O
. O

Electrophoresis O
mobility O
shift O
assay O

Template O
DNAs O
for O
in O
vitro O
RNA O
transcription O
were O
prepared O
by O
PCR O
using O
the O
wild O
- O
type O
and O
mutant O
IDDEs O
in O
the O
minigenes O
as O
templates O
and O
an O
upstream O
primer O
that O
included O
the O
T7 O
promoter O
sequence O
at O
the O
5 O
' O
end O
. O

The O
probe O
and O
competitor O
RNAs O
were O
transcribed O
by O
T7 O
RNA O
polymerase O
in O
the O
presence O
of O
[ O
alpha O
- O
32P O
] O
UTP O
and O
a O
trace O
amount O
of O
[ O
35S O
] O
UTP O
alpha O
S O
, O
respectively O
, O
using O
a O
MAXIscript O
kit O
( O
Ambion O
) O
. O

Mole O
concentrations O
of O
synthesized O
RNAs O
were O
estimated O
by O
radioactivities O
. O

Fxh O
and O
A2BP1 O
proteins O
with O
a O
myc O
tag O
were O
synthesized O
in O
vitro O
by O
using O
a O
TNT O
quick O
- O
coupled O
transcription O
/ O
translation O
system O
( O
Promega O
) O
from O
pCS3 O
+ O
MT O
constructs O
, O
which O
include O
the O
SP6 O
promoter O
. O

Binding O
reactions O
were O
carried O
out O
in O
a O
10 O
mu O
l O
mixture O
that O
contains O
10 O
mM O
HEPES O
( O
pH O
7 O
. O
9 O
) O
, O
2 O
mM O
MgCl2 O
, O
50 O
mM O
KCl O
, O
5 O
% O
glycerol O
, O
0 O
. O
5 O
mM O
DTT O
, O
5 O
mu O
g O
tRNA O
, O
1 O
mu O
l O
reticulocyte O
lysate O
reaction O
mixture O
and O
15 O
fmol O
of O
probe O
, O
on O
ice O
for O
20 O
- O
30 O
min O
. O

An O
aliquot O
of O
5 O
mu O
g O
of O
heparin O
was O
added O
to O
the O
reaction O
10 O
min O
before O
gel O
electrophoresis O
. O

The O
reaction O
mixtures O
were O
analyzed O
by O
electrophoresis O
in O
a O
6 O
% O
polyacrylamide O
gel O
using O
a O
0 O
. O
5 O
x O
TBE O
buffer O
( O
Invitrogen O
) O
. O

Cell O
culture O
and O
transfection O

The O
human B
retinoblastoma O
cell O
line O
Y79 O
was O
cultured O
and O
transfected O
with O
DNAs O
as O
described O
previously O
( O
14 O
, O
26 O
) O
. O

Total O
amounts O
of O
transfected O
DNAs O
were O
adjusted O
to O
be O
constant O
by O
addition O
of O
the O
empty O
vector O
. O

Either O
Lipofectin O
( O
Invitrogen O
) O
or O
Effectene O
transfection O
reagent O
( O
Qiagen O
) O
was O
used O
for O
the O
transfection O
. O

For O
stable O
transfection O
, O
the O
pCS3 O
+ O
MT O
expression O
constructs O
were O
co O
- O
transfected O
with O
a O
plasmid O
carrying O
a O
neomycin O
resistant O
gene O
and O
selected O
by O
0 O
. O
2 O
mM O
geneticin O
( O
Invitrogen O
) O
. O

For O
differentiation O
of O
Y79 O
cells O
, O
cells O
were O
plated O
on O
the O
poly O
- O
d O
- O
lysine O
- O
coated O
plates O
and O
then O
treated O
with O
2 O
. O
0 O
- O
2 O
. O
5 O
mM O
sodium O
butyrate O
for O
4 O
- O
5 O
days O
. O

HeLa O
cells O
were O
cultured O
as O
described O
and O
transfected O
with O
DNA O
using O
Effectene O
reagent O
( O
14 O
, O
26 O
) O
. O

Immunoblot O
analysis O

Samples O
that O
required O
both O
protein O
and O
mRNA O
analysis O
were O
split O
upon O
harvesting O
. O

Total O
cell O
proteins O
were O
subjected O
to O
SDS O
- O
PAGE O
and O
blotted O
as O
described O
previously O
( O
26 O
) O
. O

The O
primary O
antibodies O
used O
are O
monoclonal O
antibodies O
to O
a O
myc O
- O
epitope O
( O
Invitrogen O
) O
and O
green O
fluorescent O
protein O
( O
GFP O
) O
( O
Clontech O
) O
. O

Binding O
of O
antibodies O
was O
detected O
with O
the O
SuperSignal O
System O
( O
Pierce O
) O
or O
ECL O
( O
Amersham O
) O
. O

Immunofluorescent O
microscopy O

HeLa O
or O
Y79 O
cells O
grown O
in O
a O
four O
- O
chamber O
glass O
slide O
were O
transfected O
as O
described O
above O
. O

Cells O
were O
fixed O
with O
10 O
% O
formaldehyde O
24 O
- O
48 O
h O
after O
transfection O
, O
and O
permealized O
with O
0 O
. O
5 O
% O
Triton O
X O
- O
100 O
then O
blocked O
with O
5 O
% O
goat B
serum O
. O

Primary O
antibodies O
used O
were O
mouse B
anti O
- O
myc O
( O
Invitrogen O
) O
, O
rabbit B
anti O
- O
NMHC O
- O
B O
( O
29 O
) O
. O

Secondary O
antibodies O
were O
Alexa O
Fluor O
594 O
goat B
anti O
- O
mouse B
IgG O
and O
Alexa O
Fluor O
488 O
goat B
anti O
- O
rabbit B
IgG O
( O
Molecular O
Probes O
) O
. O

DAPI O
was O
used O
for O
DNA O
staining O
. O

Specimens O
were O
mounted O
in O
ProLong O
antifed O
kit O
( O
Molecular O
Probe O
) O
. O

The O
images O
were O
collected O
using O
Leica O
SP O
confocal O
microscopy O
( O
Leica O
) O
. O

RESULTS O

Tissue O
- O
dependent O
isoforms O
of O
Fxh O
and O
A2BP1 O
and O
their O
subcellular O
distribution O

The O
A2BP1 O
mRNAs O
are O
detected O
almost O
exclusively O
in O
brain O
and O
striated O
muscles O
( O
heart O
and O
skeletal O
muscles O
) O
in O
adult O
mice B
, O
whereas O
the O
Fxh O
mRNAs O
are O
expressed O
in O
a O
wide O
variety O
of O
tissues O
with O
the O
highest O
expression O
in O
brain O
and O
heart O
[ O
( O
21 O
, O
25 O
) O
and O
S O
. O
Kawamoto O
, O
unpublished O
data O
] O
. O

These O
expression O
profiles O
prompted O
us O
to O
characterize O
the O
mRNAs O
of O
A2BP1 O
and O
Fxh O
in O
brain O
and O
muscles O
. O

We O
have O
cloned O
cDNAs O
for O
two O
gene O
transcripts O
from O
brain O
, O
heart O
and O
skeletal O
muscles O
by O
RT O
- O
PCR O
and O
5 O
' O
RACE O
. O

The O
isolated O
cDNAs O
are O
schematically O
presented O
with O
genomic O
structures O
in O
Figure O
1 O
( O
for O
amino O
acid O
sequences O
see O
also O
Figure O
8 O
) O
. O

Both O
gene O
transcripts O
are O
found O
to O
undergo O
tissue O
- O
specific O
alternative O
splicing O
. O

In O
both O
cases O
, O
brain O
and O
striated O
muscles O
express O
unique O
splice O
variants O
generated O
by O
mutually O
exclusive O
splicing O
of O
exons O
B40 O
and O
M43 O
, O
respectively O
, O
which O
provide O
different O
coding O
sequences O
in O
the O
middle O
of O
the O
carboxyl O
half O
of O
the O
molecules O
. O

Southern O
blot O
analysis O
of O
the O
RT O
- O
PCR O
products O
of O
the O
Fxh O
mRNAs O
from O
the O
different O
tissues O
probed O
by O
oligonucleotides O
corresponding O
to O
B40 O
and O
M43 O
shows O
that O
M43 O
is O
exclusively O
used O
in O
heart O
and O
skeletal O
muscles O
, O
while O
B40 O
is O
predominantly O
used O
in O
brain O
( O
Figure O
2A O
) O
. O

Digestion O
of O
the O
RT O
- O
PCR O
products O
of O
the O
A2BP1 O
mRNAs O
with O
restriction O
enzymes O
unique O
to O
B40 O
and O
M43 O
demonstrates O
that O
B40 O
and O
M43 O
are O
used O
almost O
exclusively O
in O
brain O
and O
muscles O
, O
respectively O
( O
Figure O
2B O
) O
. O

A2BP1 O
contains O
an O
additional O
cassette O
type O
alternative O
exon O
A53 O
, O
consisting O
of O
53 O
nt O
. O

Inclusion O
and O
exclusion O
of O
exon O
A53 O
results O
in O
two O
different O
amino O
acid O
sequences O
at O
the O
C O
- O
terminal O
region O
owing O
to O
a O
frame O
shift O
. O

In O
the O
case O
of O
the O
A2BP1 O
gene O
, O
moreover O
, O
it O
appears O
that O
brain O
and O
striated O
muscle O
utilize O
alternative O
promoters O
, O
resulting O
in O
different O
amino O
acid O
sequences O
at O
the O
N O
- O
terminus O
. O

The O
5 O
' O
RACE O
of O
skeletal O
muscle O
mRNAs O
yielded O
essentially O
a O
single O
species O
of O
sequence O
with O
29 O
unique O
N O
- O
terminal O
amino O
acids O
. O

The O
5 O
' O
RACE O
using O
brain O
mRNAs O
; O
however O
, O
yielded O
multiple O
products O
with O
multiple O
deduced O
amino O
acid O
sequences O
, O
all O
of O
which O
differ O
from O
the O
muscle O
amino O
acid O
sequence O
at O
the O
very O
N O
- O
terminus O
. O

Here O
, O
we O
focus O
our O
analysis O
on O
a O
clone O
containing O
nine O
unique O
amino O
acids O
at O
the O
N O
- O
terminus O
, O
which O
was O
obtained O
most O
frequently O
. O

Exon O
- O
intron O
organization O
of O
Fxh O
and O
A2BP1 O
shows O
remarkable O
similarities O
and O
a O
RRM O
is O
encoded O
in O
four O
exons O
( O
Figure O
1B O
) O
. O

Of O
note O
is O
that O
the O
significant O
amounts O
of O
Fxh O
and O
A2BP1 O
mRNAs O
from O
skeletal O
muscles O
are O
missing O
a O
part O
of O
the O
RRM O
by O
exon O
skipping O
. O

Typically O
, O
as O
shown O
in O
Figure O
2C O
, O
they O
lack O
the O
93 O
nt O
exon O
that O
encodes O
RNP1 O
, O
one O
of O
the O
two O
most O
critical O
motifs O
of O
the O
RRM O
( O
30 O
) O
. O

Some O
of O
them O
lack O
almost O
the O
entire O
RRM O
( O
e O
. O
g O
. O
F402 O
in O
Figure O
1A O
) O
. O

To O
examine O
the O
subcellular O
distribution O
of O
each O
isoform O
of O
Fxh O
and O
A2BP1 O
, O
myc O
- O
tagged O
proteins O
were O
transiently O
expressed O
in O
cultured O
cells O
and O
immunostained O
with O
an O
anti O
- O
myc O
antibody O
. O

Initially O
, O
HeLa O
cells O
were O
used O
to O
investigate O
subcellular O
localization O
, O
since O
these O
cells O
are O
more O
suited O
for O
these O
studies O
. O

Representative O
confocal O
images O
are O
shown O
in O
Figure O
3A O
. O

DAPI O
and O
anti O
- O
NMHC O
- O
B O
antibodies O
serve O
as O
markers O
for O
nuclei O
and O
cytoplasm O
, O
respectively O
. O

As O
noted O
, O
the O
ratio O
of O
protein O
distributed O
between O
nuclei O
and O
cytoplasm O
differs O
among O
the O
proteins O
. O

All O
isoforms O
of O
Fxh O
have O
a O
predominant O
nuclear O
localization O
. O

The O
brain O
isoform O
of O
A2BP1 O
without O
the O
A53 O
exon O
( O
A016 O
) O
localizes O
to O
both O
nuclei O
and O
cytoplasm O
, O
whereas O
the O
other O
brain O
isoform O
with O
the O
A53 O
exon O
( O
A030 O
) O
localizes O
predominantly O
to O
cytoplasm O
with O
only O
a O
minimum O
being O
in O
the O
nuclei O
. O

The O
relative O
amounts O
of O
both O
muscle O
isoforms O
of O
A2BP1 O
( O
A713 O
and O
A715 O
) O
are O
somewhat O
between O
the O
amounts O
of O
the O
two O
brain O
isoforms O
. O

These O
data O
are O
summarized O
in O
Figure O
1A O
. O

The O
subcellular O
distribution O
of O
representative O
isoforms O
( O
F011 O
, O
A016 O
and O
A030 O
) O
was O
also O
examined O
in O
retinoblastoma O
Y79 O
cells O
, O
which O
were O
used O
as O
host O
cells O
for O
the O
transfection O
experiments O
in O
order O
to O
characterize O
splicing O
activities O
of O
Fxh O
and O
A2BP O
proteins O
( O
see O
below O
) O
. O

Although O
Y79 O
cells O
have O
a O
spherical O
shape O
and O
have O
only O
thin O
cytoplasm O
, O
exogenously O
expressed O
isoforms O
of O
Fxh O
and O
A2BP1 O
show O
a O
similar O
subcellular O
distribution O
as O
in O
HeLa O
cells O
( O
Figure O
3B O
) O
. O

F011 O
localizes O
predominantly O
to O
nuclei O
, O
A016 O
to O
both O
nuclei O
and O
cytoplasm O
and O
A030 O
predominantly O
to O
cytoplasm O
. O

Specific O
interaction O
of O
Fxh O
and O
A2BP1 O
with O
IDDE O
via O
a O
hexanucleotide O
UGCAUG O

Fxh O
and O
A2BP1 O
share O
an O
identical O
RRM O
and O
A2BP1 O
has O
been O
reported O
to O
bind O
specifically O
to O
the O
pentanucleotide O
GCAUG O
through O
this O
RRM O
( O
21 O
) O
. O

We O
have O
previously O
reported O
that O
the O
IDDE O
of O
the O
NMHC O
- O
B O
transcript O
, O
which O
is O
indispensable O
for O
the O
regulation O
of O
neural O
cell O
- O
specific O
cassette O
type O
exon O
N30 O
splicing O
, O
has O
two O
copies O
of O
GCAUG O
( O
14 O
) O
. O

Therefore O
, O
we O
investigated O
whether O
Fxh O
and O
/ O
or O
A2BP1 O
bound O
to O
the O
IDDE O
. O

Electrophoretic O
mobility O
shift O
assays O
( O
EMSAs O
) O
were O
carried O
out O
using O
labeled O
IDDE O
and O
in O
vitro O
transcribed O
and O
translated O
Fxh O
and O
A2BP1 O
, O
which O
include O
a O
myc O
- O
epitope O
. O

Since O
all O
Fxh O
and O
A2BP1 O
isoforms O
, O
except O
F402 O
and O
A704 O
, O
contain O
the O
identical O
RRM O
, O
representative O
isoforms O
were O
analyzed O
. O

The O
expression O
of O
F011 O
in O
reticulocyte O
lysate O
causes O
the O
formation O
of O
a O
RNA O
- O
protein O
complex O
whose O
migration O
shift O
distinguishes O
it O
from O
those O
of O
the O
control O
reticulocyte O
lysate O
( O
C O
in O
Figure O
4B O
, O
lanes O
3 O
and O
10 O
) O
. O

The O
unlabeled O
wild O
- O
type O
IDDE O
competes O
with O
the O
probe O
efficiently O
for O
the O
formation O
of O
the O
specific O
complex O
C O
( O
Figure O
4B O
, O
lane O
4 O
) O
. O

On O
the O
other O
hand O
, O
the O
mutant O
mc O
( O
Figure O
4A O
) O
, O
which O
has O
a O
mutation O
in O
both O
copies O
of O
UGCAUG O
, O
does O
not O
( O
Figure O
4B O
, O
lane O
7 O
) O
. O

The O
mutant O
ma O
, O
which O
has O
a O
mutation O
in O
the O
hexanucleotide O
at O
the O
5 O
' O
side O
, O
shows O
less O
efficient O
competition O
, O
compared O
with O
mb O
, O
which O
has O
a O
mutation O
in O
the O
3 O
' O
side O
of O
the O
hexanucleotide O
( O
Figure O
4B O
, O
lanes O
5 O
and O
6 O
) O
, O
indicating O
that O
the O
nucleotides O
at O
the O
5 O
' O
side O
are O
more O
important O
than O
those O
at O
the O
3 O
' O
side O
. O

The O
presence O
of O
an O
anti O
- O
myc O
antibody O
, O
but O
not O
a O
non O
- O
specific O
antibody O
, O
inhibits O
the O
formation O
of O
the O
specific O
complex O
( O
Figure O
4B O
, O
lane O
8 O
) O
. O

Synthesis O
of O
the O
full O
- O
length O
F011 O
protein O
using O
a O
reticulocyte O
lysate O
and O
specificity O
of O
the O
myc O
antibody O
for O
the O
expressed O
protein O
are O
verified O
by O
immunoblot O
analysis O
( O
Figure O
4B O
, O
lanes O
11 O
and O
12 O
) O
. O

A016 O
and O
A030 O
show O
essentially O
identical O
results O
to O
those O
with O
F011 O
( O
data O
not O
shown O
) O
. O

These O
results O
indicate O
that O
Fxh O
and O
A2BP1 O
can O
bind O
to O
the O
IDDE O
and O
that O
the O
hexanucleotide O
UGCAUG O
is O
required O
for O
their O
binding O
. O

Fxh O
and O
A2BP1 O
enhance O
N30 O
inclusion O
in O
a O
UGCAUG O
- O
dependent O
manner O

To O
study O
whether O
Fxh O
and O
A2BP1 O
regulate O
neural O
cell O
- O
specific O
splicing O
via O
binding O
to O
UGCAUG O
, O
the O
Fxh O
and O
A2BP1 O
expression O
constructs O
were O
co O
- O
transfected O
into O
retinoblastoma O
Y79 O
cells O
with O
a O
number O
of O
the O
reporter O
minigene O
constructs O
, O
which O
include O
the O
wild O
- O
type O
or O
a O
mutant O
version O
of O
UGCAUG O
in O
the O
IDDE O
( O
Figure O
4A O
) O
. O

Minigenes O
H O
and O
J O
consist O
of O
the O
exons O
E5 O
, O
N30 O
and O
E6 O
and O
their O
flanking O
introns O
with O
some O
deletions O
in O
the O
introns O
. O

The O
IDDE O
with O
or O
without O
mutations O
in O
the O
hexanucleotide O
is O
included O
or O
excluded O
between O
N30 O
and O
E6 O
. O

As O
described O
above O
, O
each O
isoform O
of O
Fxh O
and O
A2BP1 O
enters O
the O
nucleus O
to O
a O
different O
extent O
. O

To O
see O
the O
effects O
of O
different O
proteins O
on O
N30 O
splicing O
itself O
, O
independent O
of O
their O
differential O
properties O
in O
nuclear O
localization O
, O
an O
exogenous O
NLS O
was O
added O
to O
the O
expressed O
proteins O
. O

Since O
Fxh O
and O
A2BP1 O
proteins O
contain O
the O
identical O
RRM O
, O
and O
F011 O
, O
A016 O
and O
A030 O
all O
show O
the O
same O
UGCAUG O
- O
dependent O
binding O
to O
the O
IDDE O
in O
vitro O
, O
F011 O
and O
A030 O
were O
used O
for O
these O
experiments O
. O

Host O
cells O
Y79 O
at O
the O
proliferating O
stage O
exclude O
the O
N30 O
exon O
in O
~ O
90 O
% O
of O
the O
endogenous O
NMHC O
- O
B O
mRNAs O
( O
e O
. O
g O
. O
see O
Figure O
6A O
, O
lane O
1 O
) O
. O

As O
shown O
in O
Figure O
4C O
, O
the O
mRNAs O
derived O
from O
minigene O
J O
exclude O
N30 O
without O
exogenous O
expression O
of O
Fxh O
or O
A2BP1 O
in O
Y79 O
cells O
, O
similar O
to O
the O
endogenous O
NMHC O
- O
B O
mRNAs O
( O
Figure O
4C O
, O
upper O
panel O
, O
lanes O
1 O
- O
5 O
) O
. O

However O
, O
in O
the O
presence O
of O
exogenous O
expression O
of O
F011 O
and O
A030 O
, O
the O
N30 O
inclusion O
is O
increased O
( O
Figure O
4C O
, O
upper O
panel O
, O
lanes O
7 O
and O
12 O
) O
. O

The O
N30 O
inclusion O
is O
absolutely O
dependent O
on O
the O
presence O
of O
the O
IDDE O
( O
Figure O
4C O
, O
upper O
panel O
, O
lanes O
6 O
and O
11 O
) O
. O

Moreover O
, O
mutation O
of O
either O
one O
of O
the O
two O
copies O
of O
UGCAUG O
( O
ma O
, O
mb O
) O
abolishes O
the O
inclusion O
of O
N30 O
( O
Figure O
4C O
, O
upper O
panel O
, O
lanes O
8 O
- O
10 O
, O
13 O
- O
15 O
) O
. O

In O
the O
context O
of O
minigene O
H O
, O
which O
contains O
shorter O
introns O
, O
the O
larger O
extent O
of O
N30 O
inclusion O
is O
induced O
by O
either O
F011 O
or O
A030 O
overexpression O
( O
Figure O
4C O
, O
middle O
panel O
, O
lanes O
7 O
and O
12 O
) O
. O

The O
N30 O
inclusion O
of O
the O
minigene O
H O
mRNAs O
also O
depends O
on O
the O
presence O
of O
the O
IDDE O
( O
Figure O
4C O
, O
middle O
panel O
, O
lanes O
6 O
and O
11 O
) O
. O

Mutation O
of O
both O
copies O
of O
UGCAUG O
( O
mc O
) O
results O
in O
a O
complete O
loss O
of O
N30 O
inclusion O
( O
Figure O
4C O
, O
middle O
panel O
, O
lanes O
10 O
and O
15 O
) O
. O

The O
mutant O
ma O
shows O
stronger O
inhibition O
of O
N30 O
inclusion O
compared O
with O
mb O
( O
Figure O
4C O
, O
middle O
panel O
, O
lanes O
8 O
and O
9 O
) O
. O

This O
observation O
is O
consistent O
with O
the O
competition O
experiments O
of O
the O
EMSA O
shown O
in O
Figure O
4B O
, O
indicating O
that O
the O
5 O
' O
hexanucleotide O
is O
more O
important O
than O
the O
3 O
' O
hexanucleotide O
for O
binding O
of O
Fxh O
or O
A2BP1 O
to O
the O
IDDE O
as O
well O
as O
an O
activation O
of O
N30 O
splicing O
. O

Comparable O
amounts O
of O
F011 O
and O
A030 O
are O
expressed O
in O
each O
transfection O
as O
verified O
by O
immunoblots O
using O
an O
anti O
- O
myc O
antibody O
( O
Figure O
4C O
, O
lower O
panel O
, O
lanes O
6 O
- O
17 O
) O
. O

Since O
minigenes O
J O
and O
H O
lack O
a O
portion O
of O
the O
intron O
between O
exons O
N30 O
and O
E6 O
and O
, O
therefore O
, O
the O
IDDE O
is O
located O
~ O
100 O
nt O
downstream O
of O
N30 O
, O
instead O
of O
1 O
. O
5 O
kb O
as O
in O
the O
native O
gene O
, O
we O
also O
analyzed O
the O
effects O
of O
Fxh O
and O
A2BP1 O
on O
the O
N30 O
splicing O
of O
the O
wild O
- O
type O
minigene O
G O
, O
which O
includes O
full O
- O
length O
introns O
among O
E5 O
, O
N30 O
and O
E6 O
( O
Figure O
4A O
) O
. O

As O
shown O
in O
Figure O
4C O
( O
right O
panel O
) O
, O
both O
F011 O
and O
A030 O
are O
capable O
of O
promoting O
N30 O
inclusion O
, O
with O
F011 O
showing O
a O
higher O
activity O
( O
Figure O
4C O
, O
lanes O
16 O
and O
17 O
) O
. O

Although O
A030 O
can O
promote O
N30 O
inclusion O
as O
efficiently O
as O
F011 O
in O
the O
contexts O
of O
the O
minigene O
J O
and O
H O
transcripts O
, O
it O
can O
do O
so O
less O
efficiently O
than O
F011 O
in O
the O
context O
of O
the O
minigene O
G O
transcript O
. O

Interpretation O
of O
this O
observation O
will O
be O
discussed O
below O
. O

Taken O
together O
, O
Fxh O
and O
A2BP1 O
can O
activate O
N30 O
inclusion O
in O
an O
IDDE O
- O
dependent O
manner O
. O

The O
hexanucleotide O
motif O
UGCAUG O
is O
indispensable O
for O
this O
activation O
. O

Differential O
activities O
of O
alternatively O
spliced O
isoforms O
of O
Fxh O
and O
A2BP1 O
in O
promoting O
N30 O
inclusion O

Both O
Fxh O
and O
A2BP1 O
mRNAs O
are O
expressed O
in O
brain O
and O
A2BP1 O
is O
also O
expressed O
in O
striated O
muscles O
and O
Fxh O
is O
expressed O
in O
an O
even O
wider O
variety O
of O
tissues O
. O

As O
demonstrated O
above O
, O
however O
, O
both O
Fxh O
and O
A2BP1 O
transcripts O
undergo O
tissue O
- O
dependent O
alternative O
splicing O
, O
producing O
muscle O
- O
specific O
and O
brain O
- O
enriched O
isoforms O
. O

Therefore O
, O
the O
relative O
activity O
of O
individual O
isoform O
of O
Fxh O
and O
A2BP1 O
in O
promoting O
N30 O
inclusion O
was O
compared O
using O
minigene O
G O
. O

First O
, O
in O
order O
to O
evaluate O
the O
relative O
specific O
activity O
of O
each O
isoform O
in O
the O
splicing O
reaction O
separately O
from O
its O
ability O
to O
localize O
to O
nuclei O
, O
an O
exogenous O
NLS O
was O
included O
in O
the O
expressed O
protein O
to O
equalize O
the O
nuclear O
concentration O
of O
the O
expressed O
protein O
in O
these O
experiments O
. O

Essentially O
, O
all O
of O
the O
expressed O
proteins O
with O
the O
exogenous O
NLS O
are O
localized O
to O
the O
nucleus O
( O
data O
not O
shown O
) O
. O

Therefore O
, O
the O
relative O
nuclear O
concentrations O
of O
the O
expressed O
proteins O
can O
be O
estimated O
easily O
by O
immunoblots O
. O

As O
shown O
in O
immunoblots O
in O
Figure O
5A O
, O
similar O
quantities O
of O
proteins O
are O
expressed O
in O
a O
dose O
- O
dependent O
manner O
. O

Expression O
of O
the O
brain O
isoform O
F011 O
causes O
a O
dose O
- O
dependent O
increase O
in O
N30 O
inclusion O
and O
the O
extent O
of O
N30 O
inclusion O
reaches O
a O
plateau O
with O
~ O
85 O
% O
of O
the O
mRNAs O
including O
N30 O
. O

In O
contrast O
, O
inclusion O
of O
N30 O
promoted O
by O
F411 O
, O
the O
predominant O
isoform O
from O
skeletal O
muscles O
, O
reaches O
a O
plateau O
with O
only O
40 O
% O
of O
the O
mRNAs O
. O

With O
respect O
to O
A2BP1 O
, O
the O
brain O
isoform O
A030 O
shows O
higher O
activity O
in O
N30 O
inclusion O
than O
the O
muscle O
isoform O
A715 O
, O
which O
shows O
almost O
no O
activation O
, O
as O
shown O
in O
Figure O
5A O
. O

Including O
additional O
isoforms O
, O
the O
activities O
of O
individual O
isoforms O
with O
an O
exogenous O
NLS O
in O
promoting O
N30 O
inclusion O
are O
shown O
in O
Figure O
5B O
( O
lanes O
8 O
- O
13 O
) O
and O
are O
also O
summarized O
in O
Figure O
1A O
( O
nls O
) O
. O

Special O
care O
was O
taken O
to O
ensure O
that O
a O
similar O
quantity O
of O
each O
protein O
was O
expressed O
and O
, O
if O
not O
, O
different O
amounts O
were O
tested O
to O
obtain O
comparable O
expression O
. O

In O
the O
presence O
of O
the O
exogenous O
NLS O
( O
nls O
) O
, O
F011 O
and O
A016 O
show O
the O
highest O
activities O
among O
all O
isoforms O
tested O
. O

A30 O
shows O
a O
considerably O
lower O
activity O
than O
A016 O
. O

Each O
of O
the O
muscle O
isoforms O
( O
F411 O
, O
A713 O
and O
A715 O
) O
has O
a O
lower O
activity O
than O
that O
of O
their O
brain O
counterparts O
( O
F011 O
, O
A016 O
and O
A030 O
, O
respectively O
) O
. O

As O
expected O
, O
isoforms O
lacking O
a O
part O
or O
all O
of O
the O
RRM O
( O
F402 O
and O
A704 O
) O
have O
no O
activity O
for O
N30 O
inclusion O
. O

Next O
, O
the O
splicing O
activities O
of O
the O
wild O
- O
type O
proteins O
without O
an O
exogenous O
NLS O
were O
examined O
. O

Representative O
data O
are O
shown O
in O
Figure O
5B O
( O
lanes O
2 O
- O
7 O
) O
and O
the O
relative O
activities O
are O
summarized O
in O
Figure O
1A O
( O
wt O
) O
. O

The O
protein O
amounts O
detected O
by O
immunoblots O
in O
these O
experiments O
represent O
the O
total O
amounts O
of O
the O
proteins O
distributed O
to O
both O
the O
nuclei O
and O
the O
cytoplasm O
. O

The O
splicing O
activities O
of O
the O
wild O
- O
type O
proteins O
are O
consistent O
with O
the O
activities O
that O
combine O
the O
splicing O
activities O
of O
the O
proteins O
with O
the O
exogenous O
NLS O
and O
the O
activities O
of O
the O
native O
proteins O
to O
localize O
to O
nuclei O
. O

In O
the O
absence O
of O
the O
exogenous O
NLS O
( O
wt O
) O
, O
the O
brain O
isoforms O
F011 O
, O
and O
A016 O
to O
a O
lesser O
extent O
, O
are O
still O
capable O
of O
activating O
N30 O
inclusion O
efficiently O
. O

The O
muscle O
isoforms O
for O
both O
Fxh O
and O
A2BP1 O
( O
F411 O
, O
A713 O
and O
A715 O
) O
, O
as O
well O
as O
the O
brain O
isoform O
A030 O
, O
show O
only O
minimal O
activities O
. O

Therefore O
, O
F011 O
and O
A016 O
appear O
to O
have O
the O
most O
physiological O
relevance O
to O
N30 O
splicing O
activation O
. O

Overexpression O
of O
Fxh O
and O
A2BP1 O
activates O
N30 O
inclusion O
of O
endogenous O
NMHC O
- O
B O
mRNAs O

The O
human B
NMHC O
- O
B O
gene O
consists O
of O
41 O
constitutive O
exons O
and O
3 O
alternative O
exons O
. O

Its O
pre O
- O
mRNA O
is O
~ O
156 O
kb O
in O
length O
and O
it O
is O
much O
more O
complex O
than O
the O
pre O
- O
mRNA O
from O
the O
minigenes O
. O

In O
addition O
, O
the O
minigene O
pre O
- O
mRNAs O
are O
driven O
by O
a O
heterologous O
promoter O
. O

Therefore O
, O
we O
next O
examined O
if O
Fxh O
and O
A2BP1 O
were O
capable O
of O
promoting O
N30 O
inclusion O
of O
the O
endogenous O
transcript O
. O

Y79 O
cells O
were O
stably O
transfected O
with O
the O
expression O
construct O
for O
F011 O
or O
A016 O
. O

Both O
F011 O
and O
A016 O
are O
enriched O
in O
the O
brain O
and O
show O
higher O
activation O
of O
N30 O
inclusion O
in O
the O
minigene O
transcripts O
. O

mRNAs O
encoding O
endogenous O
NMHC O
- O
B O
were O
analyzed O
by O
using O
RT O
- O
PCR O
. O

As O
shown O
in O
Figure O
6 O
, O
the O
inclusion O
of O
exon O
N30 O
with O
and O
without O
another O
alternative O
exon O
, O
R18 O
, O
in O
the O
endogenous O
mRNAs O
is O
markedly O
increased O
in O
the O
clones O
which O
were O
stably O
transfected O
with O
the O
construct O
for O
F011 O
or O
A016 O
containing O
an O
exogenous O
NLS O
( O
Figure O
6 O
, O
lanes O
4 O
and O
5 O
) O
. O

Although O
to O
a O
lesser O
extent O
, O
transfection O
of O
the O
wild O
- O
type O
constructs O
without O
an O
exogenous O
NLS O
also O
results O
in O
a O
significant O
increase O
in O
N30 O
inclusion O
( O
Figure O
6 O
, O
lanes O
2 O
and O
3 O
) O
. O

Thus O
, O
exogenously O
expressed O
Fxh O
and O
A2BP1 O
are O
capable O
of O
activating O
N30 O
inclusion O
not O
only O
in O
the O
transcripts O
from O
the O
minigenes O
, O
but O
also O
in O
those O
from O
the O
native O
NMHC O
- O
B O
gene O
in O
Y79 O
cells O
. O

Fxh O
may O
cooperate O
with O
other O
factor O
( O
s O
) O
to O
promote O
N30 O
inclusion O

To O
address O
the O
role O
of O
endogenous O
Fxh O
and O
its O
potential O
interaction O
with O
other O
proteins O
in O
promoting O
N30 O
inclusion O
, O
we O
made O
use O
of O
an O
isoform O
of O
Fxh O
, O
F402 O
, O
which O
lacks O
the O
RRM O
. O

The O
mutant O
proteins O
lacking O
an O
RRM O
for O
other O
RNA O
- O
binding O
proteins O
have O
previously O
been O
reported O
to O
function O
in O
a O
dominant O
- O
negative O
fashion O
and O
inhibit O
the O
activities O
of O
the O
wild O
- O
type O
proteins O
( O
26 O
, O
31 O
) O
. O

Therefore O
, O
the O
effects O
of O
F402 O
on O
the O
N30 O
inclusion O
of O
minigene O
H O
mRNAs O
were O
examined O
in O
the O
context O
of O
the O
Y79 O
cells O
treated O
with O
butyrate O
. O

Upon O
butyrate O
treatment O
, O
as O
reported O
previously O
, O
Y79 O
cells O
enter O
in O
a O
post O
- O
mitotic O
and O
differentiated O
stage O
, O
and O
importantly O
, O
the O
endogenous O
as O
well O
as O
the O
minigene O
mRNAs O
in O
those O
cells O
include O
N30 O
to O
a O
large O
extent O
, O
unlike O
those O
in O
the O
untreated O
cells O
that O
predominantly O
exclude O
N30 O
( O
14 O
) O
. O

As O
shown O
in O
Figure O
7 O
, O
in O
the O
absence O
of O
exogenous O
expression O
of O
F402 O
, O
large O
quantities O
of O
the O
mRNAs O
derived O
from O
minigene O
H O
- O
wt O
, O
which O
contains O
the O
wild O
- O
type O
IDDE O
, O
include O
N30 O
( O
Figure O
7 O
, O
upper O
panel O
, O
lane O
4 O
) O
. O

In O
contrast O
, O
only O
small O
quantities O
of O
N30 O
inclusion O
are O
detected O
in O
the O
mRNAs O
from O
minigene O
H O
- O
mc O
, O
which O
has O
mutations O
in O
both O
copies O
of O
UGCAUG O
in O
the O
IDDE O
( O
Figure O
7 O
, O
upper O
panel O
, O
lane O
8 O
) O
. O

Therefore O
, O
the O
butyrate O
- O
treated O
Y79 O
cells O
contain O
factor O
( O
s O
) O
, O
which O
are O
capable O
of O
activating O
the O
UGCAUG O
- O
dependent O
N30 O
inclusion O
. O

Co O
- O
transfection O
of O
the O
wild O
- O
type O
minigene O
H O
- O
wt O
with O
the O
F402 O
expression O
construct O
causes O
a O
dose O
- O
dependent O
inhibition O
of O
N30 O
inclusion O
( O
Figure O
7 O
, O
lanes O
1 O
- O
3 O
) O
, O
indicating O
that O
F402 O
has O
an O
antagonistic O
effect O
on O
the O
endogenous O
factors O
with O
respect O
to O
N30 O
inclusion O
. O

Notably O
, O
the O
UGCAUG O
- O
independent O
N30 O
inclusion O
seen O
in O
the O
mutant O
minigene O
H O
- O
mc O
is O
not O
affected O
by O
the O
co O
- O
expression O
of O
F402 O
( O
Figure O
7 O
, O
lanes O
5 O
- O
7 O
) O
, O
indicating O
that O
the O
inhibitory O
activity O
of O
F402 O
depends O
on O
the O
UGCAUG O
element O
. O

The O
parallel O
experiment O
with O
the O
F011 O
expression O
construct O
does O
not O
show O
a O
significant O
effect O
on O
N30 O
splicing O
in O
either O
the O
wild O
- O
type O
or O
mutant O
minigene O
( O
Figure O
7 O
, O
lanes O
9 O
- O
16 O
) O
. O

This O
may O
be O
due O
to O
the O
fact O
that O
N30 O
inclusion O
of O
the O
wild O
- O
type O
minigene O
mRNAs O
has O
already O
reached O
a O
maximal O
level O
and O
is O
consistent O
with O
the O
idea O
that O
butyrate O
- O
treated O
Y79 O
cells O
contain O
sufficient O
amounts O
of O
protein O
( O
s O
) O
functionally O
equivalent O
to O
F011 O
, O
which O
can O
promote O
N30 O
inclusion O
in O
a O
UGCAUG O
- O
dependent O
manner O
. O

Since O
F402 O
does O
not O
bind O
to O
the O
UGCAUG O
element O
, O
and O
has O
a O
UGCAUG O
- O
dependent O
antagonistic O
effect O
on O
N30 O
splicing O
, O
it O
most O
probably O
disrupts O
protein O
- O
protein O
interactions O
of O
the O
endogenous O
proteins O
that O
are O
required O
for O
the O
UGCAUG O
- O
dependent O
activation O
of O
N30 O
splicing O
. O

Endogenous O
Fxh O
( O
and O
/ O
or O
A2BP1 O
) O
would O
be O
a O
good O
candidate O
whose O
function O
could O
be O
antagonized O
by O
exogenously O
expressed O
F402 O
. O

Thus O
, O
this O
observation O
suggests O
that O
the O
endogenous O
Fxh O
has O
an O
effect O
on O
the O
activation O
of O
N30 O
splicing O
and O
that O
other O
proteins O
cooperate O
with O
Fxh O
for O
N30 O
activation O
. O

Since O
muscle O
cells O
express O
the O
RRM O
- O
defective O
isoforms O
to O
a O
significant O
extent O
( O
Figure O
2C O
) O
and O
the O
wild O
- O
type O
F402 O
localize O
to O
nuclei O
efficiently O
, O
this O
also O
raises O
the O
possibility O
that O
the O
RRM O
- O
defective O
isoforms O
may O
have O
an O
inhibitory O
function O
on O
the O
N30 O
splicing O
in O
muscle O
cells O
. O

DISCUSSION O

Two O
major O
findings O
are O
described O
in O
this O
report O
. O

First O
, O
Fxh O
and O
A2BP1 O
facilitate O
neural O
cell O
- O
specific O
inclusion O
of O
the O
cassette O
- O
type O
exon O
via O
binding O
to O
the O
specific O
intronic O
sequence O
UGCAUG O
. O

In O
addition O
to O
a O
minigene O
model O
system O
, O
Fxh O
and O
A2BP1 O
are O
capable O
of O
facilitating O
N30 O
inclusion O
of O
the O
endogenous O
pre O
- O
mRNA O
. O

This O
result O
provides O
an O
important O
demonstration O
of O
physiological O
relevance O
and O
supports O
the O
notion O
that O
the O
NMHC O
- O
B O
pre O
- O
mRNA O
is O
likely O
to O
be O
the O
true O
target O
for O
Fxh O
or O
A2BP1 O
- O
mediated O
regulation O
. O

However O
, O
whether O
the O
endogenous O
Fxh O
or O
A2BP1 O
regulates O
endogenous O
NMHC O
- O
B O
pre O
- O
mRNA O
splicing O
needs O
to O
be O
determined O
in O
a O
future O
study O
. O

In O
vertebrates O
, O
small O
interfering O
RNAs O
and O
gene O
targeting O
strategies O
have O
recently O
been O
used O
successfully O
to O
address O
the O
roles O
of O
endogenous O
splicing O
regulators O
in O
alternative O
splicing O
of O
endogenous O
target O
pre O
- O
mRNAs O
( O
8 O
, O
32 O
- O
36 O
) O
. O

A O
second O
and O
more O
novel O
finding O
is O
the O
identification O
of O
tissue O
- O
specific O
isoforms O
of O
Fxh O
and O
A2BP1 O
with O
different O
splicing O
activities O
as O
well O
as O
different O
subcellular O
localizations O
. O

This O
finding O
raises O
the O
possibility O
that O
the O
products O
of O
the O
Fxh O
and O
A2BP1 O
genes O
can O
contribute O
to O
a O
mechanism O
as O
to O
how O
tissue O
specificity O
of O
alternative O
splicing O
is O
achieved O
. O

Many O
splicing O
factors O
are O
detected O
not O
only O
in O
the O
nuclei O
, O
but O
also O
in O
the O
cytoplasm O
( O
37 O
) O
. O

They O
are O
shuttling O
between O
the O
nucleus O
and O
the O
cytoplasm O
and O
, O
in O
some O
instances O
, O
extracellular O
stimuli O
trigger O
changes O
in O
subcellular O
distribution O
of O
these O
proteins O
. O

Such O
translocations O
have O
been O
reported O
for O
hnRNPA1 O
and O
PTB O
( O
38 O
, O
39 O
) O
. O

Moreover O
, O
a O
number O
of O
RNA O
- O
binding O
proteins O
have O
been O
demonstrated O
to O
play O
a O
role O
in O
multiple O
steps O
during O
gene O
expression O
in O
different O
subcellular O
compartments O
, O
such O
as O
pre O
- O
mRNA O
processing O
in O
nuclei O
, O
mRNA O
export O
from O
nuclei O
to O
cytoplasm O
and O
mRNA O
localization O
, O
stability O
and O
translation O
in O
cytoplasm O
( O
37 O
, O
40 O
) O
. O

Therefore O
, O
not O
surprisingly O
, O
Fxh O
and O
A2BP1 O
isoforms O
were O
found O
to O
be O
distributed O
in O
both O
the O
nuclei O
and O
the O
cytoplasm O
in O
HeLa O
and O
Y79 O
cells O
. O

However O
, O
the O
relative O
ratios O
of O
proteins O
distributed O
between O
the O
two O
subcellular O
compartments O
at O
steady O
- O
state O
differ O
among O
the O
isoforms O
. O

In O
agreement O
with O
Jin O
et O
al O
. O

( O
21 O
) O
, O
substantial O
amounts O
of O
the O
brain O
isoform O
A016 O
are O
detected O
in O
nuclei O
. O

Other O
A2BP1 O
isoforms O
, O
the O
brain O
isoform O
A030 O
and O
the O
muscle O
isoforms O
A713 O
and O
A715 O
, O
are O
only O
poorly O
detected O
in O
nuclei O
. O

This O
observation O
is O
consistent O
with O
the O
reports O
where O
endogenous O
A2BP1 O
in O
cerebellar O
Purkinje O
cells O
, O
hippocampus O
neurons O
and O
cardiac O
myocytes O
were O
shown O
to O
be O
localized O
essentially O
to O
the O
cytoplasm O
( O
23 O
, O
24 O
) O
. O

Thus O
, O
inclusion O
and O
exclusion O
of O
A53 O
and O
differences O
in O
the O
very O
N O
- O
terminal O
sequences O
results O
in O
A2BP1 O
isoforms O
with O
a O
distinct O
subcellular O
localization O
. O

It O
is O
likely O
that O
A2BP1 O
proteins O
have O
multiple O
roles O
, O
involving O
both O
nuclear O
and O
cytoplasmic O
events O
. O

In O
contrast O
, O
all O
three O
Fxh O
isoforms O
predominantly O
localized O
to O
the O
nuclei O
. O

Therefore O
, O
in O
terms O
of O
their O
localization O
, O
Fxh O
proteins O
are O
better O
candidates O
for O
regulators O
of O
the O
pre O
- O
mRNA O
splicing O
that O
takes O
place O
in O
nuclei O
. O

Of O
note O
, O
however O
, O
our O
preliminary O
results O
of O
5 O
' O
RACE O
, O
as O
well O
as O
the O
EST O
database O
, O
detect O
multiple O
5 O
' O
end O
sequences O
for O
both O
Fxh O
and O
A2BP1 O
mRNAs O
, O
which O
are O
presumably O
generated O
by O
alternative O
promoters O
and O
alternative O
splicing O
. O

The O
diversity O
of O
the O
5 O
' O
end O
cDNA O
sequences O
leads O
to O
the O
generation O
of O
a O
number O
of O
unique O
N O
- O
terminal O
amino O
acid O
sequences O
. O

Therefore O
, O
this O
study O
does O
not O
exclude O
the O
possible O
existence O
of O
other O
isoforms O
with O
different O
subcellular O
localizations O
for O
both O
Fxh O
and O
A2BP1 O
. O

Our O
study O
also O
does O
not O
exclude O
the O
possibility O
that O
some O
of O
the O
isoforms O
translocate O
between O
the O
nucleus O
and O
the O
cytoplasm O
following O
stimuli O
. O

The O
main O
aim O
of O
this O
study O
is O
to O
determine O
the O
relative O
activities O
of O
tissue O
- O
dependent O
isoforms O
of O
Fxh O
and O
A2BP1 O
in O
neural O
cell O
- O
specific O
and O
UGCAUG O
element O
- O
dependent O
alternative O
splicing O
. O

To O
obtain O
an O
indication O
of O
the O
relative O
specific O
activity O
of O
each O
isoform O
in O
transfected O
cells O
, O
the O
same O
amounts O
of O
the O
expressed O
proteins O
should O
be O
available O
for O
the O
splicing O
reaction O
in O
the O
nuclei O
. O

For O
this O
reason O
, O
an O
exogenous O
NLS O
was O
included O
in O
the O
expressed O
proteins O
. O

Essentially O
, O
all O
of O
the O
expressed O
proteins O
with O
the O
exogenous O
NLS O
localized O
to O
nuclei O
. O

Thus O
, O
the O
amounts O
of O
the O
expressed O
proteins O
determined O
by O
immunoblots O
represent O
the O
nuclear O
concentrations O
. O

The O
analysis O
using O
the O
proteins O
expressed O
with O
the O
exogenous O
NLS O
allowed O
us O
to O
compare O
directly O
the O
splicing O
activities O
of O
these O
proteins O
. O

Furthermore O
, O
this O
analysis O
also O
allowed O
us O
to O
define O
the O
critical O
regions O
of O
the O
proteins O
for O
splicing O
activation O
. O

As O
shown O
in O
Figure O
5 O
, O
the O
splicing O
activities O
of O
the O
various O
isoforms O
of O
Fxh O
and O
A2BP1 O
are O
intrinsically O
different O
, O
regardless O
of O
the O
subcellular O
localization O
properties O
of O
the O
wild O
- O
type O
proteins O
. O

Among O
the O
isoforms O
tested O
in O
this O
study O
, O
F011 O
and O
A016 O
, O
which O
include O
B40 O
, O
are O
found O
to O
have O
higher O
activities O
in O
promoting O
N30 O
inclusion O
. O

When O
the O
primary O
amino O
acid O
sequences O
, O
outside O
of O
the O
RRM O
, O
of O
these O
two O
proteins O
are O
compared O
, O
the O
C O
- O
terminal O
regions O
( O
amino O
acids O
190 O
- O
377 O
of O
F011 O
) O
show O
a O
higher O
homology O
with O
71 O
% O
identity O
, O
whereas O
the O
N O
- O
terminal O
regions O
( O
amino O
acids O
1 O
- O
112 O
of O
F011 O
) O
show O
only O
53 O
% O
identity O
. O

The O
C O
- O
terminal O
region O
includes O
four O
subregions O
of O
nearly O
identical O
stretches O
of O
amino O
acids O
( O
Figure O
8 O
, O
I O
- O
IV O
) O
. O

One O
subregion O
( O
II O
) O
includes O
13 O
amino O
acids O
encoded O
by O
exon O
B40 O
. O

Substitution O
of O
this O
subregion O
with O
exon O
M43 O
in O
Fxh O
causes O
substantial O
changes O
in O
amino O
acid O
sequences O
resulting O
in O
only O
a O
21 O
% O
identity O
in O
this O
region O
between O
F011 O
and O
F411 O
. O

Another O
subregion O
( O
IV O
) O
is O
located O
at O
the O
C O
- O
terminal O
end O
and O
A030 O
lacks O
this O
homologous O
region O
by O
the O
inclusion O
of O
exon O
A53 O
, O
which O
results O
in O
a O
frame O
shift O
. O

Since O
F411 O
and O
A030 O
show O
poor O
splicing O
activation O
compared O
with O
F011 O
and O
A016 O
, O
respectively O
, O
these O
two O
subregions O
of O
F011 O
and O
A016 O
appear O
to O
serve O
as O
activation O
domains O
, O
presumably O
by O
interacting O
with O
other O
proteins O
. O

This O
notion O
is O
supported O
by O
the O
finding O
that O
the O
RRM O
- O
lacking O
isoform O
F402 O
, O
which O
includes O
the O
same O
subregions O
II O
and O
IV O
as O
F011 O
, O
functions O
apparently O
as O
a O
dominant O
- O
negative O
mutant O
to O
the O
wild O
- O
type O
Fxh O
, O
consistent O
with O
the O
interpretation O
that O
the O
mutant O
and O
wild O
- O
type O
are O
competing O
to O
interact O
with O
other O
protein O
( O
s O
) O
. O

To O
date O
, O
Fyn O
tyrosine O
kinase O
and O
estrogen O
receptor O
- O
alpha O
have O
been O
reported O
to O
interact O
with O
Fxh O
, O
and O
ataxin O
- O
2 O
with O
A2BP1 O
( O
23 O
, O
41 O
, O
42 O
) O
. O

Whether O
these O
proteins O
participate O
in O
the O
regulation O
of O
pre O
- O
mRNA O
splicing O
is O
currently O
unknown O
. O

Of O
interest O
, O
A030 O
with O
the O
exogenous O
NLS O
activates O
N30 O
splicing O
as O
efficiently O
as O
F011 O
in O
the O
pre O
- O
mRNAs O
derived O
from O
minigenes O
J O
and O
H O
, O
which O
contain O
the O
shorter O
intron O
, O
whereas O
this O
isoform O
poorly O
activates O
N30 O
splicing O
in O
the O
pre O
- O
mRNA O
from O
minigene O
G O
, O
which O
contains O
the O
full O
- O
length O
intron O
. O

This O
observation O
implies O
that O
the O
interactions O
of O
A030 O
with O
different O
factors O
are O
required O
in O
the O
different O
pre O
- O
mRNA O
contexts O
. O

Therefore O
, O
the O
isoforms O
of O
Fxh O
and O
A2BP1 O
described O
here O
may O
have O
different O
effects O
on O
other O
UGCAUG O
- O
regulated O
alternative O
splicing O
. O

The O
involvement O
of O
the O
hexanucleotide O
UGCAUG O
in O
regulated O
alternative O
splicing O
has O
been O
experimentally O
demonstrated O
in O
a O
number O
of O
neural O
cell O
- O
specific O
, O
as O
well O
as O
other O
tissue O
- O
specific O
, O
model O
systems O
( O
13 O
- O
21 O
) O
. O

This O
hexanucleotide O
element O
plays O
a O
role O
, O
in O
most O
cases O
, O
as O
an O
enhancer O
in O
regulating O
alternative O
splicing O
of O
cassette O
- O
type O
exons O
as O
well O
as O
mutually O
exclusive O
exons O
. O

Furthermore O
, O
computational O
analysis O
has O
revealed O
that O
UGCAUG O
is O
over O
- O
represented O
in O
the O
introns O
in O
which O
splicing O
is O
regulated O
, O
compared O
with O
the O
constitutively O
spliced O
introns O
( O
43 O
) O
. O

This O
analysis O
also O
pointed O
to O
the O
UGCAUG O
element O
as O
playing O
a O
role O
in O
the O
regulation O
of O
tissue O
- O
specific O
alternative O
splicing O
in O
a O
wide O
range O
of O
tissues O
, O
but O
not O
in O
specific O
tissues O
. O

To O
date O
, O
KH O
- O
type O
splicing O
regulatory O
protein O
( O
KSRP O
) O
( O
44 O
) O
, O
A2BP1 O
( O
21 O
) O
and O
Fxh O
( O
this O
study O
) O
are O
known O
to O
be O
capable O
of O
binding O
to O
UGCAUG O
. O

KSRP O
is O
expressed O
ubiquitously O
and O
tissue O
- O
specific O
variants O
of O
this O
gene O
have O
not O
been O
described O
so O
far O
. O

In O
this O
study O
, O
we O
have O
described O
the O
existence O
of O
tissue O
- O
dependent O
isoforms O
of O
Fxh O
and O
A2BP1 O
, O
which O
, O
while O
not O
identical O
in O
some O
areas O
of O
the O
molecule O
, O
may O
contain O
the O
same O
RRM O
. O

The O
physiological O
relevance O
of O
these O
isoforms O
is O
that O
they O
have O
different O
splicing O
activities O
and O
different O
subcellular O
localizations O
. O

The O
brain O
isoforms O
promote O
N30 O
inclusion O
more O
efficiently O
than O
the O
muscle O
isoforms O
of O
both O
Fxh O
and O
A2BP1 O
. O

The O
isoforms O
lacking O
the O
RRM O
are O
normally O
expressed O
to O
a O
significant O
extent O
in O
skeletal O
muscles O
. O

This O
isoform O
is O
incapable O
of O
activating O
N30 O
splicing O
and O
, O
moreover O
, O
can O
inhibit O
N30 O
inclusion O
. O

The O
properties O
of O
these O
isoforms O
are O
consistent O
with O
Fxh O
and O
A2BP1 O
acting O
as O
regulators O
for O
N30 O
splicing O
, O
since O
N30 O
is O
included O
in O
neuronal O
cells O
, O
but O
excluded O
in O
muscles O
. O

Therefore O
, O
despite O
the O
tissue O
- O
independent O
occurrence O
of O
UGCAUG O
as O
a O
regulatory O
element O
, O
given O
the O
tissue O
- O
dependent O
isoforms O
of O
the O
UGCAUG O
- O
binding O
proteins O
( O
Fxh O
and O
A2BP1 O
) O
with O
different O
activities O
, O
the O
hexanucleotide O
UGCAUG O
could O
confer O
tissue O
specificity O
on O
regulated O
splicing O
. O

One O
of O
the O
major O
problems O
in O
understanding O
the O
mechanisms O
responsible O
for O
alternative O
pre O
- O
mRNA O
splicing O
is O
the O
manner O
in O
which O
tissue O
specificity O
is O
determined O
. O

In O
vertebrates O
, O
to O
date O
, O
only O
a O
few O
tissue O
- O
specific O
proteins O
have O
been O
identified O
as O
splicing O
regulators O
( O
8 O
- O
12 O
) O
. O

Here O
, O
we O
have O
shown O
that O
the O
tissue O
- O
dependent O
isoforms O
of O
the O
sequence O
- O
specific O
RNA O
- O
binding O
proteins O
, O
which O
themselves O
are O
generated O
by O
alternative O
splicing O
, O
have O
different O
activities O
in O
tissue O
- O
specific O
alternative O
splicing O
of O
target O
pre O
- O
mRNA O
. O

Therefore O
, O
these O
isoforms O
play O
a O
role O
in O
the O
determination O
of O
tissue O
specificity O
of O
target O
pre O
- O
mRNA O
splicing O
. O

The O
discovery O
of O
these O
tissue O
- O
dependent O
isoforms O
of O
the O
UGCAUG O
- O
binding O
proteins O
with O
different O
splicing O
activities O
adds O
an O
important O
new O
dimension O
to O
the O
molecular O
mechanisms O
responsible O
for O
regulating O
tissue O
- O
dependent O
alternative O
splicing O
mediated O
via O
UGCAUG O
. O

Mutations O
of O
PIK3CA O
in O
gastric O
adenocarcinoma O

Abstract O

Background O

Activation O
of O
the O
phosphatidylinositol O
3 O
- O
kinase O
( O
PI3K O
) O
through O
mutational O
inactivation O
of O
PTEN O
tumour O
suppressor O
gene O
is O
common O
in O
diverse O
cancer O
types O
, O
but O
rarely O
reported O
in O
gastric O
cancer O
. O

Recently O
, O
mutations O
in O
PIK3CA O
, O
which O
encodes O
the O
p110 O
alpha O
catalytic O
subunit O
of O
PI3K O
, O
have O
been O
identified O
in O
various O
human B
cancers O
, O
including O
3 O
of O
12 O
gastric O
cancers O
. O

Eighty O
percent O
of O
these O
reported O
mutations O
clustered O
within O
2 O
regions O
involving O
the O
helical O
and O
kinase O
domains O
. O

In O
vitro O
study O
on O
one O
of O
the O
" O
hot O
- O
spot O
" O
mutants O
has O
demonstrated O
it O
as O
an O
activating O
mutation O
. O

Methods O

Based O
on O
these O
data O
, O
we O
initiated O
PIK3CA O
mutation O
screening O
in O
94 O
human B
gastric O
cancers O
by O
direct O
sequencing O
of O
the O
gene O
regions O
in O
which O
80 O
% O
of O
all O
the O
known O
PIK3CA O
mutations O
were O
found O
. O

We O
also O
examined O
PIK3CA O
expression O
level O
by O
extracting O
data O
from O
the O
previous O
large O
- O
scale O
gene O
expression O
profiling O
study O
. O

Using O
Significance O
Analysis O
of O
Microarrays O
( O
SAM O
) O
, O
we O
further O
searched O
for O
genes O
that O
show O
correlating O
expression O
with O
PIK3CA O
. O

Results O

We O
have O
identified O
PIK3CA O
mutations O
in O
4 O
cases O
( O
4 O
. O
3 O
% O
) O
, O
all O
involving O
the O
previously O
reported O
hotspots O
. O

Among O
these O
4 O
cases O
, O
3 O
tumours O
demonstrated O
microsatellite O
instability O
and O
2 O
tumours O
harboured O
concurrent O
KRAS O
mutation O
. O

Data O
extracted O
from O
microarray O
studies O
showed O
an O
increased O
expression O
of O
PIK3CA O
in O
gastric O
cancers O
when O
compared O
with O
the O
non O
- O
neoplastic O
gastric O
mucosae O
( O
p O
< O
0 O
. O
001 O
) O
. O

SAM O
further O
identified O
2910 O
genes O
whose O
expression O
levels O
were O
positively O
associated O
with O
that O
of O
PIK3CA O
. O

Conclusion O

Our O
data O
suggested O
that O
activation O
of O
the O
PI3K O
signalling O
pathway O
in O
gastric O
cancer O
may O
be O
achieved O
through O
up O
- O
regulation O
or O
mutation O
of O
PIK3CA O
, O
in O
which O
the O
latter O
may O
be O
a O
consequence O
of O
mismatch O
repair O
deficiency O
. O

Background O

The O
phosphatidylinositol O
3 O
- O
kinase O
( O
PI3K O
) O
- O
AKT O
signalling O
pathway O
is O
involved O
in O
the O
regulation O
of O
diverse O
cellular O
processes O
, O
including O
cell O
growth O
, O
survival O
and O
motility O
. O

Abnormal O
activation O
of O
this O
pathway O
is O
frequently O
observed O
in O
various O
cancer O
types O
, O
leading O
to O
aberrant O
cell O
cycle O
progression O
, O
altered O
adhesion O
and O
motility O
, O
inhibition O
of O
apoptosis O
and O
induction O
of O
angiogenesis O
[ O
1 O
] O
. O

It O
has O
been O
previously O
reported O
that O
genetic O
alterations O
involving O
various O
members O
along O
this O
signalling O
pathway O
could O
lead O
to O
its O
activation O
in O
cancer O
. O

These O
include O
mutation O
, O
allelic O
loss O
or O
promoter O
methylation O
of O
the O
negative O
regulator O
PTEN O
[ O
2 O
] O
; O
or O
alternatively O
, O
chromosomal O
amplification O
or O
over O
- O
expression O
of O
the O
positive O
regulators O
PIK3CA O
[ O
3 O
- O
5 O
] O
and O
the O
various O
AKT O
kinases O
[ O
6 O
, O
7 O
] O
. O

Furthermore O
, O
changes O
in O
other O
related O
pathways O
that O
are O
commonly O
altered O
in O
cancer O
, O
such O
as O
those O
involved O
in O
growth O
factor O
stimulation O
via O
the O
G O
- O
protein O
- O
coupled O
receptors O
or O
through O
direct O
interaction O
with O
the O
activated O
form O
of O
small O
GTPase O
RAS O
, O
can O
also O
lead O
to O
PI3K O
- O
AKT O
pathway O
activation O
[ O
8 O
] O
. O

Activation O
of O
this O
pathway O
results O
in O
the O
phosphorylation O
of O
AKT O
at O
Thr O
- O
308 O
/ O
309 O
and O
Ser O
- O
473 O
/ O
474 O
. O

These O
phosphorylated O
forms O
of O
AKT O
proteins O
have O
been O
detected O
by O
Western O
blot O
or O
immunohistochemistry O
in O
various O
cancer O
types O
, O
suggesting O
the O
frequent O
activation O
of O
PI3K O
- O
AKT O
pathway O
in O
the O
carcinogenic O
process O
[ O
7 O
, O
9 O
] O
. O

Although O
genetic O
changes O
along O
the O
PI3K O
- O
AKT O
pathway O
have O
been O
repeatedly O
documented O
in O
brain O
, O
ovarian O
, O
endometrial O
, O
breast O
, O
prostate O
and O
thyroid O
cancers O
[ O
1 O
, O
2 O
] O
, O
reports O
on O
its O
mechanism O
of O
activation O
in O
gastric O
cancer O
are O
limited O
. O

Gastric O
cancer O
is O
the O
second O
most O
common O
cancer O
worldwide O
but O
its O
molecular O
basis O
of O
tumourigenesis O
is O
still O
poorly O
understood O
. O

Previous O
immunohistochemical O
study O
has O
demonstrated O
the O
presence O
of O
the O
phosphorylated O
form O
of O
AKT O
in O
78 O
% O
of O
gastric O
cancer O
[ O
10 O
] O
, O
suggesting O
that O
activation O
of O
this O
pathway O
may O
also O
be O
common O
in O
gastric O
cancer O
. O

Though O
loss O
of O
heterozygosity O
( O
LOH O
) O
involving O
the O
PTEN O
locus O
has O
been O
demonstrated O
in O
47 O
% O
of O
gastric O
cancer O
in O
a O
recent O
study O
, O
mutation O
or O
promoter O
methylation O
was O
absent O
even O
in O
cases O
with O
LOH O
[ O
11 O
] O
. O

Thus O
data O
from O
this O
study O
could O
not O
support O
the O
two O
- O
hit O
inactivation O
of O
PTEN O
in O
gastric O
cancer O
, O
while O
the O
biological O
significance O
of O
PTEN O
haploinsufficiency O
remains O
controversial O
. O

Alternatively O
, O
amplification O
of O
AKT1 O
has O
been O
reported O
in O
a O
single O
case O
of O
gastric O
cancer O
[ O
12 O
] O
, O
and O
amplification O
of O
PIK3CA O
associated O
with O
elevated O
mRNA O
levels O
has O
been O
found O
in O
36 O
% O
of O
gastric O
cancer O
[ O
11 O
] O
. O

More O
recently O
, O
Samuels O
et O
al O
. O
screened O
a O
diverse O
spectrum O
of O
human B
cancers O
for O
mutation O
in O
16 O
PI3K O
or O
PI3K O
- O
like O
genes O
and O
found O
a O
high O
frequency O
of O
somatic O
mutation O
in O
PIK3CA O
, O
which O
encodes O
the O
p110 O
alpha O
catalytic O
subunit O
. O

Major O
screening O
in O
colorectal O
cancer O
( O
CRC O
) O
identified O
PIK3CA O
mutations O
in O
74 O
out O
of O
234 O
( O
32 O
% O
) O
cases O
, O
while O
mutations O
were O
also O
noted O
in O
3 O
out O
of O
12 O
( O
25 O
% O
) O
gastric O
cancers O
. O

Reported O
mutations O
were O
mostly O
of O
missense O
type O
, O
and O
clustered O
within O
2 O
regions O
in O
the O
helical O
and O
kinase O
domains O
. O

Expression O
of O
a O
" O
hot O
- O
spot O
" O
mutant O
, O
H1047R O
, O
conferred O
a O
significant O
up O
- O
regulation O
of O
lipid O
kinase O
activity O
of O
PIK3CA O
, O
suggesting O
it O
as O
an O
activating O
mutation O
[ O
13 O
] O
. O

In O
this O
study O
, O
we O
have O
examined O
a O
series O
of O
94 O
human B
gastric O
adenocarcinomas O
for O
PIK3CA O
mutation O
. O

We O
have O
also O
examined O
PIK3CA O
expression O
level O
by O
extracting O
data O
from O
a O
large O
- O
scale O
gene O
expression O
profiling O
study O
previously O
performed O
for O
these O
cases O
[ O
14 O
, O
15 O
] O
. O

Using O
SAM O
, O
genes O
with O
significant O
correlating O
expression O
with O
PIK3CA O
have O
also O
been O
identified O
. O

Methods O

Patient B
samples O
preparation O

DNA O
samples O
used O
for O
sequencing O
were O
prepared O
from O
frozen O
tumour O
and O
non O
- O
tumour O
gastric O
mucosae O
from O
94 O
gastric O
cancer O
patients B
who O
underwent O
gastrectomy O
in O
the O
Department O
of O
Surgery O
, O
Queen O
Mary O
Hospital O
, O
The O
University O
of O
Hong O
Kong O
, O
as O
previously O
described O
[ O
16 O
] O
. O

Majority O
of O
the O
frozen O
samples O
( O
n O
= O
81 O
) O
showed O
tumour O
component O
of O
over O
70 O
% O
, O
whereas O
in O
13 O
cases O
a O
lower O
proportion O
between O
50 O
to O
70 O
% O
was O
accepted O
due O
to O
the O
tumours O
' O
inherent O
diffuse O
infiltrative O
nature O
with O
entrapment O
of O
non O
- O
neoplastic O
components O
. O

Analysis O
for O
microsatellite O
instability O
( O
MSI O
) O
, O
BRAF O
and O
KRAS O
mutation O
have O
been O
performed O
and O
reported O
previously O
[ O
16 O
] O
. O

RNA O
preparation O
and O
gene O
expression O
profiling O
using O
a O
cDNA O
microarray O
containing O
44 O
, O
500 O
cDNA O
clones O
, O
representing O
around O
30 O
, O
300 O
unique O
genes O
, O
has O
been O
performed O
and O
reported O
in O
90 O
of O
these O
tumours O
in O
comparison O
to O
22 O
non O
- O
tumour O
gastric O
mucosae O
[ O
14 O
, O
15 O
] O
. O

This O
study O
was O
approved O
by O
the O
Ethics O
Committee O
of O
the O
University O
of O
Hong O
Kong O
. O

Mutational O
screening O

Mutation O
screening O
of O
PIK3CA O
was O
performed O
for O
exons O
9 O
and O
20 O
, O
covering O
the O
mutational O
hotspots O
; O
and O
for O
exon O
18 O
, O
from O
which O
a O
mutation O
was O
found O
in O
a O
gastric O
cancer O
. O

Mutations O
in O
these O
3 O
exons O
constituted O
80 O
% O
of O
all O
PIK3CA O
mutations O
detected O
in O
the O
previous O
study O
[ O
13 O
] O
. O

PIK3CA O
intron O
- O
specific O
external O
amplification O
primers O
and O
internal O
sequencing O
primers O
were O
designed O
according O
to O
the O
previous O
study O
[ O
13 O
] O
with O
some O
modifications O
[ O
see O
Additional O
file O
1 O
] O
. O

In O
particular O
, O
primers O
for O
exon O
9 O
have O
been O
modified O
to O
avoid O
amplification O
of O
homologous O
sequences O
located O
in O
other O
chromosomes O
. O

PCR O
products O
were O
generated O
using O
the O
external O
primers O
and O
directly O
sequenced O
using O
the O
internal O
primers O
with O
the O
DYEnamic O
( O
TM O
) O
ET O
Terminator O
Cycle O
Sequencing O
Kit O
( O
Amersham O
Biosciences O
, O
Freiburg O
, O
Germany O
) O
according O
to O
the O
manufacturer O
' O
s O
instruction O
. O

Electrophoresis O
was O
performed O
in O
the O
ABI O
Prism O
( O
R O
) O
3700 O
DNA O
Analyzer O
( O
Applied O
Biosystems O
, O
Foster O
City O
, O
CA O
, O
USA O
) O
. O

For O
each O
exon O
, O
PCR O
products O
were O
generated O
from O
2 O
independent O
PCR O
reactions O
for O
sequencing O
of O
the O
forward O
and O
reverse O
strands O
. O

For O
exon O
9 O
, O
2 O
independent O
PCR O
followed O
by O
sequencing O
of O
the O
forward O
strand O
were O
performed O
. O

Analysis O
of O
the O
chromatograms O
was O
performed O
using O
the O
mutation O
analysis O
software O
Mutation O
Explorer O
( O
TM O
) O
( O
SoftGenetics O
, O
State O
College O
, O
PA O
, O
USA O
) O
. O

Extraction O
of O
expression O
data O
and O
statistical O
analysis O

Gene O
expression O
data O
were O
extracted O
from O
the O
microarray O
database O
containing O
126 O
samples O
( O
90 O
gastric O
cancers O
, O
14 O
lymph O
node O
metastasis O
and O
22 O
non O
- O
tumour O
gastric O
mucosae O
) O
based O
on O
a O
3 O
- O
fold O
signal O
above O
background O
ratio O
for O
either O
channel O
and O
with O
80 O
% O
good O
data O
[ O
14 O
] O
. O

Gene O
expression O
data O
from O
20 O
, O
336 O
cDNA O
clones O
satisfied O
this O
selection O
criteria O
and O
were O
extracted O
, O
which O
included O
a O
cDNA O
clone O
corresponding O
to O
PIK3CA O
( O
IMAGE O
clone O
number O
345430 O
, O
GenBank O
accession O
no O
. O
W72473 O
) O
. O

Expression O
data O
for O
PIK3CA O
was O
extracted O
and O
the O
differences O
in O
expression O
levels O
between O
tumour O
and O
non O
- O
tumour O
tissues O
were O
examined O
using O
the O
Student O
' O
s O
t O
- O
Test O
. O

SAM O
was O
performed O
to O
identify O
genes O
with O
significant O
correlating O
expression O
with O
PIK3CA O
[ O
17 O
] O
. O

The O
missing O
values O
in O
the O
dataset O
were O
estimated O
by O
a O
K O
- O
nearest O
neighbours O
impute O
algorithm O
using O
10 O
nearest O
neighbour O
[ O
18 O
] O
followed O
by O
5000 O
permutations O
in O
the O
SAM O
analysis O
. O

Results O

Among O
the O
94 O
gastric O
adenocarcinoma O
analysed O
, O
we O
have O
detected O
PIK3CA O
mutation O
in O
4 O
cases O
. O

Two O
cases O
harboured O
the O
mutation O
A3140G O
( O
H1047R O
) O
in O
exon O
20 O
, O
and O
the O
other O
2 O
cases O
with O
mutations O
G1624A O
( O
E542K O
) O
and O
G1633A O
( O
E545K O
) O
in O
exon O
9 O
. O

Representative O
sequence O
chromatograms O
are O
shown O
in O
figure O
1 O
. O

All O
four O
mutations O
were O
absent O
in O
the O
corresponding O
non O
- O
neoplastic O
mucosae O
and O
thus O
were O
confirmed O
as O
somatic O
mutations O
. O

Though O
the O
overall O
mutation O
frequency O
( O
4 O
. O
3 O
% O
) O
was O
lower O
than O
that O
of O
the O
previous O
study O
, O
the O
nature O
of O
the O
4 O
mutations O
found O
were O
consistent O
with O
those O
identified O
at O
the O
reported O
hotspots O
. O

In O
particular O
, O
the O
H1047R O
mutation O
has O
been O
reported O
in O
2 O
gastric O
cancers O
and O
15 O
colorectal O
cancers O
[ O
13 O
] O
. O

While O
the O
E542K O
and O
the O
E545K O
mutations O
were O
not O
found O
in O
gastric O
cancer O
in O
the O
previous O
series O
, O
a O
large O
number O
of O
colorectal O
tumours O
did O
harbour O
these O
2 O
mutations O
. O

PIK3CA O
mutation O
spectrum O
and O
their O
corresponding O
clinico O
- O
pathological O
features O
were O
listed O
in O
Table O
1 O
. O

We O
noted O
a O
higher O
tendency O
of O
high O
- O
level O
MSI O
in O
gastric O
cancers O
with O
PIK3CA O
mutations O
( O
3 O
in O
4 O
, O
75 O
% O
) O
than O
in O
those O
without O
( O
18 O
in O
90 O
, O
20 O
% O
) O
. O

Moreover O
, O
though O
the O
overall O
incidence O
of O
KRAS O
mutation O
in O
the O
studied O
population O
was O
low O
( O
8 O
in O
94 O
) O
, O
2 O
of O
the O
4 O
gastric O
cancers O
with O
PIK3CA O
mutation O
also O
harboured O
a O
KRAS O
mutation O
. O

Since O
over O
- O
expression O
of O
PIK3CA O
has O
been O
reported O
in O
gastric O
cancer O
[ O
11 O
] O
, O
we O
have O
also O
extracted O
PIK3CA O
expression O
data O
from O
our O
previous O
cDNA O
microarray O
study O
of O
these O
cases O
[ O
14 O
, O
15 O
] O
. O

We O
have O
confirmed O
that O
expression O
level O
of O
PIK3CA O
was O
significantly O
higher O
in O
gastric O
cancers O
( O
n O
= O
87 O
, O
mean O
= O
0 O
. O
099 O
, O
SD O
= O
0 O
. O
428 O
) O
when O
compared O
with O
non O
- O
neoplastic O
gastric O
mucosae O
( O
n O
= O
22 O
, O
mean O
= O
- O
0 O
. O
418 O
, O
SD O
= O
0 O
. O
426 O
; O
Student O
' O
s O
t O
- O
Test O
, O
p O
< O
0 O
. O
001 O
) O
. O

Using O
PIK3CA O
expression O
level O
as O
a O
continuous O
variable O
for O
SAM O
analysis O
[ O
17 O
] O
, O
we O
found O
2910 O
cDNA O
clones O
( O
corresponding O
to O
about O
2546 O
unique O
genes O
) O
whose O
expression O
associated O
positively O
with O
PIK3CA O
expression O
( O
median O
number O
of O
false O
significant O
= O
0 O
. O
372 O
, O
Delta O
= O
1 O
. O
107 O
) O
[ O
see O
Additional O
file O
2 O
] O
. O

Interestingly O
, O
no O
gene O
was O
found O
to O
be O
negatively O
associated O
with O
PIK3CA O
expression O
. O

Discussion O

In O
this O
study O
, O
we O
have O
reported O
the O
presence O
of O
PIK3CA O
gene O
mutation O
in O
4 O
. O
3 O
% O
of O
gastric O
cancer O
. O

A O
high O
tendency O
( O
3 O
in O
4 O
) O
of O
mismatch O
repair O
deficiency O
was O
noted O
in O
cases O
harbouring O
PIK3CA O
mutation O
. O

Though O
the O
small O
number O
of O
PIK3CA O
mutations O
in O
our O
study O
may O
not O
justify O
statistical O
claim O
of O
significance O
; O
suggestion O
of O
such O
, O
despite O
of O
its O
not O
being O
mentioned O
by O
the O
authors O
, O
can O
be O
found O
from O
a O
previous O
study O
in O
CRC O
by O
Samuels O
et O
al O
. O
. O

From O
their O
study O
of O
33 O
MSI O
and O
201 O
microsatellite O
stable O
( O
MSS O
) O
CRC O
cases O
, O
PIK3CA O
mutation O
was O
present O
in O
48 O
% O
of O
the O
MSI O
tumours O
, O
but O
only O
in O
29 O
% O
of O
the O
MSS O
tumours O
. O

A O
significant O
association O
would O
have O
been O
revealed O
if O
statistical O
analysis O
had O
been O
applied O
( O
Fisher O
' O
s O
exact O
test O
, O
p O
= O
0 O
. O
014 O
) O
[ O
13 O
] O
. O

Gastrointestinal O
tract O
cancers O
with O
MSI O
are O
known O
to O
have O
a O
different O
molecular O
pathway O
of O
tumour O
evolution O
compared O
with O
their O
MSS O
counterparts O
[ O
19 O
, O
20 O
] O
. O

This O
can O
be O
attributed O
to O
their O
propensity O
for O
frameshift O
mutations O
in O
repeat O
sequences O
, O
resulting O
in O
selective O
disruption O
of O
genes O
with O
such O
sequences O
within O
their O
coding O
regions O
. O

With O
2 O
poly O
- O
adenine O
tracts O
within O
its O
coding O
region O
, O
PTEN O
can O
be O
inactivated O
through O
frameshift O
mutations O
in O
MSI O
CRC O
, O
resulting O
in O
the O
selective O
targeting O
of O
the O
PI3K O
- O
AKT O
signalling O
pathway O
[ O
21 O
, O
22 O
] O
. O

It O
is O
also O
known O
that O
mismatch O
repair O
deficiency O
would O
lead O
to O
an O
elevated O
rate O
of O
missense O
mutation O
due O
to O
impaired O
single O
nucleotide O
mismatch O
repair O
[ O
23 O
] O
. O

Thus O
, O
the O
observed O
higher O
incidence O
of O
PIK3CA O
missense O
mutation O
in O
MSI O
colorectal O
and O
gastric O
cancers O
suggests O
yet O
another O
mechanism O
for O
the O
activation O
of O
the O
PI3K O
- O
AKT O
signalling O
pathway O
through O
mismatch O
repair O
deficiency O
. O

Our O
data O
also O
showed O
a O
higher O
tendency O
of O
KRAS O
mutation O
in O
cases O
with O
PIK3CA O
mutations O
( O
2 O
in O
4 O
) O
than O
in O
those O
without O
( O
6 O
in O
90 O
) O
. O

Yet O
again O
due O
to O
the O
low O
incidence O
of O
both O
mutations O
in O
our O
samples O
, O
statistical O
significance O
may O
not O
be O
claimed O
. O

In O
the O
study O
by O
Samuels O
et O
al O
. O
, O
some O
of O
the O
colorectal O
tumours O
with O
PIK3CA O
mutation O
also O
harboured O
KRAS O
or O
BRAF O
mutation O
[ O
13 O
] O
. O

The O
PI3K O
- O
AKT O
pathway O
is O
known O
to O
have O
a O
close O
association O
with O
the O
RAS O
- O
MEKK O
signalling O
pathway O
[ O
8 O
] O
. O

Constitutively O
active O
RAS O
can O
interact O
with O
the O
catalytic O
subunit O
of O
PI3K O
and O
lead O
to O
its O
activation O
. O

Ras O
- O
dependent O
PI3K O
activation O
contributes O
to O
the O
transforming O
phenotype O
by O
mediating O
anchorage O
- O
independent O
growth O
, O
cytoskeletal O
reorganisation O
and O
apoptosis O
evasion O
. O

It O
has O
been O
observed O
that O
genes O
involved O
in O
the O
same O
signalling O
pathway O
may O
manifest O
mutations O
in O
cancer O
cells O
in O
a O
mutually O
exclusive O
manner O
, O
presumably O
due O
to O
the O
lack O
of O
selective O
growth O
advantage O
in O
having O
a O
second O
hit O
in O
the O
already O
altered O
pathway O
. O

A O
prominent O
example O
is O
the O
mutually O
exclusive O
occurrence O
of O
the O
BRAF O
hotspot O
mutation O
( O
V600E O
) O
and O
KRAS O
mutations O
in O
colorectal O
cancer O
[ O
24 O
, O
25 O
] O
. O

However O
, O
there O
exist O
other O
examples O
of O
alterations O
in O
multiple O
components O
of O
the O
same O
signalling O
pathway O
that O
may O
lead O
to O
a O
multi O
- O
level O
modulation O
of O
its O
activity O
. O

For O
example O
, O
non O
- O
V600E O
BRAF O
mutations O
tend O
to O
occur O
together O
with O
KRAS O
mutations O
[ O
26 O
] O
, O
and O
inactivation O
of O
the O
secreted O
frizzled O
- O
related O
proteins O
( O
antagonists O
of O
WNT O
) O
by O
promoter O
methylation O
frequently O
coincides O
with O
mutations O
in O
the O
Adenomatous O
Polyposis I
Coli I
gene O
to O
achieve O
multi O
- O
level O
activation O
of O
the O
WNT O
signalling O
pathway O
in O
colorectal O
cancers O
[ O
27 O
] O
. O

Whether O
PIK3CA O
functions O
independently O
from O
RAS O
, O
or O
acts O
synergistically O
with O
RAS O
to O
produce O
additive O
effects O
on O
the O
activation O
of O
the O
same O
pathway O
awaits O
further O
clarification O
. O

By O
extracting O
data O
from O
microarray O
, O
we O
have O
confirmed O
the O
up O
- O
regulation O
of O
PIK3CA O
expression O
in O
gastric O
cancer O
tissues O
compared O
with O
the O
non O
- O
neoplastic O
gastric O
mucosae O
and O
identified O
a O
large O
number O
of O
genes O
that O
showed O
a O
significant O
positive O
correlation O
in O
expression O
level O
with O
PIK3CA O
. O

These O
genes O
participate O
in O
diverse O
cellular O
processes O
with O
177 O
as O
putative O
cell O
cycle O
- O
regulated O
genes O
[ O
28 O
] O
and O
126 O
mapped O
to O
genes O
with O
known O
functions O
in O
cell O
cycle O
regulation O
, O
cell O
proliferation O
or O
DNA O
replication O
[ O
see O
Additional O
file O
2 O
] O
. O

While O
some O
of O
these O
genes O
maybe O
induced O
by O
PIK3CA O
, O
others O
maybe O
co O
- O
ordinately O
regulated O
by O
common O
upstream O
signals O
. O

Expression O
data O
set O
at O
one O
point O
was O
limited O
in O
differentiating O
the O
above O
cause O
and O
consequence O
, O
yet O
it O
certainly O
revealed O
the O
complexity O
of O
the O
carcinogenic O
process O
and O
the O
intricate O
relationship O
of O
PIK3CA O
signalling O
with O
other O
cellular O
processes O
. O

Contrary O
to O
our O
expectation O
, O
the O
incidence O
of O
PIK3CA O
mutation O
found O
in O
the O
current O
study O
( O
4 O
% O
) O
is O
much O
lower O
compared O
with O
that O
observed O
by O
Samuel O
et O
al O
. O

( O
25 O
% O
) O
[ O
13 O
] O
. O

The O
reason O
for O
discrepancy O
may O
simply O
be O
a O
result O
of O
sample O
bias O
as O
the O
previous O
study O
involved O
only O
a O
small O
number O
of O
gastric O
cancers O
( O
n O
= O
12 O
) O
. O

However O
, O
ethnic O
differences O
can O
also O
be O
another O
possibility O
. O

The O
diverse O
pathological O
spectrum O
and O
aetiological O
factors O
of O
gastric O
cancers O
in O
different O
geographical O
locations O
may O
be O
paralleled O
by O
differences O
in O
molecular O
pathway O
of O
tumour O
development O
. O

Since O
our O
current O
study O
is O
only O
based O
on O
a O
Chinese O
population O
with O
an O
intermediate O
gastric O
cancer O
incidence O
, O
further O
studies O
involving O
patients B
from O
different O
ethnic O
groups O
will O
be O
able O
to O
address O
this O
possibility O
. O

Conclusion O

Large O
- O
scale O
screening O
of O
gastric O
adenocarcinomas O
for O
PIK3CA O
mutations O
revealed O
a O
mutation O
incidence O
of O
4 O
. O
3 O
% O
. O

Increased O
PIK3CA O
expression O
level O
was O
observed O
in O
gastric O
tumours O
compared O
with O
non O
- O
neoplastic O
mucosae O
. O

This O
increase O
in O
PIK3CA O
level O
was O
associated O
with O
the O
elevated O
expression O
of O
a O
large O
number O
of O
genes O
, O
which O
may O
constitute O
the O
upstream O
regulators O
or O
downstream O
targets O
of O
PIK3CA O
along O
the O
PI3K O
signalling O
pathway O
. O

Competing O
interests O

The O
author O
( O
s O
) O
declare O
that O
they O
have O
no O
competing O
interests O
. O

Authors O
' O
contributions O

VSWL O
carried O
out O
the O
molecular O
analysis O
, O
performed O
data O
analysis O
and O
drafted O
the O
manuscript O
. O

CWW O
, O
TLC O
, O
WZ O
assisted O
in O
the O
molecular O
analysis O
. O

KMC O
provided O
the O
clinical O
data O
. O

ASWC O
assisted O
in O
data O
analysis O
and O
edited O
the O
manuscript O
. O

SS O
and O
XC O
participated O
in O
the O
microarray O
study O
and O
data O
analysis O
. O

STY O
and O
SYL O
conceived O
of O
the O
study O
, O
participated O
in O
its O
design O
, O
coordination O
and O
data O
analysis O
, O
and O
edited O
the O
manuscript O
. O

All O
authors O
read O
and O
approved O
the O
final O
manuscript O
. O

Pre O
- O
publication O
history O

The O
pre O
- O
publication O
history O
for O
this O
paper O
can O
be O
accessed O
here O
: O

Supplementary O
Material O

Identification O
of O
kinectin O
as O
a O
novel O
Beh O
c O
et O
' O
s O
disease O
autoantigen O

Abstract O

There O
has O
been O
some O
evidence O
that O
Beh O
c O
et O
' O
s O
disease O
( O
BD O
) O
has O
a O
significant O
autoimmune O
component O
but O
the O
molecular O
identity O
of O
putative O
autoantigens O
has O
not O
been O
well O
characterized O
. O

In O
the O
initial O
analysis O
of O
the O
autoantibody O
profile O
in O
39 O
Chinese O
BD O
patients B
, O
autoantibodies O
to O
cellular O
proteins O
were O
uncovered O
in O
23 O
% O
as O
determined O
by O
immunoblotting O
. O

We O
have O
now O
identified O
one O
of O
the O
major O
autoantibody O
specificities O
using O
expression O
cloning O
. O

Serum O
from O
a O
BD O
patient B
was O
used O
as O
a O
probe O
to O
immunoscreen O
a O
lambda O
ZAP O
expression O
cDNA O
library O
. O

Candidate O
autoantigen O
cDNAs O
were O
characterized O
by O
direct O
nucleotide O
sequencing O
and O
their O
expressed O
products O
were O
examined O
for O
reactivity O
to O
the O
entire O
panel O
of O
BD O
sera O
using O
immunoprecipitation O
. O

Reactivity O
was O
also O
examined O
with O
normal O
control O
sera O
and O
disease O
control O
sera O
from O
patients B
with O
lupus O
and O
Sj O
o O
gren O
' O
s O
syndrome O
. O

Six O
independent O
candidate O
clones O
were O
isolated O
from O
the O
cDNA O
library O
screen O
and O
were O
identified O
as O
overlapping O
partial O
human B
kinectin O
cDNAs O
. O

The O
finding O
that O
kinectin O
was O
an O
autoantigen O
was O
verified O
in O
9 O
out O
of O
39 O
( O
23 O
% O
) O
BD O
patient B
sera O
by O
immunoprecipitation O
of O
the O
in O
vitro O
translation O
products O
. O

Sera O
from O
controls O
showed O
no O
reactivity O
. O

The O
significance O
of O
kinectin O
as O
a O
participant B
in O
autoimmune O
pathogenesis O
in O
BD O
and O
the O
potential O
use O
of O
autoantibody O
to O
kinectin O
in O
serodiagnostics O
are O
discussed O
. O

Introduction O

Beh O
c O
et O
' O
s O
disease O
( O
BD O
) O
is O
a O
systemic O
vasculitic O
disease O
typified O
by O
a O
triad O
of O
symptoms O
including O
recurrent O
oral O
ulcers O
, O
genital O
ulcers O
and O
uveitis O
. O

In O
addition O
, O
skin O
, O
joint O
, O
large O
vessels O
, O
nervous O
system O
and O
gastrointestinal O
systems O
may O
be O
involved O
. O

BD O
is O
a O
global O
disease O
but O
has O
the O
highest O
prevalence O
in O
the O
region O
along O
the O
ancient O
' O
Silk O
Road O
' O
in O
China O
. O

The O
etiopathogenesis O
of O
the O
disease O
remains O
unclear O
but O
microbial O
agent O
triggers O
, O
environmental O
factors O
, O
genetic O
predisposition O
, O
neutrophil O
hyperfunction O
, O
endothelial O
cell O
dysfunction O
and O
immunological O
abnormalities O
involving O
both O
T O
and O
B O
cells O
have O
been O
implicated O
. O

Increasing O
amounts O
of O
research O
evidence O
supports O
the O
possibility O
that O
it O
is O
an O
immune O
- O
mediated O
vasculitis O
, O
and O
that O
abnormal O
T O
- O
cell O
and O
B O
- O
cell O
reactions O
and O
autoantigen O
- O
driven O
autoimmunity O
play O
pivotal O
roles O
[ O
1 O
] O
. O

Systemic O
lupus O
erythematosus O
( O
SLE O
) O
is O
the O
prototypic O
systemic O
autoimmune O
rheumatic O
disease O
with O
autoantibodies O
against O
cellular O
( O
particularly O
nuclear O
) O
antigens O
, O
some O
of O
which O
are O
critically O
implicated O
in O
the O
autoimmune O
pathology O
while O
others O
provide O
valuable O
serodiagnostic O
markers O
for O
the O
disease O
. O

Unlike O
the O
picture O
in O
SLE O
and O
other O
related O
rheumatic O
diseases O
, O
in O
BD O
, O
antinuclear O
antibodies O
and O
antibodies O
to O
neutrophil O
cytoplasmic O
antigens O
etc O
. O
are O
not O
present O
. O

To O
date O
, O
since O
neither O
a O
specific O
autoantibody O
nor O
pathognomonic O
pathological O
index O
is O
available O
to O
help O
establish O
the O
diagnosis O
of O
BD O
, O
it O
is O
largely O
or O
solely O
based O
on O
clinical O
manifestations O
[ O
2 O
] O
, O
and O
a O
dilemma O
in O
diagnosis O
is O
not O
a O
rare O
occurrence O
in O
clinical O
practice O
. O

Nevertheless O
, O
since O
the O
1960s O
, O
there O
have O
been O
reports O
of O
autoantibodies O
against O
certain O
unknown O
components O
of O
human B
oral O
mucosa O
in O
sera O
of O
patients B
with O
BD O
. O

Since O
then O
, O
sporadic O
reports O
on O
findings O
of O
autoantibodies O
in O
this O
disease O
have O
been O
described O
, O
such O
as O
antibodies O
to O
retinal O
antigen O
( O
s O
) O
, O
heat O
shock O
protein O
( O
HSP O
) O
of O
some O
strains O
of O
Streptococcus B
sanguis I
cross O
- O
reactive O
with O
human B
HSP O
polypeptide O
[ O
3 O
] O
, O
antibodies O
to O
endothelial O
cell O
antigens O
( O
AECA O
) O
and O
antibodies O
to O
alpha O
- O
tropomyosin O
[ O
4 O
, O
5 O
] O
, O
attesting O
to O
the O
complicated O
humoral O
immune O
disorders O
in O
this O
disease O
. O

This O
investigation O
was O
aimed O
at O
defining O
target O
cellular O
autoantigens O
using O
time O
- O
tested O
and O
well O
- O
established O
molecular O
techniques O
. O

Immunoscreening O
of O
expression O
libraries O
using O
BD O
sera O
was O
used O
since O
this O
approach O
has O
been O
successfully O
employed O
in O
the O
characterization O
of O
many O
clinically O
relevant O
antigens O
in O
systemic O
rheumatic O
diseases O
such O
as O
SS O
- O
A O
/ O
Ro O
[ O
6 O
- O
9 O
] O
and O
SS O
- O
B O
/ O
La O
[ O
10 O
] O
antigens O
in O
Sj O
o O
gren O
' O
s O
syndrome O
( O
SjS O
) O
and O
centromere O
antigen O
CENP O
- O
B O
[ O
11 O
] O
in O
scleroderma O
. O

In O
addition O
, O
we O
have O
been O
successful O
in O
using O
this O
strategy O
to O
identify O
interesting O
autoantigens O
that O
have O
other O
biological O
significance O
. O

Examples O
of O
these O
include O
NOR90 O
/ O
hUBF O
[ O
12 O
] O
, O
p80 O
- O
coilin O
[ O
13 O
] O
, O
Golgi O
autoantigens O
[ O
14 O
- O
16 O
] O
and O
, O
more O
recently O
, O
GW182 O
[ O
17 O
] O
. O

Materials O
and O
methods O

Patients B
and O
sera O

The O
currently O
used O
empirical O
criteria O
for O
the O
diagnosis O
of O
BD O
in O
this O
study O
were O
the O
criteria O
proposed O
by O
the O
International O
Study O
Group O
for O
BD O
( O
abbreviated O
as O
' O
International O
Criteria O
' O
) O
[ O
2 O
] O
. O

The O
study O
subjects O
of O
39 O
Chinese O
BD O
patients B
comprised O
17 O
males O
and O
22 O
females O
, O
mean O
age O
37 O
+ O
/ O
- O
11 O
. O
3 O
years O
old O
, O
who O
were O
divided O
into O
two O
subgroups O
: O
25 O
typical O
BD O
patients B
( O
Group O
I O
, O
satisfying O
the O
International O
Criteria O
) O
and O
14 O
clinically O
diagnosed O
BD O
patients B
who O
had O
recurrent O
oral O
ulcers O
and O
one O
of O
the O
symptoms O
of O
genital O
ulcers O
, O
eye O
symptoms O
or O
skin O
lesions O
as O
defined O
by O
the O
International O
Criteria O
, O
as O
well O
as O
additional O
symptom O
( O
s O
) O
closely O
related O
to O
BD O
as O
listed O
in O
the O
International O
Criteria O
, O
that O
is O
, O

gastrointestinal O
ulcerations O
, O
deep O
vein O
thrombosis O
or O
arthralgia O
/ O
arthritis O
without O
evidence O
that O
the O
latter O
symptoms O
might O
be O
related O
to O
any O
other O
disease O
( O
Group O
II O
, O
defined O
as O
' O
probable O
BD O
' O
in O
this O
study O
) O
. O

Disease O
controls O
included O
10 O
patients B
with O
SLE O
and O
10 O
with O
SjS O
, O
all O
satisfying O
corresponding O
international O
classification O
criteria O
. O

All O
BD O
patients B
and O
disease O
controls O
involved O
in O
the O
study O
were O
patients B
treated O
at O
the O
Rheumatology O
Department O
of O
Ren O
Ji O
Hospital O
, O
Shanghai O
, O
China O
, O
where O
their O
clinical O
data O
and O
serum O
samples O
were O
collected O
. O

Twenty O
normal O
control O
sera O
were O
randomly O
selected O
from O
healthy O
blood O
donors O
working O
in O
the O
same O
hospital O
. O

This O
study O
was O
approved O
by O
the O
institution O
review O
board O
of O
Ren O
Ji O
Hospital O
which O
is O
affiliated O
with O
Shanghai O
Second O
Medical O
University O
, O
and O
each O
patient B
involved O
gave O
informed O
consent O
. O

All O
serum O
samples O
were O
preserved O
at O
- O
20 O
degrees O
C O
or O
- O
70 O
degrees O
C O
until O
use O
. O

Cell O
lines O
and O
cell O
extracts O

HeLa O
( O
ATCC O
CCL O
2 O
. O
2 O
) O
and O
T24 O
( O
human B
transitional O
cell O
bladder O
carcinoma O
) O
were O
obtained O
from O
the O
American O
Type O
Culture O
Collection O
( O
Rockville O
, O
MD O
, O
USA O
) O
. O

A O
bovine B
aortic O
endothelial O
cell O
line O
was O
kindly O
provided O
by O
Dr O
Eugene O
G O
Levin O
from O
the O
Scripps O
Research O
Institute O
( O
La O
Jolla O
, O
CA O
, O
USA O
) O
. O

Cells O
were O
cultured O
in O
DMEM O
containing O
10 O
% O
calf B
serum O
, O
harvested O
and O
extracted O
in O
Buffer O
A O
( O
150 O
mM O
NaCl O
, O
10 O
mM O
Tris O
- O
HCl O
, O
pH7 O
. O
2 O
, O
0 O
. O
5 O
% O
Nonidet O
P O
- O
40 O
) O
with O
protease O
inhibitor O
( O
Complete O
( O
TM O
) O
; O
Boehringer O
Mannheim O
, O
Indianapolis O
, O
IN O
, O
USA O
) O
. O

For O
the O
preparation O
of O
whole O
cell O
extract O
, O
10 O
volumes O
of O
Laemmli O
gel O
sample O
buffer O
[ O
18 O
] O
were O
added O
to O
the O
cell O
pellet O
, O
boiled O
for O
3 O
min O
and O
stored O
at O
- O
20 O
degrees O
C O
until O
use O
. O

Western O
blot O

Whole O
cell O
lysates O
from O
bovine B
aortic O
endothelial O
cell O
, O
HeLa O
and O
T24 O
cells O
were O
resolved O
individually O
by O
discontinuous O
7 O
. O
5 O
% O
gel O
SDS O
- O
PAGE O
according O
to O
Laemmli O
' O
s O
method O
[ O
18 O
] O
. O

Immunoblotting O
was O
performed O
as O
described O
by O
Towbin O
et O
al O
. O

[ O
19 O
] O
with O
modifications O
. O

Nitrocellulose O
strips O
were O
blocked O
with O
3 O
% O
nonfat O
milk O
in O
PBS O
containing O
0 O
. O
05 O
% O
Tween O
- O
20 O
( O
PBS O
- O
T O
) O
and O
then O
incubated O
with O
BD O
patient B
sera O
and O
normal O
control O
sera O
( O
1 O
: O
100 O
dilution O
) O
at O
room O
temperature O
for O
1 O
h O
. O

Filters O
were O
washed O
extensively O
with O
PBS O
- O
T O
to O
remove O
any O
unbound O
antibodies O
. O

Bound O
antibodies O
were O
detected O
with O
polyvalent O
, O
peroxidase O
- O
conjugated O
goat B
anti O
- O
human B
Ig O
and O
visualized O
by O
incubating O
the O
nitrocellulose O
strips O
in O
chemiluminescent O
reagents O
( O
NEN O
Life O
Science O
Products O
Inc O
. O
, O
Boston O
, O
MA O
, O
USA O
) O
and O
exposing O
to O
Kodak O
XAR O
- O
5 O
films O
. O

Screening O
of O
phage O
cDNA O
expression O
library O
with O
antibody O
probes O

Serum O
from O
a O
BD O
patient B
showing O
the O
highest O
antibody O
titer O
in O
immunoblotting O
was O
selected O
as O
a O
probe O
and O
used O
at O
a O
dilution O
of O
1 O
: O
300 O
for O
initial O
immunoscreening O
of O
approximately O
106 O
recombinants O
from O
a O
T24 O
cDNA O
expression O
library O
. O

The O
latter O
was O
constructed O
in O
lambda O
ZAPExpress O
vector O
( O
Stratagene O
, O
La O
Jolla O
, O
CA O
, O
USA O
) O
and O
screened O
as O
previously O
described O
[ O
20 O
- O
22 O
] O
. O

All O
screenings O
were O
performed O
on O
duplicate O
isopropyl O
beta O
- O
D O
- O
thiogalactoside O
( O
IPTG O
) O
pre O
- O
impregnated O
nitrocellulose O
filters O
, O
and O
immunoreactive O
clones O
were O
detected O
by O
chemiluminescence O
. O

Positive O
phages O
were O
subsequently O
plaque O
purified O
to O
100 O
% O
by O
two O
repeated O
rounds O
of O
screening O
at O
low O
plaque O
densities O
. O

Before O
screening O
the O
cDNA O
library O
, O
the O
BD O
serum O
was O
extensively O
adsorbed O
against O
bacteria O
and O
wild O
- O
type O
lambda O
ZAP O
phage O
mixture O
to O
reduce O
background O
binding O
. O

Analysis O
of O
candidate O
cDNAs O

Purified O
candidate O
plaques O
were O
subcloned O
in O
vivo O
into O
pBK O
- O
CMV O
plasmids O
using O
ExAssist O
( O
TM O
) O
helper O
phage O
as O
recommended O
in O
the O
manufacturer O
' O
s O
instructions O
( O
Stratagene O
) O
. O

The O
recombinant O
pBK O
- O
CMV O
plasmids O
were O
then O
purified O
using O
QIAprep O
Spin O
Minprep O
Kit O
( O
Qiagen O
, O
Valencia O
, O
CA O
, O
USA O
) O
. O

Restriction O
enzyme O
digestion O
of O
plasmids O
with O
EcoRI O
and O
XhoI O
and O
electrophoresis O
in O
a O
standard O
1 O
. O
0 O
% O
agarose O
gel O
was O
used O
to O
analyze O
the O
length O
of O
cDNA O
insert O
of O
each O
candidate O
plasmid O
. O

The O
complete O
nucleotide O
sequence O
was O
determined O
using O
Bigdye O
terminator O
sequencing O
and O
a O
semi O
- O
automated O
sequencer O
model O
377 O
( O
ABI O
, O
Foster O
City O
, O
CA O
, O
USA O
) O
. O

Both O
nucleotide O
and O
deduced O
amino O
acid O
sequences O
were O
analyzed O
for O
similarity O
with O
known O
sequences O
using O
BLAST O
search O
[ O
23 O
] O
and O
ExPASy O
Proteomics O
tools O
. O

Secondary O
structure O
analysis O
for O
coiled O
- O
coil O
motifs O
was O
conducted O
with O
the O
software O
program O
COILS O
[ O
24 O
] O
. O

Immunoprecipitation O
of O
in O
vitro O
translation O
products O

Candidate O
cDNA O
clones O
were O
used O
as O
templates O
for O
in O
vitro O
transcription O
and O
translation O
and O
the O
products O
were O
used O
as O
substrates O
for O
immunoprecipitation O
to O
confirm O
the O
specificity O
of O
reaction O
with O
BD O
sera O
. O

In O
brief O
, O
1 O
mu O
g O
of O
the O
pBK O
- O
CMV O
plasmid O
identified O
in O
the O
screening O
outlined O
above O
was O
added O
as O
template O
in O
a O
50 O
- O
mu O
l O
reaction O
for O
the O
coupled O
in O
vitro O
transcription O
and O
translation O
reaction O
with O
a O
rabbit B
reticulocyte O
lysate O
system O
( O
Promega O
, O
Madison O
, O
WI O
, O
USA O
) O
in O
the O
presence O
of O
35S O
- O
methionine O
( O
Trans O
- O
35S O
label O
; O
ICN O
Biochemicals O
, O
Costa O
Mesa O
, O
CA O
, O
USA O
) O
and O
RNasin O
( O
R O
) O
Ribonuclease O
Inhibitor O
( O

Stratagene O
) O
as O
recommended O
by O
the O
manufacturer O
( O
Promega O
) O
. O

Translation O
was O
carried O
out O
at O
30 O
degrees O
C O
for O
1 O
. O
5 O
h O
. O

Products O
were O
analyzed O
in O
a O
12 O
. O
5 O
% O
gel O
SDS O
- O
PAGE O
and O
stored O
at O
- O
80 O
degrees O
C O
for O
further O
immunoprecipitation O
analysis O
. O

The O
in O
vitro O
translation O
proteins O
were O
examined O
for O
reactivity O
by O
sera O
using O
immunoprecipitation O
described O
[ O
8 O
, O
25 O
] O
. O

Results O
and O
discussion O

Autoantibody O
detection O
in O
sera O
from O
BD O
patients B

Initial O
examination O
of O
a O
group O
of O
39 O
BD O
patients B
using O
indirect O
immunofluorescence O
( O
IIF O
) O
on O
a O
HEp O
- O
2 O
cell O
substrate O
did O
not O
yield O
any O
characteristic O
nuclear O
or O
cytoplasmic O
staining O
patterns O
. O

BD O
is O
thought O
by O
some O
to O
be O
a O
vasculitic O
disease O
involving O
pathophysiology O
of O
endothelial O
cells O
, O
and O
antibody O
to O
endothelial O
cell O
antigen O
( O
AECA O
) O
has O
been O
reported O
. O

Reports O
on O
the O
prevalence O
of O
AECA O
have O
varied O
largely O
and O
alpha O
- O
enolase O
was O
reported O
as O
one O
of O
the O
putative O
target O
antigens O
[ O
26 O
] O
. O

In O
this O
study O
, O
the O
use O
of O
bovine B
aortic O
endothelial O
cells O
as O
substrate O
for O
IIF O
did O
not O
provide O
any O
additional O
data O
. O

However O
, O
Western O
blot O
analysis O
of O
the O
BD O
sera O
began O
to O
show O
some O
interesting O
autoreactivity O
using O
cell O
lysates O
from O
both O
HeLa O
and O
bovine B
aortic O
endothelial O
cells O
. O

HeLa O
cells O
were O
initially O
used O
for O
this O
analysis O
because O
they O
are O
commonly O
used O
in O
the O
laboratory O
as O
Western O
blot O
substrate O
. O

Fig O
. O

1 O
illustrates O
the O
common O
reactivity O
to O
49 O
kDa O
and O
120 O
kDa O
proteins O
in O
the O
endothelial O
cell O
lysates O
. O

These O
antigens O
were O
also O
detected O
in O
HeLa O
and O
T24 O
cells O
; O
the O
latter O
cell O
line O
was O
analyzed O
because O
our O
laboratory O
at O
The O
Scripps O
Research O
Institute O
has O
produced O
an O
excellent O
expression O
cDNA O
library O
from O
the O
T24 O
line O
and O
the O
positive O
result O
with O
the O
T24 O
cell O
extracts O
allowed O
us O
to O
screen O
the O
T24 O
library O
. O

Ig O
isotype O
analysis O
showed O
that O
all O
reactivity O
was O
largely O
IgG O
antibodies O
. O

Since O
the O
49 O
kDa O
and O
120 O
kDa O
bands O
were O
observed O
in O
cell O
extracts O
from O
bovine B
as O
well O
as O
human B
cell O
lines O
, O
these O
autoantigens O
might O
be O
evolutionarily O
conserved O
. O

In O
total O
, O
nine O
out O
of O
39 O
BD O
sera O
( O
23 O
% O
) O
had O
autoantibody O
to O
the O
49 O
kDa O
antigen O
and O
eight O
( O
20 O
% O
) O
to O
the O
120 O
kDa O
antigen O
. O

Four O
BD O
sera O
( O
10 O
% O
) O
reacted O
with O
both O
proteins O
. O

Additionally O
, O
sera O
that O
showed O
common O
reactivity O
to O
the O
120 O
kDa O
protein O
also O
demonstrated O
a O
common O
band O
that O
migrated O
at O
~ O
150 O
kDa O
, O
although O
it O
appeared O
weaker O
than O
the O
120 O
kDa O
band O
. O

These O
antigens O
appeared O
to O
have O
different O
molecular O
weights O
than O
those O
of O
the O
known O
autoantigens O
in O
systemic O
rheumatic O
diseases O
. O

In O
addition O
, O
other O
reactive O
bands O
were O
detected O
but O
they O
were O
not O
as O
commonly O
shared O
as O
the O
49 O
kDa O
and O
120 O
kDa O
bands O
. O

The O
49 O
kDa O
protein O
was O
shown O
to O
be O
distinct O
from O
48 O
kDa O
SS O
- O
B O
/ O
La O
or O
50 O
kDa O
Jo O
- O
1 O
proteins O
( O
Fig O
. O
1 O
) O
. O

The O
120 O
kDa O
antigen O
was O
also O
shown O
to O
migrate O
differently O
from O
alanyl O
tRNA O
synthetase O
in O
another O
Western O
blot O
analysis O
( O
data O
not O
shown O
) O
and O
did O
not O
share O
any O
apparent O
crossreactive O
epitopes O
with O
the O
49 O
kDa O
antigen O
. O

Western O
blot O
analyses O
of O
20 O
normal O
control O
sera O
did O
not O
show O
the O
reactivities O
observed O
with O
BD O
sera O
. O

In O
order O
to O
further O
characterize O
these O
autoreactivities O
, O
a O
serum O
sample O
from O
the O
Group O
I O
definitive O
BD O
patients B
with O
the O
strongest O
reactivity O
to O
49 O
kDa O
and O
120 O
kDa O
antigens O
( O
Fig O
. O
1 O
, O
lane O
2 O
) O
was O
selected O
as O
antibody O
probe O
for O
expression O
library O
screening O
. O

Kinectin O
identified O
as O
a O
novel O
BD O
autoantigen O

After O
screening O
500 O
, O
000 O
clones O
from O
the O
T24 O
cell O
lambda O
ZAPExpress O
expression O
library O
, O
seven O
immunoreactive O
clones O
were O
isolated O
and O
plaque O
purified O
in O
two O
to O
three O
rounds O
to O
achieve O
100 O
% O
homogeneity O
. O

The O
cDNA O
inserts O
were O
subcloned O
in O
vivo O
into O
pBK O
- O
CMV O
plasmids O
, O
analyzed O
by O
restriction O
digestion O
using O
EcoRI O
and O
XhoI O
enzymes O
, O
and O
submitted O
to O
direct O
nucleotide O
sequencing O
across O
the O
polylinker O
arms O
. O

The O
cDNA O
inserts O
represented O
six O
independent O
clones O
designated O
BD41 O
( O
identical O
to O
BD44 O
) O
, O
BD481 O
, O
BD42 O
, O
BD47 O
, O
BD482 O
and O
BD49 O
. O

Their O
identities O
were O
established O
as O
overlapping O
partial O
cDNAs O
of O
human B
kinectin O
, O
ranging O
from O
1 O
. O
9 O
kb O
to O
3 O
kb O
( O
Fig O
. O
2a O
) O
. O

The O
full O
- O
length O
human B
kinectin O
( O
GenBank O
accession O
number O
NM O
_ O
182926 O
[ O
27 O
] O
) O
has O
4 O
, O
816 O
bases O
containing O
an O
open O
reading O
frame O
coding O
1 O
, O
357 O
amino O
acid O
residues O
with O
molecular O
mass O
156 O
kDa O
. O

All O
six O
cDNAs O
lacked O
the O
5 O
' O
portion O
of O
the O
kinectin O
sequence O
to O
different O
degrees O
but O
spanned O
a O
sequence O
of O
kinectin O
that O
extended O
to O
the O
3 O
' O
- O
untranslated O
region O
. O

Secondary O
structure O
analysis O
of O
kinectin O
protein O
using O
the O
program O
COILS O
identified O
a O
long O
region O
of O
alpha O
- O
helical O
coiled O
- O
coil O
domain O
that O
extended O
from O
amino O
acid O
residue O
327 O
to O
the O
C O
- O
terminus O
( O
Fig O
. O
2a O
, O
hatched O
boxes O
) O
. O

In O
vitro O
coupled O
transcription O
and O
translation O
of O
BD44 O
and O
BD42 O
clones O
directed O
the O
synthesis O
of O
[ O
35S O
] O
- O
methionine O
- O
labeled O
polypeptides O
that O
migrated O
at O
95 O
and O
60 O
kDa O
, O
respectively O
, O
in O
addition O
to O
smaller O
polypeptides O
( O
Fig O
. O
2b O
) O
. O

These O
products O
had O
predicted O
molecular O
weights O
of O
103 O
kDa O
and O
75 O
kDa O
. O

Kinectin O
was O
initially O
identified O
in O
chick B
embryo O
brain O
microsome O
as O
an O
integral O
membrane O
protein O
anchored O
in O
endoplasmic O
reticulum O
and O
involved O
in O
kinesin O
- O
driven O
vesicle O
motility O
along O
microtubules O
[ O
28 O
, O
29 O
] O
. O

Kinectin O
consists O
of O
a O
120 O
- O
kDa O
and O
a O
160 O
- O
kDa O
polypeptide O
interacting O
through O
the O
alpha O
- O
helical O
coiled O
- O
coil O
domain O
to O
form O
a O
heterodimer O
[ O
30 O
] O
. O

The O
full O
- O
length O
kinectin O
is O
the O
160 O
kDa O
polypeptide O
containing O
an O
N O
- O
terminal O
transmembrane O
helix O
followed O
by O
a O
bipartite O
nuclear O
localization O
sequence O
and O
two O
C O
- O
terminal O
leucine O
zipper O
motifs O
. O

We O
presume O
that O
the O
120 O
kDa O
polypeptide O
detected O
in O
Western O
blot O
( O
Fig O
. O
1 O
) O
is O
the O
truncated O
version O
of O
the O
160 O
- O
kDa O
polypeptide O
, O
lacking O
the O
N O
- O
terminal O
first O
232 O
amino O
acids O
[ O
30 O
] O
. O

The O
N O
- O
terminus O
of O
the O
160 O
- O
kDa O
polypeptide O
consists O
of O
a O
transmembrane O
domain O
that O
anchors O
kinectin O
to O
endoplasmic O
reticulum O
[ O
30 O
, O
31 O
] O
. O

This O
120 O
kDa O
polypeptide O
is O
probably O
the O
predominant O
form O
detected O
in O
the O
Western O
blot O
analysis O
( O
Fig O
. O
1 O
) O
because O
of O
its O
preferential O
solubility O
due O
to O
the O
omission O
of O
the O
N O
- O
terminal O
transmembrane O
domain O
. O

Other O
functions O
for O
kinectin O
have O
been O
reported O
. O

Yeast B
two O
- O
hybrid O
screen O
studies O
from O
several O
laboratories O
have O
revealed O
the O
interaction O
of O
the O
Rho O
family O
of O
GTPase O
with O
kinectin O
, O
and O
have O
shown O
the O
functional O
links O
among O
RhoG O
, O
kinectin O
and O
kinesin O
, O
with O
kinectin O
as O
a O
key O
effector O
of O
RhoG O
microtubule O
- O
dependent O
cellular O
activity O
[ O
32 O
] O
. O

Kinectin O
was O
also O
identified O
as O
an O
important O
constituent O
of O
integrin O
- O
based O
adhesion O
complexes O
, O
which O
link O
integrins O
to O
the O
cytoskeleton O
and O
recruit O
signaling O
molecules O
[ O
33 O
] O
. O

A O
new O
study O
reported O
that O
a O
kinectin O
isoform O
lacking O
a O
major O
portion O
of O
the O
kinesin O
- O
binding O
domain O
is O
very O
probably O
the O
most O
conservative O
form O
of O
kinectin O
; O
it O
does O
not O
bind O
kinesin O
but O
act O
as O
a O
membrane O
anchor O
for O
the O
translation O
elongation O
factor O
- O
1 O
delta O
in O
the O
endoplasmic O
reticulum O
[ O
34 O
] O
. O

Prevalence O
and O
specificity O
of O
anti O
- O
kinectin O
autoantibodies O

The O
in O
vitro O
[ O
35S O
] O
- O
methionine O
- O
labeled O
translation O
product O
of O
BD44 O
, O
representing O
the O
largest O
recombinant O
kinectin O
fragment O
available O
, O
was O
used O
as O
the O
antigen O
substrate O
in O
an O
immunoprecipitation O
assay O
. O

Out O
of O
39 O
BD O
patient B
sera O
, O
nine O
( O
23 O
% O
) O
recognized O
the O
BD44 O
translation O
product O
( O
Fig O
. O
3 O
) O
, O
whereas O
sera O
from O
20 O
normal O
controls O
, O
10 O
SLE O
and O
10 O
SjS O
patients B
did O
not O
show O
reactivity O
. O

Among O
the O
nine O
anti O
- O
kinectin O
positive O
patients B
, O
six O
( O
6 O
/ O
25 O
, O
24 O
% O
) O
were O
from O
Group O
I O
( O
definitive O
BD O
) O
including O
the O
BD O
patient B
whose O
serum O
was O
used O
in O
the O
immunoscreening O
of O
expression O
cDNA O
library O
, O
and O
three O
( O
3 O
/ O
14 O
, O
21 O
. O
4 O
% O
) O
patients B
were O
from O
the O
Group O
II O
( O
probable O
BD O
) O
in O
this O
study O
. O

According O
to O
the O
Fisher O
Exact O
Probability O
calculation O
( O
P O
= O
1 O
. O
00 O
) O
, O
there O
is O
no O
statistically O
significant O
difference O
for O
antibody O
to O
kinectin O
between O
the O
two O
groups O
. O

The O
combined O
data O
substantiated O
the O
finding O
that O
kinectin O
is O
an O
autoantigen O
that O
can O
be O
recognized O
by O
sera O
from O
23 O
% O
of O
Chinese O
BD O
patients B
in O
this O
study O
with O
at O
least O
one O
immunoreactive O
region O
or O
autoepitope O
residing O
within O
the O
BD44 O
encoded O
polypeptide O
. O

Currently O
, O
there O
are O
more O
than O
six O
diagnostic O
/ O
classification O
criteria O
for O
BD O
, O
among O
which O
the O
International O
Criteria O
have O
been O
applied O
most O
extensively O
due O
to O
its O
relatively O
high O
sensitivity O
( O
91 O
% O
) O
and O
specificity O
( O
96 O
% O
) O
[ O
2 O
] O
. O

As O
discussed O
above O
, O
differential O
diagnosis O
of O
BD O
might O
be O
confusing O
in O
clinical O
practice O
since O
no O
specific O
laboratory O
test O
is O
available O
, O
and O
some O
patients B
may O
have O
symptoms O
and O
signs O
strongly O
suggestive O
of O
BD O
but O
do O
not O
fully O
satisfy O
the O
International O
Criteria O
, O
as O
in O
the O
Group O
II O
( O
probable O
BD O
) O
patients B
in O
our O
study O
group O
. O

A O
number O
of O
investigators O
have O
pointed O
out O
that O
a O
comprehensive O
analysis O
of O
the O
clinical O
data O
for O
a O
given O
patient B
is O
very O
important O
for O
correct O
clinical O
diagnosis O
of O
BD O
, O
and O
that O
classification O
/ O
diagnosis O
criteria O
, O
including O
the O
International O
Criteria O
, O
should O
be O
followed O
but O
should O
not O
be O
exclusive O
. O

The O
observation O
that O
three O
out O
of O
14 O
patients B
in O
the O
probable O
BD O
group O
also O
had O
antibody O
to O
kinectin O
and O
the O
similar O
percentage O
of O
positive O
reactors O
between O
this O
group O
and O
Group O
I O
( O
21 O
. O
4 O
% O
versus O
24 O
% O
) O
supports O
this O
notion O
. O

The O
further O
use O
of O
non O
- O
clinical O
parameters O
such O
as O
immunological O
biomarkers O
as O
adjuncts O
to O
identify O
BD O
patients B
could O
be O
of O
help O
in O
the O
classification O
of O
this O
disease O
entity O

While O
our O
work O
was O
ongoing O
, O
anti O
- O
kinectin O
antibodies O
were O
reported O
in O
sera O
from O
patients B
with O
hepatocellular O
carcinoma O
( O
HCC O
) O
[ O
35 O
, O
36 O
] O
and O
aplastic O
anemia O
[ O
37 O
, O
38 O
] O
. O

The O
first O
HCC O
report O
[ O
35 O
] O
identified O
kinectin O
as O
a O
tumor O
- O
associated O
antigen O
from O
the O
screening O
of O
an O
autologous O
cDNA O
library O
constructed O
from O
the O
cancer O
of O
a O
30 O
- O
year O
- O
old O
patient B
from O
Guangxi O
, O
China O
. O

This O
report O
stated O
that O
four O
out O
of O
five O
HCC O
patients B
tested O
were O
positive O
for O
anti O
- O
kinectin O
antibody O
[ O
35 O
] O
. O

In O
2004 O
, O
another O
laboratory O
also O
reported O
the O
cloning O
of O
kinectin O
as O
a O
tumor O
- O
associated O
antigen O
from O
a O
( O
presumably O
) O
different O
30 O
- O
year O
- O
old O
Chinese O
HCC O
patient B
[ O
36 O
] O
. O

In O
contrast O
, O
anti O
- O
kinectin O
antibodies O
were O
not O
detected O
in O
other O
studies O
of O
HCC O
patients B
associated O
with O
our O
laboratory O
[ O
39 O
, O
40 O
] O
. O

The O
reports O
of O
anti O
- O
kinectin O
antibodies O
in O
aplastic O
anemia O
are O
also O
very O
interesting O
[ O
37 O
, O
38 O
] O
. O

The O
initial O
report O
by O
Hirano O
et O
al O
. O
identified O
kinectin O
by O
screening O
an O
aplastic O
anemia O
patient B
for O
candidate O
antigens O
using O
a O
Clontech O
human B
fetal O
liver O
cDNA O
expression O
library O
and O
it O
was O
concluded O
that O
seven O
out O
of O
18 O
aplastic O
anemia O
patients B
were O
positive O
for O
anti O
- O
kinectin O
while O
none O
of O
the O
normal O
or O
disease O
controls O
had O
this O
antibody O
[ O
37 O
] O
. O

In O
their O
recent O
report O
, O
Hirano O
et O
al O
. O
reported O
that O
anti O
- O
kinectin O
antibodies O
were O
found O
in O
39 O
% O
of O
aplastic O
anemia O
patients B
from O
the O
United O
States O
but O
only O
in O
three O
out O
of O
30 O
( O
10 O
% O
) O
cases O
in O
Japan O
[ O
38 O
] O
. O

In O
our O
study O
reported O
here O
, O
kinectin O
antibodies O
were O
only O
detected O
in O
BD O
patients B
and O
not O
in O
normal O
controls O
and O
SLE O
and O
SjS O
disease O
controls O
. O

None O
of O
the O
BD O
patients B
with O
anti O
- O
kinectin O
had O
signs O
of O
HCC O
or O
aplastic O
anemia O
at O
the O
time O
of O
diagnosis O
and O
at O
up O
to O
4 O
years O
of O
follow O
- O
up O
. O

Mapping O
of O
epitope O
( O
s O
) O
recognized O
by O
anti O
- O
kinectin O
antibodies O
may O
shed O
light O
on O
the O
question O
of O
whether O
different O
autoepitopes O
reside O
within O
the O
kinectin O
molecule O
recognized O
by O
sera O
from O
different O
diseases O
. O

Kinectin O
- O
a O
new O
member O
of O
coiled O
- O
coil O
cytoplasmic O
autoantigens O

We O
have O
recently O
reviewed O
the O
literature O
on O
the O
growing O
number O
of O
cytoplasmic O
autoantigens O
rich O
in O
alpha O
- O
helical O
coiled O
- O
coil O
domains O
as O
typified O
from O
our O
study O
of O
Golgi O
autoantigens O
[ O
41 O
] O
. O

Golgi O
autoantigens O
are O
generally O
high O
molecular O
weight O
proteins O
between O
100 O
and O
350 O
kDa O
and O
rich O
in O
coiled O
- O
coil O
domains O
in O
the O
central O
region O
with O
non O
- O
coiled O
- O
coil O
or O
globular O
domains O
at O
both O
N O
and O
C O
termini O
. O

Golgi O
autoantigens O
are O
displayed O
on O
the O
cytoplasmic O
face O
of O
the O
Golgi O
complex O
and O
are O
not O
localized O
to O
apoptotic O
blebs O
during O
apoptosis O
[ O
42 O
] O
. O

Giantin O
, O
the O
highest O
molecular O
weight O
Golgi O
autoantigen O
reported O
, O
is O
the O
predominant O
target O
of O
human B
anti O
- O
Golgi O
complex O
antibodies O
and O
multiple O
non O
- O
cross O
- O
reactive O
epitopes O
have O
been O
mapped O
spanning O
the O
350 O
kDa O
protein O
[ O
43 O
] O
. O

Other O
high O
molecular O
weight O
autoantigens O
with O
similar O
features O
have O
been O
reported O
in O
cytoplasmic O
and O
mitotic O
organelles O
suggesting O
that O
these O
selected O
proteins O
become O
autoimmunogenic O
based O
on O
their O
subcellular O
association O
and O
molecular O
features O
[ O
41 O
] O
. O

For O
example O
, O
in O
the O
endosomal O
compartment O
, O
the O
two O
known O
autoantigens O
are O
early O
endosomal O
protein O
EEA1 O
( O
180 O
kDa O
) O
[ O
44 O
] O
and O
CLIP O
- O
170 O
( O
170 O
kDa O
) O
[ O
45 O
] O
. O

There O
is O
also O
a O
series O
of O
centrosomal O
autoantigens O
identified O
as O
coiled O
- O
coil O
- O
rich O
proteins O
including O
pericentrin O
, O
a O
220 O
kDa O
protein O
[ O
46 O
] O
, O
ninein O
, O
a O
protein O
with O
alternatively O
spliced O
products O
of O
245 O
and O
249 O
kDa O
[ O
47 O
] O
, O
Cep250 O
( O
250 O
kDa O
) O
and O
Cep110 O
( O
110 O
kDa O
) O
[ O
48 O
] O
. O

Centromere O
autoantigens O
have O
been O
described O
but O
the O
two O
interesting O
ones O
related O
to O
this O
discussion O
are O
CENP O
- O
E O
[ O
49 O
] O
and O
CENP O
- O
F O
[ O
50 O
] O
; O
both O
are O
high O
molecular O
weight O
proteins O
( O
312 O
to O
400 O
kDa O
) O
and O
have O
the O
same O
type O
of O
overall O
structure O
as O
discussed O
above O
. O

NuMA O
is O
another O
large O
coiled O
- O
coil O
protein O
located O
at O
the O
mitotic O
spindle O
pole O
and O
is O
the O
most O
common O
target O
autoantigen O
in O
sera O
with O
mitotic O
spindle O
apparatus O
staining O
[ O
51 O
] O
. O

Non O
- O
muscle O
myosin O
( O
~ O
200 O
kDa O
) O
is O
a O
cytoskeletal O
autoantigen O
[ O
52 O
] O
that O
falls O
in O
the O
same O
group O
of O
high O
molecular O
weight O
and O
coiled O
- O
coil O
- O
rich O
autoantigens O
. O

These O
endosomal O
, O
centrosomal O
, O
mitotic O
apparatus O
and O
intracellular O
autoantigens O
are O
, O
like O
the O
golgins O
, O
proteins O
with O
high O
molecular O
weights O
and O
an O
overall O
high O
content O
of O
coiled O
- O
coil O
domains O
. O

The O
combination O
of O
these O
two O
physical O
features O
in O
autoantigens O
may O
contribute O
to O
the O
induction O
and O
production O
of O
autoimmune O
antibodies O
in O
certain O
disease O
states O
. O

Kinectin O
is O
an O
integral O
membrane O
protein O
largely O
confined O
to O
the O
endoplasmic O
reticulum O
[ O
28 O
, O
31 O
] O
and O
it O
fits O
into O
this O
new O
category O
of O
autoantigens O
that O
are O
large O
coiled O
- O
coil O
rich O
proteins O
( O
> O
= O
100 O
kDa O
) O
in O
the O
cytoplasm O
. O

Conclusion O

Here O
we O
report O
the O
detection O
of O
kinectin O
autoantibody O
in O
23 O
% O
of O
Chinese O
patients B
with O
BD O
. O

The O
identity O
of O
kinectin O
as O
a O
BD O
- O
related O
autoantigen O
has O
not O
been O
reported O
to O
date O
. O

Autoantibody O
reaction O
against O
kinectin O
in O
BD O
observed O
in O
this O
study O
further O
confirms O
the O
autoimmune O
involvement O
in O
BD O
and O
may O
provide O
new O
inroads O
into O
elucidating O
the O
immunopathogenesis O
of O
the O
disease O
. O

In O
an O
effort O
to O
clarify O
the O
association O
of O
BD O
with O
antibody O
to O
kinectin O
, O
it O
is O
essential O
to O
measure O
antibody O
to O
kinectin O
in O
larger O
patient B
populations O
including O
both O
BD O
, O
probable O
BD O
and O
important O
autoimmune O
rheumatic O
diseases O
such O
as O
SLE O
, O
SjS O
, O
rheumatoid O
arthritis O
etc O
. O
, O
as O
well O
as O
those O
diseases O
not O
easily O
differentiated O
from O
BD O
, O
such O
as O
recurrent O
aphthous O
oral O
ulcer O
, O
Reiter O
' O
s O
syndrome O
, O
inflammatory O
bowel O
diseases O
etc O
. O

On O
the O
other O
hand O
, O
further O
analysis O
of O
the O
association O
of O
anti O
- O
kinectin O
antibody O
with O
different O
manifestations O
or O
disease O
' O
subtypes O
' O
of O
BD O
is O
another O
important O
project O
. O

Anti O
- O
kinectin O
is O
clearly O
only O
one O
of O
the O
antigen O
- O
antibody O
systems O
identified O
because O
there O
were O
many O
other O
antibodies O
observed O
in O
the O
Western O
blot O
analysis O
of O
BD O
sera O
. O

Using O
other O
sera O
for O
immunoscreening O
would O
probably O
lead O
to O
the O
identification O
of O
other O
potentially O
important O
antigen O
- O
antibody O
systems O
. O

Abbreviations O

AECA O
= O
antibody O
to O
endothelial O
cell O
antigen O
; O
BD O
= O
Beh O
c O
et O
' O
s O
disease O
; O
DMEM O
= O
Dulbecco O
' O
s O
modified O
Eagle O
' O
s O
medium O
; O
HCC O
= O
hepatocellular O
carcinoma O
; O
HSP O
= O
heat O
shock O
protein O
; O
IIF O
= O
indirect O
immunofluorescence O
; O
PBS O
= O
phosphate O
buffered O
saline O
; O
SjS O
= O
Sj O
o O
gren O
' O
s O
syndrome O
; O
SLE O
= O
systemic O
lupus I
erythematosus I
. O

Competing O
interests O

The O
authors O
declare O
that O
they O
have O
no O
competing O
interests O
. O

Authors O
' O
contributions O

YL O
performed O
the O
study O
and O
drafted O
the O
manuscript O
. O

PY O
provided O
technical O
help O
throughout O
the O
study O
. O

SLC O
and O
EMT O
conceived O
the O
study O
, O
participated O
in O
the O
design O
and O
helped O
in O
the O
analysis O
of O
the O
data O
. O

EKLC O
participated O
in O
the O
design O
of O
the O
study O
, O
interpreted O
data O
and O
helped O
to O
draft O
the O
manuscript O
. O

All O
authors O
read O
and O
approved O
the O
final O
manuscript O
. O

EndoNet O
: O
an O
information O
resource O
about O
endocrine O
networks O

Abstract O

EndoNet O
is O
a O
new O
database O
that O
provides O
information O
about O
the O
components O
of O
endocrine O
networks O
and O
their O
relations O
. O

It O
focuses O
on O
the O
endocrine O
cell O
- O
to O
- O
cell O
communication O
and O
enables O
the O
analysis O
of O
intercellular O
regulatory O
pathways O
in O
humans B
. O

In O
the O
EndoNet O
data O
model O
, O
two O
classes O
of O
components O
span O
a O
bipartite O
directed O
graph O
. O

One O
class O
represents O
the O
hormones O
( O
in O
the O
broadest O
sense O
) O
secreted O
by O
defined O
donor O
cells O
. O

The O
other O
class O
consists O
of O
the O
acceptor O
or O
target O
cells O
expressing O
the O
corresponding O
hormone O
receptors O
. O

The O
identity O
and O
anatomical O
environment O
of O
cell O
types O
, O
tissues O
and O
organs O
is O
defined O
through O
references O
to O
the O
CYTOMER O
( O
R O
) O
ontology O
. O

With O
the O
EndoNet O
user O
interface O
, O
it O
is O
possible O
to O
query O
the O
database O
for O
hormones O
, O
receptors O
or O
tissues O
and O
to O
combine O
several O
items O
from O
different O
search O
rounds O
in O
one O
complex O
result O
set O
, O
from O
which O
a O
network O
can O
be O
reconstructed O
and O
visualized O
. O

For O
each O
entity O
, O
a O
detailed O
characteristics O
page O
is O
available O
. O

Some O
well O
- O
established O
endocrine O
pathways O
are O
offered O
as O
showcases O
in O
the O
form O
of O
predefined O
result O
sets O
. O

These O
sets O
can O
be O
used O
as O
a O
starting O
point O
for O
a O
more O
complex O
query O
or O
for O
obtaining O
a O
quick O
overview O
. O

The O
EndoNet O
database O
is O
accessible O
at O
. O

INTRODUCTION O

Theoretical O
analyses O
in O
the O
post O
- O
sequencing O
era O
, O
in O
particular O
in O
the O
context O
of O
systems O
biology O
approaches O
, O
increasingly O
investigate O
the O
properties O
of O
all O
kinds O
of O
pathways O
and O
networks O
such O
as O
metabolic O
and O
signaling O
pathways O
. O

It O
is O
commonly O
accepted O
that O
we O
need O
formal O
descriptions O
of O
these O
networks O
to O
make O
systematic O
use O
of O
the O
overwhelming O
body O
of O
facts O
gathered O
over O
decades O
of O
laboratory O
work O
both O
in O
narrow O
or O
global O
scale O
. O

Corresponding O
databases O
have O
been O
created O
and O
are O
available O
now O
for O
metabolic O
networks O
( O
KEGG O
) O
( O
1 O
, O
2 O
) O
, O
protein O
interaction O
networks O
( O
BIND O
and O
DIP O
) O
( O
3 O
, O
4 O
) O
and O
signaling O
pathways O
( O
CSNDB O
, O
Patika O
and O
TRANSPATH O
( O
R O
) O
) O
( O
5 O
- O
8 O
) O
, O
just O
to O
name O
a O
few O
. O

So O
far O
, O
however O
, O
their O
main O
focus O
is O
on O
intracellular O
processes O
. O

Intercellular O
signaling O
is O
addressed O
only O
insofar O
as O
usually O
the O
pathways O
modeled O
start O
with O
extracellular O
ligands O
, O
and O
as O
there O
exist O
catalogs O
and O
databases O
about O
secreted O
proteins O
, O
or O
the O
' O
secretome O
' O
, O
of O
certain O
systems O
( O
9 O
- O
12 O
) O
. O

This O
shortcoming O
is O
part O
of O
the O
more O
comprehensive O
problem O
, O
the O
genotype O
- O
phenotype O
gap O
: O
from O
a O
certain O
genotype O
, O
we O
are O
able O
to O
infer O
a O
' O
molecular O
phenotype O
' O
, O
but O
for O
correlating O
it O
with O
a O
more O
complex O
phenotype O
such O
as O
a O
biological O
process O
, O
or O
even O
a O
certain O
disease O
and O
its O
clinical O
appearance O
, O
we O
still O
depend O
largely O
on O
the O
mere O
description O
of O
an O
observed O
correlation O
. O

There O
is O
no O
way O
to O
infer O
such O
a O
phenotype O
through O
all O
the O
different O
layers O
of O
increasing O
complexity O
between O
genomic O
DNA O
sequences O
and O
the O
physiological O
function O
of O
whole O
organs O
and O
their O
interplay O
within O
an O
organism O
. O

There O
may O
be O
principal O
barriers O
preventing O
such O
an O
inference O
across O
different O
complexity O
levels O
, O
but O
even O
to O
explore O
these O
limits O
, O
we O
have O
to O
make O
attempts O
to O
bridge O
the O
genotype O
- O
phenotype O
gap O
. O

We O
have O
to O
do O
the O
next O
step O
towards O
modeling O
intercellular O
networks O
that O
are O
inextricably O
linked O
to O
the O
physiology O
of O
multicellular O
organisms O
( O
13 O
) O
. O

Being O
one O
of O
the O
most O
complex O
constructs O
in O
the O
body O
, O
the O
endocrine O
system O
comprises O
numerous O
cells O
and O
tissues O
that O
secrete O
hormones O
which O
pass O
through O
the O
body O
, O
activate O
specific O
receptors O
of O
target O
cells O
and O
initiate O
there O
multiple O
intracellular O
signaling O
pathways O
. O

Here O
, O
we O
present O
a O
new O
database O
, O
EndoNet O
, O
which O
provides O
information O
about O
the O
components O
of O
endocrine O
networks O
and O
their O
relations O
, O
and O
enables O
the O
analysis O
of O
intercellular O
regulatory O
pathways O
in O
humans B
. O

The O
EndoNet O
database O
is O
accessible O
at O
. O

RESULTS O

The O
biological O
schema O
of O
endocrine O
actions O

Development O
and O
function O
of O
different O
organs O
as O
well O
as O
the O
response O
of O
a O
whole O
multicellular O
organism O
to O
its O
environment O
is O
coordinated O
through O
a O
complex O
communication O
system O
between O
specialized O
cells O
that O
are O
part O
of O
its O
organs O
. O

This O
communication O
is O
mostly O
mediated O
by O
hormones O
. O

In O
a O
broader O
sense O
, O
this O
functional O
class O
of O
biomolecules O
also O
comprises O
growth O
factors O
, O
cytokines O
, O
chemokines O
and O
other O
signal O
transmitters O
. O

This O
generic O
view O
is O
supported O
, O
for O
instance O
, O
by O
the O
definition O
given O
for O
' O
hormone O
activity O
' O
by O
Gene O
Ontology O
( O
GO O
) O
( O
14 O
) O
: O
' O
Any O
substance O
formed O
in O
very O
small O
amounts O
in O
one O
specialized O
organ O
or O
group O
of O
cells O
and O
carried O
( O
sometimes O
in O
the O
bloodstream O
) O
to O
another O
organ O
or O
group O
of O
cells O
, O
in O
the O
same O
organism O
, O
upon O
which O
it O
has O
a O
specific O
regulatory O
action O
' O
. O

This O
definition O
is O
also O
broad O
enough O
to O
include O
modes O
of O
hormonal O
actions O
as O
diverse O
as O
endocrine O
, O
paracrine O
and O
autocrine O
effects O
. O

Accordingly O
, O
with O
the O
term O
' O
hormone O
' O
one O
might O
refer O
to O
any O
extracellular O
substance O
that O
induces O
specific O
responses O
in O
target O
cell O
and O
helps O
to O
coordinate O
growth O
, O
differentiation O
, O
gene O
expression O
and O
metabolic O
activities O
of O
various O
cells O
, O
tissues O
and O
organs O
in O
multicellular O
organisms O
( O
15 O
) O
. O

Hormones O
can O
be O
classified O
based O
on O
their O
chemical O
nature O
, O
solubility O
, O
the O
distance O
over O
which O
the O
signal O
acts O
and O
so O
on O
( O
15 O
, O
16 O
) O
. O

From O
the O
viewpoint O
of O
genome O
- O
phenotype O
relations O
, O
it O
is O
reasonable O
to O
distinguish O
between O
polypeptide O
, O
thus O
, O
genome O
- O
encoded O
hormones O
, O
on O
one O
hand O
, O
and O
those O
low O
- O
molecular O
weight O
hormones O
such O
as O
steroids O
, O
with O
only O
the O
machinery O
of O
their O
synthesis O
being O
genome O
- O
encoded O
, O
on O
the O
other O
hand O
. O

Another O
classification O
of O
hormones O
, O
which O
seems O
to O
be O
overlapping O
with O
the O
previous O
one O
refers O
to O
the O
intracellular O
location O
of O
their O
receptors O
and O
, O
thus O
, O
how O
the O
subsequent O
signal O
is O
further O
transduced O
: O
membrane O
- O
bound O
receptors O
usually O
trigger O
more O
or O
less O
complex O
signaling O
cascades O
towards O
the O
nucleus O
, O
whereas O
nuclear O
receptors O
, O
mainly O
bound O
by O
low O
- O
molecular O
weight O
hormones O
, O
have O
a O
very O
short O
signaling O
pathway O
downstream O
since O
they O
act O
as O
transcription O
factors O
themselves O
( O
15 O
, O
16 O
) O
. O

In O
intercellular O
communication O
, O
we O
can O
basically O
differentiate O
between O
two O
kinds O
of O
cells O
: O
donor O
cells O
which O
synthesize O
and O
secrete O
a O
hormone O
, O
and O
acceptor O
cells O
which O
express O
a O
hormone O
receptor O
( O
Figure O
1a O
) O
. O

Donor O
cells O
become O
active O
under O
the O
influence O
of O
an O
external O
, O
mostly O
environmental O
, O
stimulus O
. O

In O
the O
acceptor O
cell O
, O
binding O
of O
the O
hormone O
to O
a O
receptor O
triggers O
an O
intracellular O
signal O
transduction O
cascade O
with O
different O
kinds O
of O
end O
nodes O
and O
effects O
: O
transcription O
factors O
affecting O
the O
gene O
expression O
program O
of O
the O
acceptor O
cell O
, O
metabolic O
enzymes O
controlling O
the O
cell O
' O
s O
metabolism O
, O
structural O
components O
which O
define O
the O
acceptor O
' O
s O
morphological O
features O
, O
or O
components O
of O
the O
secretory O
apparatus O
regulating O
the O
release O
of O
other O
extracellular O
molecules O
. O

If O
synthesis O
and O
secretion O
of O
another O
hormone O
is O
among O
the O
effects O
exerted O
by O
receptor O
activation O
, O
the O
acceptor O
is O
turned O
into O
a O
donor O
cell O
, O
thus O
becoming O
an O
internal O
node O
of O
the O
organism O
' O
s O
endocrine O
network O
. O

Acceptor O
cells O
which O
do O
not O
become O
producers O
of O
another O
hormone O
are O
called O
' O
terminal O
target O
cells O
' O
of O
the O
endocrine O
network O
, O
but O
finally O
constitute O
the O
overall O
physiological O
effect O
of O
the O
respective O
hormonal O
pathway O
, O
or O
simply O
the O
phenotype O
( O
Figure O
1a O
) O
. O

The O
EndoNet O
data O
model O

In O
the O
EndoNet O
data O
model O
, O
two O
classes O
of O
entities O
- O
- O
hormones O
( O
in O
the O
broadest O
sense O
) O
and O
their O
acceptor O
or O
target O
cells O
expressing O
the O
corresponding O
receptors O
- O
- O
span O
a O
bipartite O
directed O
graph O
. O

Since O
one O
and O
the O
same O
hormone O
may O
be O
secreted O
by O
multiple O
cell O
types O
( O
donor O
cells O
) O
, O
each O
such O
secretion O
event O
is O
represented O
by O
a O
hormone O
node O
on O
its O
own O
. O

Similarly O
, O
each O
cell O
type O
known O
to O
express O
a O
hormone O
receptor O
( O
acceptor O
cell O
) O
leads O
to O
an O
individual O
node O
. O

The O
graph O
' O
s O
edges O
represent O
hormone O
transport O
and O
binding O
to O
a O
receptor O
( O
intercellular O
edges O
) O
, O
on O
one O
hand O
, O
and O
triggering O
or O
inhibition O
of O
hormone O
secretion O
by O
a O
receptor O
activated O
by O
hormone O
binding O
( O
intracellular O
edges O
) O
, O
on O
the O
other O
hand O
. O

Optionally O
, O
an O
edge O
representing O
the O
transport O
of O
a O
hormone O
can O
be O
subdivided O
by O
introducing O
the O
transport O
medium O
( O
usually O
blood O
) O
as O
an O
additional O
, O
intermediary O
node O
. O

Thus O
, O
in O
the O
conceptual O
schema O
of O
the O
EndoNet O
database O
( O
Figure O
1b O
) O
, O
the O
links O
between O
cells O
/ O
organs O
and O
hormone O
define O
donor O
cells O
( O
' O
D O
' O
) O
, O
those O
between O
cells O
/ O
organs O
and O
receptors O
acceptor O
cells O
( O
' O
A O
' O
) O
. O

If O
an O
acceptor O
cell O
synthesizes O
another O
hormone O
in O
response O
to O
an O
incoming O
signal O
, O
it O
becomes O
an O
internal O
node O
in O
the O
emerging O
hormonal O
network O
. O

In O
EndoNet O
, O
the O
pathway O
between O
a O
hormone O
receptor O
expressed O
in O
an O
acceptor O
cell O
and O
a O
hormone O
synthesized O
in O
the O
same O
cell O
( O
intracellular O
edge O
) O
is O
handled O
as O
a O
black O
box O
. O

In O
case O
of O
genome O
- O
encoded O
peptide O
hormones O
, O
cross O
- O
references O
to O
entries O
in O
the O
TRANSPATH O
( O
R O
) O
database O
, O
which O
describe O
the O
signaling O
cascade O
starting O
from O
the O
hormone O
' O
s O
receptor O
and O
ending O
at O
the O
hormone O
' O
s O
gene O
, O
are O
provided O
, O
if O
available O
. O

Datasets O
on O
non O
- O
peptide O
hormones O
will O
in O
future O
be O
enriched O
by O
a O
specific O
metabolic O
add O
- O
on O
which O
will O
include O
references O
to O
the O
databases O
KEGG O
( O
1 O
) O
and O
BRENDA O
( O
17 O
) O
, O
allowing O
for O
further O
characterization O
of O
the O
steps O
performed O
during O
the O
hormone O
' O
s O
synthesis O
and O
the O
regulation O
of O
both O
activity O
and O
expression O
of O
the O
enzymes O
involved O
. O

By O
now O
, O
the O
EndoNet O
structure O
already O
allows O
for O
including O
descriptions O
of O
the O
physiological O
effects O
induced O
by O
hormone O
binding O
( O
see O
below O
, O
Future O
Developments O
) O
. O

The O
contents O
of O
EndoNet O

In O
the O
present O
version O
of O
EndoNet O
, O
and O
as O
a O
first O
approach O
, O
we O
consider O
the O
endocrine O
( O
hormonal O
) O
network O
of O
the O
human B
body O
. O

For O
each O
molecule O
( O
hormone O
or O
receptor O
) O
, O
a O
primary O
name O
and O
synonyms O
are O
given O
. O

In O
case O
of O
peptide O
hormones O
, O
the O
sequence O
of O
the O
processed O
polypeptide O
, O
rather O
than O
that O
of O
the O
protein O
precursor O
, O
is O
specified O
. O

For O
a O
multimeric O
protein O
hormone O
, O
the O
subunit O
composition O
as O
well O
as O
the O
sequences O
of O
all O
subunits O
are O
stored O
; O
the O
same O
holds O
true O
for O
hormone O
receptors O
. O

Additionally O
, O
all O
peptide O
hormone O
and O
receptor O
datasets O
have O
links O
to O
HumanPSD B
( O
TM O
) O
( O
18 O
) O
and O
to O
the O
Swiss O
- O
Prot O
database O
, O
The O
structures O
of O
non O
- O
peptide O
hormones O
can O
be O
accessed O
through O
the O
corresponding O
hyperlinks O
to O
the O
KEGG O
COMPOUND O
section O
. O

Finally O
, O
all O
molecules O
may O
have O
links O
to O
the O
TRANSPATH O
( O
R O
) O
database O
. O

As O
described O
, O
EndoNet O
utilizes O
data O
about O
the O
tissues O
from O
which O
hormones O
are O
secreted O
and O
in O
which O
receptors O
are O
expressed O
to O
define O
donor O
and O
acceptor O
cells O
, O
respectively O
. O

The O
identity O
and O
anatomical O
environment O
of O
cell O
types O
, O
tissues O
and O
organs O
is O
defined O
through O
references O
to O
the O
CYTOMER O
( O
R O
) O
ontology O
( O
19 O
, O
20 O
) O
; O
in O
the O
numerous O
cases O
where O
the O
receptors O
are O
ubiquitously O
expressed O
, O
just O
the O
root O
term O
' O
human B
body O
' O
is O
linked O
. O

Data O
on O
whether O
a O
hormone O
' O
s O
synthesis O
is O
triggered O
or O
inhibited O
by O
another O
hormone O
through O
its O
respective O
receptor O
in O
a O
particular O
cell O
was O
obtained O
by O
manual O
selection O
from O
textbooks O
[ O
e O
. O
g O
. O
( O
16 O
) O
, O
] O
, O
monographies O
( O
21 O
) O
, O
original O
literature O
, O
the O
EST O
library O
information O
and O
the O
linked O
databases O
( O
TRANSPATH O
( O
R O
) O
, O
HumanPSD B
( O
TM O
) O
and O
Swiss O
- O
Prot O
) O
. O

The O
contents O
of O
EndoNet O
are O
summarized O
in O
Table O
1 O
. O

Web O
interface O
, O
queries O
and O
visualization O

EndoNet O
can O
be O
accessed O
through O
the O
WWW O
via O
a O
JSP O
- O
based O
web O
interface O
. O

Hormones O
, O
receptors O
and O
tissues O
can O
be O
queried O
for O
their O
names O
, O
and O
detailed O
information O
on O
all O
identified O
components O
is O
available O
through O
individual O
characteristics O
pages O
. O

Each O
hormone O
' O
s O
individual O
entry O
page O
displays O
its O
source O
and O
target O
tissues O
( O
donor O
and O
acceptor O
cells O
) O
as O
well O
as O
its O
receptors O
, O
along O
with O
some O
molecular O
data O
. O

Similarly O
, O
each O
receptor O
entry O
exhibits O
the O
tissues O
in O
which O
the O
receptor O
is O
expressed O
, O
and O
the O
hormones O
it O
interacts O
with O
. O

Finally O
, O
each O
tissue O
entry O
lists O
the O
hormone O
receptors O
that O
are O
found O
in O
, O
as O
well O
as O
the O
hormones O
that O
are O
synthesized O
and O
secreted O
by O
the O
tissue O
. O

It O
is O
also O
indicated O
whether O
the O
corresponding O
tissue O
exerts O
gender O
- O
specific O
properties O
; O
additional O
information O
based O
on O
the O
CYTOMER O
( O
R O
) O
ontology O
is O
available O
through O
the O
corresponding O
link O
on O
the O
tissue O
detail O
page O
. O

Instead O
of O
searching O
for O
a O
name O
of O
a O
hormone O
, O
one O
can O
also O
browse O
the O
hierarchical O
hormone O
classification O
featured O
by O
EndoNet O
( O
available O
at O
the O
' O
Search O
' O
page O
) O
. O

Each O
query O
result O
can O
be O
used O
as O
starting O
point O
for O
an O
extended O
query O
. O

Different O
items O
of O
interest O
can O
be O
selected O
and O
added O
to O
a O
common O
result O
set O
. O

Since O
several O
search O
results O
can O
be O
combined O
, O
it O
is O
possible O
to O
create O
sets O
with O
multiple O
search O
parameters O
. O

At O
any O
step O
of O
this O
incremental O
retrieval O
process O
, O
the O
sets O
of O
hormones O
, O
receptors O
and O
tissues O
obtained O
so O
far O
can O
be O
used O
as O
starting O
points O
for O
reconstructing O
a O
network O
by O
a O
depth O
- O
first O
graph O
traversal O
algorithm O
. O

The O
maximum O
number O
of O
steps O
can O
be O
selected O
separately O
for O
the O
upstream O
and O
downstream O
part O
of O
the O
reconstruction O
process O
. O

Subsequently O
, O
the O
graph O
will O
be O
displayed O
using O
a O
Graphviz O
- O
based O
visualization O
method O
[ O
( O
22 O
) O
, O
] O
. O

In O
the O
resulting O
image O
, O
hormones O
and O
receptors O
are O
represented O
as O
nodes O
grouped O
together O
into O
subgraphs O
representing O
the O
tissues O
( O
cells O
/ O
organs O
) O
they O
are O
secreted O
from O
or O
expressed O
in O
, O
respectively O
( O
Figure O
2 O
) O
. O

Autocrine O
loops O
( O
donor O
and O
acceptor O
cell O
being O
identical O
) O
are O
treated O
specially O
for O
visualization O
. O

On O
demand O
, O
the O
hormones O
' O
transport O
media O
can O
be O
included O
in O
the O
visualization O
of O
intercellular O
edges O
, O
enabling O
the O
user O
to O
choose O
between O
different O
complexities O
of O
output O
. O

Intracellular O
edges O
, O
which O
connect O
a O
receptor O
to O
a O
hormone O
, O
represent O
the O
influence O
of O
a O
receptor O
' O
s O
activation O
on O
the O
secretion O
of O
a O
hormone O
from O
the O
same O
cell O
and O
are O
displayed O
differently O
, O
depending O
on O
whether O
this O
influence O
is O
triggering O
or O
inhibitory O
in O
nature O
. O

The O
graph O
is O
displayed O
as O
a O
clickable O
image O
map O
, O
linking O
each O
entity O
to O
its O
detailed O
characteristics O
page O
, O
thus O
making O
the O
database O
entries O
accessible O
from O
the O
graphical O
overview O
of O
a O
network O
, O
too O
. O

The O
graph O
is O
available O
in O
two O
different O
formats O
: O
PNG O
and O
SVG O
. O

While O
virtually O
every O
browser O
supports O
PNG O
( O
a O
' O
pixel O
' O
or O
' O
bitmap O
' O
format O
) O
, O
only O
a O
few O
of O
them O
provide O
a O
zoom O
functionality O
for O
bitmap O
pictures O
. O

Scalable O
vector O
graphics O
, O
( O
SVG O
) O
provides O
more O
functionality O
( O
including O
perfect O
image O
quality O
throughout O
all O
zoom O
factors O
) O
, O
but O
until O
now O
most O
browsers O
do O
not O
support O
SVG O
natively O
and O
require O
an O
SVG O
plugin O
( O
) O
for O
displaying O
this O
vector O
- O
based O
format O
. O

Some O
well O
- O
established O
endocrine O
pathways O
are O
offered O
as O
showcases O
in O
the O
form O
of O
predefined O
result O
sets O
. O

For O
instance O
, O
sets O
representing O
the O
hypothalamic O
- O
hypophyseal O
axis O
with O
a O
focus O
on O
either O
thyroid O
hormones O
, O
adrenal O
hormones O
, O
growth O
hormones O
or O
prolactin O
are O
provided O
. O

These O
predefined O
sets O
can O
be O
used O
for O
obtaining O
a O
quick O
overview O
or O
as O
starting O
points O
for O
more O
complex O
queries O
. O

FUTURE O
DEVELOPMENTS O

Among O
the O
important O
improvements O
of O
EndoNet O
which O
we O
are O
currently O
working O
on O
is O
the O
possibility O
to O
represent O
intercellular O
communication O
at O
different O
levels O
of O
the O
hierarchical O
organization O
of O
organs O
, O
tissues O
and O
cells O
in O
the O
organism O
, O
as O
well O
as O
to O
distinguish O
between O
such O
communications O
in O
male O
and O
female O
organisms O
. O

These O
options O
will O
be O
introduced O
by O
a O
tighter O
integration O
with O
the O
CYTOMER O
- O
based O
ontology O
( O
19 O
, O
20 O
) O
. O

The O
next O
step O
will O
be O
to O
expand O
the O
contents O
of O
EndoNet O
towards O
the O
details O
of O
the O
processes O
occurring O
in O
the O
transport O
medium O
, O
usually O
the O
blood O
. O

Since O
not O
all O
hormones O
are O
transported O
as O
free O
molecules O
and O
some O
hydrophobic O
hormones O
( O
e O
. O
g O
. O
steroids O
and O
thyroids O
) O
need O
to O
be O
bound O
to O
specific O
carrier O
proteins O
, O
proper O
description O
of O
such O
transporters O
and O
their O
interaction O
with O
the O
corresponding O
hormones O
will O
be O
required O
. O

It O
is O
planned O
to O
involve O
quantitative O
data O
about O
the O
regular O
or O
pathological O
levels O
of O
the O
hormones O
, O
the O
overall O
kinetics O
of O
each O
hormone O
in O
the O
blood O
( O
monotonous O
decay O
, O
oscillating O
concentrations O
, O
increase O
in O
response O
to O
certain O
stimuli O
, O
etc O
. O
) O
, O
its O
turnover O
and O
metabolic O
products O
, O
etc O
. O

That O
will O
allow O
utilization O
of O
EndoNet O
contents O
for O
diagnostic O
purposes O
. O

In O
future O
, O
the O
EndoNet O
data O
model O
will O
be O
extended O
in O
order O
to O
incorporate O
external O
stimuli O
( O
e O
. O
g O
. O
light O
) O
and O
physiological O
states O
( O
stress O
, O
age O
, O
etc O
. O
) O
in O
a O
formalized O
manner O
, O
allowing O
to O
determine O
whether O
or O
not O
and O
in O
which O
quantity O
a O
hormone O
will O
be O
secreted O
under O
the O
given O
circumstances O
. O

EndoNet O
will O
then O
link O
the O
physiological O
effects O
of O
hormones O
with O
the O
intracellular O
molecular O
processes O
leading O
to O
its O
synthesis O
and O
secretion O
in O
the O
donor O
cells O
, O
and O
to O
the O
effects O
on O
its O
acceptor O
cells O
. O

At O
many O
places O
of O
these O
intracellular O
and O
intercellular O
networks O
, O
genetically O
determined O
aberrations O
may O
cause O
specific O
, O
sometimes O
pathological O
phenotypes O
. O

Thus O
, O
EndoNet O
will O
enable O
to O
bridge O
the O
gap O
between O
known O
genotypes O
and O
their O
molecular O
and O
clinical O
phenotypes O
in O
this O
area O
of O
medical O
research O
and O
its O
applications O
. O

DISCUSSION O

At O
its O
present O
state O
, O
EndoNet O
provides O
a O
high O
coverage O
of O
molecules O
that O
are O
conventionally O
considered O
as O
hormones O
as O
well O
as O
other O
molecules O
that O
are O
involved O
in O
intercellular O
communication O
, O
such O
as O
growth O
factors O
, O
lymphokines O
and O
chemokines O
and O
their O
known O
receptors O
. O

The O
aim O
of O
the O
database O
is O
to O
provide O
a O
useful O
resource O
for O
studying O
the O
principal O
features O
of O
hormonal O
networks O
in O
a O
comprehensive O
way O
, O
as O
it O
was O
done O
more O
exemplarily O
in O
the O
past O
for O
these O
kinds O
of O
networks O
( O
23 O
) O
, O
but O
was O
done O
globally O
for O
many O
other O
intracellular O
network O
types O
, O
such O
as O
metabolic O
, O
protein O
interaction O
and O
transcription O
networks O
[ O
reviewed O
in O
( O
24 O
, O
25 O
) O
] O
. O

EndoNet O
database O
certainly O
is O
not O
yet O
complete O
but O
will O
grow O
rapidly O
. O

Gender O
differences O
in O
factors O
influencing O
insulin O
resistance O
in O
elderly O
hyperlipemic O
non O
- O
diabetic O
subjects O

Abstract O

Background O

The O
increase O
in O
the O
prevalence O
of O
insulin O
resistance O
- O
related O
metabolic O
syndrome O
, O
a O
disorder O
that O
greatly O
increases O
the O
risk O
of O
diabetes O
, O
heart O
attack O
and O
stroke O
, O
is O
alarming O
. O

One O
of O
the O
most O
frequent O
and O
early O
symptoms O
of O
metabolic O
syndrome O
is O
hypertriglyceridemia O
. O

We O
examined O
the O
gender O
differences O
between O
various O
metabolic O
factors O
related O
to O
insulin O
resistance O
in O
elderly O
non O
- O
diabetic O
men B
and O
postmenopausal O
women B
of O
comparable O
age O
suffering O
from O
hypertriglyceridemia O
, O
and O
compared O
them O
with O
healthy O
subjects O
of O
equal O
age O
. O

Results O

The O
indexes O
of O
insulin O
resistance O
HOMA O
IR O
and O
QUICKI O
were O
significantly O
higher O
in O
both O
hyperlipemic O
men B
and O
women B
than O
in O
controls O
; O
95 O
% O
confidence O
limits O
of O
hyperlipemic O
subjects O
did O
not O
overlap O
with O
controls O
. O

In O
both O
normolipemic O
and O
hyperlipemic O
men B
and O
women B
serum O
leptin O
correlated O
significantly O
with O
insulin O
resistance O
, O
while O
HDL O
- O
cholesterol O
correlated O
inversely O
with O
HOMA O
- O
IR O
only O
in O
women B
( O
both O
normo O
- O
and O
hyperlipemic O
) O
, O
and O
serum O
tumor O
necrosis O
factor O
alpha O
( O
TNF O
alpha O
) O
only O
in O
hyperlipemic O
women B
. O

According O
to O
results O
of O
multiple O
regression O
analysis O
with O
HOMA O
- O
IR O
as O
a O
dependent O
variable O
, O
leptin O
played O
a O
significant O
role O
in O
determining O
insulin O
resistance O
in O
both O
genders O
, O
but O
- O
aside O
from O
leptin O
- O
triglycerides O
, O
TNF O
alpha O
and O
decreased O
HDL O
- O
cholesterol O
were O
significant O
determinants O
in O
women B
, O
while O
body O
mass O
index O
and O
decreased O
HDL O
- O
cholesterol O
were O
significant O
determinants O
in O
men B
. O

The O
coefficient O
of O
determination O
( O
R2 O
) O
of O
HOMA O
IR O
by O
above O
mentioned O
metabolic O
variables O
was O
in O
women B
above O
60 O
% O
, O
in O
men B
only O
about O
40 O
% O
. O

Conclusion O

The O
significant O
role O
of O
serum O
leptin O
in O
determination O
of O
insulin O
resistance O
in O
both O
elderly O
men B
and O
postmenopausal O
women B
of O
equal O
age O
was O
confirmed O
. O

However O
, O
the O
study O
also O
revealed O
significant O
gender O
differences O
: O
in O
women B
a O
strong O
influence O
of O
triglycerides O
, O
TNF O
alpha O
and O
decreased O
HDL O
- O
cholesterol O
, O
in O
men B
only O
a O
mild O
role O
of O
BMI O
and O
decreased O
HDL O
- O
cholesterol O
. O

Background O

In O
association O
with O
pandemic O
obesity O
the O
prevalence O
of O
the O
insulin O
resistance O
- O
related O
metabolic O
syndrome O
is O
constantly O
growing O
[ O
1 O
] O
. O

As O
a O
consequence O
of O
this O
fact O
, O
type O
2 O
diabetes O
mellitus O
and O
cardiovascular O
mortality O
occurs O
in O
much O
younger O
age O
groups O
[ O
2 O
] O
. O

A O
typical O
hyperlipemia O
, O
consisting O
of O
an O
increase O
of O
serum O
triglycerides O
and O
a O
decrease O
of O
serum O
HDL O
- O
cholesterol O
, O
is O
a O
characteristic O
and O
an O
early O
symptom O
of O
this O
syndrome O
[ O
3 O
] O
. O

With O
increasing O
age O
, O
body O
mass O
index O
( O
BMI O
) O
and O
adiposity O
, O
insulin O
sensitivity O
declines O
and O
the O
number O
of O
cardiovascular O
risk O
factors O
increases O
in O
both O
genders O
[ O
4 O
- O
6 O
] O
. O

It O
was O
repeatedly O
demonstrated O
that O
plasma O
concentration O
of O
leptin O
- O
a O
hormone O
produced O
mainly O
by O
adipose O
tissue O
- O
is O
substantially O
higher O
in O
all O
age O
groups O
of O
women B
than O
in O
men B
[ O
7 O
- O
10 O
] O
. O

This O
may O
be O
caused O
by O
different O
size O
and O
/ O
or O
distribution O
of O
fat O
tissue O
compartments O
influenced O
by O
hormones O
: O
estrogens O
stimulate O
, O
whereas O
testosterone O
inhibits O
leptin O
secretion O
. O

In O
women B
subcutaneous O
fat O
mass O
prevails O
- O
and O
during O
augmentation O
of O
overweight O
it O
increases O
- O
while O
in O
men B
intra O
- O
abdominal O
fat O
mass O
prevails O
[ O
11 O
- O
13 O
] O
. O

Subcutaneous O
fat O
in O
particular O
serves O
as O
a O
substantial O
source O
of O
tumor O
necrosis O
factor O
alpha O
( O
TNF O
alpha O
) O
, O
which O
represents O
one O
of O
the O
factors O
that O
interfere O
with O
insulin O
signal O
transduction O
into O
the O
cells O
[ O
14 O
- O
16 O
] O
. O

Leptin O
, O
TNF O
alpha O
and O
some O
other O
factors O
are O
abundantly O
expressed O
in O
adipose O
tissue O
and O
contribute O
to O
the O
insulin O
resistance O
that O
accompanies O
overweight O
and O
obesity O
. O

Leptin O
correlates O
positively O
with O
hyperinsulinemia O
, O
BMI O
, O
fat O
mass O
and O
hypertriglyceridemia O
, O
respectively O
, O
and O
correlates O
inversely O
with O
HDL O
- O
cholesterol O
and O
lean O
body O
mass O
[ O
17 O
- O
25 O
] O
. O

The O
incidence O
and O
mortality O
of O
ischemic O
heart O
disease O
and O
of O
other O
consequences O
of O
atherosclerosis O
increases O
with O
age O
in O
both O
genders O
, O
especially O
after O
the O
age O
of O
sixty O
. O

In O
premenopausal O
women B
, O
however O
, O
the O
incidence O
of O
these O
disorders O
is O
considerably O
less O
frequent O
than O
in O
men B
of O
appropriate O
age O
. O

After O
the O
menopause O
the O
prevalence O
of O
metabolic O
syndrome O
and O
cardiovascular O
mortality O
in O
women B
gradually O
increases O
, O
attaining O
values O
comparable O
to O
men B
at O
about O
the O
age O
of O
70 O
[ O
2 O
, O
26 O
] O
. O

Paradoxically O
, O
it O
takes O
place O
at O
the O
time O
when O
serum O
leptin O
concentration O
in O
women B
has O
relatively O
decreased O
[ O
27 O
, O
28 O
] O
. O

The O
aim O
of O
this O
study O
was O
to O
analyze O
the O
interrelations O
between O
several O
metabolic O
variables O
and O
factors O
related O
to O
insulin O
resistance O
in O
groups O
of O
both O
normal O
and O
hyperlipemic O
postmenopausal O
women B
and O
men B
of O
appropriate O
age O
, O
and O
to O
attempt O
to O
elucidate O
the O
gender O
differences O
and O
some O
pathophysiologic O
mechanisms O
of O
these O
differences O
. O

We O
compared O
homeostatic O
indexes O
of O
insulin O
resistance O
HOMA O
IR O
and O
QUICKI O
, O
serum O
lipid O
and O
insulin O
parameters O
, O
uric O
acid O
, O
leptin O
and O
TNF O
alpha O
between O
groups O
of O
subjects O
without O
apparent O
symptoms O
of O
metabolic O
syndrome O
, O
and O
groups O
showing O
mild O
hypertriglyceridemia O
with O
decreased O
HDL O
- O
cholesterol O
. O

In O
addition O
, O
serum O
concentration O
of O
the O
heart O
fraction O
of O
fatty O
acid O
binding O
protein O
( O
hFATP O
) O
was O
explored O
as O
a O
factor O
that O
might O
reflect O
the O
regulative O
role O
of O
PPAR O
gamma O
in O
lipid O
homeostasis O
[ O
29 O
, O
30 O
] O
, O
and O
serum O
IgG O
anticardiolipin O
( O
ACL O
- O
IgG O
) O
was O
investigated O
as O
an O
indirect O
indicator O
of O
oxidized O
lipid O
fractions O
related O
to O
atherosclerotic O
complications O
[ O
31 O
, O
32 O
] O
. O

Methods O

Subjects O

The O
study O
was O
carried O
out O
on O
70 O
out O
- O
patients B
of O
the O
Metabolic O
Center O
at O
the O
hospital O
in O
Sternberk O
, O
Czech O
Republic O
. O

From O
these O
, O
40 O
patients B
( O
20 O
men B
and O
20 O
women B
) O
were O
selected O
with O
mild O
hyperlipidemia O
, O
i O
. O
e O
. O
with O
plasma O
triglyceride O
concentration O
exceeding O
2 O
. O
0 O
mmol O
/ O
l O
, O
total O
cholesterol O
exceeding O
6 O
. O
0 O
mmol O
/ O
l O
, O
LDL O
cholesterol O
exceeding O
4 O
. O
0 O
mmol O
/ O
l O
, O
and O
with O
HDL O
cholesterol O
concentration O
in O
men B
under O
1 O
. O
0 O
mmol O
/ O
l O
, O
and O
in O
women B
under O
1 O
. O
2 O
mmol O
/ O
l O
. O

These O
groups O
were O
denominated O
as O
" O
hyperlipemic O
" O
. O

Two O
other O
groups O
( O
10 O
men B
and O
20 O
women B
) O
with O
approximately O
normal O
serum O
values O
of O
these O
variables O
were O
taken O
as O
" O
controls O
" O
. O

The O
average O
age O
in O
men B
was O
59 O
. O
1 O
+ O
/ O
- O
10 O
. O
6 O
y O
, O
and O
in O
women B
59 O
. O
4 O
+ O
/ O
- O
10 O
. O
1 O
y O
, O
respectively O
. O

The O
differences O
between O
lipid O
parameters O
of O
hyperlipemic O
and O
control O
groups O
were O
highly O
statistically O
significant O
, O
while O
the O
age O
differences O
were O
insignificant O
( O
see O
Table O
1 O
) O
. O

None O
of O
the O
patients B
had O
clinically O
apparent O
diabetes O
mellitus O
, O
but O
some O
of O
the O
hyperlipemic O
patients B
exerted O
impaired O
glucose O
tolerance O
or O
impaired O
fasting O
glucose O
( O
values O
between O
6 O
. O
1 O
and O
7 O
. O
0 O
mmol O
/ O
l O
, O
or O
between O
6 O
. O
1 O
and O
7 O
. O
8 O
mmol O
/ O
l O
, O
respectively O
) O
. O

None O
of O
the O
patients B
was O
treated O
with O
insulin O
, O
peroral O
antidiabetics O
or O
antihyperlipemic O
drugs O
; O
some O
of O
them O
were O
treated O
with O
antihypertensive O
therapy O
. O

No O
signs O
of O
major O
clinical O
or O
laboratory O
symptoms O
of O
other O
diseases O
were O
present O
in O
any O
group O
of O
the O
explored O
patients B
. O

Blood O
samples O
were O
obtained O
in O
the O
morning O
via O
a O
venipuncture O
after O
overnight O
fasting O
. O

After O
clotting O
the O
serum O
was O
separated O
and O
stored O
at O
- O
20 O
degrees O
until O
used O
. O

An O
informed O
consent O
was O
obtained O
from O
all O
probands O
. O

Body O
mass O
indexes O
( O
BMI O
) O
, O
defined O
as O
weight O
in O
kilograms O
divided O
by O
the O
square O
of O
height O
in O
meters O
, O
were O
calculated O
. O

Biochemical O
methods O

Serum O
leptin O
concentrations O
were O
measured O
by O
a O
sandwich O
ELISA O
test O
kit O
( O
Human B
Leptin O
ELISA O
, O
BioVendor O
Laboratory O
Medicine O
, O
Inc O
, O
Czech O
Republic O
) O
. O

Its O
sensitivity O
limit O
was O
0 O
. O
2 O
ng O
/ O
ml O
, O
intraassay O
CV O
6 O
. O
1 O
% O
at O
the O
level O
of O
7 O
. O
5 O
ng O
/ O
m O
, O
inter O
- O
assay O
CV O
8 O
. O
5 O
% O
at O
the O
level O
of O
4 O
. O
8 O
ng O
/ O
ml O
. O

Tetramethylbenzidine O
was O
used O
as O
a O
substrate O
; O
quality O
controls O
were O
human B
based O
. O

Several O
other O
hormones O
and O
peptides O
were O
estimated O
by O
routine O
immunochemical O
tests O
: O
insulin O
, O
C O
- O
peptide O
, O
TNF O
alpha O
( O
IMMULITE O
, O
Diagnostic O
Products O
Corporation O
, O
Los O
Angeles O
, O
CA O
, O
U O
. O
S O
. O
A O
. O
) O
, O
proinsulin O
intact O
( O
DAKO O
, O
Denmark O
) O
, O
IgG O
anticardiolipin O
( O
ACL O
- O
IgG O
, O
IMMCO O
Diagnostics O
, O
Buffalo O
, O
NY O
, O
U O
. O
S O
. O
A O
. O
) O
and O
heart O
fatty O
acid O
binding O
protein O
( O
hFABP O
, O
Hbt O
HUMAN B
H O
- O
FABP O
, O

HyCult O
Biotechnology O
, O
Uden O
, O
the O
Netherlands O
) O
. O

Serum O
concentration O
of O
glucose O
, O
total O
cholesterol O
, O
triglycerides O
, O
HDL O
- O
cholesterol O
, O
LDL O
- O
cholesterol O
, O
Apoprotein O
B O
and O
uric O
acid O
were O
measured O
on O
a O
ILAB O
- O
600 O
biochemical O
analyzer O
( O
Instrumentation O
Laboratory O
, O
Lexington O
, O
Ma O
, O
U O
. O
S O
. O
A O
. O
) O
using O
BioVendor O
sets O
. O

All O
samples O
were O
processed O
and O
examined O
according O
to O
principles O
of O
good O
laboratory O
practice O
and O
under O
constant O
intralaboratory O
and O
external O
quality O
control O
. O

The O
homeostatic O
indexes O
of O
insulin O
resistance O
( O
HOMA O
IR O
and O
QUICKI O
) O
were O
calculated O
according O
to O
the O
homeostasis O
model O
of O
assessment O
[ O
33 O
- O
35 O
] O
as O
follows O
: O

HOMA O
IR O
= O
fasting O
insulin O
( O
mu O
U O
/ O
ml O
) O
* O
fasting O
glucose O
( O
mmol O
/ O
l O
) O
/ O
22 O
. O
5 O
; O

QUICKI O
= O
1 O
/ O
[ O
log O
fasting O
insulin O
( O
mu O
U O
/ O
ml O
) O
+ O
log O
fasting O
glucose O
( O
mg O
/ O
100 O
ml O
) O
] O
. O

Statistics O

Statistical O
analysis O
was O
performed O
using O
the O
Version O
6 O
SAS O
/ O
STAT O
software O
( O
SAS O
Institute O
, O
Inc O
. O
, O
Cary O
, O
NC O
, O
U O
. O
S O
. O
A O
. O
) O
. O

The O
Shapiro O
- O
Wilks O
tests O
were O
used O
in O
testing O
the O
normality O
of O
distribution O
. O

Some O
of O
the O
data O
obtained O
were O
not O
normally O
distributed O
. O

The O
statistical O
significance O
of O
differences O
between O
the O
means O
in O
the O
hyperlipemic O
and O
control O
groups O
were O
evaluated O
using O
the O
unpaired O
Student O
' O
s O
T O
- O
test O
in O
the O
case O
of O
normal O
distribution O
of O
data O
sets O
, O
and O
using O
the O
Kolmogorov O
- O
Smirnov O
test O
when O
at O
least O
in O
one O
of O
the O
data O
sets O
the O
normal O
distribution O
was O
excluded O
. O

Spearman O
' O
s O
rank O
- O
order O
correlation O
was O
used O
for O
correlation O
analysis O
. O

Multiple O
regression O
analysis O
was O
performed O
using O
HOMA O
IR O
indexes O
of O
insulin O
resistance O
as O
dependent O
variables O
, O
and O
other O
metabolic O
and O
hormonal O
factors O
( O
lipid O
parameters O
, O
BMI O
, O
leptin O
, O
TNF O
alpha O
, O
hFABP O
, O
ACL O
- O
IgG O
) O
as O
independent O
variables O
. O

The O
so O
- O
called O
step O
- O
down O
regression O
model O
was O
used O
to O
select O
dominant O
independent O
variables O
. O

Various O
four O
- O
member O
groups O
of O
independent O
( O
explanatory O
) O
variables O
were O
used O
for O
the O
analysis O
and O
the O
non O
- O
zero O
intercept O
was O
taken O
into O
account O
. O

The O
independent O
variables O
were O
then O
dropped O
, O
one O
at O
a O
time O
; O
at O
each O
stage O
one O
variable O
making O
the O
least O
contribution O
to O
the O
dependent O
variable O
( O
i O
. O
e O
. O
that O
showed O
the O
least O
p O
- O
value O
in O
the O
test O
of O
the O
regression O
coefficient O
being O
zero O
) O
was O
excluded O
. O

The O
coefficient O
of O
determination O
R2 O
, O
which O
can O
be O
viewed O
as O
a O
percentage O
explaining O
the O
total O
variance O
, O
was O
simultaneously O
monitored O
. O

A O
great O
drop O
in O
R2 O
after O
excluding O
some O
independent O
variable O
enabled O
selection O
of O
those O
independent O
variables O
that O
could O
be O
thought O
to O
be O
the O
most O
important O
determinants O
of O
the O
dependent O
variable O
. O

Results O

Table O
1 O
demonstrates O
mean O
parameters O
in O
individual O
groups O
of O
subjects O
matched O
according O
to O
sex O
, O
lipid O
parameters O
and O
age O
. O

While O
the O
age O
of O
all O
four O
groups O
did O
not O
differ O
substantially O
, O
the O
concentrations O
of O
total O
serum O
cholesterol O
, O
triglycerides O
, O
HDL O
- O
cholesterol O
and O
LDL O
- O
cholesterol O
differ O
very O
significantly O
in O
both O
male O
and O
female O
hyperlipemic O
groups O
as O
compared O
with O
controls O
. O

In O
addition O
, O
the O
concentration O
of O
triglycerides O
in O
control O
women B
was O
significantly O
higher O
than O
in O
control O
men B
, O
the O
concentration O
of O
triglycerides O
in O
hyperlipemic O
women B
was O
lower O
than O
in O
hyperlipemic O
men B
, O
and O
the O
concentration O
of O
HDL O
- O
cholesterol O
in O
hyperlipemic O
women B
was O
very O
significantly O
higher O
when O
compared O
with O
hyperlipemic O
men B
. O

Table O
2 O
shows O
the O
values O
of O
other O
metabolic O
and O
insulin O
parameters O
, O
factors O
related O
to O
insulin O
resistance O
and O
indexes O
of O
insulin O
resistance O
, O
respectively O
. O

Body O
mass O
indexes O
and O
uric O
acid O
concentration O
were O
significantly O
higher O
in O
hyperlipemic O
men B
as O
compared O
to O
controls O
, O
but O
not O
in O
hyperlipemic O
women B
. O

Uric O
acid O
concentration O
was O
substantially O
lower O
in O
hyperlipemic O
women B
than O
in O
hyperlipemic O
men B
. O

Plasma O
concentrations O
of O
glycemia O
, O
insulin O
and O
intact O
proinsulin O
were O
significantly O
higher O
in O
both O
hyperlipemic O
men B
and O
women B
as O
compared O
with O
controls O
of O
identical O
gender O
, O
while O
the O
concentration O
of O
leptin O
increased O
only O
in O
hyperlipemic O
men B
. O

However O
, O
serum O
leptin O
concentrations O
of O
both O
control O
and O
hyperlipemic O
women B
were O
significantly O
higher O
than O
in O
corresponding O
groups O
of O
men B
. O

Serum O
concentrations O
of O
TNF O
alpha O
, O
hFABP O
and O
ACL O
- O
IgG O
in O
hyperlipemic O
groups O
of O
both O
men B
and O
women B
were O
not O
significantly O
different O
from O
control O
groups O
. O

On O
the O
other O
hand O
, O
the O
indexes O
of O
insulin O
resistance O
HOMA O
IR O
and O
QUICKI O
differed O
very O
significantly O
in O
hyperlipemic O
groups O
of O
both O
men B
and O
women B
as O
compared O
with O
corresponding O
control O
groups O
, O
more O
distinctly O
in O
women B
. O

From O
Fig O
. O

1 O
, O
presenting O
95 O
% O
confidence O
limits O
of O
insulin O
resistance O
indexes O
HOMA O
IR O
and O
QUICKI O
, O
we O
concluded O
that O
in O
groups O
of O
hyperlipemic O
patients B
of O
both O
genders O
the O
insulin O
resistance O
was O
substantially O
higher O
than O
in O
control O
groups O
; O
the O
groups O
did O
not O
overlap O
each O
other O
. O

In O
Table O
3 O
the O
results O
of O
Spearman O
' O
s O
correlations O
between O
insulin O
resistance O
index O
HOMA O
IR O
and O
various O
metabolic O
parameters O
are O
presented O
. O

In O
the O
control O
group O
of O
men B
, O
positive O
significant O
correlation O
between O
HOMA O
IR O
and O
serum O
leptin O
concentration O
, O
and O
inverse O
significant O
correlation O
between O
HOMA O
IR O
and O
ACL O
IgG O
, O
respectively O
, O
were O
found O
. O

In O
the O
control O
group O
of O
women B
, O
the O
significance O
of O
Spearman O
' O
s O
correlation O
between O
HOMA O
IR O
and O
leptin O
was O
more O
expressive O
; O
inverse O
correlation O
between O
HOMA O
IR O
and O
HDL O
- O
cholesterol O
was O
also O
present O
. O

In O
the O
hyperlipemic O
group O
of O
men B
, O
the O
significance O
of O
the O
correlation O
between O
HOMA O
IR O
was O
more O
expressive O
in O
relation O
to O
the O
control O
group O
, O
and O
no O
significant O
correlation O
between O
HOMA O
IR O
and O
ACL O
IgG O
was O
found O
. O

In O
the O
hyperlipemic O
group O
of O
women B
, O
however O
, O
the O
significance O
of O
Spearman O
' O
s O
correlation O
between O
HOMA O
IR O
and O
serum O
leptin O
concentration O
weakened O
, O
the O
inverse O
correlation O
between O
HOMA O
IR O
and O
HDL O
- O
cholesterol O
remained O
approximately O
unchanged O
, O
and O
a O
positive O
correlation O
between O
HOMA O
IR O
and O
serum O
concentration O
of O
TNF O
alpha O
appeared O
. O

Table O
4 O
shows O
results O
of O
multiple O
regression O
analysis O
, O
when O
data O
from O
both O
control O
and O
hyperlipemic O
groups O
of O
each O
gender O
were O
judged O
together O
. O

HOMA O
IR O
was O
considered O
as O
a O
dependent O
variable O
and O
differently O
changed O
constellations O
of O
metabolic O
and O
other O
factors O
were O
taken O
as O
independent O
variables O
. O

In O
men B
, O
BMI O
and O
leptin O
seemed O
to O
play O
a O
main O
role O
in O
influencing O
the O
insulin O
resistance O
index O
HOMA O
IR O
, O
while O
TGL O
, O
ACL O
IgG O
and O
LDL O
- O
cholesterol O
didn O
' O
t O
play O
any O
significant O
role O
( O
see O
left O
columns O
of O
Table O
4 O
) O
. O

The O
decreasing O
of O
HDL O
- O
cholesterol O
concentration O
may O
also O
have O
some O
influence O
( O
see O
a O
significant O
drop O
of O
R2 O
after O
exclusion O
of O
this O
factor O
in O
Table O
4A O
, O
4B O
) O
. O

But O
in O
the O
presence O
of O
leptin O
in O
the O
group O
of O
independent O
factors O
, O
the O
drop O
of O
R2 O
after O
exclusion O
of O
HDL O
- O
cholesterol O
from O
these O
factors O
was O
minimal O
( O
see O
Table O
4C O
) O
. O

On O
the O
other O
hand O
, O
after O
the O
exclusion O
of O
TNF O
alpha O
from O
the O
group O
of O
independent O
variables O
( O
see O
Table O
4B O
, O
4D O
) O
the O
value O
of O
R2 O
has O
unexpectedly O
risen O
, O
which O
could O
reflect O
the O
interference O
of O
TNF O
alpha O
with O
factors O
increasing O
the O
insulin O
resistance O
. O

In O
women B
( O
see O
right O
columns O
of O
Table O
4 O
) O
, O
the O
maximal O
values O
of O
R2 O
were O
achieved O
with O
combination O
of O
independent O
variables O
containing O
TGL O
, O
leptin O
and O
HDL O
- O
cholesterol O
( O
about O
60 O
% O
influence O
on O
HOMA O
IR O
! O
- O
see O
Table O
4A O
, O
4B O
, O
4C O
) O
. O

TNF O
alpha O
seemed O
to O
play O
quite O
a O
different O
role O
than O
in O
men B
: O
after O
exclusion O
of O
this O
factor O
from O
the O
group O
of O
independent O
factors O
R2significantly O
decreased O
( O
see O
Table O
4B O
, O
4D O
) O
. O

In O
contrast O
to O
men B
, O
the O
role O
of O
BMI O
seemed O
to O
be O
minimal O
. O

As O
in O
men B
, O
the O
role O
of O
ACL O
IgG O
and O
LDL O
- O
cholesterol O
in O
influencing O
HOMA O
IR O
was O
negligible O
, O
but O
in O
contrast O
to O
men B
, O
hFABP O
might O
play O
a O
certain O
role O
in O
this O
process O
( O
see O
Table O
4D O
) O
. O

Generally O
, O
the O
insulin O
resistance O
( O
represented O
by O
HOMA O
IR O
) O
was O
in O
men B
much O
less O
influenced O
by O
metabolic O
variables O
than O
in O
women B
; O
while O
in O
women B
in O
some O
combinations O
of O
dependent O
variables O
R2 O
reached O
64 O
% O
, O
in O
men B
the O
maximal O
value O
of O
R2 O
was O
only O
39 O
% O
. O

Discussion O

In O
our O
previous O
paper O
[ O
36 O
] O
, O
the O
mean O
value O
of O
HOMA O
IR O
in O
healthy O
subjects O
of O
both O
genders O
and O
of O
age O
comparable O
with O
our O
controls O
was O
1 O
. O
57 O
+ O
/ O
- O
0 O
. O
87 O
, O
and O
the O
mean O
value O
of O
index O
QUICKI O
0 O
. O
366 O
+ O
/ O
- O
0 O
. O
029 O
, O
respectively O
. O

These O
values O
, O
as O
well O
as O
the O
95 O
% O
confidence O
limits O
, O
correspond O
to O
values O
found O
in O
controls O
in O
this O
study O
. O

In O
accordance O
with O
many O
previous O
papers O
, O
serum O
concentrations O
of O
leptin O
in O
women B
( O
both O
control O
and O
hyperlipemic O
) O
were O
substantially O
higher O
than O
in O
men B
. O

In O
the O
control O
group O
of O
women B
the O
correlation O
between O
leptin O
and O
HOMA O
IR O
was O
highly O
significant O
. O

However O
, O
in O
hyperlipemic O
women B
the O
significance O
of O
this O
correlation O
lessened O
, O
because O
HOMA O
IR O
increased O
considerably O
( O
and O
significantly O
) O
but O
serum O
concentration O
of O
leptin O
only O
slightly O
( O
insignificantly O
) O
. O

In O
men B
the O
significance O
of O
correlations O
between O
serum O
leptin O
and O
HOMA O
IR O
was O
high O
and O
approximately O
the O
same O
in O
both O
the O
control O
and O
hyperlipemic O
groups O
, O
because O
the O
values O
of O
HOMA O
IR O
as O
well O
as O
serum O
leptin O
have O
nearly O
doubled O
in O
hyperlipemic O
in O
relation O
to O
control O
groups O
. O

In O
non O
- O
hyperlipemic O
postmenopausal O
women B
the O
high O
concentration O
of O
serum O
leptin O
was O
not O
associated O
with O
higher O
insulin O
resistance O
: O
HOMA O
IR O
did O
not O
differ O
substantially O
from O
men B
. O

A O
significant O
increase O
of O
insulin O
resistance O
in O
hyperlipemic O
women B
was O
associated O
by O
only O
slight O
and O
insignificant O
increase O
of O
leptin O
concentration O
. O

According O
to O
Spearman O
' O
s O
correlations O
, O
an O
increase O
of O
serum O
TNF O
alpha O
and O
/ O
or O
a O
decrease O
of O
HDL O
- O
cholesterol O
might O
also O
play O
a O
distinct O
role O
in O
this O
respect O
. O

( O
see O
Table O
3 O
) O
. O

In O
contrast O
to O
women B
, O
in O
hyperlipemic O
men B
the O
increase O
of O
insulin O
resistance O
index O
was O
approximately O
proportional O
with O
the O
increase O
of O
leptin O
concentration O
. O

Multiple O
regression O
analysis O
affirmed O
the O
importance O
of O
leptin O
serum O
in O
increasing O
of O
insulin O
resistance O
in O
both O
genders O
. O

In O
men B
, O
only O
BMI O
and O
HDL O
- O
cholesterol O
from O
other O
factors O
studied O
seemed O
to O
play O
a O
certain O
role O
, O
but O
the O
maximal O
values O
of O
influencing O
HOMA O
IR O
reached O
only O
39 O
% O
, O
with O
leptin O
and O
BMI O
being O
the O
more O
important O
factors O
. O

On O
the O
other O
hand O
, O
in O
women B
the O
maximal O
determination O
of O
HOMA O
IR O
as O
high O
as O
60 O
% O
was O
registered O
in O
combination O
of O
serum O
leptin O
, O
TGL O
and O
decreased O
HDL O
- O
cholesterol O
as O
independent O
factors O
; O
the O
role O
of O
BMI O
was O
insignificant O
. O

It O
is O
not O
known O
how O
leptin O
is O
regulated O
. O

A O
strong O
correlation O
between O
plasma O
leptin O
and O
fasting O
insulin O
undoubtedly O
exists O
, O
but O
hyperleptinemia O
in O
both O
obese O
and O
lean O
humans B
is O
not O
likely O
the O
result O
of O
hyperinsulinemia O
[ O
37 O
] O
. O

A O
relationship O
between O
leptin O
and O
insulin O
dependent O
on O
sex O
or O
BMI O
was O
reported O
, O
but O
relationship O
between O
triglyceride O
concentrations O
and O
leptin O
was O
independent O
of O
sex O
, O
BMI O
, O
and O
insulin O
[ O
18 O
, O
24 O
] O
. O

Hyperleptinemia O
, O
as O
an O
early O
sign O
of O
obesity O
, O
was O
closely O
linked O
to O
subcutaneous O
fat O
mass O
[ O
39 O
, O
40 O
] O
. O

Percentage O
of O
body O
fat O
has O
been O
shown O
to O
be O
the O
strongest O
predictor O
of O
leptin O
levels O
even O
in O
lean O
women B
[ O
41 O
] O
. O

Leptin O
was O
highly O
correlated O
with O
percentage O
of O
body O
fat O
and O
with O
fat O
mass O
in O
adults O
irrespective O
of O
gender O
and O
age O
; O
however O
, O
the O
mean O
determinant O
of O
leptin O
plasma O
concentration O
in O
men B
and O
postmenopausal O
women B
was O
BMI O
, O
while O
in O
premenopausal O
women B
it O
was O
only O
the O
fat O
mass O
[ O
42 O
] O
. O

These O
findings O
contrast O
with O
our O
results O
showing O
minimal O
influence O
of O
BMI O
on O
HOMA O
IR O
in O
postmenopausal O
women B
. O

All O
factors O
mentioned O
are O
connected O
with O
fat O
tissue O
: O
leptin O
and O
TNF O
alpha O
are O
directly O
produced O
chiefly O
by O
adipocytes O
, O
BMI O
growth O
is O
obviously O
accompanied O
by O
fat O
mass O
increase O
, O
and O
the O
typical O
hypertriglyceridemia O
associated O
with O
a O
decrease O
of O
HDL O
- O
cholesterol O
goes O
along O
with O
obesity O
and O
fat O
mass O
growth O
. O

The O
gender O
differences O
in O
circulating O
leptin O
were O
best O
explained O
by O
percentage O
of O
body O
fat O
and O
- O
inversely O
- O
by O
lean O
body O
mass O
[ O
25 O
] O
. O

In O
both O
genders O
the O
intra O
- O
abdominal O
fat O
correlated O
with O
insulin O
resistance O
, O
while O
the O
subcutaneous O
fat O
correlated O
with O
circulating O
leptin O
[ O
11 O
, O
12 O
] O
. O

In O
men B
obesity O
led O
to O
a O
prevalent O
increase O
of O
intra O
- O
abdominal O
fat O
, O
while O
in O
women B
of O
subcutaneous O
fat O
[ O
13 O
] O
. O

Influences O
of O
different O
compartments O
of O
adipose O
tissues O
could O
elucidate O
the O
variability O
of O
correlations O
between O
insulin O
resistance O
and O
high O
leptin O
concentrations O
in O
lean O
and O
obese O
subjects O
of O
both O
genders O
[ O
43 O
] O
. O

In O
our O
non O
- O
hyperlipemic O
postmenopausal O
women B
the O
content O
of O
subcutaneous O
fat O
mass O
might O
be O
higher O
than O
in O
non O
- O
hyperlipemic O
men B
of O
appropriate O
age O
, O
which O
indicated O
a O
higher O
serum O
concentration O
of O
leptin O
. O

However O
, O
the O
insulin O
resistance O
- O
related O
to O
intra O
- O
abdominal O
fat O
mass O
- O
did O
not O
differ O
from O
men B
. O

The O
significant O
increase O
of O
insulin O
resistance O
and O
leptin O
concentration O
in O
hyperlipemic O
men B
might O
reflect O
the O
growing O
content O
of O
both O
subcutaneous O
and O
intra O
- O
abdominal O
fat O
mass O
( O
see O
the O
significant O
increase O
of O
BMI O
) O
. O

In O
hyperlipemic O
women B
the O
significant O
increase O
of O
insulin O
resistance O
accompanying O
only O
minimal O
insignificant O
increase O
of O
leptin O
could O
be O
caused O
by O
prevalent O
growing O
of O
intra O
- O
abdominal O
fat O
mass O
. O

In O
elderly O
postmenopausal O
women B
, O
an O
association O
between O
leptin O
and O
plasma O
lipoprotein O
concentration O
was O
found O
which O
depended O
on O
adiposity O
[ O
17 O
] O
, O
and O
inverse O
correlations O
between O
serum O
leptin O
and O
HDL O
- O
cholesterol O
were O
described O
[ O
44 O
] O
. O

In O
our O
study O
, O
insulin O
resistance O
in O
women B
seemed O
to O
be O
more O
notably O
than O
in O
men B
influenced O
by O
lipid O
disorders O
, O
i O
. O
e O
. O
positively O
by O
serum O
triglycerides O
and O
inversely O
by O
HDL O
- O
cholesterol O
. O

These O
findings O
might O
be O
important O
in O
considering O
the O
concept O
of O
treatment O
of O
insulin O
resistance O
- O
related O
disorders O
in O
postmenopausal O
women B
. O

The O
significant O
role O
of O
TNF O
alpha O
in O
insulin O
resistance O
, O
caused O
by O
inhibiting O
the O
transduction O
of O
insulin O
signaling O
and O
by O
down O
- O
regulation O
of O
glucose O
transporter O
GLUT O
- O
4 O
and O
insulin O
receptor O
substrate O
- O
1 O
, O
has O
been O
repeatedly O
confirmed O
[ O
45 O
- O
48 O
] O
. O

Our O
results O
supported O
these O
findings O
unambiguously O
only O
in O
women B
, O
while O
in O
men B
TNF O
alpha O
seemed O
paradoxically O
to O
interfere O
with O
other O
factors O
- O
mainly O
BMI O
and O
leptin O
- O
in O
influencing O
insulin O
resistance O
, O
thus O
playing O
a O
quite O
different O
role O
. O

Previously O
it O
was O
found O
[ O
46 O
] O
that O
correlation O
between O
serum O
TNF O
alpha O
on O
the O
one O
side O
, O
and O
insulin O
, O
HOMA O
IR O
, O
serum O
triglycerids O
, O
respectively O
, O
on O
the O
other O
side O
, O
was O
substantially O
more O
significant O
in O
women B
than O
in O
men B
. O

Serum O
concentration O
of O
TNF O
alpha O
in O
patients B
with O
type O
2 O
diabetes O
of O
both O
genders O
correlated O
only O
with O
the O
quantity O
of O
intra O
- O
abdominal O
fat O
compartment O
[ O
50 O
] O
. O

Visceral O
obesity O
correlated O
with O
plasmatic O
aldosterone O
and O
with O
insulin O
resistance O
only O
in O
premenopausal O
women B
, O
but O
not O
in O
men B
[ O
51 O
] O
. O

From O
all O
these O
data O
we O
might O
support O
our O
above O
mentioned O
conclusion O
- O
that O
rising O
of O
insulin O
resistance O
in O
hyperlipemic O
women B
was O
associated O
with O
an O
increase O
of O
intra O
- O
abdominal O
fat O
, O
because O
this O
fat O
mass O
in O
particular O
is O
a O
source O
of O
TNF O
alpha O
, O
which O
interfered O
with O
insulin O
sensitivity O
only O
in O
women B
. O

We O
came O
to O
this O
conclusion O
irrespective O
of O
the O
finding O
that O
the O
increase O
of O
serum O
TNF O
alpha O
in O
hyperlipemic O
women B
was O
statistically O
insignificant O
; O
results O
of O
Spearman O
' O
s O
correlation O
( O
Table O
3 O
) O
and O
multiple O
regression O
analysis O
confirm O
a O
distinct O
role O
of O
this O
factor O
. O

In O
hyperlipemic O
men B
not O
only O
the O
serum O
concentration O
of O
TNF O
alpha O
has O
decreased O
instead O
of O
increasing O
, O
but O
according O
to O
multiple O
regression O
analysis O
it O
played O
a O
quite O
different O
role O
in O
influencing O
insulin O
sensitivity O
, O
interfering O
with O
factors O
that O
determined O
insulin O
resistance O
( O
leptin O
and O
BMI O
) O
. O

In O
the O
control O
group O
of O
men B
IgG O
anticardiolipin O
was O
inversely O
correlated O
to O
HOMA O
IR O
. O

The O
significance O
of O
this O
finding O
is O
not O
clear O
. O

These O
antibodies O
indicate O
vascular O
and O
thrombotic O
complications O
and O
oxidative O
modification O
of O
lipoproteins O
[ O
52 O
, O
53 O
] O
and O
may O
represent O
an O
increased O
risk O
of O
atherogenic O
and O
inflammatory O
complications O
. O

In O
this O
case O
, O
however O
, O
their O
growing O
might O
be O
connected O
with O
an O
increase O
in O
insulin O
sensitivity O
. O

Anyway O
, O
ACL O
IgG O
evidently O
did O
not O
participate O
significantly O
in O
influencing O
the O
increase O
of O
insulin O
resistance O
associated O
with O
hyperlipidemia O
, O
although O
other O
anti O
- O
cardiolipin O
correlations O
could O
be O
masked O
by O
the O
relatively O
large O
inter O
- O
individual O
variations O
in O
this O
parameter O
. O

Neither O
serum O
concentration O
of O
hFABP O
, O
a O
factor O
ensuring O
transmembrane O
transport O
and O
oxidative O
metabolisation O
of O
long O
- O
chain O
fatty O
acids O
[ O
54 O
, O
55 O
] O
, O
was O
significantly O
changed O
in O
hyperlipemic O
and O
insulin O
resistant O
subjects O
of O
both O
genders O
. O

This O
factor O
was O
very O
weakly O
associated O
only O
with O
HOMA O
IR O
in O
women B
( O
see O
Table O
4D O
) O
, O
indicating O
that O
enhanced O
metabolisation O
of O
fatty O
acids O
in O
cells O
might O
to O
some O
degree O
contribute O
to O
insulin O
resistance O
. O

Conclusions O

In O
postmenopausal O
women B
as O
well O
as O
in O
men B
of O
approximately O
equal O
age O
serum O
leptin O
plays O
a O
significant O
role O
as O
an O
important O
determinant O
of O
insulin O
resistance O
. O

In O
addition O
to O
this O
factor O
, O
in O
women B
the O
grade O
of O
insulin O
resistance O
is O
very O
considerably O
influenced O
by O
serum O
triglycerides O
, O
tumor O
necrosis O
factor O
alpha O
, O
and O
by O
decreased O
concentration O
of O
HDL O
- O
cholesterol O
, O
while O
in O
men B
only O
a O
mild O
influence O
of O
BMI O
and O
decreased O
HDL O
- O
cholesterol O
is O
observed O
. O

These O
findings O
are O
explained O
as O
a O
consequence O
of O
gender O
- O
related O
differences O
in O
adipose O
tissue O
composition O
and O
/ O
or O
distribution O
in O
both O
normal O
- O
weight O
and O
over O
- O
weight O
subjects O
and O
should O
be O
taken O
into O
account O
in O
treatment O
of O
patients B
with O
metabolic O
risk O
factors O
of O
cardiovascular O
diseases O
. O

List O
of O
abbreviations O

HDL O
- O
Cholesterol O
= O
high O
- O
density O
cholesterol O

LDL O
- O
cholesterol O
= O
low O
- O
density O
cholesterol O

HOMA O
IR O
= O
Homeostasis O
Assessment O
of O
Insulin O
Resistance O

= O
fasting O
insulin O
( O
mu O
U O
/ O
ml O
) O
* O
fasting O
glucose O
( O
mmol O
/ O
l O
) O
/ O
22 O
, O
5 O

QUICKI O
= O
1 O
/ O
[ O
log O
fasting O
insulin O
( O
mu O
U O
/ O
ml O
) O
+ O
log O
fasting O
glucose O
( O
mg O
/ O
100 O
ml O
) O
] O

TNF O
alpha O
= O
tumor O
necrosis O
factor O
alpha O

BMI O
= O
body O
mass O
index O

R2 O
= O
coefficient O
of O
determination O

hFABP O
= O
heart O
fatty O
acid O
binding O
protein O

ACL O
- O
IgG O
= O
IgG O
fraction O
of O
anticardiolipin O

TGL O
= O
triglycerides O

GLUT O
- O
4 O
= O
glucose O
transporter O
- O
4 O

PPAR O
gamma O
= O
Peroxisome O
Proliferator O
- O
Associated O
Receptor O
gamma O

CV O
= O
coefficient O
of O
variation O

Authors O
' O
contributions O

Dr O
. O
Radka O
Lichnovsk O
a O
collected O
the O
clinical O
material O
, O
performed O
analysis O
of O
biochemical O
values O
and O
edited O
the O
manuscript O
. O

Dr O
. O
Simona O
Gwozdziewiczov O
a O
performed O
analysis O
of O
clinical O
and O
biochemical O
data O
and O
edited O
the O
manuscript O
. O

Prof O
. O

Jir O
i O
Hreb O
i O
cek O
initiated O
the O
study O
, O
participated O
in O
its O
design O
and O
coordination O
, O
and O
wrote O
and O
edited O
the O
manuscript O
. O

Alexithymia O
and O
anxiety O
in O
female O
chronic O
pain O
patients B

Abstract O

Objectives O

Alexithymia O
is O
highly O
prevalent O
among O
chronic O
pain O
patients B
. O

Pain O
is O
a O
remarkable O
cause O
for O
high O
levels O
of O
chronic O
anxiety O
. O

The O
purpose O
of O
this O
study O
was O
to O
investigate O
the O
prevalence O
of O
alexithymia O
and O
to O
determine O
anxiety O
levels O
among O
DSM O
- O
IV O
somatoform O
pain O
disorder O
( O
chronic O
pain O
) O
female O
patients B
and O
to O
examine O
the O
relationship O
between O
alexithymia O
and O
the O
self O
- O
reporting O
of O
pain O
. O

Methods O

Thirty O
adult O
females O
( O
mean O
age O
: O
34 O
, O
63 O
+ O
/ O
- O
10 O
, O
62 O
years O
) O
, O
who O
applied O
to O
the O
outpatient O
psychiatry O
clinic O
at O
a O
public O
hospital O
with O
the O
diagnosis O
of O
chronic O
pain O
disorder O
( O
DSM O
- O
IV O
) O
, O
were O
included O
in O
the O
study O
. O

Thirty O
seven O
healthy O
females O
( O
mean O
age O
: O
34 O
, O
46 O
+ O
/ O
- O
7 O
, O
43 O
years O
) O
, O
who O
matched O
for O
sociodemographic O
features O
with O
the O
patient B
group O
, O
consisted O
the O
control O
group O
. O

A O
sociodemographic O
data O
form O
, O
26 O
- O
item O
Toronto O
Alexithymia O
Scale O
( O
TAS O
- O
26 O
) O
, O
Spielberger O
Trait O
Anxiety O
Inventory O
( O
STAI O
) O
were O
administered O
to O
each O
subject O
and O
information O
was O
obtained O
on O
several O
aspects O
of O
the O
patients B
' O
pain O
, O
including O
intensity O
( O
measured O
by O
VAS O
) O
, O
and O
duration O
. O

Results O

Chronic O
pain O
patients B
were O
found O
significantly O
more O
alexithymic O
than O
controls O
. O

There O
was O
a O
positive O
correlation O
between O
TAS O
- O
26 O
scores O
and O
the O
duration O
of O
pain O
. O

The O
alexithymic O
and O
nonalexithymic O
group O
did O
not O
differ O
in O
their O
perception O
of O
pain O
. O

Neither O
positive O
correlation O
nor O
significant O
difference O
was O
found O
between O
alexithymia O
and O
trait O
anxiety O
in O
pain O
patients B
. O

Discussion O

Alexithymia O
may O
be O
important O
in O
addressing O
the O
diversity O
of O
subjective O
factors O
involved O
in O
pain O
. O

The O
conceptualization O
of O
alexithymia O
as O
a O
personality O
trait O
as O
well O
as O
a O
secondary O
state O
reaction O
is O
underlined O
by O
our O
data O
. O

Background O

The O
original O
definition O
of O
alexithymia O
is O
the O
inability O
to O
identify O
and O
use O
verbal O
language O
to O
describe O
feelings O
[ O
1 O
, O
2 O
] O
. O

Alexithymia O
has O
been O
associated O
with O
a O
variety O
of O
psychiatric O
disorders O
as O
well O
as O
physical O
illness O
[ O
3 O
- O
10 O
] O
. O

As O
a O
measure O
, O
Toronto O
Alexithymia O
Scale O
was O
significantly O
correlated O
with O
the O
measures O
of O
the O
tendency O
to O
experience O
and O
report O
physical O
signs O
and O
symptoms O
[ O
11 O
] O
. O

Several O
studies O
have O
found O
a O
high O
prevalence O
of O
alexithymia O
in O
pain O
patients B
. O

Chronic O
pain O
patients B
frequently O
exhibit O
many O
of O
the O
core O
features O
of O
alexithymia O
, O
such O
as O
problems O
in O
identifying O
and O
describing O
subjective O
feelings O
, O
impoverished O
imaginative O
abilities O
, O
and O
excessive O
preoccupation O
with O
physical O
symptoms O
and O
external O
events O
. O

Although O
several O
studies O
have O
found O
a O
high O
prevalence O
of O
alexithymia O
in O
pain O
patients B
, O
the O
way O
alexithymia O
may O
possibly O
influence O
pain O
experience O
is O
still O
unclear O
[ O
12 O
, O
13 O
] O
. O

DSM O
- O
IV O
- O
TR O
defines O
pain O
disorder O
as O
the O
presence O
of O
pain O
that O
is O
" O
the O
predominant O
focus O
of O
clinical O
attention O
" O
[ O
14 O
] O
. O

In O
chronic O
pain O
disorder O
, O
patients B
complain O
of O
chronic O
pain O
, O
for O
which O
no O
physical O
etiology O
could O
be O
found O
or O
the O
underlying O
disorder O
is O
insufficient O
in O
explaining O
the O
symptoms O
. O

The O
pain O
causes O
clinically O
significant O
distress O
or O
impairment O
in O
social O
, O
occupational O
, O
or O
other O
important O
areas O
of O
functioning O
. O

Psychological O
factors O
are O
judged O
to O
have O
an O
important O
role O
in O
the O
onset O
, O
severity O
, O
exacerbation O
, O
or O
maintenance O
of O
the O
pain O
[ O
15 O
] O
. O

The O
alexithymic O
person B
' O
s O
difficulty O
in O
identifying O
and O
describing O
feelings O
may O
increase O
symptom O
reporting O
by O
several O
mechanisms O
. O

Consequently O
, O
due O
to O
the O
difficulty O
to O
experience O
and O
express O
emotions O
, O
alexithymia O
has O
been O
linked O
with O
somatosensory O
amplification O
, O
which O
is O
the O
tendency O
to O
focus O
on O
benign O
somatic O
sensations O
. O

Alexithymic O
subjects O
are O
considered O
to O
focus O
on O
somatic O
manifestations O
of O
emotional O
arousal O
, O
resulting O
in O
misinterpretation O
of O
somatic O
sensations O
as O
signs O
of O
physical O
illness O
[ O
12 O
, O
13 O
, O
16 O
] O
. O

Accordingly O
, O
previous O
studies O
have O
found O
evidence O
of O
an O
association O
between O
alexithymia O
and O
the O
development O
of O
functional O
somatic O
symptoms O
, O
as O
seen O
in O
patients B
with O
somatoform O
disorders O
. O

On O
the O
other O
hand O
, O
alexithymia O
may O
also O
occur O
as O
a O
secondary O
state O
reaction O
in O
response O
to O
severe O
and O
chronic O
medical O
illness O
[ O
17 O
- O
21 O
] O
. O

Based O
on O
previous O
findings O
, O
these O
factors O
are O
worth O
receiving O
more O
attention O
in O
terms O
of O
clinical O
research O
. O

The O
purpose O
of O
the O
present O
study O
was O
to O
investigate O
the O
prevalence O
of O
alexithymia O
among O
DSM O
- O
IV O
somatoform O
pain O
disorder O
( O
chronic O
pain O
) O
female O
patients B
and O
to O
examine O
the O
relationship O
between O
alexithymia O
and O
the O
self O
- O
reporting O
of O
pain O
in O
this O
group O
of O
patients B
. O

Besides O
, O
the O
study O
searched O
for O
the O
anxiety O
levels O
of O
chronic O
pain O
patients B
with O
or O
without O
alexithymia O
. O

Materials O
and O
methods O

Sample O

The O
sample O
consisted O
of O
30 O
females O
who O
applied O
to O
the O
outpatient O
psychiatry O
clinic O
at O
a O
public O
hospital O
and O
who O
met O
DSM O
- O
IV O
diagnostic O
criteria O
for O
chronic O
pain O
disorder O
. O

Patients B
with O
concomitant O
psychiatric O
disorders O
, O
such O
as O
major O
depression O
, O
anxiety O
disorders O
and O
somatoform O
disorders O
other O
than O
pain O
disorder O
were O
excluded O
. O

Patients B
either O
directly O
applied O
to O
the O
psychiatry O
clinic O
themselves O
or O
were O
referred O
for O
psychiatric O
assessment O
from O
another O
outpatient O
clinic O
, O
mainly O
physical O
medicine O
and O
rehabilitation O
. O

After O
complete O
description O
of O
the O
study O
, O
written O
informed O
consent O
was O
obtained O
from O
each O
subject O
. O

The O
control O
group O
was O
37 O
healthy O
females O
, O
who O
matched O
for O
age O
, O
and O
education O
with O
the O
subjects O
. O

All O
subjects O
participated O
voluntarily O
in O
the O
study O
and O
gave O
consent O
after O
the O
procedure O
had O
been O
fully O
explained O
to O
them O
. O

The O
mean O
age O
of O
the O
patients B
and O
the O
healthy O
controls O
was O
34 O
, O
63 O
+ O
/ O
- O
10 O
, O
62 O
( O
range O
: O
16 O
- O
62 O
) O
and O
34 O
, O
46 O
+ O
/ O
- O
7 O
, O
43 O
( O
range O
: O
22 O
- O
57 O
) O
years O
and O
the O
educational O
level O
was O
6 O
, O
13 O
+ O
/ O
- O
3 O
, O
03 O
( O
range O
: O
5 O
- O
11 O
) O
and O
6 O
, O
59 O
+ O
/ O
- O
2 O
, O
9 O
( O
range O
: O
5 O
- O
14 O
) O
years O
, O
respectively O
. O

There O
were O
no O
significant O
differences O
between O
the O
two O
groups O
with O
respect O
to O
age O
( O
t O
= O
0 O
, O
79 O
, O
df O
= O
65 O
, O
P O
> O
0 O
, O
05 O
) O
, O
educational O
level O
( O
t O
= O
1 O
, O
02 O
, O
df O
= O
65 O
, O
P O
> O
0 O
, O
05 O
) O
, O
and O
marital O
status O
( O
x2 O
= O
0 O
, O
51 O
, O
df O
= O
1 O
, O
P O
> O
0 O
, O
05 O
) O
. O

Measures O

A O
detailed O
sociodemographic O
data O
form O
was O
used O
for O
all O
subjects O
. O

All O
participants B
were O
applied O
Structured O
Clinical O
Interview O
for O
DSM O
- O
IV O
( O
SCID O
- O
I O
) O
[ O
22 O
] O
, O
Turkish O
version O
[ O
23 O
] O
. O

Regarding O
the O
pain O
assessment O
, O
information O
was O
first O
obtained O
on O
several O
aspects O
of O
the O
patients B
' O
pain O
, O
such O
as O
intensity O
, O
and O
duration O
. O

Pain O
intensity O
was O
measured O
by O
Visual O
Analogue O
Scale O
( O
VAS O
) O
, O
using O
a O
horizontal O
10 O
- O
cm O
line O
with O
the O
statement O
' O
no O
pain O
at O
all O
' O
at O
the O
extreme O
left O
- O
hand O
end O
and O
' O
the O
worst O
possible O
pain O
' O
or O
' O
unbearable O
' O
at O
the O
right O
- O
hand O
extreme O
. O

VAS O
is O
scored O
by O
measuring O
the O
distance O
from O
the O
end O
of O
the O
scale O
indicating O
absence O
of O
pain O
( O
or O
no O
distress O
or O
no O
pain O
relief O
) O
to O
the O
place O
marked O
by O
the O
patient B
[ O
24 O
] O
. O

The O
psychometric O
scales O
used O
in O
the O
study O
were O
the O
26 O
- O
item O
Toronto O
Alexithymia O
Scale O
( O
TAS O
- O
26 O
] O
and O
the O
Trait O
Anxiety O
Inventory O
( O
STAI O
) O
, O
which O
were O
both O
validated O
in O
Turkish O
population O
studies O
[ O
25 O
- O
28 O
] O
. O

TAS O
is O
a O
psychometrically O
well O
validated O
and O
reliable O
instrument O
in O
the O
assessment O
of O
alexithymia O
. O
TAS O
has O
been O
validated O
in O
Turkish O
studies O
as O
a O
true O
or O
false O
scale O
. O

Twenty O
- O
six O
items O
are O
scored O
either O
as O
1 O
or O
0 O
and O
the O
higher O
scores O
indicate O
higher O
degrees O
of O
alexithymia O
. O

TAS O
has O
an O
interval O
consistency O
of O
0 O
. O
65 O
[ O
Kuder O
- O
Richardson O
) O
and O
test O
- O
retest O
reliability O
is O
r O
= O
0 O
. O
71 O
, O
p O
< O
0 O
. O
01 O
in O
Turkish O
reliability O
and O
validity O
study O
. O

The O
sample O
was O
divided O
into O
nonalexithymic O
and O
alexityhmic O
groups O
, O
with O
the O
recommended O
cut O
- O
off O
score O
of O
11 O
[ O
27 O
] O
. O

Spielberger O
Trait O
Anxiety O
Inventory O
( O
STAI O
) O
is O
one O
of O
the O
two O
sections O
of O
the O
Spielberger O
Anxiety O
Inventory O
( O
the O
other O
, O
measuring O
state O
anxiety O
) O
. O

' O
Trait O
anxiety O
' O
has O
been O
defined O
as O
anxiety O
proneness O
, O
that O
is O
, O
the O
tendency O
to O
respond O
to O
situations O
perceived O
as O
threatening O
with O
elevations O
in O
the O
intensity O
of O
state O
anxiety O
[ O
26 O
] O
. O

Statistical O
analysis O

In O
order O
to O
determine O
the O
relative O
importance O
of O
a O
number O
of O
factors O
in O
pain O
disorders O
, O
we O
used O
both O
correlation O
analyses O
. O

The O
alexithymic O
and O
nonalexithymic O
groups O
were O
compared O
using O
the O
independent O
sample O
t O
- O
tests O
on O
scores O
of O
psychological O
tests O
. O

The O
statistical O
procedure O
, O
which O
was O
carried O
out O
by O
a O
SPSS O
package O
program O
for O
Windows O
using O
Chi O
- O
square O
, O
Fisher O
' O
s O
exact O
test O
, O
two O
tailed O
t O
test O
and O
Pearson O
correlation O
coefficients O
, O
was O
also O
used O
to O
determine O
group O
differences O
( O
alexithymics O
versus O
nonalexithymics O
) O
in O
sociodemographic O
variables O
and O
various O
aspects O
of O
pain O
. O

Results O

In O
the O
chronic O
pain O
group O
, O
56 O
. O
7 O
% O
of O
patients B
( O
n O
= O
17 O
) O
had O
a O
score O
greater O
than O
11 O
on O
the O
TAS O
- O
26 O
, O
and O
were O
considered O
alexithymic O
. O

The O
mean O
TAS O
- O
26 O
score O
of O
the O
alexithymic O
group O
( O
n O
= O
17 O
) O
was O
17 O
. O
88 O
+ O
/ O
- O
3 O
. O
43 O
and O
the O
nonalexithymic O
group O
( O
n O
= O
13 O
) O
was O
8 O
. O
39 O
+ O
/ O
- O
2 O
. O
02 O
. O

Age O
( O
t O
= O
1 O
, O
38 O
, O
df O
= O
28 O
, O
p O
> O
0 O
, O
18 O
) O
, O
education O
( O
t O
= O
- O
0 O
, O
21 O
, O
df O
= O
28 O
, O
p O
> O
0 O
, O
16 O
) O
and O
marital O
status O
( O
x2 O
= O
0 O
, O
27 O
, O
df O
= O
1 O
, O
p O
> O
0 O
, O
87 O
) O
were O
not O
associated O
with O
alexithymia O
( O
Table O
1 O
) O
. O

In O
the O
control O
group O
, O
24 O
, O
3 O
% O
of O
patients B
( O
n O
= O
9 O
) O
were O
alexithymic O
according O
to O
TAS O
- O
26 O
. O

The O
mean O
TAS O
- O
26 O
score O
of O
the O
alexithymic O
group O
( O
n O
= O
9 O
) O
was O
13 O
, O
82 O
+ O
/ O
- O
1 O
, O
93 O
and O
the O
nonalexithymic O
group O
( O
n O
= O
28 O
) O
was O
10 O
, O
33 O
+ O
/ O
- O
0 O
, O
86 O
. O

Alexithymia O
was O
not O
associated O
with O
age O
( O
t O
= O
- O
1 O
, O
08 O
, O
df O
= O
35 O
, O
p O
> O
0 O
, O
29 O
) O
, O
educational O
level O
( O
t O
= O
1 O
, O
1 O
, O
df O
= O
35 O
, O
p O
> O
0 O
, O
28 O
) O
, O
or O
marital O
status O
( O
x2 O
= O
0 O
, O
74 O
, O
df O
= O
1 O
, O
p O
> O
0 O
, O
79 O
) O
or O
anxiety O
levels O
in O
the O
control O
subjects O
( O
Table O
1 O
) O
. O

The O
duration O
and O
severity O
of O
pain O
, O
TAS O
- O
26 O
scores O
, O
and O
STAI O
scores O
of O
the O
female O
pain O
patients B
are O
shown O
in O
Table O
2 O
. O

Comparison O
of O
the O
alexithymics O
with O
nonalexithymics O
on O
either O
the O
severity O
of O
pain O
or O
pain O
duration O
showed O
no O
statistical O
significance O
( O
t O
= O
0 O
, O
64 O
, O
df O
= O
28 O
, O
p O
> O
0 O
, O
52 O
, O
t O
= O
2 O
, O
05 O
, O
df O
= O
28 O
, O
p O
> O
0 O
, O
05 O
, O
respectively O
) O
. O

TAS O
- O
26 O
score O
and O
duration O
of O
pain O
were O
found O
positively O
correlated O
( O
r O
= O
0 O
, O
50 O
, O
n O
= O
30 O
, O
p O
> O
0 O
, O
005 O
) O
. O

STAI O
( O
trait O
) O
scores O
of O
the O
alexithymics O
in O
the O
pain O
group O
did O
not O
significantly O
differ O
from O
the O
nonlalexithymics O
( O
t O
= O
0 O
, O
06 O
, O
df O
= O
28 O
, O
p O
> O
0 O
, O
95 O
) O
and O
besides O
, O
TAS O
- O
26 O
and O
STAI O
scores O
were O
not O
correlated O
( O
r O
= O
0 O
, O
06 O
, O
p O
> O
0 O
, O
72 O
) O
. O

In O
summary O
, O
there O
are O
three O
points O
to O
be O
emphasized O
. O

First O
, O
chronic O
pain O
patients B
were O
found O
significantly O
more O
alexithymic O
than O
controls O
( O
56 O
, O
7 O
% O
to O
24 O
, O
3 O
% O
) O
. O

Second O
, O
a O
positive O
correlation O
was O
observed O
between O
TAS O
- O
26 O
scores O
and O
duration O
of O
pain O
. O

Third O
, O
neither O
positive O
correlation O
nor O
significant O
difference O
was O
found O
between O
alexithymia O
and O
trait O
anxiety O
in O
pain O
patients B
. O

Discussion O

The O
results O
of O
the O
present O
study O
suggest O
that O
patients B
with O
chronic O
pain O
disorder O
are O
more O
alexithymic O
than O
individuals O
with O
no O
pain O
. O

This O
finding O
is O
consistent O
with O
results O
obtained O
with O
earlier O
measures O
of O
alexithymia O
[ O
11 O
- O
13 O
] O
. O

Although O
they O
may O
share O
common O
clinical O
features O
, O
alexithymia O
and O
somatoform O
pain O
are O
independent O
constructs O
. O

Alexithymia O
may O
be O
a O
consequence O
to O
the O
effects O
of O
severe O
physical O
symptoms O
, O
such O
as O
a O
reduced O
quality O
of O
life O
and O
limitations O
in O
daily O
activities O
. O

Besides O
, O
alexithymia O
may O
be O
conceptualized O
as O
a O
personality O
trait O
as O
well O
as O
a O
secondary O
state O
reaction O
[ O
2 O
, O
3 O
, O
15 O
- O
17 O
] O
. O

In O
this O
study O
, O
the O
question O
investigated O
was O
whether O
alexithymia O
has O
any O
correlation O
with O
the O
duration O
or O
severity O
of O
the O
pain O
itself O
. O

There O
were O
no O
significant O
differences O
between O
alexithymic O
and O
nonalexitymic O
patients B
on O
self O
reports O
of O
current O
pain O
severity O
. O

This O
is O
in O
accordance O
with O
Cox O
' O
s O
study O
[ O
1994 O
] O
in O
which O
it O
was O
further O
pointed O
out O
that O
alexithymic O
patients B
were O
found O
to O
use O
significantly O
more O
verbal O
descriptors O
of O
pain O
compared O
to O
nonalexithymic O
patients B
[ O
13 O
] O
. O

In O
our O
study O
, O
pain O
intensity O
was O
only O
evaluated O
by O
using O
VAS O
. O

One O
problem O
in O
trying O
to O
measure O
the O
intensity O
of O
pain O
is O
the O
lack O
of O
an O
objective O
way O
. O

Pain O
is O
a O
subjective O
experience O
and O
each O
patient B
may O
communicate O
in O
a O
different O
way O
, O
verbally O
or O
nonverbally O
[ O
29 O
] O
. O

Patients B
in O
this O
sample O
were O
sufferers O
of O
chronic O
pain O
, O
who O
had O
already O
chosen O
an O
approved O
way O
of O
expressing O
their O
distress O
. O

Since O
this O
is O
true O
regardless O
of O
alexithymia O
, O
alexithymic O
groups O
and O
nonalexithymic O
groups O
in O
this O
sample O
showed O
no O
difference O
on O
pain O
severity O
. O

The O
positive O
correlation O
between O
alexithymia O
and O
the O
duration O
of O
pain O
in O
this O
sample O
supports O
the O
assumption O
of O
a O
two O
- O
way O
hypothesis O
. O

It O
is O
often O
assumed O
that O
pain O
can O
be O
caused O
by O
alexithymic O
personality O
traits O
and O
also O
that O
severe O
and O
chronic O
pain O
may O
cause O
emotional O
change O
. O

One O
of O
the O
limitations O
of O
this O
study O
is O
that O
because O
of O
the O
cross O
- O
sectional O
design O
, O
we O
are O
unable O
to O
draw O
conclusions O
about O
the O
direction O
of O
causality O
between O
alexithymia O
and O
pain O
. O

The O
duration O
of O
the O
patients B
' O
pain O
could O
approximately O
be O
determined O
, O
yet O
the O
preexisting O
level O
of O
alexithymia O
was O
not O
known O
. O

In O
the O
usual O
absence O
of O
internal O
stimuli O
, O
alexithymic O
person B
may O
be O
expected O
to O
maintain O
an O
external O
focus O
of O
attention O
, O
such O
as O
pain O
. O

Symptom O
chronicity O
may O
force O
the O
alexithymic O
person B
to O
attent O
to O
and O
amplify O
this O
somatic O
sensation O
. O

Difficulties O
in O
the O
ability O
to O
identify O
and O
differentiate O
emotions O
and O
somatic O
experiences O
are O
core O
features O
of O
the O
alexithymic O
construct O
. O

Therefore O
, O
alexithymic O
patients B
might O
be O
expected O
to O
differ O
from O
nonalexithymic O
ones O
in O
their O
anxiety O
levels O
. O

Yet O
, O
in O
our O
pain O
group O
alexithymic O
patients B
showed O
no O
significant O
difference O
from O
the O
nonalexithymics O
on O
trait O
anxiety O
. O

Besides O
, O
alexithymia O
and O
anxiety O
were O
not O
correlated O
at O
all O
. O

The O
reasons O
may O
be O
lying O
in O
the O
specific O
characteristics O
of O
this O
patient B
group O
itself O
. O

The O
study O
included O
patients B
suffering O
from O
chronic O
symptoms O
; O
with O
an O
average O
of O
7 O
, O
44 O
+ O
/ O
- O
6 O
, O
82 O
years O
of O
pain O
in O
the O
alexithymic O
and O
3 O
, O
31 O
+ O
/ O
- O
2 O
, O
79 O
years O
in O
the O
nonalexithymic O
groups O
. O

Persistency O
of O
any O
physical O
symptom O
may O
bring O
along O
alexithymia O
as O
a O
coping O
strategy O
. O

In O
their O
paper O
, O
Crook O
and O
Tunks O
( O
1988 O
) O
examined O
the O
types O
of O
coping O
strategies O
used O
by O
persistent O
pain O
sufferers O
and O
addressed O
to O
the O
importance O
to O
alter O
their O
attitudes O
and O
behavior O
that O
tend O
toward O
catastrophizing O
, O
avoidance O
and O
withdrawal O
, O
rather O
than O
simply O
concentrate O
on O
trying O
to O
teach O
them O
techniques O
for O
' O
coping O
with O
stress O
' O
to O
help O
persistent O
pain O
sufferers O
[ O
30 O
] O
. O

Sufferers O
of O
chronic O
symptoms O
in O
this O
sample O
were O
members O
of O
a O
subgroup O
who O
have O
been O
seeking O
medical O
care O
for O
a O
long O
time O
and O
besides O
given O
the O
chance O
of O
being O
referred O
to O
a O
psychiatrist O
. O

Therefore O
, O
alexithymic O
or O
not O
, O
their O
anxiety O
might O
have O
induced O
unique O
coping O
strategies O
and O
illness O
behavior O
. O

Alexithymia O
may O
be O
important O
in O
addressing O
the O
diversity O
of O
subjective O
factors O
involved O
in O
pain O
[ O
31 O
] O
. O

It O
is O
not O
known O
whether O
it O
should O
be O
addressed O
in O
the O
treatment O
of O
pain O
patients B
, O
but O
a O
high O
level O
of O
alexithymia O
may O
effect O
the O
nature O
of O
assessment O
. O

In O
summary O
, O
the O
conceptualization O
of O
alexithymia O
as O
a O
personality O
trait O
as O
well O
as O
a O
secondary O
state O
reaction O
is O
underlined O
by O
our O
data O
. O

However O
, O
regarding O
the O
cross O
- O
sectional O
design O
of O
this O
study O
, O
only O
limited O
conclusions O
can O
be O
drawn O
about O
the O
nature O
of O
the O
causal O
relationship O
between O
alexithymia O
and O
chronic O
pain O
. O

Therefore O
, O
future O
longitudinal O
studies O
assessing O
the O
cause O
of O
alexithymic O
characteristics O
are O
required O
to O
fully O
elucidate O
the O
concepts O
of O
primary O
and O
secondary O
alexithymia O
. O

Molecular O
polymorphism O
, O
differentiation O
and O
introgression O
in O
the O
period O
gene O
between O
Lutzomyia B
intermedia I
and O
Lutzomyia B
whitmani I

Abstract O

Background O

Lutzomyia B
intermedia I
and O
Lutzomyia B
whitmani I
( O
Diptera B
: O
Psychodidae I
) O
are O
important O
and O
very O
closely O
related O
vector O
species O
of O
cutaneous O
leishmaniasis O
in O
Brazil O
, O
which O
are O
distinguishable O
by O
a O
few O
morphological O
differences O
. O

There O
is O
evidence O
of O
mitochondrial O
introgression O
between O
the O
two O
species O
but O
it O
is O
not O
clear O
whether O
gene O
flow O
also O
occurs O
in O
nuclear O
genes O
. O

Results O

We O
analyzed O
the O
molecular O
variation O
within O
the O
clock O
gene O
period O
( O
per O
) O
of O
these O
two O
species O
in O
five O
different O
localities O
in O
Eastern O
Brazil O
. O

AMOVA O
and O
Fst O
estimates O
showed O
no O
evidence O
for O
geographical O
differentiation O
within O
species O
. O

On O
the O
other O
hand O
, O
the O
values O
were O
highly O
significant O
for O
both O
analyses O
between O
species O
. O

The O
two O
species O
show O
no O
fixed O
differences O
and O
a O
higher O
number O
of O
shared O
polymorphisms O
compared O
to O
exclusive O
mutations O
. O

In O
addition O
, O
some O
haplotypes O
that O
are O
" O
typical O
" O
of O
one O
species O
were O
found O
in O
some O
individuals O
of O
the O
other O
species O
suggesting O
either O
the O
persistence O
of O
old O
polymorphisms O
or O
the O
occurrence O
of O
introgression O
. O

Two O
tests O
of O
gene O
flow O
, O
one O
based O
on O
linkage O
disequilibrium O
and O
a O
MCMC O
analysis O
based O
on O
coalescence O
, O
suggest O
that O
the O
two O
species O
might O
be O
exchanging O
alleles O
at O
the O
per O
locus O
. O

Conclusion O

Introgression O
might O
be O
occurring O
between O
L B
. I
intermedia I
and O
L B
. I
whitmani O
in O
period O
, O
a O
gene O
controlling O
behavioral O
rhythms O
in O
Drosophila B
. O

This O
result O
raises O
the O
question O
of O
whether O
similar O
phenomena O
are O
occurring O
at O
other O
loci O
controlling O
important O
aspects O
of O
behavior O
and O
vectorial O
capacity O
. O

Background O

The O
Phlebotominae B
sand O
flies O
Lutzomyia B
intermedia I
Lutz O
& O
Neiva O
1912 O
and O
Lutzomyia B
whitmani O
Antunes O
& O
Coutinho O
1912 O
are O
vectors O
of O
cutaneous O
leishmaniasis O
in O
Brazil O
. O

These O
are O
closely O
related O
species O
that O
can O
be O
only O
distinguished O
by O
a O
few O
morphological O
differences O
[ O
1 O
] O
and O
both O
show O
high O
anthropophily O
and O
reported O
natural O
infections O
with O
Leishmania O
in O
different O
regions O
of O
Brazil O
[ O
2 O
] O
. O

Despite O
their O
importance O
as O
vectors O
, O
only O
a O
handful O
of O
studies O
have O
been O
carried O
out O
in O
these O
two O
species O
using O
molecular O
techniques O
[ O
3 O
- O
6 O
] O
. O

One O
of O
the O
most O
important O
findings O
from O
an O
epidemiological O
perspective O
is O
the O
evidence O
obtained O
for O
introgression O
between O
the O
two O
species O
using O
mitochondrial O
DNA O
[ O
4 O
] O
. O

This O
was O
particularly O
interesting O
because O
apparently O
, O
only O
lineages O
of O
L B
. I
whitmani I
sympatric O
with O
L B
. I
intermedia I
have O
been O
involved O
in O
cutaneous O
leishmaniasis O
transmission O
in O
the O
peridomestic O
environment O
[ O
4 O
] O
, O
which O
suggests O
that O
genes O
controlling O
aspects O
of O
vectorial O
capacity O
could O
be O
passing O
from O
one O
species O
to O
the O
other O
. O

In O
fact O
, O
mitochondrial O
introgression O
has O
been O
reported O
in O
other O
sand O
fly O
species O
[ O
7 O
, O
8 O
] O
suggesting O
that O
might O
be O
a O
common O
phenomenon O
in O
these O
insect O
vectors O
. O

However O
, O
because O
mitochondrial O
genes O
can O
introgress O
relatively O
easily O
between O
closely O
related O
species O
[ O
9 O
] O
, O
it O
becomes O
important O
to O
examine O
whether O
introgression O
can O
occur O
with O
nuclear O
genes O
. O

The O
Drosophila B
period O
( O
per O
) O
gene O
homologue O
was O
isolated O
in O
sand O
flies O
by O
Peixoto O
et O
al O
. O

[ O
10 O
] O
. O

This O
circadian O
clock O
gene O
was O
originally O
identified O
using O
mutagenesis O
by O
Konopka O
and O
Benzer O
[ O
11 O
] O
, O
but O
is O
also O
known O
to O
control O
the O
differences O
in O
the O
" O
lovesong O
" O
rhythms O
between O
D B
. I
melanogaster I
and O
D B
. I
simulans I
[ O
12 O
] O
, O
that O
are O
important O
to O
the O
sexual O
isolation O
between O
these O
two O
species O
[ O
13 O
- O
15 O
] O
. O

In O
addition O
, O
per O
was O
implicated O
in O
the O
control O
of O
species O
- O
specific O
circadian O
mating O
rhythms O
in O
Drosophila B
and O
Bractocera O
, O
which O
might O
also O
constitute O
a O
reproductive O
isolation O
mechanism O
[ O
16 O
- O
18 O
] O
. O

Thus O
per O
may O
possibly O
represent O
an O
example O
of O
a O
Drosophila B
speciation O
gene O
[ O
19 O
] O
, O
and O
in O
fact O
it O
has O
been O
used O
as O
a O
molecular O
marker O
in O
a O
number O
of O
speciation O
and O
evolutionary O
studies O
, O
not O
only O
in O
Drosophila B
( O
reviewed O
in O
[ O
20 O
] O
) O
but O
also O
in O
other O
insects O
( O
e O
. O
g O
. O
[ O
21 O
] O
) O
including O
sand O
flies O
[ O
22 O
- O
24 O
] O
. O

Because O
per O
controls O
the O
circadian O
clock O
in O
different O
insects O
[ O
25 O
] O
, O
it O
is O
almost O
certainly O
involved O
in O
the O
rhythms O
of O
activity O
and O
biting O
of O
sand O
flies O
[ O
26 O
] O
, O
which O
are O
very O
important O
to O
leishmaniasis O
transmission O
. O

In O
addition O
, O
per O
might O
be O
involved O
in O
reproductive O
isolation O
in O
sand O
flies I
, O
via O
mating O
rhythms O
, O
or O
via O
their O
" O
lovesongs O
" O
[ O
2 O
, O
27 O
] O
. O
per O
is O
thus O
a O
particularly O
interesting O
marker O
, O
among O
the O
few O
available O
, O
for O
an O
introgression O
analysis O
in O
L B
. I
intermedia I
and O
L B
. I
whitmani I
. O

Evidence O
for O
introgression O
in O
per O
might O
suggest O
that O
gene O
flow O
between O
these O
two O
vector O
species O
is O
occurring O
at O
other O
genes O
controlling O
important O
aspects O
of O
behavior O
and O
vectorial O
capacity O
. O

It O
might O
also O
suggest O
that O
per O
does O
not O
have O
a O
strong O
role O
in O
their O
reproductive O
isolation O
. O

In O
the O
current O
study O
, O
we O
analyzed O
the O
molecular O
variation O
within O
the O
per O
gene O
of O
L B
. I
intermedia I
and O
L B
. I
whitmani O
in O
five O
different O
localities O
in O
Eastern O
Brazil O
. O

Results O

Polymorphism O
and O
divergence O
between O
L B
. I
intermedia I
and O
L O
. I
whitmani O

A O
total O
of O
68 O
sequences O
from O
L B
. I
intermedia I
and O
53 O
from O
L B
. I
whitmani I
homologue O
to O
a O
fragment O
of O
the O
period O
gene O
were O
analyzed O
from O
populations O
of O
five O
localities O
in O
Eastern O
Brazil O
( O
Fig O
1 O
) O
. O

The O
alignment O
of O
72 O
variable O
sites O
is O
shown O
in O
Fig O
2 O
. O

Although O
most O
of O
the O
changes O
are O
either O
synonymous O
or O
occur O
within O
the O
58 O
bp O
intron O
, O
non O
- O
synonymous O
substitutions O
are O
observed O
causing O
9 O
amino O
acid O
differences O
among O
the O
sequences O
( O
Fig O
2 O
) O
. O

Table O
1 O
shows O
the O
number O
of O
sequences O
of O
each O
population O
of O
the O
two O
species O
, O
the O
number O
of O
polymorphic O
sites O
( O
S O
) O
and O
the O
estimates O
of O
molecular O
polymorphism O
theta O
( O
based O
on O
the O
total O
number O
of O
mutations O
) O
and O
pi O
. O

Table O
1 O
also O
shows O
the O
Tajima O
' O
s O
[ O
28 O
] O
and O
Fu O
& O
Li O
' O
s O
[ O
29 O
] O
statistics O
. O

Within O
each O
species O
, O
all O
populations O
present O
similar O
levels O
of O
polymorphism O
with O
the O
exception O
of O
L B
. I
whitmani O
from O
Ilh O
e O
us O
, O
which O
seems O
to O
be O
less O
polymorphic O
than O
the O
others O
. O

This O
population O
was O
also O
the O
only O
one O
presenting O
a O
significant O
value O
in O
the O
Fu O
& O
Li O
test O
but O
only O
at O
the O
5 O
% O
level O
. O

Finally O
, O
the O
last O
column O
of O
Table O
1 O
presents O
the O
recombination O
estimator O
gamma O
[ O
30 O
] O
indicating O
that O
both O
species O
show O
evidence O
of O
intragenic O
recombination O
in O
the O
per O
gene O
. O

To O
investigate O
the O
level O
of O
intra O
and O
interspecific O
differences O
, O
initially O
an O
AMOVA O
was O
carried O
out O
as O
shown O
in O
Table O
2 O
. O

The O
results O
show O
a O
non O
- O
significant O
within O
species O
and O
a O
significant O
between O
species O
molecular O
variation O
at O
the O
per O
locus O
. O

Table O
3 O
shows O
a O
more O
detailed O
analysis O
of O
the O
intraspecific O
differentiation O
among O
populations O
of O
L B
. I
intermedia I
and O
L O
. O
whitmani O
. O

None O
of O
the O
pairwise O
and O
overall O
fixation O
indexes O
( O
Fst O
) O
are O
significant O
in O
the O
case O
of O
L B
. I
intermedia I
and O
only O
one O
( O
Posse O
x O
Ilh O
e O
us O
) O
has O
a O
borderline O
significant O
value O
in O
L O
. O
whitmani O
. O

The O
results O
therefore O
show O
that O
no O
significant O
geographical O
heterogeneity O
was O
detected O
among O
the O
populations O
of O
the O
two O
species O
. O

The O
estimated O
number O
of O
migrants O
per O
generation O
, O
based O
on O
the O
overall O
Fst O
values O
, O
is O
20 O
. O
683 O
for O
L B
. I
intermedia I
and O
23 O
. O
125 O
for O
L O
. O
whitmani O
. O

Table O
4 O
shows O
measures O
for O
DNA O
divergence O
between O
species O
( O
Dxy O
and O
Da O
) O
, O
as O
well O
as O
the O
Fst O
and O
Nm O
values O
considering O
each O
species O
as O
a O
unique O
population O
. O

Dxy O
is O
the O
average O
number O
of O
nucleotide O
substitutions O
per O
site O
between O
alleles O
from O
two O
different O
populations O
and O
Da O
is O
the O
number O
of O
net O
nucleotide O
substitutions O
between O
two O
populations O
. O

Table O
4 O
also O
shows O
the O
number O
of O
polymorphisms O
exclusive O
for O
each O
species O
( O
Sint O
and O
Swhit O
) O
, O
the O
number O
of O
shared O
polymorphisms O
( O
Ss O
) O
and O
the O
number O
of O
fixed O
differences O
( O
Sf O
) O
between O
species O
. O

As O
one O
can O
note O
, O
there O
is O
a O
high O
number O
of O
shared O
polymorphisms O
between O
species O
, O
and O
no O
fixed O
differences O
between O
them O
suggesting O
either O
the O
persistence O
of O
ancestral O
polymorphisms O
or O
the O
occurrence O
of O
introgression O
. O

In O
fact O
, O
there O
is O
one O
shared O
haplotype O
between O
the O
two O
species O
( O
IPO13 O
, O
WPO10 O
and O
WPO19 O
) O
and O
three O
L O
. O
whitmani O
sequences O
( O
WAC02 O
, O
WPO13 O
and O
WPO14 O
) O
which O
show O
only O
one O
nucleotide O
difference O
to O
" O
typical O
" O
L B
. I
intermedia I
haplotypes O
( O
see O
also O
below O
) O
. O

Genealogy O
of O
period O
sequences O

A O
phylogenetic O
analysis O
of O
the O
period O
gene O
sequences O
from O
L B
. I
intermedia I
and O
L B
. I
whitmani O
was O
carried O
out O
with O
the O
Minimum O
Evolution O
method O
using O
the O
Kimura O
2 O
- O
parameter O
distance O
( O
Fig O
3 O
) O
. O

A O
sequence O
from O
L B
. I
umbratilis I
, O
a O
related O
species O
from O
the O
same O
subgenus O
Nyssomyia O
, O
was O
used O
as O
outgroup O
[ O
24 O
] O
. O

The O
tree O
shows O
L B
. I
intermedia I
and O
L O
. O
whitmani O
as O
non O
- O
monophyletic O
. O

However O
, O
despite O
the O
low O
bootstrap O
values O
, O
which O
are O
below O
50 O
% O
in O
most O
cases O
, O
there O
is O
a O
large O
group O
that O
contains O
most O
L O
. I
intermedia I
sequences O
and O
a O
second O
large O
group O
with O
most O
L O
. O
whitmani O
sequences O
. O

A O
few O
other O
sequences O
are O
clustered O
outside O
these O
two O
main O
groups O
. O

It O
is O
interesting O
to O
note O
that O
there O
are O
three O
L B
. I
whitmani I
alleles O
( O
WAC2 O
, O
WPO13 O
and O
WPO14 O
) O
inside O
L B
. I
intermedia I
main O
group O
, O
as O
well O
as O
one O
L B
. I
intermedia I
allele O
( O
ICP16 O
) O
inside O
the O
L B
. I
whitmani I
main O
group O
. O

In O
addition O
, O
a O
second O
L B
. I
intermedia I
allele O
( O
IPO13 O
) O
is O
a O
shared O
haplotype O
between O
the O
two O
species O
as O
mentioned O
above O
. O

Again O
, O
the O
results O
suggest O
either O
the O
persistence O
of O
ancestral O
polymorphisms O
or O
the O
occurrence O
of O
introgression O
between O
the O
two O
species O
. O

Very O
similar O
results O
were O
obtained O
using O
the O
maximum O
likelihood O
algorithm O
as O
implemented O
in O
PAUP O
4 O
. O
0b10 O
software O
[ O
31 O
] O
( O
data O
not O
shown O
) O
. O

As O
mentioned O
before O
, O
there O
is O
evidence O
of O
intragenic O
recombination O
in O
the O
per O
gene O
fragment O
of O
both O
species O
( O
see O
Table O
1 O
) O
and O
for O
that O
reason O
the O
bifurcating O
tree O
shown O
in O
Fig O
3 O
has O
to O
be O
viewed O
with O
caution O
, O
as O
different O
regions O
of O
the O
gene O
might O
have O
different O
phylogenetic O
histories O
[ O
32 O
] O
. O

Therefore O
, O
we O
constructed O
Minimum O
Evolution O
trees O
with O
the O
two O
most O
polymorphic O
non O
- O
recombining O
blocks O
of O
the O
per O
gene O
fragment O
identified O
using O
the O
Hudson O
and O
Kaplan O
[ O
33 O
] O
method O
available O
in O
the O
DNAsp O
4 O
. O
1 O
program O
[ O
34 O
] O
. O

We O
did O
not O
observed O
major O
changes O
in O
the O
genealogy O
of O
the O
L B
. I
intermedia I
and O
L O
. O
whitmani O
per O
sequences O
, O
especially O
regarding O
the O
five O
haplotypes O
( O
ICP16 O
, O
IPO13 O
, O
WAC2 O
, O
WPO13 O
and O
WPO14 O
) O
that O
clearly O
cluster O
with O
sequences O
of O
the O
other O
species O
( O
data O
not O
shown O
) O
. O

Finally O
, O
a O
haplotype O
network O
was O
estimated O
from O
per O
sequences O
using O
statistical O
parsimony O
, O
as O
described O
by O
Templeton O
et O
al O
. O

[ O
35 O
] O
and O
implemented O
in O
the O
TCS1 O
. O
21 O
software O
[ O
36 O
] O
( O
Fig O
4 O
) O
. O

A O
small O
number O
of O
ambiguities O
were O
resolved O
as O
suggested O
by O
Crandall O
and O
Templeton O
[ O
37 O
] O
. O

The O
haplotype O
network O
shows O
connections O
between O
sequences O
from O
each O
species O
, O
separating O
most O
of O
the O
sequences O
of O
L B
. I
intermedia I
and O
L O
. O
whitmani O
in O
two O
groups O
. O

No O
intraspecific O
geographical O
structuring O
was O
found O
. O

Once O
again O
, O
some O
of O
the O
L O
. O
whitmani O
sequences O
( O
WAC2 O
, O
WAC10 O
, O
WPO13 O
and O
WPO14 O
) O
appear O
more O
closely O
related O
to O
L B
. I
intermedia I
haplotypes O
. O

In O
addition O
, O
one O
L B
. I
intermedia I
allele O
( O
ICP16 O
) O
is O
connected O
by O
a O
small O
number O
of O
mutations O
to O
some O
of O
the O
main O
L O
. O
whitmani O
haplotypes O
and O
IPO13 O
is O
a O
shared O
haplotype O
between O
the O
two O
species O
. O

These O
results O
confirm O
the O
same O
putative O
introgressed O
sequences O
indicated O
by O
the O
phylogenetic O
reconstructions O
. O

LD O
test O
of O
introgression O

We O
tested O
the O
hypothesis O
of O
gene O
flow O
between O
L B
. I
intermedia I
and O
L O
. I
whitmani O
using O
a O
method O
based O
on O
linkage O
disequilibrium O
( O
LD O
) O
developed O
by O
Machado O
et O
al O
. O

[ O
38 O
] O
. O

In O
this O
test O
, O
x O
is O
the O
difference O
between O
the O
average O
LD O
found O
among O
all O
pairs O
of O
shared O
polymorphisms O
( O
DSS O
) O
between O
the O
two O
species O
and O
the O
average O
LD O
among O
all O
pairs O
of O
sites O
for O
which O
one O
member O
is O
a O
shared O
polymorphism O
and O
the O
other O
is O
an O
exclusive O
polymorphism O
( O
DSX O
) O
. O

In O
case O
of O
gene O
flow O
x O
should O
tend O
to O
be O
positive O
[ O
see O
[ O
38 O
] O
for O
more O
details O
] O
. O

Because O
of O
limitations O
on O
the O
total O
number O
of O
sequences O
that O
could O
be O
handled O
by O
the O
WH O
program O
we O
could O
not O
perform O
the O
simulations O
with O
all O
sequences O
. O

Therefore O
, O
we O
carried O
out O
the O
LD O
test O
of O
introgression O
between O
each O
pair O
of O
sympatric O
populations O
of O
L B
. I
intermedia I
and O
L O
. O
whitmani O
from O
the O
localities O
of O
Posse O
, O
Afonso O
Claudio O
and O
Corte O
de O
Pedra O
. O

The O
input O
files O
were O
prepared O
using O
the O
values O
of O
recombination O
and O
linkage O
disequilibrium O
calculated O
by O
the O
SITES O
program O
[ O
30 O
] O
for O
each O
population O
( O
data O
not O
shown O
) O
. O

Although O
no O
significant O
values O
were O
found O
for O
the O
smaller O
samples O
of O
Afonso O
Claudio O
and O
Corte O
de O
Pedra O
, O
the O
results O
( O
Table O
5 O
) O
present O
evidence O
for O
introgression O
in O
the O
period O
gene O
in O
both O
directions O
( O
from O
L B
. I
intermedia I
to O
L O
. O
whitmani O
and O
vice O
- O
versa O
) O
in O
the O
locality O
of O
Posse O
. O

Isolation O
with O
Migration O
model O

To O
further O
examine O
the O
gene O
flow O
between O
L B
. I
intermedia I
and O
L O
. O
whitmani O
we O
used O
the O
IM O
software O
[ O
39 O
] O
. O

The O
Isolation O
with O
Migration O
model O
has O
six O
demographic O
parameters O
that O
include O
two O
migration O
rates O
, O
one O
for O
each O
population O
. O

The O
IM O
software O
estimates O
the O
posterior O
probability O
for O
each O
of O
the O
model O
parameters O
, O
fitting O
the O
Isolation O
with O
Migration O
model O
to O
the O
data O
. O

One O
of O
the O
assumptions O
of O
this O
model O
is O
that O
the O
loci O
studied O
do O
not O
have O
internal O
recombination O
. O

Therefore O
, O
we O
identified O
four O
different O
non O
- O
recombining O
blocks O
of O
our O
fragment O
of O
per O
, O
which O
were O
then O
treated O
as O
different O
loci O
in O
the O
analysis O
. O

The O
four O
- O
gametes O
test O
[ O
33 O
] O
implemented O
in O
DnaSP4 O
. O
1 O
was O
used O
for O
the O
identification O
of O
possible O
recombination O
events O
. O

Since O
the O
program O
estimates O
parameters O
for O
a O
pair O
of O
closely O
related O
populations O
or O
species O
, O
all O
sequences O
of O
each O
species O
were O
used O
in O
the O
analysis O
as O
a O
single O
population O
. O

We O
performed O
MCMC O
runs O
using O
the O
IM O
software O
with O
different O
seed O
numbers O
, O
in O
order O
to O
guarantee O
convergence O
of O
the O
sample O
. O

Maximum O
likelihood O
estimates O
of O
migration O
parameters O
revealed O
a O
non O
- O
zero O
value O
for O
both O
species O
, O
m1 O
= O
1 O
. O
398 O
and O
m2 O
= O
1 O
. O
014 O
( O
m1 O
- O
from O
L B
. I
whitmani I
towards O
L B
. I
intermedia I
; O
m2 O
- O
from O
L B
. I
whitmani I
towards O
L B
. I
intermedia I
) O
. O

Fig O
5 O
shows O
the O
posterior O
distributions O
for O
migration O
rates O
and O
reveals O
a O
null O
probability O
for O
the O
absence O
of O
migration O
from O
L O
. O
whitmani O
towards O
L B
. I
intermedia I
. O

In O
addition O
, O
the O
absence O
of O
migration O
in O
the O
opposite O
direction O
is O
not O
included O
in O
the O
95 O
% O
confidence O
interval O
( O
values O
range O
from O
0 O
. O
222 O
to O
8 O
. O
898 O
) O
, O
thus O
supporting O
the O
presence O
of O
migration O
in O
both O
directions O
. O

The O
conversion O
of O
the O
migration O
rate O
estimate O
to O
population O
migration O
rate O
per O
generation O
( O
m1 O
and O
m2 O
) O
is O
not O
accurate O
when O
the O
population O
size O
is O
based O
on O
a O
single O
locus O
. O

However O
, O
the O
average O
of O
the O
migrant O
number O
per O
generation O
for O
both O
species O
was O
very O
close O
to O
the O
Nm O
estimate O
based O
on O
Fst O
values O
( O
Nm O
~ O
0 O
. O
49 O
in O
Table O
4 O
, O
m1 O
~ O
0 O
. O
52 O
and O
m2 O
~ O
0 O
. O
34 O
) O
. O

Discussion O

There O
is O
some O
evidence O
that O
L B
. I
intermedia I
and O
L O
. I
whitmani I
might O
represent O
sibling O
- O
species O
complexes O
in O
Brazil O
. O

Lutzomyia B
neivai I
Pinto O
1926 O
, O
a O
sibling O
of O
L B
. I
intermedia I
is O
found O
in O
parts O
of O
Southern O
and O
Western O
Brazil O
and O
some O
other O
countries O
of O
South O
America O
[ O
40 O
] O
. O

The O
present O
study O
did O
not O
include O
populations O
of O
this O
species O
. O

In O
the O
case O
of O
L O
. O
whitmani O
, O
mitochondrial O
data O
[ O
3 O
, O
6 O
] O
indicates O
three O
main O
lineages O
in O
Brazil O
: O
an O
Amazonian O
group O
, O
a O
North O
- O
South O
group O
and O
a O
Northeast O
group O
. O

We O
did O
not O
find O
strong O
evidence O
of O
a O
geographical O
differentiation O
in O
the O
period O
gene O
among O
populations O
of O
L B
. I
whitmani O
although O
one O
of O
the O
pairwise O
Fst O
comparisons O
( O
Posse O
x O
Ilh O
e O
us O
) O
was O
significant O
at O
the O
5 O
% O
level O
. O

When O
we O
compare O
L B
. I
intermedia I
and O
L B
. I
whitmani I
, O
we O
find O
a O
highly O
significant O
Fst O
value O
( O
0 O
. O
3373 O
) O
, O
which O
is O
however O
smaller O
than O
that O
observed O
for O
the O
period O
gene O
between O
sympatric O
siblings O
of O
Lutzomyia B
longipalpis I
( O
Fst O
= O
0 O
. O
3952 O
) O
[ O
23 O
] O
, O
a O
complex O
of O
cryptic O
species O
that O
are O
vectors O
of O
American B
visceral I
leishmaniasis I
. O

Therefore O
, O
despite O
the O
presence O
of O
diagnostic O
morphological O
characters O
to O
identify O
L B
. I
intermedia I
and O
L O
. O
whitmani O
[ O
1 O
] O
the O
level O
of O
molecular O
divergence O
in O
period O
is O
not O
as O
high O
as O
the O
cryptic O
L B
. I
longipalpis I
siblings O
. O

Even O
though O
it O
is O
hard O
to O
distinguish O
introgression O
from O
the O
persistence O
of O
ancestral O
polymorphisms O
, O
a O
test O
of O
gene O
flow O
based O
on O
the O
signature O
introgression O
leaves O
on O
the O
patterns O
of O
linkage O
disequilibrium O
[ O
38 O
] O
as O
well O
as O
simulations O
that O
fit O
the O
" O
Isolation O
with O
Migration O
" O
model O
to O
the O
data O
suggest O
that O
L B
. I
intermedia I
and O
L B
. I
whitmani O
might O
be O
exchanging O
alleles O
at O
the O
per O
locus O
. O

This O
is O
further O
supported O
by O
the O
presence O
of O
shared O
haplotypes O
between O
the O
two O
species O
in O
Posse O
and O
very O
similar O
sequences O
in O
all O
sympatric O
populations O
. O

There O
is O
mounting O
evidence O
that O
introgression O
plays O
a O
major O
role O
in O
the O
evolution O
of O
closely O
related O
insect O
vector O
species O
. O

Introgression O
among O
vectors O
may O
have O
important O
epidemiological O
consequences O
. O

Gene O
flow O
in O
loci O
that O
affect O
vectorial O
capacity O
, O
such O
as O
those O
controlling O
host O
preference O
and O
susceptibility O
to O
parasite O
infection O
, O
can O
change O
the O
transmission O
patterns O
and O
consequently O
make O
the O
disease O
control O
a O
harder O
task O
. O

Introgression O
of O
genes O
that O
control O
adaptation O
to O
particular O
types O
of O
environment O
can O
also O
have O
a O
major O
impact O
on O
the O
spread O
of O
vector O
- O
borne O
diseases O
as O
was O
proposed O
for O
the O
major O
African O
malaria O
vector O
Anopheles B
gambiae I
[ O
41 O
] O
. O

The O
same O
can O
be O
said O
about O
genes O
controlling O
insecticide O
resistance O
. O

For O
example O
, O
Weill O
et O
al O
. O

[ O
42 O
] O
found O
a O
kdr O
mutation O
responsible O
for O
pyrethroid O
resistance O
in O
the O
Mopti O
form O
of O
Anopheles B
gambiae I
, O
a O
normally O
susceptible O
taxon O
of O
this O
species O
complex O
. O

Sequence O
analysis O
reveals O
that O
this O
resistant O
allele O
probably O
originates O
through O
introgression O
from O
the O
Savanna O
form O
. O

Although O
L O
. I
intermedia I
and O
L O
. O
whitmani O
are O
closely O
related O
and O
only O
distinguished O
by O
a O
few O
morphological O
differences O
, O
they O
do O
show O
differentiation O
in O
some O
other O
important O
traits O
. O

For O
example O
, O
in O
Posse O
, O
one O
of O
the O
localities O
we O
studied O
, O
the O
two O
species O
show O
differences O
in O
abundance O
during O
the O
year O
. O

L B
. I
intermedia I
is O
more O
abundant O
in O
the O
summer O
while O
L B
. I
whitmani O
is O
more O
frequent O
in O
the O
winter O
months O
[ O
2 O
] O
. O

They O
also O
show O
differences O
in O
microhabitat O
preferences O
, O
L B
. I
intermedia I
being O
more O
common O
in O
the O
peridomestic O
area O
while O
L O
. O
whitmani O
is O
found O
mainly O
in O
the O
surrounding O
forest O
[ O
2 O
] O
. O

In O
addition O
, O
the O
two O
species O
show O
marked O
differences O
in O
their O
tendencies O
to O
bite O
humans B
in O
the O
early O
morning O
, O
with O
L O
. O
whitmani O
showing O
higher O
feeding O
rates O
than O
L B
. I
intermedia I
[ O
26 O
] O
. O

Therefore O
, O
despite O
the O
evidence O
of O
introgression O
in O
the O
period O
gene O
in O
this O
locality O
, O
there O
are O
important O
ecological O
and O
behavioral O
differences O
between O
the O
two O
species O
in O
Posse O
suggesting O
that O
gene O
flow O
is O
probably O
rather O
limited O
in O
loci O
controlling O
these O
traits O
. O

Hence O
, O
it O
is O
yet O
not O
clear O
whether O
introgression O
has O
played O
an O
important O
role O
in O
the O
evolution O
of O
L B
. I
intermedia I
and O
L O
. O
whitmani O
. O

Further O
work O
with O
other O
genes O
might O
help O
clarify O
the O
issue O
. O

Conclusion O

Evidence O
for O
introgression O
between O
L B
. I
intermedia I
and O
L B
. I
whitmani I
obtained O
using O
mitochondrial O
DNA O
[ O
4 O
] O
seems O
to O
be O
corroborated O
by O
our O
data O
on O
the O
period O
gene O
, O
a O
nuclear O
marker O
. O

Nevertheless O
, O
considering O
that O
period O
is O
potentially O
involved O
in O
reproductive O
isolation O
and O
might O
be O
, O
therefore O
, O
less O
prone O
to O
introgression O
than O
the O
" O
average O
" O
gene O
[ O
43 O
] O
, O
it O
is O
possible O
that O
much O
higher O
levels O
of O
gene O
flow O
between O
the O
two O
species O
occur O
at O
other O
genes O
. O

It O
might O
, O
on O
the O
other O
hand O
, O
suggest O
that O
this O
behavioral O
gene O
, O
or O
at O
least O
the O
fragment O
we O
analyzed O
, O
did O
not O
play O
a O
role O
in O
speciation O
between O
L B
. I
intermedia I
and O
L O
. O
whitmani O
. O

In O
fact O
the O
same O
has O
been O
suggested O
for O
some O
Drosophila B
species I
[ O
44 O
] O
despite O
per O
' O
s O
role O
controlling O
lovesong O
and O
mating O
rhythm O
differences O
between O
D B
. I
melanogaster I
and O
D B
. I
simulans I
[ O
13 O
- O
16 O
] O
. O

Although O
the O
evidence O
for O
introgression O
in O
the O
per O
gene O
between O
L B
. I
intermedia I
and O
L O
. O
whitmani O
is O
not O
overwhelming O
, O
it O
does O
indicate O
the O
need O
to O
extend O
this O
analysis O
to O
other O
loci O
in O
the O
future O
. O

We O
are O
currently O
isolating O
new O
molecular O
markers O
in O
the O
two O
species O
to O
carry O
out O
a O
multi O
- O
locus O
approach O
[ O
39 O
] O
that O
might O
help O
determining O
how O
much O
variation O
in O
gene O
flow O
and O
differentiation O
there O
is O
across O
the O
genome O
of O
these O
two O
very O
important O
leishmaniasis O
vectors O
. O

Methods O

Sand O
fly O
samples O

Sand O
fly O
samples O
used O
in O
this O
work O
were O
all O
the O
F1 O
generation O
from O
wild O
collected O
females O
from O
the O
Brazilian O
localities O
of O
Posse O
( O
Petr O
o O
polis O
, O
Rio O
de O
Janeiro O
State O
, O
22 O
degrees O
30 O
' O
S O
43 O
degrees O
10 O
' O
W O
) O
, O
Jacarepagu O
a O
( O
Rio O
de O
Janeiro O
, O
Rio O
de O
Janeiro O
State O
, O
22 O
degrees O
55 O
' O
S O
43 O
degrees O
21 O
' O
W O
) O
, O
Afonso O
Claudio O
( O
Esp O
i O
rito O
Santo O
State O
, O
20 O
degrees O
04 O
' O
S O
41 O
degrees O
07 O
' O
W O
) O
, O
Corte O
de O
Pedra O
( O
Presidente O
Tancredo O
Neves O
, O
Bahia O
State O
, O
13 O
degrees O
27 O
' O
S O
39 O
degrees O

25 O
' O
W O
) O
and O
Ilh O
e O
us O
( O
Bahia O
State O
, O
14 O
degrees O
50 O
' O
S O
39 O
degrees O
06 O
' O
W O
) O
. O

L O
. O
intermedia O
and O
L O
. O
whitmani O
were O
identified O
according O
to O
Young O
and O
Duncan O
[ O
1 O
] O
. O

The O
progeny O
of O
each O
wild O
caught O
female O
was O
raised O
separately O
according O
to O
Souza O
et O
al O
. O

[ O
45 O
] O
and O
only O
one O
F1 O
male O
of O
each O
female O
was O
used O
for O
the O
molecular O
analysis O
, O
which O
included O
68 O
individuals O
of O
L B
. I
intermedia I
( O
12 O
from O
Afonso O
Claudio O
, O
18 O
from O
Posse O
, O
20 O
from O
Corte O
de O
Pedra O
and O
18 O
from O
Jacarepagu O
a O
) O
and O
51 O
individuals O
of O
L B
. I
whitmani O
( O
12 O
from O
Afonso O
Claudio O
, O
17 O
from O
Posse O
, O
3 O
from O
Corte O
de O
Pedra O
and O
19 O
from O
Ilh O
e O
us O
) O
. O

Note O
that O
, O
although O
the O
distribution O
of O
the O
two O
species O
shows O
considerable O
overlap O
in O
Eastern O
Brazil O
, O
in O
many O
localities O
only O
one O
species O
is O
found O
or O
is O
far O
more O
abundant O
than O
the O
other O
. O

There O
are O
also O
seasonal O
and O
microhabitat O
differences O
in O
abundance O
between O
them O
in O
areas O
of O
sympatry O
[ O
2 O
] O
. O

DNA O
methods O

Genomic O
DNA O
was O
prepared O
according O
to O
Jowett O
[ O
46 O
] O
with O
slight O
modifications O
and O
the O
PCR O
was O
carried O
out O
for O
30 O
cycles O
at O
95 O
degrees O
C O
for O
30 O
sec O
, O
60 O
degrees O
C O
for O
30 O
sec O
and O
72 O
degrees O
C O
for O
30 O
sec O
, O
using O
Abgene O
, O
Amersham O
Biosciences O
or O
Biotools O
reagents O
according O
to O
manufacturers O
directions O
. O

The O
per O
primer O
sequences O
are O
: O
5llper2 O
: O
5 O
' O
- O
AGCATCCTTTTGTAGCAAAC O
- O
3 O
' O
( O
forward O
) O
and O
3llper2 O
: O
5 O
' O
- O
TCAGATGAACTCTTGCTGTC O
- O
3 O
' O
( O
reverse O
) O
. O

These O
primers O
amplify O
a O
486 O
bp O
fragment O
of O
the O
sand O
fly I
per O
gene O
homologue O
that O
includes O
part O
of O
the O
PAS O
/ O
CLD O
domain O
, O
an O
intron O
( O
58 O
bp O
) O
and O
the O
beginning O
of O
the O
perS O
domain O
[ O
24 O
] O
. O

The O
amplified O
fragments O
were O
cloned O
using O
the O
pMOSBlue O
blunt O
ended O
cloning O
kit O
( O
Amersham O
Biosciences O
) O
and O
plasmid O
DNA O
preparation O
was O
carried O
out O
using O
the O
" O
Flexiprep O
" O
Kit O
( O
Amersham O
Biosciences O
) O
. O

Cloned O
PCR O
fragments O
were O
sequenced O
at O
Funda O
c O
a O
o O
Oswaldo O
Cruz O
and O
at O
University O
of O
Leicester O
using O
ABI O
377 O
sequencers O
. O

With O
the O
exception O
of O
two O
L O
. O
whitmani O
individuals O
from O
Corte O
de O
Pedra O
( O
see O
below O
) O
, O
only O
one O
sequence O
of O
each O
sand O
fly O
( O
representing O
one O
of O
the O
two O
possible O
alleles O
) O
was O
used O
in O
the O
analysis O
but O
an O
average O
of O
three O
sequences O
per O
individual O
were O
obtained O
in O
order O
to O
check O
possible O
PCR O
induced O
mutations O
. O

In O
addition O
, O
PCR O
fragments O
were O
also O
sequenced O
directly O
in O
some O
cases O
for O
the O
same O
reason O
. O

In O
the O
case O
of O
the O
two O
L O
. O
whitmani O
mentioned O
above O
6 O
and O
9 O
clones O
were O
sequenced O
respectively O
from O
specimens O
WCP01 O
and O
WCP03 O
to O
determine O
both O
alleles O
simply O
to O
increase O
the O
size O
of O
this O
small O
sample O
. O

Negative O
controls O
were O
performed O
for O
all O
amplification O
reactions O
. O

In O
addition O
, O
PCR O
, O
cloning O
and O
sequencing O
were O
repeated O
for O
two O
individuals O
to O
confirm O
putative O
introgressed O
sequences O
and O
to O
exclude O
the O
possibility O
that O
they O
were O
the O
result O
of O
PCR O
contamination O
. O

Finally O
, O
for O
at O
least O
two O
individuals O
with O
putative O
introgressed O
sequences O
, O
we O
could O
define O
the O
other O
allele O
from O
additional O
clones O
( O
not O
included O
in O
the O
analysis O
) O
, O
which O
showed O
to O
be O
typical O
of O
the O
species O
, O
indicating O
no O
identification O
problems O
. O

The O
sequences O
were O
submitted O
to O
GenBank O
( O
accession O
numbers O
AY927062 O
to O
AY927182 O
) O
. O

Sequence O
analyses O

The O
preliminary O
sequence O
editing O
was O
carried O
out O
using O
the O
Wisconsin O
Package O
Version O
9 O
. O
1 O
, O
Genetics O
Computer O
Group O
( O
GCG O
) O
, O
Madison O
, O
and O
ClustalX O
[ O
47 O
] O
was O
used O
to O
perform O
the O
multiple O
alignment O
. O

Analyses O
of O
population O
polymorphisms O
and O
differentiation O
between O
populations O
were O
carried O
out O
using O
DNAsp4 O
. O
1 O
[ O
34 O
] O
and O
ProSeq O
[ O
48 O
] O
softwares O
, O
while O
Arlequin O
v O
. O

2 O
. O
0 O
[ O
49 O
] O
was O
used O
for O
an O
analysis O
of O
molecular O
variance O
( O
AMOVA O
) O
between O
populations O
. O

The O
Minimum O
Evolution O
phylogenetic O
tree O
was O
constructed O
using O
MEGA O
3 O
. O
1 O
software O
[ O
50 O
] O
. O

The O
haplotype O
network O
was O
estimated O
using O
TCS1 O
. O
21 O
[ O
36 O
] O
. O

Recombination O
and O
linkage O
disequilibrium O
analyses O
were O
performed O
using O
the O
DNAsp4 O
. O
1 O
and O
SITES O
program O
[ O
30 O
] O
. O

Linkage O
disequilibrium O
simulations O
were O
carried O
out O
by O
the O
WH O
program O
[ O
51 O
, O
52 O
] O
and O
Markov O
Chain O
Monte O
Carlo O
( O
MCMC O
) O
simulations O
of O
the O
isolation O
with O
migration O
model O
were O
performed O
using O
the O
algorithm O
implemented O
in O
the O
IM O
program O
[ O
39 O
] O
. O

Authors O
' O
contributions O

CJM O
generate O
and O
analyzed O
all O
the O
data O
and O
drafted O
the O
manuscript O
. O

NAS O
and O
CAC O
collected O
and O
maintained O
sand O
fly O
samples O
. O

CPK O
helped O
to O
write O
the O
manuscript O
and O
supervised O
CJM O
during O
her O
stay O
in O
Leicester O
. O

AAP O
is O
the O
principal O
investigator O
, O
participated O
in O
its O
design O
and O
coordination O
, O
and O
helped O
to O
write O
the O
manuscript O
. O

All O
authors O
read O
and O
approved O
the O
final O
manuscript O
. O

Presence O
of O
antibodies O
against O
cyclic O
citrullinated O
peptides O
in O
patients B
with O
' O
rhupus O
' O
: O
a O
cross O
- O
sectional O
study O

Abstract O

' O
Rhupus O
' O
is O
a O
rare O
condition O
sharing O
features O
of O
rheumatoid O
arthritis O
( O
RA O
) O
and O
systemic O
lupus O
erythematosus O
( O
SLE O
) O
. O

If O
rhupus O
is O
a O
distinctive O
entity O
, O
an O
overlap O
between O
RA O
and O
SLE O
or O
a O
subset O
of O
SLE O
is O
currently O
debated O
. O

This O
study O
was O
performed O
to O
explore O
the O
prevalence O
of O
antibodies O
against O
cyclic O
citrullinated O
peptides O
( O
anti O
- O
CCP O
antibodies O
) O
in O
rhupus B
. O

Patients B
meeting O
American O
College O
of O
Rheumatology O
criteria O
for O
RA O
, O
SLE O
, O
or O
both O
were O
included O
. O

Clinical O
and O
radiographic O
features O
were O
recorded O
and O
sera O
were O
searched O
for O
anti O
- O
CCP O
antibodies O
, O
rheumatoid O
factor O
, O
antinuclear O
antibodies O
, O
anti O
- O
extractable O
nuclear O
antigens O
, O
and O
antibodies O
against O
double O
- O
stranded O
DNA O
( O
anti O
- O
dsDNA O
antibodies O
) O
. O

Seven O
patients B
for O
each O
group O
were O
included O
. O

Clinical O
and O
serological O
features O
for O
RA O
or O
SLE O
were O
similar O
between O
rhupus O
and O
RA O
patients B
, O
and O
between O
rhupus O
and O
SLE O
patients B
, O
respectively O
. O

Values O
for O
anti O
- O
CCP O
antibodies O
obtained O
were O
significantly O
( O
p O
< O
0 O
. O
05 O
) O
higher O
in O
RA O
( O
6 O
/ O
7 O
) O
and O
rhupus O
( O
4 O
/ O
7 O
) O
than O
in O
SLE O
patients B
( O
0 O
/ O
7 O
) O
and O
healthy O
subjects O
( O
0 O
/ O
7 O
) O
. O

Our O
data O
support O
the O
possibility O
that O
rhupus O
is O
an O
overlap O
between O
RA O
and O
SLE O
, O
because O
highly O
specific O
autoantibodies O
for O
RA O
( O
anti O
- O
CCP O
) O
and O
for O
SLE O
( O
anti O
- O
dsDNA O
and O
anti O
- O
Sm O
) O
are O
detected O
in O
coexistence O
. O

Introduction O

The O
clinical O
coexistence O
of O
rheumatoid O
arthritis O
( O
RA O
) O
and O
systemic O
lupus O
erythematosus O
( O
SLE O
) O
was O
first O
described O
in O
1969 O
by O
Kantor O
and O
was O
termed O
' O
rhupus O
syndrome O
' O
by O
Schur O
( O
both O
cited O
in O
[ O
1 O
] O
) O
. O

Since O
then O
, O
fewer O
than O
100 O
cases O
of O
rhupus O
have O
been O
published O
[ O
1 O
- O
3 O
] O
. O

In O
an O
epidemiological O
study O
including O
about O
7 O
, O
000 O
new O
patients B
, O
the O
prevalence O
of O
RA O
was O
15 O
% O
and O
for O
SLE O
it O
was O
8 O
. O
9 O
% O
. O

The O
expected O
coincidence O
of O
RA O
and O
SLE O
by O
chance O
would O
therefore O
be O
1 O
. O
2 O
% O
. O

However O
, O
the O
observed O
prevalence O
of O
rhupus O
was O
0 O
. O
09 O
% O
, O
less O
than O
one O
- O
tenth O
of O
that O
expected O
[ O
1 O
] O
. O

Previous O
reports O
have O
shown O
that O
the O
patients B
with O
rhupus O
display O
an O
array O
of O
autoantibodies O
including O
anti O
- O
double O
- O
stranded O
DNA O
( O
anti O
- O
dsDNA O
) O
, O
anti O
- O
Sm O
( O
both O
highly O
specific O
for O
SLE O
) O
, O
anti O
- O
SSA O
, O
anti O
- O
SSB O
, O
anti O
- O
ribonucleoprotein O
, O
antinuclear O
antibodies O
( O
ANA O
) O
, O
anti O
- O
cardiolipins O
, O
and O
rheumatoid O
factor O
( O
RF O
) O
[ O
1 O
, O
2 O
] O
. O

However O
, O
no O
study O
has O
yet O
been O
performed O
to O
investigate O
the O
presence O
of O
antibodies O
against O
cyclic O
citrullinated O
peptides O
( O
anti O
- O
CCP O
antibodies O
) O
, O
which O
have O
a O
specificity O
for O
RA O
of O
96 O
to O
98 O
% O
( O
for O
second O
- O
generation O
assays O
( O
anti O
- O
CCP2 O
) O
) O
[ O
4 O
, O
5 O
] O
. O

Recent O
data O
have O
confirmed O
that O
these O
antibodies O
are O
rarely O
if O
ever O
present O
in O
other O
autoimmune O
diseases O
such O
as O
SLE O
, O
Sj O
o O
gren O
' O
s O
syndrome O
( O
SS O
) O
, O
scleroderma O
and O
myositis O
[ O
6 O
] O
. O

Nowadays O
, O
it O
is O
a O
matter O
of O
debate O
whether O
rhupus O
is O
a O
clinically O
and O
immunologically O
distinctive O
entity O
[ O
2 O
] O
, O
a O
true O
overlap O
between O
SLE O
and O
RA O
[ O
7 O
] O
, O
or O
a O
subgroup O
of O
patients B
with O
lupus O
[ O
8 O
] O
. O

This O
descriptive O
, O
cross O
- O
sectional O
study O
was O
performed O
to O
investigate O
the O
frequency O
of O
anti O
- O
CCP O
antibodies O
in O
a O
cohort O
of O
patients B
with O
rhupus O
. O

Materials O
and O
methods O

We O
included O
all O
patients B
fulfilling O
American O
College O
of O
Rheumatology O
( O
ACR O
) O
classification O
criteria O
for O
both O
RA O
[ O
9 O
] O
and O
SLE O
[ O
10 O
] O
who O
belonged O
to O
our O
cohorts O
of O
patients B
with O
RA O
and O
with O
SLE O
. O

Comparisons O
were O
made O
with O
age O
- O
and O
gender O
- O
matched O
patients B
with O
RA O
and O
with O
SLE O
, O
and O
healthy O
subjects O
. O

The O
study O
was O
approved O
by O
the O
local O
ethics O
committee O
, O
and O
informed O
consent O
was O
obtained O
. O

Serum O
samples O
were O
obtained O
and O
stored O
at O
- O
75 O
degrees O
C O
until O
use O
. O

Sera O
were O
analyzed O
for O
anti O
- O
CCP2 O
antibodies O
by O
ELISA O
( O
Inova O
Diagnostics O
, O
San O
Diego O
, O
CA O
, O
USA O
) O
with O
a O
cutoff O
value O
of O
60 O
U O
/ O
ml O
. O

Fine O
antinuclear O
reactivities O
( O
ELISA O
; O
Inova O
Diagnostics O
) O
, O
RF O
( O
nephelometry O
) O
, O
ANA O
( O
indirect O
immunofluorescence O
on O
HEp O
- O
2 O
slides O
) O
, O
and O
anti O
- O
dsDNA O
( O
indirect O
immunofluorescence O
on O
Crithidia O
luciliae O
substrate O
) O
antibodies O
were O
also O
determined O
. O

Except O
for O
healthy O
individuals O
, O
standard O
radiographs O
of O
hands O
were O
available O
. O

For O
statistical O
analysis O
, O
ANOVA O
and O
the O
Mann O
- O
Whitney O
U O
test O
were O
performed O
as O
appropriate O
with O
GraphPad O
Prism O
4 O
. O
0 O
software O
( O
GraphPad O
Inc O
, O
San O
Diego O
, O
CA O
, O
USA O
) O
. O

Results O

Seven O
female O
patients B
with O
a O
median O
age O
of O
44 O
years O
( O
range O
25 O
to O
64 O
) O
met O
our O
inclusion O
criteria O
. O

The O
major O
clinical O
and O
laboratory O
findings O
are O
presented O
in O
Table O
1 O
. O

Healthy O
individuals O
and O
all O
patients B
belonged O
to O
cohorts O
from O
the O
same O
ethnic O
group O
( O
Hispanic O
mestizo O
) O
. O

No O
differences O
in O
demographic O
data O
were O
found O
between O
groups O
. O

Mean O
ACR O
criteria O
for O
SLE O
were O
5 O
. O
57 O
( O
range O
4 O
to O
8 O
) O
in O
the O
SLE O
group O
, O
and O
5 O
. O
57 O
( O
4 O
to O
8 O
) O
in O
the O
rhupus O
group O
. O

In O
the O
same O
way O
, O
mean O
ACR O
criteria O
for O
RA O
were O
6 O
( O
4 O
to O
7 O
) O
in O
the O
RA O
group O
, O
and O
5 O
. O
14 O
( O
4 O
to O
6 O
) O
for O
the O
patients B
with O
rhupus O
. O

In O
all O
patients B
with O
rhupus O
, O
RA O
was O
presented O
as O
the O
initial O
disease O
, O
as O
has O
been O
described O
previously O
[ O
2 O
] O
. O

In O
accordance O
with O
another O
report O
, O
in O
two O
patients B
the O
disease O
started O
during O
their O
childhood O
as O
juvenile O
chronic O
arthritis O
[ O
1 O
] O
. O

Anti O
- O
CCP O
antibodies O
were O
found O
in O
four O
of O
seven O
( O
57 O
% O
) O
patients B
with O
rhupus O
, O
and O
in O
six O
of O
seven O
( O
86 O
% O
) O
patients B
with O
RA O
, O
whereas O
neither O
patients B
with O
SLE O
nor O
healthy O
individuals O
showed O
reactivity O
. O

When O
the O
concentrations O
in O
each O
group O
were O
compared O
, O
a O
statistical O
significant O
difference O
between O
groups O
was O
found O
( O
ANOVA O
, O
p O
< O
0 O
. O
05 O
) O
. O

The O
mean O
concentration O
of O
anti O
- O
CCP O
antibodies O
was O
584 O
U O
/ O
ml O
( O
range O
0 O
to O
1 O
, O
237 O
) O
in O
patients B
with O
rhupus O
( O
Figure O
1 O
) O
, O
875 O
U O
/ O
ml O
( O
0 O
to O
1 O
, O
178 O
) O
in O
the O
RA O
group O
( O
not O
significant O
compared O
with O
rhupus O
) O
, O
1 O
U O
/ O
ml O
( O
0 O
to O
10 O
) O
for O
SLE O
individuals O
( O
p O
< O
0 O
. O
05 O
compared O
with O
rhupus O
) O
, O
and O
0 O
U O
/ O
ml O
( O
0 O
to O
2 O
) O
for O
healthy O
controls O
( O
p O
< O
0 O
. O
05 O
compared O
with O
rhupus B
) O
. O

Two O
of O
three O
patients B
with O
rhupus O
who O
were O
negative O
for O
anti O
- O
CCP O
antibodies O
were O
also O
negative O
for O
anti O
- O
dsDNA O
antibodies O
, O
RF O
and O
anti O
- O
extractable O
nuclear O
antigen O
antibodies O
, O
although O
both O
patients B
met O
RA O
and O
SLE O
classification O
criteria O
, O
including O
ANA O
and O
erosive O
arthritis O
. O

Differences O
in O
ANA O
, O
anti O
- O
dsDNA O
and O
anti O
- O
extractable O
nuclear O
antigen O
autoantibodies O
between O
patients B
with O
rhupus O
and O
those O
with O
SLE O
were O
not O
found O
. O

We O
also O
found O
no O
difference O
in O
the O
prevalence O
of O
RF O
between O
patients B
with O
rhupus O
and O
those O
with O
RA O
. O

Surprisingly O
, O
one O
healthy O
subject O
was O
positive O
for O
RF O
, O
ANA O
and O
anti O
- O
SSA O
antibodies O
, O
although O
she O
was O
asymptomatic O
and O
no O
features O
of O
any O
disease O
were O
found O
. O

Discussion O

The O
close O
association O
between O
different O
type O
II O
human B
leukocyte O
antigen O
( O
HLA O
) O
molecules O
and O
the O
risk O
of O
RA O
is O
well O
established O
. O

These O
major O
histocompatibility O
complex O
( O
MHC O
) O
class O
II O
molecules O
share O
the O
same O
amino O
acid O
sequence O
( O
QKRAA O
or O
QRRAA O
) O
in O
positions O
69 O
to O
74 O
of O
the O
beta O
- O
chain O
, O
namely O
the O
' O
shared O
epitope O
' O
. O

Recent O
works O
have O
demonstrated O
that O
this O
' O
shared O
epitope O
' O
preferentially O
binds O
peptides O
containing O
the O
non O
- O
standard O
amino O
acid O
citrulline O
( O
deiminated O
arginine O
) O
[ O
11 O
] O
. O

In O
addition O
, O
an O
abnormally O
increased O
function O
of O
the O
enzyme O
peptidylarginine O
deiminase O
4 O
( O
PAD4 O
; O
responsible O
for O
the O
deimination O
of O
arginine O
) O
and O
an O
elevated O
anti O
- O
CCP O
autoantibody O
production O
in O
patients B
with O
RA O
have O
been O
demonstrated O
[ O
12 O
] O
. O

These O
facts O
have O
built O
the O
first O
bridge O
between O
cellular O
and O
humoral O
autoimmunity O
in O
a O
major O
rheumatic O
disease O
, O
supporting O
a O
pathogenetic O
role O
for O
an O
abnormal O
metabolism O
of O
citrulline O
in O
the O
development O
of O
RA O
[ O
13 O
, O
14 O
] O
. O

Patients B
with O
SLE O
are O
often O
part O
of O
the O
control O
group O
when O
determining O
the O
specificity O
of O
anti O
- O
CCP O
antibodies O
for O
RA O
[ O
15 O
] O
, O
although O
some O
studies O
have O
been O
performed O
specifically O
on O
patients B
with O
SLE O
. O

These O
studies O
contribute O
some O
clues O
to O
the O
role O
of O
anti O
- O
CCP O
antibodies O
in O
rhupus B
. O

Mediwake O
and O
colleagues O
[ O
16 O
] O
, O
in O
a O
study O
exploring O
the O
predictive O
value O
of O
anti O
- O
CCP O
antibodies O
to O
distinguish O
erosive O
arthritis O
in O
SLE O
, O
found O
ten O
patients B
( O
out O
of O
231 O
) O
with O
erosive O
arthritis O
, O
two O
of O
whom O
had O
anti O
- O
CCP O
antibodies O
. O

In O
concord O
with O
this O
, O
Hoffman O
and O
colleagues O
[ O
15 O
] O
demonstrate O
that O
three O
patients B
with O
erosive O
arthritis O
, O
included O
in O
a O
cohort O
of O
235 O
patients B
with O
SLE O
, O
were O
positive O
for O
anti O
- O
CCP O
antibodies O
. O

These O
authors O
suggest O
that O
the O
presence O
of O
anti O
- O
CCP O
antibodies O
can O
predispose O
for O
a O
chronic O
RA O
- O
like O
arthritis O
in O
patients B
with O
SLE O
. O

Additionally O
Weissman O
and O
colleagues O
[ O
17 O
] O
demonstrated O
that O
patients B
with O
SLE O
can O
display O
radiographic O
abnormalities O
similar O
to O
those O
of O
RA O
, O
although O
the O
presence O
of O
marginal O
erosions O
is O
a O
rare O
finding O
. O

In O
the O
present O
study O
we O
demonstrate O
that O
the O
patients B
with O
rhupus O
show O
a O
very O
similar O
arthritis O
pattern O
( O
including O
erosive O
disease O
) O
and O
similar O
autoantibody O
production O
( O
RF O
and O
anti O
- O
CCP O
antibodies O
) O
to O
those O
in O
patients B
with O
RA O
. O

In O
addition O
, O
patients B
with O
rhupus O
display O
a O
clinical O
and O
serological O
profile O
indistinguishable O
from O
patients B
with O
SLE O
. O

Moreover O
, O
the O
presence O
of O
other O
coexistent O
autoimmune O
diseases O
was O
similar O
in O
all O
groups O
of O
patients B
( O
two O
patients B
with O
rhupus O
, O
three O
patients B
with O
RA O
, O
and O
three O
patients B
with O
SLE O
also O
had O
SS O
) O
. O

We O
found O
high O
titers O
of O
anti O
- O
CCP O
antibodies O
in O
four O
of O
seven O
( O
57 O
% O
) O
patients B
with O
rhupus O
, O
a O
frequency O
similar O
to O
that O
reported O
for O
RA O
[ O
4 O
] O
. O

This O
finding O
, O
together O
with O
the O
clinical O
similarity O
, O
supports O
the O
contention O
that O
rhupus O
belongs O
to O
the O
RA O
spectrum O
. O

The O
high O
prevalence O
of O
anti O
- O
CCP O
antibodies O
in O
RA O
found O
in O
our O
study O
could O
be O
explained O
by O
a O
selection O
bias O
because O
only O
patients B
with O
RA O
with O
an O
aggressive O
disease O
( O
namely O
erosive O
arthritis O
and O
RF O
+ O
) O
were O
included O
. O

In O
contrast O
, O
the O
mean O
ACR O
criterion O
for O
SLE O
was O
similar O
between O
patients B
with O
rhupus O
and O
those O
with O
SLE O
, O
including O
the O
' O
robust O
' O
features O
of O
SLE O
such O
as O
renal O
and O
neurological O
involvement O
, O
and O
anti O
- O
dsDNA O
and O
anti O
- O
Sm O
antibodies O
. O

These O
clinical O
and O
serological O
features O
shared O
between O
patients B
with O
rhupus O
and O
those O
with O
SLE O
also O
place O
rhupus O
in O
the O
SLE O
spectrum O
. O

Titration O
of O
anti O
- O
CCP O
antibodies O
in O
the O
rhupus O
group O
clearly O
shows O
a O
bimodal O
distribution O
, O
suggesting O
the O
existence O
of O
two O
different O
subpopulations O
. O

Because O
of O
the O
small O
number O
of O
patients B
, O
we O
are O
unable O
to O
define O
the O
differential O
features O
underlying O
each O
subset O
. O

However O
, O
two O
of O
three O
patients B
negative O
for O
anti O
- O
CCP O
antibodies O
were O
also O
negative O
for O
both O
RF O
and O
anti O
- O
dsDNA O
antibodies O
. O

Conclusion O

On O
the O
basis O
of O
the O
presence O
of O
shared O
clinical O
features O
of O
RA O
( O
mainly O
erosive O
arthritis O
) O
and O
SLE O
( O
including O
renal O
and O
neurological O
involvement O
) O
along O
with O
the O
presence O
of O
anti O
- O
dsDNA O
and O
anti O
- O
CCP O
autoantibodies O
in O
our O
patients B
with O
rhupus O
, O
our O
findings O
strongly O
support O
the O
contention O
that O
rhupus O
is O
a O
true O
overlap O
between O
RA O
and O
SLE O
, O
not O
merely O
a O
part O
of O
the O
clinical O
spectrum O
of O
the O
articular O
involvement O
seen O
in O
SLE O
. O

Moreover O
, O
on O
the O
basis O
of O
the O
mean O
ACR O
criteria O
for O
both O
diseases O
, O
we O
have O
confirmed O
that O
patients B
with O
rhupus O
have O
more O
RA O
- O
associated O
and O
less O
SLE O
- O
associated O
damage O
, O
an O
issue O
that O
has O
been O
suggested O
previously O
[ O
2 O
] O
. O

To O
our O
knowledge O
, O
this O
is O
the O
first O
report O
exploring O
the O
prevalence O
of O
anti O
- O
CCP O
antibodies O
specifically O
in O
patients B
with O
rhupus B
. O

More O
studies O
are O
needed O
to O
expand O
the O
pathogenetic O
knowledge O
of O
this O
overlap O
syndrome O
. O

Abbreviations O

ANA O
= O
antinuclear O
antibodies O
; O
anti O
- O
CCP O
antibodies O
= O
antibodies O
against O
cyclic O
citrullinated O
peptides O
; O
anti O
- O
dsDNA O
antibodies O
= O
antibodies O
against O
double O
- O
stranded O
DNA O
; O
ELISA O
= O
enzyme O
- O
linked O
immunosorbent O
assay O
; O
RA O
= O
rheumatoid O
arthritis O
; O
RF O
= O
rheumatoid O
factor O
; O
SLE O
= O
systemic O
lupus I
erythematosus I
; O
SS O
= O
Sj O
o O
gren O
' O
s O
syndrome O
. O

Competing O
interests O

The O
authors O
declare O
that O
they O
have O
no O
competing O
interests O
. O

Authors O
' O
contributions O

LA O
- O
G O
participated O
in O
the O
conception O
and O
design O
of O
the O
experiments O
, O
in O
the O
acquisition O
, O
analysis O
and O
interpretation O
of O
data O
, O
and O
was O
involved O
in O
drafting O
the O
manuscript O
. O

RS O
performed O
the O
immunoassays O
. O

RM O
- O
V O
participated O
in O
the O
analysis O
and O
interpretation O
of O
data O
and O
performed O
the O
statistical O
analysis O
. O

LG O
- O
G O
participated O
in O
the O
analysis O
and O
interpretation O
of O
data O
. O

AV O
participated O
in O
the O
recruitment O
of O
patients B
and O
the O
acquisition O
of O
data O
. O

RB O
participated O
in O
the O
interpretation O
of O
data O
, O
revising O
the O
manuscript O
for O
intellectual O
content O
and O
giving O
the O
final O
approval O
of O
the O
version O
to O
be O
published O
. O

All O
authors O
read O
and O
approved O
the O
final O
manuscript O
. O

Identification O
of O
a O
human B
peripheral O
blood O
monocyte O
subset O
that O
differentiates O
into O
osteoclasts O

Abstract O

Increased O
bone O
resorption O
mediated O
by O
osteoclasts O
causes O
various O
diseases O
such O
as O
osteoporosis O
and O
bone O
erosion O
in O
rheumatoid O
arthritis O
( O
RA O
) O
. O

Osteoclasts O
are O
derived O
from O
the O
monocyte O
/ O
macrophage O
lineage O
, O
but O
the O
precise O
origin O
remains O
unclear O
. O

In O
the O
present O
study O
, O
we O
show O
that O
the O
purified O
CD16 O
- O
human B
peripheral O
blood O
monocyte O
subset O
, O
but O
not O
the O
CD16 O
+ O
monocyte O
subset O
, O
differentiates O
into O
osteoclast O
by O
stimulation O
with O
receptor O
activator O
of O
NF O
- O
kappa O
B O
ligand O
( O
RANKL O
) O
in O
combination O
with O
macrophage O
colony O
- O
stimulating O
factor O
( O
M O
- O
CSF O
) O
. O

Integrin O
- O
beta O
3 O
mRNA O
and O
the O
integrin O
- O
alpha O
v O
beta O
3 O
heterodimer O
were O
only O
expressed O
on O
CD16 O
- O
monocytes O
, O
when O
they O
were O
stimulated O
with O
RANKL O
+ O
M O
- O
CSF O
. O

Downregulation O
of O
beta O
3 O
- O
subunit O
expression O
by O
small O
interfering O
RNA O
targeting O
beta O
3 O
abrogated O
osteoclastogenesis O
from O
the O
CD16 O
- O
monocyte O
subset O
. O

In O
contrast O
, O
the O
CD16 O
+ O
monocyte O
subset O
expressed O
larger O
amounts O
of O
tumor O
necrosis O
factor O
alpha O
and O
IL O
- O
6 O
than O
the O
CD16 O
- O
subset O
, O
which O
was O
further O
enhanced O
by O
RANKL O
stimulation O
. O

Examination O
of O
RA O
synovial O
tissue O
showed O
accumulation O
of O
both O
CD16 O
+ O
and O
CD16 O
- O
macrophages O
. O

Our O
results O
suggest O
that O
peripheral O
blood O
monocytes O
consist O
of O
two O
functionally O
heterogeneous O
subsets O
with O
distinct O
responses O
to O
RANKL O
. O

Osteoclasts O
seem O
to O
originate O
from O
CD16 O
- O
monocytes O
, O
and O
integrin O
beta O
3 O
is O
necessary O
for O
osteoclastogenesis O
. O

Blockade O
of O
accumulation O
and O
activation O
of O
CD16 O
- O
monocytes O
could O
therefore O
be O
a O
beneficial O
approach O
as O
an O
anti O
- O
bone O
resorptive O
therapy O
, O
especially O
for O
RA O
. O

Introduction O

Rheumatoid O
arthritis O
( O
RA O
) O
is O
an O
autoimmune O
disease O
characterized O
by O
chronic O
inflammation O
and O
proliferation O
of O
the O
synovium O
in O
multiple O
joints O
. O

A O
large O
number O
of O
inflammatory O
cells O
, O
including O
T O
cells O
, O
B O
cells O
, O
macrophages O
and O
dendritic O
cells O
, O
accumulate O
in O
the O
affected O
synovium O
, O
and O
these O
inflammatory O
cells O
, O
together O
with O
fibroblast O
- O
like O
synoviocytes O
, O
express O
various O
cytokines O
, O
such O
as O
tumor O
necrosis O
factor O
alpha O
( O
TNF O
alpha O
) O
, O
IL O
- O
6 O
and O
receptor O
activator O
of O
NF O
- O
kappa O
B O
ligand O
( O
RANKL O
) O
, O
which O
are O
known O
to O
induce O
differentiation O
and O
activation O
of O
osteoclasts O
. O

The O
inflammatory O
synovial O
tissue O
, O
known O
as O
pannus O
, O
invades O
the O
articular O
bone O
and O
causes O
focal O
bone O
erosion O
, O
which O
is O
the O
hallmark O
of O
RA O
. O

Histopathologically O
, O
osteoclasts O
are O
present O
at O
the O
interface O
of O
the O
pannus O
and O
bone O
. O

Interestingly O
, O
the O
deletion O
of O
RANKL O
or O
c O
- O
Fos O
gene O
, O
which O
is O
important O
for O
osteoclastogenesis O
, O
results O
in O
minimal O
bone O
destruction O
in O
mouse B
models O
of O
arthritis O
[ O
1 O
, O
2 O
] O
. O

Furthermore O
, O
other O
studies O
indicated O
that O
inhibition O
of O
osteoclastogenesis O
by O
osteoprotegerin O
, O
a O
decoy O
receptor O
for O
RANKL O
, O
limits O
bone O
destruction O
in O
experimental O
models O
of O
arthritis O
. O

These O
studies O
suggest O
that O
osteoclasts O
are O
involved O
in O
focal O
bone O
erosion O
in O
RA O
[ O
3 O
] O
. O

Osteoclasts O
are O
derived O
from O
the O
monocyte O
/ O
macrophage O
lineage O
. O

It O
is O
reported O
that O
osteoclast O
precursors O
reside O
in O
human B
peripheral O
blood O
monocytes O
[ O
4 O
, O
5 O
] O
. O

A O
marked O
increase O
of O
the O
circulating O
osteoclast O
precursors O
was O
demonstrated O
in O
patients B
with O
erosive O
psoriatic O
arthritis O
as O
well O
as O
in O
arthritic O
TNF O
alpha O
transgenic O
mice B
[ O
6 O
, O
7 O
] O
. O

It O
was O
also O
shown O
that O
peripheral O
monocytes O
differentiate O
into O
osteoclasts O
when O
seeded O
on O
RANKL O
/ O
osteoclast O
differentiation O
factor O
- O
producing O
RA O
synovial O
fibroblasts O
[ O
8 O
] O
. O

In O
addition O
, O
RA O
synovial O
macrophages O
thought O
to O
originate O
from O
peripheral O
blood O
monocytes O
were O
shown O
to O
differentiate O
into O
osteoclasts O
[ O
9 O
, O
10 O
] O
. O

Monocytes O
are O
therefore O
involved O
not O
only O
in O
synovial O
inflammation O
, O
but O
also O
in O
bone O
remodeling O
as O
potential O
precursors O
for O
synovial O
macrophages O
and O
osteoclasts O
. O

Human B
peripheral O
blood O
monocytes O
consist O
of O
two O
major O
subsets O
, O
CD16 O
+ O
and O
CD16 O
- O
, O
comprising O
5 O
- O
10 O
% O
and O
90 O
- O
95 O
% O
of O
the O
monocytes O
, O
respectively O
. O

These O
two O
subsets O
exhibit O
different O
chemotaxis O
activities O
and O
potential O
of O
cytokine O
production O
[ O
11 O
, O
12 O
] O
. O

Moreover O
, O
activation O
of O
the O
Toll O
- O
like O
receptor O
induces O
distinct O
subsets O
, O
CD1b O
+ O
dendritic O
cells O
and O
DC O
- O
SIGN O
+ O
( O
dendritic O
cell O
- O
specific O
C O
- O
type O
lectin O
ICAM O
- O
3 O
- O
grabbing O
nonintegrin O
) O
macrophages O
from O
CD16 O
+ O
and O
CD16 O
- O
monocytes O
, O
respectively O
[ O
13 O
] O
. O

It O
has O
not O
been O
revealed O
, O
however O
, O
which O
monocyte O
subset O
develops O
into O
osteoclasts O
. O

In O
the O
present O
study O
, O
we O
determined O
the O
human B
peripheral O
blood O
monocyte O
subset O
that O
differentiates O
into O
osteoclasts O
, O
and O
revealed O
that O
each O
subset O
exhibits O
a O
different O
response O
for O
osteoclastogenic O
stimuli O
. O

Materials O
and O
methods O

Purification O
of O
peripheral O
blood O
monocytes O

Peripheral O
blood O
monocytes O
from O
healthy O
donors O
were O
collected O
using O
Ficoll O
- O
Conray O
( O
Imuuno O
- O
Biological O
Laboratories O
, O
Gunma O
, O
Japan O
) O
gradient O
centrifugation O
. O

Negative O
selection O
of O
monocytes O
was O
performed O
using O
MACS O
microbeads O
( O
Miltenyi O
Biotec O
, O
Auburn O
, O
CA O
, O
USA O
) O
according O
to O
the O
protocol O
supplied O
by O
the O
manufacturer O
. O

The O
purified O
monocytes O
were O
separated O
into O
two O
subsets O
, O
CD16 O
+ O
and O
CD16 O
- O
monocytes O
, O
using O
CD16 O
MicroBeads O
( O
Miltenyi O
Biotec O
) O
. O

Flow O
cytometry O
analysis O
using O
FITC O
- O
conjugated O
mouse B
anti O
- O
CD14 O
mAb O
( O
MY4 O
; O
Bechman O
Coulter O
, O
Fullerton O
, O
CA O
, O
USA O
) O
and O
phycoerythin O
- O
conjugated O
mouse B
anti O
- O
CD16 O
mAb O
( O
3G8 O
; O
BD O
Biosciences O
, O
San O
Jose O
, O
CA O
, O
USA O
) O
showed O
that O
the O
purities O
of O
the O
CD16 O
+ O
and O
CD16 O
- O
monocytes O
were O
more O
than O
90 O
% O
and O
92 O
% O
, O
respectively O
. O

For O
the O
other O
experiment O
, O
monocytes O
were O
purified O
using O
CD14 O
MicroBeads O
( O
Miltenyi O
Biotec O
) O
, O
and O
then O
stained O
either O
with O
FITC O
- O
conjugated O
mouse B
anti O
- O
CD33 O
mAb O
( O
MY9 O
; O
Bechman O
Coulter O
) O
or O
phycoerythin O
- O
conjugated O
mouse B
anti O
- O
CD16 O
mAb O
( O
3G8 O
) O
. O

Cell O
sorting O
of O
the O
stained O
cells O
was O
performed O
using O
a O
FACS O
Vantage O
cytometer O
( O
BD O
Biosciences O
) O
or O
a O
MoFlo O
cell O
sorter O
( O
Dako O
, O
Glostrup O
, O
Denmark O
) O
. O

Osteoclast O
differentiation O

Purified O
CD16 O
+ O
and O
CD16 O
- O
monocytes O
( O
5 O
x O
104 O
cells O
/ O
well O
) O
were O
incubated O
in O
96 O
- O
well O
plates O
in O
alpha O
MEM O
( O
Sigma O
, O
St O
Louis O
, O
MO O
, O
USA O
) O
with O
heat O
- O
inactivated O
10 O
% O
fetal O
bovine B
serum O
( O
FBS O
) O
( O
Sigma O
) O
or O
with O
Ultra O
- O
Low O
IgG O
FBS O
( O
IgG O
< O
5 O
mu O
g O
/ O
ml O
; O
Invitrogen O
, O
Carlsbad O
, O
CA O
, O
USA O
) O
, O
and O
where O
indicated O
with O
M O
- O
CSF O
+ O
RANKL O
( O
Peprotech O
, O
Rocky O
Hill O
, O
NJ O
, O
USA O

) O
. O

For O
the O
other O
experiments O
, O
varied O
numbers O
of O
CD16 O
+ O
monocytes O
( O
1 O
x O
103 O
, O
2 O
. O
5 O
x O
103 O
, O
5 O
x O
103 O
) O
were O
mixed O
with O
CD16 O
- O
monocytes O
( O
5 O
x O
104 O
cells O
/ O
well O
) O
, O
and O
were O
cultured O
in O
96 O
- O
well O
plates O
in O
alpha O
MEM O
with O
heat O
- O
inactivated O
10 O
% O
FBS O
. O

The O
medium O
was O
replaced O
with O
fresh O
medium O
3 O
days O
later O
, O
and O
after O
incubation O
for O
7 O
days O
the O
cells O
were O
stained O
for O
tartrate O
- O
resistant O
acid O
phosphatase O
( O
TRAP O
) O
expression O
using O
a O
commercial O
kit O
( O
Hokudo O
, O
Sapporo O
, O
Japan O
) O
. O

The O
number O
of O
TRAP O
- O
positive O
multinucleated O
cells O
( O
MNC O
) O
in O
three O
randomly O
selected O
fields O
examined O
at O
100 O
x O
magnification O
or O
the O
total O
number O
of O
TRAP O
- O
positive O
MNC O
per O
well O
was O
counted O
under O
light O
microscopy O
. O

Resorption O
assay O

Monocytes O
were O
seeded O
onto O
plates O
coated O
with O
calcium O
phosphate O
thin O
films O
( O
Biocoat O
Osteologic O
; O
BD O
Biosciences O
) O
and O
were O
incubated O
with O
RANKL O
( O
40 O
ng O
/ O
ml O
) O
+ O
M O
- O
CSF O
( O
25 O
ng O
/ O
ml O
) O
for O
7 O
days O
. O

The O
cells O
were O
then O
lysed O
in O
bleach O
solution O
( O
6 O
% O
NaOCl O
, O
5 O
. O
2 O
% O
NaCl O
) O
. O

The O
resorption O
lacunae O
were O
examined O
under O
light O
microscopy O
. O

Enzyme O
- O
linked O
immunosorbent O
assay O

Purified O
monocytes O
were O
cultured O
in O
96 O
- O
well O
plates O
where O
indicated O
either O
with O
RANKL O
or O
M O
- O
CSF O
for O
24 O
hours O
. O

Concentrations O
of O
TNF O
alpha O
and O
IL O
- O
6 O
in O
the O
culture O
supernatant O
were O
measured O
with O
an O
ELISA O
kit O
( O
BioSourse O
International O
, O
Camarillo O
, O
CA O
, O
USA O
) O
. O

For O
experiments O
of O
matrix O
metalloproteinase O
( O
MMP O
) O
- O
9 O
and O
TRAP O
- O
5b O
, O
culture O
supernatants O
were O
collected O
on O
day O
7 O
and O
the O
concentrations O
of O
these O
enzymes O
were O
measured O
using O
an O
MMP O
- O
9 O
ELISA O
kit O
( O
Amersham O
Biosciences O
, O
Piscataway O
, O
NJ O
, O
USA O
) O
or O
a O
TRAP O
- O
5b O
ELISA O
kit O
( O
Suomen O
, O
Turku O
, O
Finland O
) O
. O

Reverse O
transcriptase O
- O
polymerase O
chain O
reaction O

Monocytes O
( O
1 O
x O
106 O
cells O
/ O
well O
) O
were O
cultured O
in O
six O
- O
well O
plates O
with O
M O
- O
CSF O
alone O
or O
with O
M O
- O
CSF O
+ O
RANKL O
for O
3 O
days O
. O

Total O
RNA O
was O
extracted O
using O
RNeasy O
Micro O
( O
Qiagen O
, O
Valencia O
, O
CA O
, O
USA O
) O
. O

The O
RNA O
was O
then O
treated O
with O
DNase O
I O
( O
Qiagen O
) O
. O

The O
oligo O
( O
dT O
) O
- O
primed O
cDNA O
was O
synthesized O
using O
Superscript O
II O
reverse O
transcriptase O
( O
Invitrogen O
) O
. O

The O
amount O
of O
cDNA O
for O
amplification O
was O
adjusted O
by O
the O
amount O
of O
RNA O
measured O
by O
an O
optical O
density O
meter O
and O
also O
by O
beta O
- O
actin O
or O
GAPDH O
PCR O
products O
. O

One O
microliter O
of O
cDNA O
was O
amplified O
in O
a O
50 O
mu O
l O
final O
volume O
containing O
25 O
pmol O
appropriate O
primer O
pair O
, O
10 O
pmol O
each O
of O
the O
four O
deoxynucleotide O
triphosphates O
, O
and O
5 O
units O
FastStart O
Taq O
DNA O
Polymerase O
( O
Roche O
, O
Manheim O
, O
Germany O
) O
in O
a O
thermal O
cycler O
( O
PTC O
- O
200 O
; O
MJ O
GeneWorks O
, O
Waltham O
, O
MA O
, O
USA O
) O
. O

The O
PCR O
conditions O
were O
25 O
- O
40 O
cycles O
of O
denaturation O
( O
95 O
degrees O
C O
for O
30 O
s O
) O
, O
annealing O
( O
60 O
- O
62 O
degrees O
C O
for O
1 O
min O
) O
and O
extension O
( O
72 O
degrees O
C O
for O
1 O
min O
) O
. O

The O
sequences O
of O
the O
primers O
are O
presented O
in O
Table O
1 O
. O

The O
PCR O
products O
were O
separated O
by O
electrophoresis O
through O
2 O
% O
agarose O
gel O
. O

Western O
immunoblot O
analysis O

Purified O
monocytes O
were O
cultured O
for O
3 O
days O
in O
the O
presence O
of O
40 O
ng O
/ O
ml O
M O
- O
CSF O
with O
or O
without O
25 O
ng O
/ O
ml O
RANKL O
. O

Cells O
were O
lysed O
in O
RIPA O
Lysis O
buffer O
( O
upstate O
, O
Lake O
Placid O
, O
NY O
, O
USA O
) O
containing O
protease O
inhibitors O
( O
Roche O
) O
for O
15 O
min O
at O
4 O
degrees O
C O
. O

A O
total O
of O
20 O
mu O
g O
protein O
was O
boiled O
in O
the O
presence O
of O
6 O
x O
sodium O
dodecyl O
sulfate O
sample O
buffer O
, O
and O
was O
separated O
on O
7 O
. O
5 O
% O
or O
10 O
% O
sodium O
dodecyl O
sulfate O
- O
polyacrylamide O
gel O
( O
ATTO O
, O
Tokyo O
, O
Japan O
) O
. O

Proteins O
were O
then O
electrotransferred O
to O
a O
polyvinylidene O
fluoride O
microporous O
membrane O
( O
Millipore O
, O
Billerica O
, O
MA O
, O
USA O
) O
in O
a O
semidry O
system O
. O

Membranes O
were O
incubated O
in O
10 O
% O
skim O
milk O
prepared O
in O
phosphate O
- O
buffered O
saline O
( O
PBS O
) O
containing O
0 O
. O
1 O
% O
Tween O
20 O
, O
and O
were O
subjected O
to O
immunoblotting O
. O

Antibodies O
used O
were O
goat B
anti O
- O
RANK O
antibody O
( O
Techne O
Corporation O
, O
Minneapolis O
, O
MN O
, O
USA O
) O
, O
goat B
anti O
- O
c O
- O
fms O
antibody O
( O
R O
& O
D O
systems O
, O
Minneapolis O
, O
MN O
, O
USA O
) O
, O
and O
mouse B
anti O
- O
beta O
- O
actin O
mAb O
( O
AC O
- O
15 O
; O
Sigma O
) O
. O

Peroxidase O
- O
conjugated O
rabbit B
anti O
- O
goat B
IgG O
antibody O
( O
Dako O
) O
or O
peroxidase O
- O
conjugated O
rabbit B
anti O
- O
mouse B
IgG O
antibody O
( O
Dako O
) O
was O
used O
as O
the O
second O
antibody O
. O

The O
signals O
were O
visualized O
by O
chemiluminescence O
reagent O
( O
ECL O
; O
Amersham O
Biocsiences O
, O
Little O
Chalfont O
, O
UK O
) O
. O

Cell O
surface O
expression O
of O
c O
- O
fms O

The O
following O
mAbs O
were O
used O
for O
analysis O
of O
c O
- O
fms O
expression O
: O
Alexa O
647 O
- O
conjugated O
anti O
- O
CD14 O
mAb O
( O
UCHM1 O
; O
Serotec O
, O
Oxford O
, O
UK O
) O
, O
FITC O
- O
conjugated O
anti O
- O
CD16 O
mAb O
( O
3G8 O
; O
Bechman O
Coulter O
) O
and O
phycoerythin O
- O
conjugated O
anti O
- O
c O
- O
fms O
mAb O
( O
61708 O
; O
R O
& O
D O
systems O
) O
. O

Alexa O
647 O
- O
conjugated O
mouse B
IgG2a O
( O
Serotec O
) O
, O
FITC O
- O
conjugated O
mouse B
IgG1 O
( O
BD O
Biosciences O
) O
and O
phycoerythin O
- O
conjugated O
mouse B
IgG1 O
( O
Bechman O
Coulter O
) O
were O
used O
as O
isotype O
controls O
. O

Peripheral O
blood O
monocytes O
( O
1 O
x O
105 O
cells O
) O
were O
incubated O
with O
1 O
mu O
g O
human B
IgG O
for O
15 O
minutes O
, O
and O
were O
then O
stained O
with O
three O
fluorochrome O
- O
labeled O
mAbs O
for O
45 O
minutes O
on O
ice O
. O

The O
stained O
cells O
were O
analyzed O
with O
a O
FACS O
Calibur O
( O
BD O
Biosciences O
) O
. O

Immunofluorescent O
staining O

Monocytes O
( O
8 O
x O
104 O
cells O
/ O
well O
) O
were O
allowed O
to O
adhere O
on O
96 O
- O
well O
plates O
overnight O
or O
were O
cultured O
with O
M O
- O
CSF O
and O
RANKL O
for O
2 O
- O
4 O
days O
. O

The O
cells O
were O
fixed O
in O
acetone O
and O
then O
stained O
with O
anti O
- O
alpha O
v O
beta O
3 O
mAb O
( O
LM609 O
; O
Chemicon O
, O
Temecula O
, O
CA O
, O
USA O
) O
or O
mouse B
IgG1 O
( O
11711 O
; O
R O
& O
D O
Systems O
) O
as O
an O
isotype O
- O
matched O
control O
. O

Alexa O
fluor546 O
- O
conjugated O
goat B
anti O
- O
mouse B
IgG1 O
antibody O
( O
Molecular O
Probes O
, O
Eugene O
, O
OR O
, O
USA O
) O
was O
used O
as O
the O
second O
antibody O
. O

TOTO O
- O
3 O
( O
Molecular O
Probes O
) O
was O
used O
for O
nuclear O
staining O
. O

Flow O
cytometric O
analysis O
of O
p38 O
MAPK O
and O
ERK1 O
/ O
2 O
phosphorylation O

Purified O
monocytes O
were O
cultured O
in O
the O
presence O
of O
25 O
ng O
/ O
ml O
M O
- O
CSF O
for O
3 O
days O
, O
and O
were O
either O
left O
unstimulated O
or O
were O
stimulated O
with O
40 O
ng O
/ O
ml O
RANKL O
at O
37 O
degrees O
C O
. O

Stimulations O
were O
stopped O
by O
adding O
an O
equal O
volume O
of O
PhosFlow O
Fix O
Buffer O
I O
solution O
( O
BD O
Biosciences O
) O
to O
the O
cell O
culture O
. O

After O
incubation O
for O
10 O
minutes O
at O
37 O
degrees O
C O
, O
the O
cells O
were O
permeabilized O
by O
washing O
twice O
at O
room O
temperature O
in O
PhosFlow O
Perm O
/ O
Wash O
Buffer O
I O
( O
BD O
Biosciences O
) O
. O

A O
total O
of O
1 O
x O
105 O
cells O
was O
then O
Fc O
blocked O
with O
1 O
mu O
g O
human B
IgG O
for O
15 O
minutes O
, O
and O
was O
stained O
with O
Alexa O
Fluor O
647 O
- O
conjugated O
mAb O
either O
to O
phospho O
- O
p38 O
MAPK O
( O
T180 O
/ O
Y182 O
) O
or O
to O
phospho O
- O
ERK1 O
/ O
2 O
( O
T202 O
/ O
Y204 O
) O
( O
BD O
Biosciences O
) O
for O
30 O
minutes O
at O
room O
temperature O
. O

Alexa O
Fluor O
647 O
- O
conjugated O
mouse B
IgG1 O
( O
BD O
Biosciences O
) O
was O
used O
as O
an O
isotype O
control O
. O

The O
cells O
were O
washed O
in O
PhosFlow O
Perm O
/ O
Wash O
Buffer O
I O
, O
and O
were O
analyzed O
by O
flow O
cytometry O
, O
as O
already O
described O
. O

RNA O
interference O

RNA O
oligonucleotides O
( O
iGENE O
, O
Tsukuba O
, O
Japan O
) O
were O
designed O
based O
on O
the O
algorithm O
that O
incorporates O
single O
nucleotide O
polymorphism O
and O
homology O
screening O
to O
ensure O
a O
target O
- O
specific O
RNA O
interference O
effect O
. O

The O
following O
sense O
and O
antisense O
oligonucleotides O
were O
used O
: O
integrin O
beta O
3 O
, O
5 O
' O
- O
GCU O
UCA O
AUG O
AGG O
AAG O
UGA O
AGA O
AGC O
A O
- O
AG O
and O
3 O
' O
- O
UA O
- O
CGA O
AGU O
UAC O
UCC O
UUC O
ACU O
UCU O
UCG O
U O
; O
randomized O
control O
, O
5 O
' O
- O
CGA O
UUC O
GCU O
AGA O
CCG O
GCU O
UCA O
UUG O
C O
- O
AG O
and O
3 O
' O
- O
UA O
- O
GCU O
AAG O
CGA O
UCU O
GGC O
CGA O
AGU O
AAC O
G O
; O
and O
lamin O

, O
5 O
' O
- O
GAG O
GAA O
CUG O
GAC O
UUC O
CAG O
AAG O
AAC O
A O
- O
AG O
and O
3 O
' O
- O
UA O
- O
CUC O
CUU O
GAC O
CUG O
AAG O
GUC O
UUC O
UUG O
U O
. O

CD16 O
- O
monocytes O
( O
8 O
x O
104 O
cells O
/ O
well O
) O
were O
incubated O
in O
96 O
- O
well O
plates O
in O
optimem O
( O
Invitrogen O
) O
. O

After O
1 O
hour O
, O
siRNAs O
were O
transfected O
into O
the O
cells O
using O
oligofectamine O
( O
Qiagen O
) O
based O
on O
the O
method O
recommended O
by O
the O
manufacturer O
. O

After O
2 O
hours O
, O
the O
cells O
were O
washed O
once O
with O
PBS O
, O
followed O
by O
the O
addition O
of O
alpha O
MEM O
supplemented O
with O
10 O
% O
FBS O
, O
M O
- O
CSF O
and O
RANKL O
. O

After O
a O
2 O
- O
day O
incubation O
, O
the O
beta O
3 O
mRNA O
expression O
was O
analyzed O
by O
RT O
- O
PCR O
with O
different O
PCR O
cycles O
, O
as O
described O
earlier O
. O

Immunofluorescent O
staining O
for O
the O
alpha O
v O
beta O
3 O
heterodimer O
was O
also O
performed O
as O
described O
above O
, O
and O
numbers O
of O
alpha O
v O
beta O
3 O
- O
positive O
cells O
were O
counted O
in O
randomly O
selected O
three O
fields O
at O
100 O
x O
magnification O
. O

Seven O
days O
after O
the O
transfection O
of O
siRNAs O
, O
the O
number O
of O
TRAP O
- O
positive O
MNC O
in O
five O
fields O
examined O
at O
100 O
x O
magnification O
was O
counted O
under O
light O
microscopy O
. O

Inhibition O
of O
osteoclastogenesis O
with O
cyclic O
RGDfV O
peptide O

CD16 O
- O
monocytes O
were O
incubated O
in O
96 O
- O
well O
plates O
with O
M O
- O
CSF O
+ O
RANKL O
for O
2 O
days O
. O

A O
medium O
containing O
either O
cyclic O
RGDfV O
peptide O
( O
Arg O
- O
Gly O
- O
Asp O
- O
D O
- O
Phe O
- O
Val O
) O
( O
Calbiochem O
, O
San O
Diego O
, O
CA O
, O
USA O
) O
or O
dimethyl O
sulfoxide O
was O
then O
added O
. O

After O
incubation O
for O
a O
further O
5 O
days O
, O
the O
number O
of O
TRAP O
- O
positive O
MNC O
in O
five O
fields O
examined O
at O
100 O
x O
magnification O
was O
counted O
under O
light O
microscopy O
. O

Immunohistochemistry O

Synovial O
tissue O
samples O
were O
obtained O
during O
total O
knee O
joint O
replacement O
surgery O
from O
four O
RA O
patients B
. O

Signed O
consent O
forms O
were O
obtained O
before O
the O
operation O
. O

The O
experimental O
protocol O
was O
approved O
by O
the O
ethics O
committee O
of O
the O
Tokyo O
Medical O
and O
Dental O
University O
. O

RA O
was O
diagnosed O
according O
to O
the O
American O
College O
of O
Rheumatology O
criteria O
[ O
14 O
] O
. O

Double O
immunofluorescent O
staining O
for O
CD68 O
and O
CD16 O
antigens O
was O
conducted O
on O
optimal O
cutting O
temperature O
- O
embedded O
sections O
of O
frozen O
synovial O
samples O
. O

Eight O
- O
micrometer O
- O
thick O
cryostat O
sections O
of O
RA O
synovium O
were O
fixed O
in O
acetone O
for O
3 O
minutes O
and O
were O
then O
rehydrated O
in O
PBS O
for O
5 O
minutes O
. O

The O
samples O
were O
incubated O
in O
5 O
mu O
g O
/ O
ml O
proteinase O
K O
( O
Roche O
) O
, O
50 O
mM O
ethylenediamine O
tetraacetic O
acid O
, O
100 O
mM O
Tris O
- O
HCl O
, O
pH O
8 O
. O
0 O
, O
for O
15 O
minutes O
at O
room O
temperature O
followed O
by O
a O
wash O
in O
PBS O
. O

The O
samples O
were O
then O
blocked O
with O
10 O
% O
goat B
serum O
in O
PBS O
for O
60 O
minutes O
at O
room O
temperature O
, O
and O
were O
incubated O
with O
anti O
- O
CD16 O
mAb O
( O
3G8 O
; O
Immunotech O
, O
Marseille O
, O
France O
) O
or O
mouse B
IgG1 O
( O
11711 O
) O
as O
an O
isotype O
- O
matched O
control O
in O
1 O
% O
bovine B
serum O
albumin O
/ O
PBS O
for O
60 O
minutes O
at O
room O
temperature O
. O

The O
samples O
were O
then O
washed O
three O
times O
in O
PBS O
, O
for O
5 O
minutes O
each O
, O
and O
incubated O
with O
Alexa O
fluor546 O
- O
conjugated O
goat B
anti O
- O
mouse B
IgG1 O
antibody O
( O
Molecular O
Probes O
) O
in O
1 O
% O
bovine B
serum O
albumin O
/ O
PBS O
for O
60 O
minutes O
at O
room O
temperature O
. O

The O
samples O
were O
then O
sequentially O
stained O
for O
CD68 O
antigen O
in O
a O
manner O
similar O
to O
that O
used O
for O
CD16 O
staining O
. O

The O
samples O
were O
stained O
with O
anti O
- O
CD68 O
mAb O
( O
PGM1 O
; O
Immunotech O
) O
or O
mouse B
IgG3 O
( O
6A3 O
; O
MBL O
, O
Nagoya O
, O
Japan O
) O
followed O
by O
labeling O
with O
Alexa O
fluor488 O
- O
conjugated O
goat B
anti O
- O
mouse B
IgG3 O
antibody O
( O
Molecular O
Probes O
) O
. O

The O
samples O
were O
examined O
by O
confocal O
laser O
scanning O
microscope O
( O
Olympus O
, O
Tokyo O
, O
Japan O
) O
. O

Statistical O
analysis O

Data O
are O
expressed O
as O
the O
mean O
+ O
/ O
- O
standard O
error O
of O
the O
mean O
. O

A O
nonpaired O
Student O
' O
s O
t O
test O
was O
used O
for O
comparison O
, O
using O
the O
StatView O
program O
( O
Abacus O
Concepts O
, O
Berkeley O
, O
CA O
, O
USA O
) O
. O

P O
< O
0 O
. O
05 O
was O
considered O
statistically O
significant O
. O

Results O

Induction O
of O
osteoclasts O
from O
CD16 O
- O
peripheral O
blood O
monocytes O

To O
identify O
the O
monocyte O
subset O
that O
differentiates O
into O
osteoclasts O
, O
we O
examined O
osteoclast O
formation O
from O
CD16 O
+ O
and O
CD16 O
- O
human B
peripheral O
blood O
monocytes O
. O

The O
monocyte O
subsets O
were O
purified O
using O
magnetic O
beads O
. O

Incubation O
with O
M O
- O
CSF O
alone O
did O
not O
induce O
osteoclast O
formation O
from O
either O
subset O
( O
Figure O
1a O
) O
. O

Culture O
with O
M O
- O
CSF O
+ O
RANKL O
induced O
a O
significant O
number O
of O
TRAP O
- O
positive O
MNC O
from O
the O
CD16 O
- O
subset O
, O
whereas O
only O
few O
CD16 O
+ O
monocytes O
differentiated O
into O
TRAP O
- O
positive O
MNC O
( O
Figure O
1a O
, O
b O
) O
. O

We O
then O
assessed O
the O
bone O
resorptive O
ability O
by O
culturing O
cells O
on O
calcium O
phosphate O
- O
coated O
plates O
with O
M O
- O
CSF O
+ O
RANKL O
. O

Resorption O
lacunae O
were O
detected O
only O
in O
the O
CD16 O
- O
subset O
( O
Figure O
1c O
) O
, O
indicating O
the O
TRAP O
- O
positive O
CD16 O
- O
- O
derived O
MNC O
possessed O
the O
osteoclast O
phenotype O
. O

Similar O
results O
were O
obtained O
using O
purified O
monocytes O
by O
FACS O
sorting O
( O
purities O
: O
CD16 O
+ O
, O
96 O
% O
; O
CD16 O
- O
, O
97 O
% O
) O
. O

The O
number O
of O
TRAP O
- O
positive O
MNC O
induced O
were O
36 O
+ O
/ O
- O
3 O
cells O
/ O
well O
and O
348 O
+ O
/ O
- O
13 O
cells O
/ O
well O
from O
CD16 O
+ O
and O
CD16 O
- O
monocytes O
, O
respectively O
. O

To O
exclude O
the O
possibility O
that O
the O
anti O
- O
CD16 O
antibody O
used O
for O
isolation O
of O
CD16 O
+ O
monocytes O
inhibits O
osteoclast O
formation O
, O
we O
separated O
the O
two O
subsets O
, O
CD33low O
monocytes O
and O
CD33high O
monocytes O
, O
using O
anti O
- O
CD33 O
mAb O
and O
a O
fluorescent O
cell O
sorter O
, O
since O
it O
was O
reported O
that O
CD33low O
monocytes O
correspond O
to O
CD16 O
+ O
, O
and O
that O
CD33high O
correspond O
to O
CD16 O
- O
monocytes O
[ O
15 O
] O
. O

On O
average O
, O
the O
CD33low O
population O
contained O
CD16 O
- O
( O
10 O
. O
2 O
% O
) O
/ O
CD16 O
+ O
( O
89 O
. O
8 O
% O
) O
monocytes O
, O
and O
the O
CD33high O
population O
contained O
CD16 O
- O
( O
86 O
. O
3 O
% O
) O
/ O
CD16 O
+ O
( O
13 O
. O
7 O
% O
) O
monocytes O
. O

Culture O
with O
M O
- O
CSF O
+ O
RANKL O
induced O
TRAP O
- O
positive O
MNC O
from O
CD33high O
monocytes O
, O
whereas O
no O
or O
few O
CD33low O
monocytes O
differentiated O
into O
TRAP O
- O
positive O
MNC O
( O
CD33low O
vs O
CD33high O
, O
2 O
+ O
/ O
- O
1 O
vs O
192 O
+ O
/ O
- O
71 O
cells O
/ O
well O
; O
n O
= O
3 O
) O
. O

TRAP O
- O
5b O
and O
MMP O
- O
9 O
in O
the O
culture O
supernatants O
, O
both O
of O
which O
are O
known O
to O
be O
produced O
by O
osteoclasts O
, O
were O
measured O
by O
ELISA O
. O

The O
concentrations O
of O
both O
enzymes O
were O
significantly O
higher O
in O
the O
culture O
supernatant O
of O
CD16 O
- O
monocytes O
than O
in O
that O
of O
CD16 O
+ O
monocytes O
( O
Figure O
1d O
) O
. O

These O
results O
suggest O
that O
the O
CD16 O
- O
peripheral O
blood O
monocyte O
subset O
, O
but O
not O
the O
CD16 O
+ O
subset O
, O
differentiate O
into O
osteoclasts O
by O
incubation O
with O
RANKL O
+ O
M O
- O
CSF O
. O

CD16 O
+ O
monocytes O
do O
not O
affect O
the O
osteoclastogenesis O
from O
CD16 O
- O
monocytes O

To O
examine O
whether O
CD16 O
+ O
monocytes O
affect O
osteoclastogenesis O
from O
CD16 O
- O
monocytes O
, O
varied O
numbers O
of O
CD16 O
+ O
monocytes O
were O
mixed O
with O
CD16 O
- O
monocytes O
( O
5 O
x O
104 O
cells O
/ O
well O
) O
, O
and O
were O
cultured O
for O
7 O
days O
in O
the O
presence O
of O
M O
- O
CSF O
+ O
RANKL O
. O

The O
number O
of O
TRAP O
- O
positive O
MNC O
was O
not O
altered O
by O
the O
presence O
of O
CD16 O
+ O
monocytes O
( O
Figure O
2 O
) O
. O

The O
results O
indicated O
that O
CD16 O
+ O
monocytes O
did O
not O
hamper O
or O
enhance O
the O
osteoclastogenesis O
from O
CD16 O
- O
monocytes O
. O

Differences O
in O
cytokine O
production O
by O
RANKL O
- O
stimulated O
or O
M O
- O
CSF O
- O
stimulated O
CD16 O
+ O
and O
CD16 O
- O
monocytes O

To O
compare O
the O
biological O
response O
of O
CD16 O
+ O
and O
CD16 O
- O
subsets O
with O
either O
RANKL O
or O
M O
- O
CSF O
stimulation O
, O
we O
measured O
the O
amount O
of O
TNF O
alpha O
and O
IL O
- O
6 O
production O
after O
exposure O
of O
cells O
to O
various O
concentrations O
of O
RANKL O
or O
M O
- O
CSF O
with O
an O
ELISA O
. O

Without O
RANKL O
the O
CD16 O
+ O
subset O
produced O
a O
significant O
amount O
of O
TNF O
alpha O
and O
IL O
- O
6 O
, O
whereas O
the O
CD16 O
- O
subset O
produced O
undetectable O
levels O
( O
Figure O
3a O
) O
. O

RANKL O
stimulation O
increased O
TNF O
alpha O
production O
from O
both O
subsets O
in O
a O
dose O
- O
dependent O
manner O
, O
although O
the O
CD16 O
+ O
subset O
produced O
more O
TNF O
alpha O
than O
did O
the O
CD16 O
- O
subset O
. O

RANKL O
stimulation O
also O
enhanced O
IL O
- O
6 O
production O
from O
the O
CD16 O
+ O
subset O
, O
but O
not O
from O
the O
CD16 O
- O
subset O
. O

M O
- O
CSF O
stimulation O
increased O
TNF O
alpha O
and O
IL O
- O
6 O
production O
from O
both O
subsets O
, O
although O
the O
CD16 O
+ O
subset O
produced O
more O
than O
the O
CD16 O
- O
subset O
( O
Figure O
3b O
) O
. O

These O
results O
suggest O
that O
CD16 O
+ O
monocytes O
also O
respond O
both O
to O
RANKL O
and O
M O
- O
CSF O
stimulation O
, O
although O
such O
stimulation O
does O
not O
result O
in O
differentiation O
into O
osteoclasts O
. O

CD16 O
+ O
monocytes O
were O
also O
noted O
to O
express O
higher O
amounts O
of O
inflammatory O
cytokines O
compared O
with O
CD16 O
- O
monocytes O
with O
or O
without O
RANKL O
or O
M O
- O
CSF O
stimulation O
. O

Comparison O
of O
expression O
levels O
of O
molecules O
involved O
in O
osteoclastogenesis O
between O
CD16 O
+ O
and O
CD16 O
- O
monocytes O

Diverse O
molecules O
are O
involved O
in O
RANKL O
/ O
RANK O
and O
its O
costimulatory O
signal O
transduction O
pathways O
[ O
16 O
] O
. O

The O
different O
response O
to O
RANKL O
+ O
M O
- O
CSF O
stimulation O
between O
the O
CD16 O
+ O
monocyte O
subset O
and O
the O
CD16 O
- O
monocytes O
subset O
might O
be O
explained O
by O
the O
expression O
profiles O
of O
these O
molecules O
. O

We O
therefore O
examined O
the O
mRNA O
levels O
of O
the O
following O
molecules O
: O
receptor O
activator O
of O
NF O
- O
kappa O
B O
( O
RANK O
) O
, O
the O
receptor O
for O
RANKL O
; O
c O
- O
fms O
, O
the O
receptor O
for O
M O
- O
CSF O
; O
tumor O
necrosis O
factor O
receptor O
- O
associated O
factor O
6 O
( O
TRAF O
- O
6 O
) O
, O
the O
adaptor O
protein O
for O
RANK O
; O
c O
- O
Fos O
and O
nuclear O
factor O
of O
activated O
T O
cells O
c1 O
( O
NFATc1 O
) O
, O
transcription O
factors O
that O
are O
essential O
for O
osteoclastogenesis O
; O
DNAX O
- O
activation O
protein O
12 O
( O
DAP12 O
) O
and O
Fc O
receptor O

gamma O
chain O
( O
FcR O
gamma O
) O
, O
adaptor O
proteins O
known O
to O
deliver O
costimulatory O
signals O
in O
RANKL O
- O
induced O
osteoclastogenesis O
; O
signal O
regulatory O
protein O
beta O
1 O
( O
SIRP O
- O
beta O
1 O
) O
, O
triggering O
receptor O
expressed O
on O
myeloid O
cells O
2 O
( O
TREM O
- O
2 O
) O
and O
osteoclast O
- O
associated O
receptor O
( O
OSCAR O
) O
, O
transmembrane O
receptors O
that O
associate O
with O
either O
DAP12 O
or O
FcR O
gamma O
; O
and O
alpha O
v O
and O
beta O
3 O
, O
integrins O
known O
to O
be O
expressed O
as O
the O
alpha O
v O
beta O
3 O
heterodimer O
on O

osteoclasts O
. O

The O
mRNA O
levels O
of O
RANK O
, O
c O
- O
fms O
, O
TRAF O
- O
6 O
, O
DAP12 O
and O
SIRP O
- O
beta O
1 O
under O
the O
baseline O
condition O
( O
no O
stimulation O
) O
varied O
between O
the O
donors O
; O
however O
, O
we O
did O
not O
find O
consistent O
differences O
in O
the O
mRNA O
levels O
of O
these O
molecules O
between O
the O
CD16 O
+ O
monocyte O
subset O
and O
the O
CD16 O
- O
monocyte O
subset O
among O
three O
to O
six O
donors B
( O
Figure O
4a O
) O
. O

The O
mRNA O
levels O
of O
other O
molecules O
, O
apart O
from O
integrin O
beta O
3 O
, O
were O
similar O
between O
the O
two O
subsets O
under O
the O
no O
- O
stimulation O
condition O
. O

Although O
the O
mRNA O
levels O
of O
RANK O
, O
c O
- O
fms O
, O
DAP12 O
, O
FcR O
gamma O
, O
TREM O
- O
2 O
and O
OSCAR O
increased O
in O
response O
to O
M O
- O
CSF O
alone O
or O
M O
- O
CSF O
+ O
RANKL O
in O
both O
subsets O
, O
the O
expression O
levels O
were O
not O
significantly O
different O
between O
the O
two O
subsets O
. O

Expressions O
of O
TRAF O
- O
6 O
, O
c O
- O
Fos O
and O
SIRP O
- O
beta O
1 O
mRNA O
did O
not O
change O
following O
stimulation O
with O
M O
- O
CSF O
+ O
RANKL O
. O

Of O
note O
, O
the O
expression O
of O
NFATc1 O
mRNA O
was O
enhanced O
by O
M O
- O
CSF O
+ O
RANKL O
treatment O
only O
in O
the O
CD16 O
- O
subset O
. O

Furthermore O
, O
expression O
of O
integrin O
alpha O
v O
in O
both O
subsets O
was O
enhanced O
by O
M O
- O
CSF O
with O
or O
without O
RANKL O
; O
however O
, O
the O
expression O
level O
was O
greater O
in O
the O
CD16 O
- O
subset O
. O

It O
was O
noted O
that O
integrin O
- O
beta O
3 O
mRNA O
was O
detected O
only O
in O
the O
CD16 O
- O
subset O
and O
was O
increased O
by O
M O
- O
CSF O
+ O
RANKL O
stimulation O
, O
but O
not O
by O
M O
- O
CSF O
alone O
. O

The O
protein O
expression O
of O
RANK O
under O
the O
baseline O
condition O
was O
weakly O
detected O
in O
both O
subsets O
, O
and O
the O
levels O
were O
varied O
between O
donors O
by O
western O
immunoblotting O
. O

The O
protein O
expression O
of O
c O
- O
fms O
was O
weakly O
detected O
in O
unstimulated O
CD16 O
+ O
monocytes O
, O
but O
not O
in O
CD16 O
- O
monocytes O
( O
Figure O
4b O
) O
. O

Flow O
cytometry O
analysis O
of O
c O
- O
fms O
in O
fresh O
monocytes O
, O
however O
, O
showed O
that O
both O
subsets O
express O
the O
molecule O
on O
the O
cell O
surface O
( O
Figure O
4c O
) O
. O

Expressions O
of O
both O
RANK O
and O
c O
- O
fms O
were O
upregulated O
by O
M O
- O
CSF O
alone O
and O
by O
M O
- O
CSF O
+ O
RANKL O
, O
and O
we O
did O
not O
find O
consistent O
differences O
in O
the O
protein O
levels O
of O
these O
molecules O
between O
the O
two O
monocyte O
subsets O
. O

The O
profiles O
of O
expression O
levels O
of O
molecules O
involved O
in O
RANKL O
/ O
RANK O
and O
its O
costimulatory O
pathways O
are O
similar O
between O
the O
two O
subsets O
, O
except O
for O
NFATc1 O
, O
integrin O
alpha O
v O
and O
integrin O
beta O
3 O
. O

We O
therefore O
assumed O
that O
the O
distinct O
induction O
of O
NFATc1 O
, O
integrin O
alpha O
v O
and O
integrin O
beta O
3 O
in O
response O
to O
RANKL O
stimulation O
among O
the O
two O
monocyte O
subsets O
might O
explain O
the O
differences O
in O
their O
abilities O
to O
differentiate O
into O
osteoclasts O
. O

RANKL O
stimulation O
induces O
alpha O
v O
beta O
3 O
expression O
on O
CD16 O
- O
monocytes O

The O
integrin O
- O
beta O
3 O
subunit O
binds O
to O
integrin O
alpha O
v O
only O
and O
is O
expressed O
as O
the O
heterodimeric O
protein O
alpha O
v O
beta O
3 O
on O
monocytes O
and O
osteoclasts O
[ O
17 O
] O
. O

We O
examined O
the O
expression O
of O
alpha O
v O
beta O
3 O
on O
CD16 O
+ O
and O
CD16 O
- O
monocytes O
by O
immunofluorescent O
staining O
. O

Neither O
unstimulated O
nor O
M O
- O
CSF O
- O
stimulated O
monocyte O
subsets O
expressed O
alpha O
v O
beta O
3 O
( O
Figure O
4d O
and O
data O
not O
shown O
) O
. O

After O
48 O
and O
72 O
hours O
of O
treatment O
with O
M O
- O
CSF O
+ O
RANKL O
, O
alpha O
v O
beta O
3 O
- O
positive O
mononuclear O
cells O
were O
observed O
in O
CD16 O
- O
monocyte O
cultures O
but O
not O
in O
CD16 O
+ O
monocyte O
cultures O
. O

At O
96 O
hours O
, O
both O
alpha O
v O
beta O
3 O
- O
positive O
mononuclear O
cells O
and O
multinucleated O
cells O
were O
present O
in O
the O
CD16 O
- O
monocyte O
culture O
. O

The O
results O
indicated O
that O
alpha O
v O
beta O
3 O
was O
selectively O
expressed O
on O
CD16 O
- O
monocytes O
in O
the O
presence O
of O
M O
- O
CSF O
+ O
RANKL O
, O
and O
the O
expression O
was O
revealed O
before O
the O
cells O
differentiate O
into O
typical O
multinucleated O
osteoclasts O
. O

RANKL O
activates O
ERK O
and O
p38 O
kinases O
only O
in O
CD16 O
- O
monocytes O

Since O
ERK O
and O
p38 O
MAPK O
are O
essential O
in O
RANKL O
- O
induced O
osteoclastogenesis O
[ O
18 O
- O
20 O
] O
, O
we O
next O
examined O
whether O
these O
kinases O
were O
activated O
differently O
in O
CD16 O
+ O
monocytes O
and O
in O
CD16 O
- O
monocytes O
. O

Purified O
monocytes O
were O
precultured O
with O
25 O
ng O
/ O
ml O
M O
- O
CSF O
for O
3 O
days O
to O
enhance O
RANK O
expression O
, O
and O
were O
then O
treated O
with O
RANKL O
. O

The O
RANKL O
treatment O
induced O
phosphorylation O
of O
both O
ERK O
and O
p38 O
MAPK O
in O
CD16 O
- O
monocytes O
at O
5 O
minutes O
postexposure O
, O
although O
the O
p38 O
MAPK O
phosphorylation O
was O
weak O
. O

Both O
phosphorylations O
declined O
to O
a O
basal O
level O
within O
20 O
minutes O
( O
Figure O
5 O
) O
. O

In O
contrast O
, O
ERK O
and O
p38 O
MAPK O
were O
not O
detectably O
phosphorylated O
in O
CD16 O
+ O
monocytes O
with O
RANKL O
. O

siRNA O
targeting O
integrin O
beta O
3 O
inhibits O
osteoclastogenesis O
from O
CD16 O
- O
monocytes O

The O
integrin O
- O
beta O
3 O
cytoplasmic O
domain O
is O
essential O
for O
activation O
of O
intracellular O
signals O
from O
alpha O
v O
beta O
3 O
heterodimers O
[ O
17 O
] O
. O

We O
therefore O
examined O
the O
involvement O
of O
alpha O
v O
beta O
3 O
in O
RANKL O
+ O
M O
- O
CSF O
- O
induced O
osteoclastogenesis O
in O
human B
CD16 O
- O
monocytes O
using O
siRNA O
targeting O
the O
integrin O
- O
beta O
3 O
subunit O
. O

The O
integrin O
- O
beta O
3 O
siRNA O
or O
control O
randomized O
siRNA O
were O
transfected O
into O
CD16 O
- O
monocytes O
. O

At O
48 O
hours O
post O
- O
transfection O
, O
we O
determined O
the O
integrin O
- O
beta O
3 O
mRNA O
level O
and O
alpha O
v O
beta O
3 O
heterodimer O
protein O
expression O
. O

The O
integrin O
- O
beta O
3 O
mRNA O
level O
was O
reduced O
in O
the O
integrin O
- O
beta O
3 O
siRNA O
- O
transfected O
monocytes O
compared O
with O
control O
siRNA O
- O
transfected O
monocytes O
( O
Figure O
6a O
) O
. O

The O
alpha O
v O
beta O
3 O
heterodimer O
expression O
was O
evaluated O
by O
immunofluorescent O
staining O
. O

The O
number O
of O
alpha O
v O
beta O
3 O
- O
positive O
cells O
was O
significantly O
decreased O
in O
integrin O
- O
beta O
3 O
siRNA O
- O
transfected O
monocytes O
compared O
with O
that O
in O
control O
siRNA O
( O
Figure O
6b O
) O
. O

After O
7 O
days O
of O
incubation O
, O
the O
number O
of O
TRAP O
- O
positive O
MNC O
was O
counted O
. O

Transfection O
with O
integrin O
- O
beta O
3 O
siRNA O
significantly O
reduced O
the O
number O
of O
TRAP O
- O
positive O
MNC O
in O
a O
dose O
- O
dependent O
manner O
compared O
with O
control O
siRNA O
transfection O
( O
Figure O
6c O
) O
. O

In O
addition O
, O
the O
use O
of O
siRNA O
directed O
toward O
a O
different O
site O
of O
integrin O
- O
beta O
3 O
mRNA O
also O
inhibited O
osteoclast O
formation O
from O
CD16 O
- O
monocytes O
( O
data O
not O
shown O
) O
. O

On O
the O
other O
hand O
, O
siRNA O
that O
targeted O
lamin O
, O
which O
was O
used O
as O
a O
negative O
control O
, O
did O
not O
inhibit O
the O
induction O
of O
osteoclasts O
( O
data O
not O
shown O
) O
. O

These O
results O
indicate O
the O
importance O
of O
integrin O
beta O
3 O
in O
RANKL O
- O
induced O
osteoclast O
formation O
from O
CD16 O
- O
peripheral O
blood O
monocytes O
. O

Cyclic O
RGDfV O
peptide O
inhibits O
the O
osteoclastogenesis O
from O
CD16 O
- O
monocytes O

Integrin O
alpha O
v O
beta O
3 O
recognizes O
a O
common O
tripeptide O
sequence O
, O
RGD O
( O
Arg O
- O
Gly O
- O
Asp O
) O
, O
which O
is O
present O
in O
bone O
matrix O
proteins O
such O
as O
vitronectin O
and O
fibronectin O
[ O
21 O
] O
. O

Cyclic O
RGDfV O
peptide O
( O
Arg O
- O
Gly O
- O
Asp O
- O
D O
- O
Phe O
- O
Val O
) O
inhibits O
binding O
of O
the O
RGD O
- O
containing O
molecules O
to O
alpha O
v O
beta O
3 O
[ O
22 O
] O
. O

To O
investigate O
the O
role O
of O
ligand O
binding O
to O
the O
alpha O
v O
beta O
3 O
heterodimer O
in O
the O
osteoclastogenesis O
, O
we O
examined O
whether O
cyclic O
RGDfV O
peptide O
inhibits O
the O
formation O
of O
osteoclasts O
. O

Cyclic O
RGDfV O
peptide O
significantly O
reduced O
the O
number O
of O
TRAP O
- O
positive O
MNC O
in O
a O
dose O
- O
dependent O
manner O
( O
Figure O
6d O
) O
. O

The O
results O
imply O
possible O
involvement O
of O
ligand O
bindings O
to O
alpha O
v O
beta O
3 O
in O
the O
osteoclastogenesis O
. O

Knockdown O
of O
integrin O
beta O
3 O
did O
not O
affect O
the O
expression O
of O
NFATc1 O
mRNA O

In O
the O
next O
step O
, O
we O
determined O
whether O
integrin O
- O
beta O
3 O
- O
siRNA O
- O
induced O
inhibition O
of O
the O
osteoclastogenesis O
reflects O
downregulation O
of O
NFATc1 O
, O
which O
is O
a O
key O
transcription O
factor O
in O
osteoclastogenesis O
[ O
23 O
] O
. O

For O
this O
purpose O
, O
we O
compared O
NFATc1 O
mRNA O
levels O
between O
integrin O
beta O
3 O
and O
control O
siRNA O
- O
transfected O
monocytes O
. O

Interestingly O
, O
integrin O
- O
beta O
3 O
knockdown O
did O
not O
alter O
the O
NFATc1 O
mRNA O
level O
( O
Figure O
7 O
) O
, O
suggesting O
that O
signal O
transduction O
mediated O
by O
integrin O
beta O
3 O
does O
not O
affect O
the O
expression O
of O
NFATc1 O
. O

Detection O
of O
CD16 O
+ O
and O
CD16 O
- O
macrophages O
in O
synovium O
of O
RA O
patients B

RA O
synovial O
macrophages O
are O
derived O
from O
peripheral O
blood O
monocytes O
, O
and O
their O
recruitment O
into O
the O
synovium O
is O
facilitated O
by O
various O
adhesion O
molecules O
and O
chemokines O
[ O
24 O
] O
. O

To O
analyze O
CD16 O
expression O
on O
synovial O
macrophages O
, O
RA O
synovial O
tissues O
were O
double O
- O
stained O
for O
CD16 O
and O
a O
macrophage O
marker O
, O
CD68 O
. O

CD16 O
- O
/ O
CD68 O
+ O
macrophages O
were O
widespread O
in O
the O
synovium O
. O

Although O
less O
frequent O
, O
CD16 O
+ O
/ O
CD68 O
+ O
macrophages O
were O
also O
observed O
both O
in O
the O
synovial O
intima O
and O
subintima O
( O
Figure O
8 O
) O
. O

The O
presence O
of O
two O
subsets O
of O
macrophages O
, O
CD16 O
+ O
and O
CD16 O
- O
, O
in O
RA O
synovium O
indicates O
that O
both O
CD16 O
+ O
and O
CD16 O
- O
peripheral O
blood O
monocytes O
are O
recruited O
into O
the O
synovium O
. O

Discussion O

Human B
peripheral O
blood O
monocytes O
are O
a O
heterogeneous O
population O
, O
and O
they O
are O
divided O
into O
two O
subsets O
based O
on O
the O
expression O
of O
CD16 O
. O

The O
CD16 O
+ O
and O
CD16 O
- O
monocyte O
subsets O
show O
functional O
differences O
in O
migration O
, O
cytokine O
production O
and O
differentiation O
into O
macrophages O
or O
dendritic O
cells O
[ O
11 O
- O
13 O
, O
15 O
] O
. O

We O
focused O
on O
the O
heterogeneity O
of O
the O
monocytes O
, O
and O
the O
primary O
question O
addressed O
in O
this O
study O
was O
which O
monocyte O
subset O
could O
be O
the O
source O
of O
osteoclasts O
. O

The O
results O
demonstrated O
that O
CD16 O
- O
peripheral O
blood O
monocytes O
, O
but O
not O
CD16 O
+ O
monocytes O
, O
differentiated O
in O
vitro O
into O
osteoclasts O
by O
treatment O
with O
RANKL O
+ O
M O
- O
CSF O
. O

To O
investigate O
the O
molecular O
mechanisms O
of O
the O
different O
response O
to O
RANKL O
and O
the O
differentiation O
into O
osteoclasts O
between O
CD16 O
+ O
and O
CD16 O
- O
monocytes O
, O
we O
examined O
the O
expression O
of O
molecules O
known O
to O
be O
involved O
in O
osteoclastogenesis O
. O

The O
expression O
profiles O
of O
integrin O
alpha O
v O
, O
integrin O
beta O
3 O
and O
NFATc1 O
were O
different O
between O
the O
two O
subsets O
. O

Integrin O
alpha O
v O
beta O
3 O
heterodimer O
was O
expressed O
only O
on O
RANKL O
and O
M O
- O
CSF O
- O
stimulated O
CD16 O
- O
monocytes O
. O

It O
is O
known O
that O
alpha O
v O
beta O
3 O
expressed O
on O
osteoclasts O
is O
important O
in O
bone O
resorption O
as O
well O
as O
in O
attachment O
of O
osteoclasts O
to O
the O
bone O
matrix O
[ O
25 O
] O
. O

It O
was O
recently O
reported O
that O
bone O
marrow O
macrophages O
of O
integrin O
- O
beta O
3 O
- O
deficient O
mice B
could O
not O
differentiate O
into O
mature O
osteoclasts O
in O
vitro O
, O
suggesting O
that O
alpha O
v O
beta O
3 O
is O
involved O
not O
only O
in O
activation O
, O
but O
also O
in O
differentiation O
, O
of O
osteoclasts O
in O
mice B
[ O
26 O
, O
27 O
] O
. O

The O
authors O
also O
showed O
that O
alpha O
v O
beta O
3 O
and O
c O
- O
fms O
share O
a O
common O
intracellular O
signaling O
pathway O
, O
including O
the O
activation O
of O
ERK O
and O
the O
induction O
of O
c O
- O
Fos O
[ O
27 O
] O
, O
both O
of O
which O
are O
essential O
for O
osteoclastogenesis O
[ O
28 O
, O
29 O
] O
. O

In O
addition O
, O
it O
was O
reported O
that O
echistatin O
, O
an O
alpha O
v O
beta O
3 O
antagonist O
, O
inhibited O
osteoclast O
formation O
of O
mouse B
bone O
marrow O
cells O
[ O
30 O
] O
. O

In O
accordance O
with O
these O
reports O
, O
our O
data O
showed O
that O
knockdown O
of O
integrin O
- O
beta O
3 O
expression O
resulted O
in O
downregulation O
of O
the O
alpha O
v O
beta O
3 O
heterodimer O
, O
and O
abrogated O
osteoclastogenesis O
from O
human B
peripheral O
blood O
CD16 O
- O
monocytes O
. O

We O
also O
showed O
that O
blocking O
of O
adhesive O
ligands O
to O
bind O
to O
alpha O
v O
beta O
3 O
by O
RGDfV O
peptide O
inhibited O
osteoclast O
formation O
from O
CD16 O
- O
monocytes O
. O

Taken O
together O
, O
the O
process O
of O
ligand O
binding O
to O
alpha O
v O
beta O
3 O
may O
be O
involved O
in O
the O
osteoclastogenesis O
. O

Blockade O
of O
alpha O
v O
beta O
3 O
could O
therefore O
be O
a O
therapeutically O
beneficial O
approach O
to O
modulate O
osteoclastogenesis O
. O

Indeed O
, O
integrin O
alpha O
v O
beta O
3 O
antagonists O
effectively O
treated O
osteoporosis O
in O
mice B
, O
rats B
and O
humans B
, O
and O
protected O
bone O
destruction O
in O
rat B
adjuvant O
- O
induced O
arthritis O
in O
vivo O
[ O
31 O
- O
34 O
] O
. O

Of O
note O
, O
it O
is O
reported O
that O
patients B
with O
Iraqi O
- O
Jewish O
- O
type O
Glanzmann O
thrombasthenia O
who O
are O
deficient O
in O
integrin O
beta O
3 O
do O
not O
develop O
osteopetrosis O
because O
of O
the O
upregulation O
of O
alpha O
2 O
beta O
1 O
expression O
on O
osteoclasts O
, O
although O
the O
bone O
- O
resorptive O
ability O
of O
the O
osteoclasts O
was O
decreased O
in O
vitro O
[ O
35 O
] O
. O

The O
function O
of O
alpha O
v O
beta O
3 O
in O
vivo O
in O
osteoclast O
formation O
and O
resorptive O
function O
could O
therefore O
be O
partially O
compensated O
by O
other O
integrins O
. O

Although O
all O
the O
multinucleated O
osteoclasts O
expressed O
alpha O
v O
beta O
3 O
( O
Figure O
4d O
) O
[ O
36 O
] O
, O
a O
small O
number O
of O
M O
- O
CSF O
+ O
RANKL O
- O
stimulated O
mononuclear O
CD16 O
- O
monocytes O
expressed O
alpha O
v O
beta O
3 O
( O
Figure O
4d O
) O
. O

Multinucleated O
osteoclasts O
are O
formed O
by O
fusion O
of O
osteoclast O
precursor O
cells O
[ O
37 O
] O
. O

It O
was O
reported O
that O
alpha O
v O
beta O
3 O
is O
involved O
in O
the O
migration O
of O
osteoclast O
precursors O
[ O
30 O
] O
. O

The O
alpha O
v O
beta O
3 O
- O
positive O
cells O
could O
therefore O
be O
forced O
to O
migrate O
by O
the O
ligands O
and O
may O
fuse O
with O
closed O
alpha O
v O
beta O
3 O
- O
negative O
cells O
. O

Alternatively O
, O
only O
alpha O
v O
beta O
3 O
- O
positive O
cells O
may O
be O
fused O
with O
each O
other O
. O

It O
is O
possible O
to O
consider O
that O
signaling O
from O
CD16 O
by O
anti O
- O
CD16 O
mAb O
- O
coated O
magnetic O
beads O
, O
which O
were O
used O
for O
the O
cell O
separation O
, O
or O
by O
IgG O
contained O
in O
FBS O
might O
inhibit O
osteoclastogenesis O
from O
CD16 O
+ O
monocytes O
. O

We O
therefore O
separated O
the O
two O
subsets O
using O
anti O
- O
CD33 O
mAb O
and O
a O
fluorescent O
cell O
sorter O
, O
and O
stimulated O
the O
cells O
with O
M O
- O
CSF O
+ O
RANKL O
. O

The O
results O
showed O
that O
CD33low O
monocytes O
, O
which O
correspond O
to O
CD16 O
+ O
monocytes O
, O
still O
could O
not O
differentiate O
into O
osteoclasts O
. O

CD16 O
is O
a O
heterodimer O
consisting O
of O
Fc O
gamma O
IIIa O
and O
Fc O
gamma O
, O
and O
has O
low O
affinity O
for O
the O
Fc O
region O
of O
IgG O
. O

Aggregation O
of O
CD16 O
by O
immune O
complexes O
leads O
to O
transmission O
of O
activating O
signals O
via O
the O
immunoreceptor O
tyrosine O
- O
based O
activation O
motif O
in O
the O
gamma O
chain O
[ O
38 O
] O
. O

We O
also O
assessed O
osteoclastogenesis O
from O
the O
two O
monocyte O
subsets O
using O
IgG O
- O
depleted O
bovine B
serum O
. O

Even O
in O
the O
IgG O
- O
free O
medium O
, O
CD16 O
- O
monocytes O
but O
not O
CD16 O
+ O
monocytes O
differentiated O
into O
osteoclasts O
( O
data O
not O
shown O
) O
. O

We O
could O
therefore O
exclude O
the O
possibility O
that O
signal O
transduction O
through O
CD16 O
inhibits O
osteoclastogenesis O
from O
CD16 O
+ O
monocytes O
. O

NFATc1 O
is O
a O
key O
transcription O
factor O
in O
osteoclastogenesis O
[ O
16 O
] O
. O

In O
the O
present O
study O
, O
stimulation O
with O
M O
- O
CSF O
+ O
RANKL O
increased O
the O
NFATc1 O
mRNA O
expression O
in O
the O
CD16 O
- O
subset O
only O
, O
similar O
to O
integrin O
alpha O
v O
and O
integrin O
beta O
3 O
. O

The O
differences O
in O
NFATc1 O
induction O
might O
therefore O
also O
explain O
the O
difference O
in O
osteoclastogenesis O
between O
the O
two O
monocyte O
subsets O
. O

It O
is O
of O
interest O
that O
knockdown O
of O
integrin O
beta O
3 O
did O
not O
lower O
the O
mRNA O
level O
of O
NFATc1 O
. O

This O
result O
supports O
the O
notion O
that O
NFATc1 O
is O
located O
upstream O
of O
integrin O
- O
beta O
3 O
expression O
[ O
39 O
] O
. O

It O
is O
also O
possible O
that O
parallel O
activation O
of O
two O
signaling O
pathways O
mediated O
by O
integrin O
beta O
3 O
and O
NFATc1 O
contributes O
to O
osteoclastogenesis O
independently O
or O
cooperatively O
. O

Further O
studies O
are O
needed O
to O
determine O
the O
mechanisms O
of O
integrin O
beta O
3 O
involvement O
in O
RANKL O
/ O
RANK O
- O
mediated O
osteoclast O
differentiation O
. O

It O
has O
been O
demonstrated O
that O
MAPK O
families O
, O
ERK O
and O
p38 O
MAPK O
, O
were O
activated O
by O
RANKL O
- O
induced O
intracellular O
signalings O
in O
osteoclasts O
and O
osteoclast O
precursors O
[ O
18 O
, O
19 O
] O
. O

In O
addition O
, O
these O
kinases O
are O
involved O
in O
the O
differentiation O
of O
osteoclasts O
[ O
20 O
] O
. O

We O
showed O
that O
RANKL O
stimulation O
induced O
phosphorylation O
of O
ERK O
and O
p38 O
MAPK O
only O
in O
CD16 O
- O
monocytes O
. O

It O
is O
suggested O
that O
differential O
activation O
of O
these O
kinases O
may O
partially O
explain O
the O
distinct O
properties O
of O
the O
two O
monocyte O
subsets O
upon O
RANKL O
stimulation O
. O

Our O
results O
showed O
that O
CD16 O
+ O
monocytes O
produce O
higher O
levels O
of O
inflammatory O
cytokines O
including O
TNF O
alpha O
and O
IL O
- O
6 O
compared O
with O
CD16 O
- O
monocytes O
. O

These O
results O
are O
consistent O
with O
the O
previous O
report O
showing O
that O
CD16 O
+ O
monocytes O
produced O
larger O
amounts O
of O
TNF O
alpha O
upon O
lipopolysaccharide O
or O
lipopeptide O
stimulation O
than O
did O
CD16 O
- O
monocytes O
[ O
40 O
] O
. O

Interestingly O
, O
we O
showed O
that O
stimulation O
either O
with O
RANKL O
or O
M O
- O
CSF O
upregulated O
the O
TNF O
alpha O
and O
IL O
- O
6 O
production O
by O
CD16 O
+ O
monocytes O
. O

A O
marked O
increase O
of O
CD16 O
+ O
monocytes O
in O
peripheral O
blood O
is O
reported O
in O
inflammatory O
diseases O
, O
such O
as O
infection O
, O
malignancy O
, O
Kawasaki O
disease O
and O
RA O
[ O
41 O
- O
44 O
] O
. O

Taken O
together O
, O
CD16 O
+ O
monocytes O
may O
be O
an O
important O
source O
of O
inflammatory O
cytokines O
. O

In O
mice B
, O
peripheral O
blood O
Ly O
- O
6Chigh O
monocytes O
, O
which O
are O
thought O
to O
correspond O
to O
human B
CD16 O
- O
monocytes O
, O
increase O
in O
inflammatory O
conditions O
, O
and O
these O
cells O
are O
recruited O
into O
sites O
of O
inflammation O
[ O
45 O
] O
. O

In O
contrast O
, O
Ly O
- O
6Clow O
monocytes O
, O
which O
are O
thought O
to O
correspond O
to O
human B
CD16 O
+ O
, O
migrate O
into O
noninflamed O
tissues O
[ O
12 O
] O
. O

These O
data O
on O
mouse B
monocytes O
seem O
to O
be O
in O
contrast O
to O
the O
data O
on O
human B
monocytes O
, O
which O
show O
expansion O
of O
CD16 O
+ O
monocytes O
in O
inflammatory O
conditions O
where O
they O
produce O
larger O
amounts O
of O
inflammatory O
cytokines O
. O

At O
present O
, O
it O
is O
not O
clear O
whether O
mouse B
monocyte O
subsets O
, O
Ly O
- O
6Clow O
/ O
Ly O
- O
6Chigh O
, O
represent O
human B
monocyte O
subsets O
, O
CD16 O
+ O
/ O
CD16 O
- O
monocytes O
, O
and O
whether O
the O
biologic O
functions O
of O
mouse B
monocytes O
are O
analogous O
to O
those O
of O
human B
monocytes O
. O

In O
mice B
, O
blood O
monocytes O
newly O
released O
from O
the O
bone O
marrow O
are O
exclusively O
Ly O
- O
6Chigh O
and O
the O
level O
of O
Ly O
- O
6C O
is O
downregulated O
while O
in O
circulation O
[ O
45 O
] O
. O

It O
is O
thus O
suggested O
that O
in O
mice B
the O
two O
monocyte O
subsets O
differing O
in O
Ly O
- O
6C O
expression O
represent O
different O
stages O
in O
the O
maturation O
pathway O
. O

In O
the O
human B
, O
transition O
from O
CD16 O
- O
monocytes O
to O
CD16 O
+ O
monocytes O
is O
observed O
upon O
culture O
with O
IL O
- O
10 O
, O
M O
- O
CSF O
and O
transforming O
growth O
factor O
beta O
in O
vitro O
[ O
42 O
, O
46 O
] O
. O

Similar O
to O
mouse B
monocytes O
, O
therefore O
, O
human B
peripheral O
blood O
CD16 O
- O
monocytes O
may O
also O
maturate O
into O
CD16 O
+ O
monocytes O
. O

It O
is O
reported O
that O
a O
significant O
number O
of O
RA O
synovial O
cells O
in O
the O
intima O
express O
CD16 O
, O
suggesting O
that O
CD16 O
+ O
cells O
are O
synovial O
macrophages O
[ O
47 O
] O
. O

We O
confirmed O
that O
both O
CD16 O
+ O
and O
CD16 O
- O
macrophages O
accumulate O
in O
the O
RA O
synovium O
by O
double O
- O
color O
immunohistochemical O
staining O
for O
CD68 O
and O
CD16 O
. O

A O
number O
of O
chemokines O
are O
abundantly O
expressed O
in O
the O
RA O
synovium O
[ O
24 O
, O
48 O
] O
. O

Among O
these O
cytokines O
, O
MCP O
- O
1 O
, O
MIP O
- O
1 O
alpha O
, O
SDF O
- O
1 O
, O
RANTES O
and O
fractalkine O
can O
induce O
migration O
of O
CD16 O
- O
monocytes O
in O
vitro O
( O
[ O
11 O
, O
12 O
] O
and O
unpublished O
data O
) O
. O

On O
the O
other O
hand O
, O
migration O
of O
CD16 O
+ O
monocytes O
is O
induced O
only O
by O
fractalkine O
. O

These O
chemokines O
therefore O
seem O
to O
play O
an O
important O
role O
in O
recruitment O
of O
CD16 O
+ O
and O
CD16 O
- O
monocytes O
from O
the O
circulating O
pool O
into O
the O
RA O
synovium O
. O

The O
osteoclast O
inducers O
are O
also O
produced O
in O
the O
RA O
synovium O
. O

RANKL O
is O
expressed O
by O
synovial O
fibroblasts O
and O
activated O
T O
cells O
[ O
49 O
- O
51 O
] O
, O
while O
M O
- O
CSF O
is O
expressed O
on O
RA O
synovial O
macrophages O
and O
fibroblasts O
[ O
52 O
, O
53 O
] O
. O

TNF O
alpha O
and O
IL O
- O
6 O
, O
which O
are O
mainly O
expressed O
on O
RA O
synovial O
macrophages O
and O
fibroblasts O
, O
respectively O
, O
could O
also O
enhance O
osteoclast O
differentiation O
[ O
54 O
] O
. O

Collectively O
, O
it O
is O
probable O
that O
the O
recruited O
CD16 O
- O
monocytes O
/ O
macrophages O
differentiate O
into O
osteoclasts O
in O
the O
RA O
synovium O
, O
and O
contribute O
to O
bone O
destruction O
. O

On O
the O
other O
hand O
, O
CD16 O
+ O
monocytes O
/ O
macrophages O
might O
also O
be O
involved O
in O
RA O
pathogenesis O
by O
producing O
inflammatory O
cytokines O
including O
TNF O
alpha O
and O
IL O
- O
6 O
. O

Since O
TNF O
alpha O
and O
IL O
- O
6 O
enhance O
osteoclast O
formation O
[ O
54 O
, O
55 O
] O
, O
CD16 O
+ O
monocytes O
/ O
macrophages O
may O
also O
contribute O
to O
osteoclastogenesis O
in O
RA O
synovium O
. O

Conclusion O

We O
have O
shown O
that O
human B
peripheral O
blood O
monocytes O
consist O
of O
two O
functionally O
heterogeneous O
subsets O
with O
distinct O
response O
to O
osteoclastogenic O
stimuli O
. O

Osteoclasts O
seem O
to O
originate O
from O
CD16 O
- O
monocytes O
, O
and O
integrin O
beta O
3 O
is O
necessary O
for O
the O
osteoclastogenesis O
. O

The O
blockade O
of O
accumulation O
and O
activation O
of O
CD16 O
- O
monocytes O
could O
therefore O
be O
a O
beneficial O
approach O
as O
an O
anti O
- O
bone O
resorptive O
therapy O
, O
especially O
for O
RA O
. O

Abbreviations O

DAP O
= O
DNAX O
- O
activation O
protein O
; O
ELISA O
= O
enzyme O
- O
linked O
immunosorbent O
assay O
; O
FBS O
= O
fetal O
bovine B
serum O
; O
FcR O
gamma O
= O
Fc O
receptor O
gamma O
chain O
; O
IL O
= O
interleukin O
; O
FITC O
= O
fluorescein O
isothiocianate O
; O
mAb O
, O
monoclonal O
antibody O
; O
M O
- O
CSF O
= O
macrophage O
colony O
- O
stimulating O
factor O
; O
MEM O
= O
modified O
Eagle O
' O
s O
medium O
; O
MMP O
= O
matrix O
metalloproteinase O
; O
MNC O
= O
multinucleated O
cells O
; O
NF O
= O
nuclear O
factor O

; O
OSCAR O
= O
osteoclast O
- O
associated O
receptor O
; O
PBS O
= O
phosphate O
- O
buffered O
saline O
; O
PCR O
= O
polymerase O
chain O
reaction O
; O
RA O
= O
rheumatoid O
arthritis O
; O
RANK O
= O
receptor O
activator O
of O
NF O
- O
kappa O
B O
; O
RANKL O
= O
receptor O
activator O
of O
NF O
- O
kappa O
B O
ligand O
; O
RT O
= O
reverse O
transcriptase O
; O
siRNA O
= O
small O
interfering O
RNA O
; O
SIRP O
- O
beta O
1 O
= O
signal O
regulatory O
protein O
- O
beta O
1 O
; O
TNF O
alpha O
= O
tumor O
necrosis O
factor O
alpha O
; O
TRAF O
= O
tumor O
necrosis O
factor O

receptor O
- O
associated O
factor O
; O
TRAP O
= O
tartrate O
- O
resistant O
acid O
phosphatase O
; O
TREM O
= O
triggering O
receptor O
expressed O
on O
myeloid O
cells O
. O

Competing O
interests O

The O
authors O
declare O
that O
they O
have O
no O
competing O
interests O
. O

Authors O
' O
contributions O

YK O
participated O
in O
the O
design O
of O
the O
study O
, O
carried O
out O
the O
experiments O
and O
statistical O
analysis O
, O
and O
drafted O
the O
manuscript O
. O

KH O
and O
KT O
participated O
in O
the O
design O
of O
the O
study O
and O
its O
coordination O
. O

TN O
and O
NM O
conceived O
of O
the O
study O
, O
participated O
in O
its O
design O
and O
coordination O
, O
and O
helped O
to O
draft O
the O
manuscript O
. O

All O
authors O
read O
and O
approved O
the O
final O
manuscript O
. O

Embryonic O
sympathoblasts O
transiently O
express O
TrkB O
in O
vivo O
and O
proliferate O
in O
response O
to O
brain O
- O
derived O
neurotrophic O
factor O
in O
vitro O

Abstract O

Background O

Nerve O
growth O
factor O
and O
neurotrophin O
- O
3 O
are O
involved O
in O
the O
development O
of O
sympathetic O
neurons O
; O
however O
, O
whether O
brain O
derived O
neurotrophic O
factor O
also O
plays O
a O
role O
is O
not O
known O
. O

The O
purpose O
of O
this O
study O
was O
to O
determine O
whether O
BDNF O
and O
its O
receptor O
, O
TrkB O
, O
are O
expressed O
during O
the O
development O
of O
paravertebral O
sympathetic O
ganglia O
in O
vivo O
and O
to O
determine O
the O
effect O
of O
BDNF O
in O
vitro O
. O

Results O

As O
neural O
crest O
cells O
coalesce O
to O
form O
sympathetic O
ganglia O
, O
TrkB O
- O
positive O
cells O
are O
seen O
in O
both O
chicken B
and O
mouse B
embryos O
. O

In O
chicken B
embryos O
, O
TrkB O
- O
expressing O
cells O
first O
appear O
at O
Hamburger O
- O
Hamilton O
Stage O
( O
St O
) O
27 O
and O
they O
co O
- O
express O
HNK O
- O
1 O
, O
confirming O
that O
they O
are O
migrating O
neural O
crest O
cells O
. O

The O
TrkB O
- O
positive O
cells O
lack O
neural O
markers O
at O
this O
stage O
; O
however O
, O
they O
migrate O
with O
other O
neurally O
differentiating O
cells O
that O
are O
TrkA O
and O
TrkC O
- O
positive O
. O

By O
St O
. O

29 O
/ O
30 O
, O
TrkB O
- O
positive O
cells O
begin O
to O
express O
the O
neural O
specific O
markers O
Hu O
C O
/ O
D O
and O
Islet O
- O
1 O
; O
eventually O
, O
all O
TrkB O
positive O
cells O
commence O
neural O
differentiation O
. O

By O
St O
. O

34 O
, O
TrkB O
and O
TrkC O
staining O
are O
lost O
. O

BDNF O
transcript O
expression O
parallels O
that O
of O
TrkB O
. O

In O
the O
mouse B
, O
TrkB O
- O
positive O
cells O
surround O
newly O
formed O
sympathetic O
ganglia O
and O
a O
small O
number O
of O
TrkB O
positive O
cells O
that O
co O
- O
express O
tyrosine O
hydroxylase O
are O
seen O
within O
ganglia O
between O
E13 O
. O
5 O
- O
15 O
. O

In O
cell O
culture O
, O
many O
cells O
from O
St O
. O

29 O
- O
30 O
chicken B
lumbar O
sympathetic O
ganglia O
express O
neural O
markers O
and O
are O
dividing O
, O
indicating O
that O
they O
are O
sympathoblasts O
. O

Sympathoblasts O
and O
neurons O
require O
both O
nerve O
growth O
factor O
and O
neurotrophin O
- O
3 O
for O
survival O
. O

BDNF O
increases O
the O
number O
of O
cells O
expressing O
neural O
markers O
in O
culture O
by O
increasing O
number O
of O
cells O
that O
incorporate O
bromodeoxyuridine O
. O

In O
contrast O
, O
most O
TrkB O
- O
positive O
sympathetic O
cells O
in O
vivo O
are O
not O
actively O
proliferating O
between O
E6 O
- O
E8 O
. O

Conclusion O

Developing O
paravertebral O
sympathetic O
ganglia O
in O
avian O
and O
murine B
embryos O
contain O
a O
subpopulation O
of O
sympathoblasts O
that O
transiently O
express O
TrkB O
and O
ultimately O
commence O
neuronal O
differentiation O
. O

These O
TrkB O
expressing O
sympathoblasts O
are O
not O
actively O
dividing O
in O
vivo O
; O
yet O
, O
when O
placed O
in O
vitro O
, O
will O
divide O
in O
response O
to O
BDNF O
. O

This O
suggests O
that O
the O
availability O
of O
BDNF O
in O
vivo O
fails O
to O
reach O
a O
threshold O
necessary O
to O
induce O
proliferation O
. O

We O
suggest O
that O
excess O
TrkB O
stimulation O
of O
sympathoblasts O
in O
vivo O
may O
lead O
to O
the O
genesis O
of O
neuroblastoma O
. O

Background O

Neural O
crest O
cells O
destined O
to O
become O
paravertebral O
sympathetic O
neurons O
proliferate O
and O
differentiate O
during O
migration O
and O
gangliogenesis O
. O

In O
chicken B
embryos O
, O
migrating O
neural O
crest O
cells O
express O
catecholamines O
at O
Hamburger O
/ O
Hamilton O
Stage O
( O
St O
. O
) O
19 O
, O
and O
these O
cells O
form O
the O
primary O
sympathetic O
chain O
dorsolateral O
to O
the O
aorta O
at O
St O
. O

22 O
( O
E3 O
. O
5 O
) O
[ O
1 O
] O
. O

Between O
St O
. O

23 O
( O
E4 O
) O
and O
St O
. O

28 O
( O
E6 O
) O
, O
these O
cells O
disperse O
and O
undergo O
a O
secondary O
migration O
to O
form O
the O
paravertebral O
sympathetic O
chain O
that O
resides O
ventral O
to O
the O
spinal O
cord O
and O
dorsal O
root O
ganglion O
[ O
1 O
] O
. O

After O
ganglia O
coalesce O
, O
sympathoblasts O
express O
markers O
of O
neuronal O
differentiation O
, O
such O
as O
Q211 O
and O
tyrosine O
hydroxylase O
( O
TH O
) O
, O
at O
a O
time O
when O
they O
also O
incorporate O
[ O
3H O
] O
- O
thymidine O
[ O
2 O
] O
. O

Time O
lapse O
photography O
has O
shown O
that O
cultured O
E15 O
. O
5 O
- O
E16 O
. O
5 O
sympathetic O
neurons O
from O
rat B
embryos O
extend O
axons O
while O
they O
divide O
[ O
3 O
- O
5 O
] O
. O

Although O
proliferation O
appears O
to O
be O
an O
important O
process O
to O
expand O
the O
sympathetic O
neuron O
population O
during O
differentiation O
, O
the O
mechanisms O
that O
guide O
sympathoblast O
proliferation O
have O
not O
been O
identified O
. O

The O
development O
of O
sympathetic O
neurons O
is O
guided O
by O
neurotrophins O
. O

Neurotrophin O
- O
3 O
( O
NT O
- O
3 O
) O
binds O
to O
its O
receptor O
, O
TrkC O
, O
to O
promote O
the O
survival O
of O
cultured O
sympathoblasts O
from O
early O
lumbar O
paravertebral O
ganglia O
[ O
6 O
] O
. O

Nerve O
growth O
factor O
( O
NGF O
) O
signals O
through O
its O
receptor O
, O
TrkA O
, O
to O
promote O
the O
survival O
of O
sympathetic O
neurons O
upon O
target O
innervation O
[ O
7 O
] O
. O

There O
are O
severe O
sympathetic O
defects O
in O
the O
superior O
cervical O
ganglion O
of O
individual O
NT O
- O
3 O
and O
NGF O
knockout O
mice B
[ O
8 O
- O
10 O
] O
. O

Furthermore O
, O
there O
is O
no O
additional O
cell O
death O
in O
the O
superior O
cervical O
ganglion O
of O
NT O
- O
3 O
and O
NGF O
double O
knockout O
mouse B
embryos O
, O
suggesting O
that O
all O
of O
the O
neurons O
are O
dependent O
on O
both O
neurotrophins O
for O
survival O
[ O
11 O
] O
. O

There O
is O
also O
an O
increase O
in O
sympathetic O
neuron O
cell O
death O
in O
TrkA O
knockout O
mice B
[ O
12 O
] O
. O

However O
, O
in O
TrkB O
and O
BDNF O
knockout O
mice B
, O
there O
is O
no O
apparent O
phenotype O
in O
the O
superior O
cervical O
ganglion O
, O
and O
there O
is O
little O
evidence O
that O
TrkB O
or O
BDNF O
is O
expressed O
in O
sympathetic O
ganglia O
. O

Thus O
, O
it O
is O
generally O
thought O
that O
TrkB O
and O
BDNF O
have O
little O
or O
no O
roles O
in O
guiding O
the O
development O
of O
sympathetic O
neurons O
. O

In O
addition O
to O
their O
developmental O
functions O
, O
neurotrophin O
receptors O
regulate O
cell O
behavior O
in O
neuroblastoma O
, O
a O
tumor O
found O
in O
sympathetic O
ganglia O
and O
adrenal O
medulla O
. O

Tumors O
that O
express O
TrkA O
often O
spontaneously O
regress O
, O
while O
those O
that O
express O
TrkB O
and O
its O
ligand O
, O
brain O
- O
derived O
neurotrophic O
factor O
( O
BDNF O
) O
, O
grow O
aggressively O
, O
are O
invasive O
, O
and O
fail O
to O
respond O
to O
chemotherapeutic O
agents O
[ O
13 O
] O
. O

The O
presence O
of O
TrkA O
in O
neuroblastoma O
tumors O
is O
consistent O
with O
its O
expression O
in O
developing O
sympathetic O
neurons O
, O
and O
suggests O
that O
regressive O
neuroblastoma O
tumors O
arise O
from O
early O
sympathetic O
neurons O
that O
express O
TrkA O
. O

The O
function O
of O
TrkB O
in O
early O
sympathetic O
development O
is O
unknown O
, O
which O
makes O
understanding O
the O
etiology O
of O
aggressive O
neuroblastoma O
tumors O
difficult O
. O

Based O
on O
its O
function O
in O
neuroblastoma O
tumors O
, O
we O
hypothesize O
that O
BDNF O
and O
TrkB O
expression O
in O
differentiating O
sympathoblasts O
is O
responsible O
for O
expanding O
the O
neuronal O
population O
through O
proliferation O
. O

We O
sought O
to O
determine O
whether O
BDNF O
and O
TrkB O
are O
involved O
in O
sympathetic O
development O
. O

We O
report O
that O
during O
early O
embryonic O
development O
, O
TrkB O
is O
expressed O
in O
a O
subset O
of O
differentiating O
sympathoblasts O
in O
both O
avian O
and O
murine B
embryos O
. O

We O
also O
find O
that O
BDNF O
promotes O
the O
proliferation O
of O
TrkB O
- O
positive O
sympathoblasts O
in O
cell O
culture O
. O

However O
, O
the O
majority O
of O
TrkB O
positive O
cells O
in O
vivo O
fail O
to O
take O
up O
bromodeoxyuridine O
( O
BrdU O
) O
over O
a O
24 O
hr O
period O
, O
suggesting O
that O
endogenous O
BDNF O
concentrations O
do O
not O
reach O
a O
threshold O
necessary O
to O
stimulate O
proliferation O
of O
sympathoblasts O
. O

Shortly O
after O
all O
of O
the O
TrkB O
positive O
cells O
commence O
neuronal O
differentiation O
, O
TrkB O
immunoreactivity O
is O
lost O
. O

These O
results O
suggest O
that O
prolonged O
expression O
and O
/ O
or O
activation O
of O
TrkB O
signaling O
at O
these O
early O
stages O
may O
be O
an O
early O
event O
triggering O
the O
formation O
of O
neuroblastoma O
. O

Results O

TrkB O
is O
expressed O
during O
migration O
of O
neural O
crest O
cells O
to O
sympathetic O
ganglia O

We O
first O
determined O
whether O
TrkB O
is O
expressed O
in O
neural O
crest O
- O
derived O
cells O
in O
the O
region O
ventral O
to O
the O
spinal O
cord O
and O
dorsal O
root O
ganglia O
where O
sympathetic O
ganglia O
coalesce O
between O
Hamburger O
/ O
Hamilton O
Stages O
( O
St O
. O
) O
25 O
- O
28 O
/ O
29 O
. O

To O
identify O
cells O
that O
have O
commenced O
neuronal O
differentiation O
, O
transverse O
sections O
of O
the O
lumbar O
spinal O
column O
region O
were O
stained O
with O
antibodies O
against O
Hu O
C O
/ O
D O
[ O
14 O
] O
, O
a O
neuronal O
- O
specific O
RNA O
- O
binding O
protein O
, O
or O
Islet O
- O
1 O
, O
a O
transcription O
factor O
found O
in O
sympathetic O
neurons O
[ O
15 O
] O
. O

We O
found O
that O
Hu O
C O
/ O
D O
and O
Islet O
- O
1 O
are O
expressed O
in O
the O
same O
cells O
both O
in O
vivo O
and O
in O
vitro O
throughout O
sympathetic O
development O
. O

In O
experiments O
done O
between O
St O
. O

25 O
and O
28 O
, O
Islet O
- O
1 O
staining O
appeared O
weaker O
than O
Hu O
C O
/ O
D O
staining O
, O
and O
thus O
we O
used O
Hu O
C O
/ O
D O
to O
identify O
differentiating O
neurons O
at O
these O
stages O
. O

At O
later O
stages O
, O
Islet O
- O
1 O
was O
used O
to O
facilitate O
the O
identification O
of O
neurons O
because O
of O
the O
nuclear O
location O
of O
its O
immunoreactivity O
. O

Cells O
expressing O
Hu O
C O
/ O
D O
are O
first O
detected O
at O
St O
. O

25 O
ventral O
to O
the O
spinal O
cord O
and O
dorsal O
root O
ganglion O
and O
lateral O
to O
the O
dorsal O
aorta O
( O
Figure O
1A O
, O
1B O
) O
. O

By O
St O
. O

26 O
, O
the O
number O
of O
cells O
that O
express O
Hu O
C O
/ O
D O
in O
this O
region O
increases O
dramatically O
( O
Figure O
1C O
) O
. O

TrkB O
- O
expressing O
cells O
first O
appear O
at O
St O
. O

27 O
in O
the O
same O
region O
and O
are O
adjacent O
to O
Hu O
C O
/ O
D O
- O
positive O
cells O
( O
Figure O
1D O
, O
2A O
, O
2H O
) O
. O

TrkB O
- O
positive O
cells O
co O
- O
localize O
with O
a O
neural O
crest O
marker O
, O
HNK O
- O
1 O
( O
Figures O
2B O
, O
2C O
, O
2D O
) O
. O

The O
Hu O
C O
/ O
D O
- O
positive O
cells O
in O
this O
region O
are O
likely O
to O
be O
sympathetic O
neurons O
, O
since O
they O
appear O
in O
the O
region O
where O
sympathetic O
ganglia O
form O
and O
express O
tyrosine O
hydroxylase O
, O
a O
rate O
- O
limiting O
enzyme O
in O
the O
synthesis O
of O
catecholamines O
( O
Figures O
2E O
, O
2F O
, O
2G O
) O
. O

At O
St O
. O

28 O
/ O
29 O
, O
the O
cells O
begin O
to O
coalesce O
ventral O
to O
the O
dorsal O
root O
ganglion O
and O
the O
Hu O
C O
/ O
D O
- O
positive O
cells O
and O
TrkB O
- O
positive O
cells O
remain O
as O
two O
separate O
cell O
populations O
( O
Figure O
1E O
) O
; O
however O
, O
shortly O
afterwards O
, O
all O
of O
the O
TrkB O
- O
positive O
cells O
begin O
to O
express O
Islet O
- O
1 O
( O
Figure O
3B O
) O
and O
Hu O
C O
/ O
D O
( O
data O
not O
shown O
) O
. O

Developmental O
regulation O
of O
TrkA O
, O
TrkB O
, O
TrkC O
, O
and O
BDNF O
expression O

In O
contrast O
to O
TrkB O
, O
the O
other O
neurotrophin O
receptors O
, O
TrkA O
and O
TrkC O
, O
are O
co O
- O
expressed O
in O
both O
Hu O
C O
/ O
D O
- O
positive O
and O
Hu O
C O
/ O
D O
- O
negative O
cells O
at O
St O
. O

27 O
( O
Figures O
2I O
, O
2J O
) O
. O

We O
find O
that O
approximately O
30 O
% O
of O
the O
Islet O
- O
1 O
- O
positive O
cells O
express O
TrkA O
( O
Figure O
3A O
) O
, O
while O
50 O
% O
express O
TrkB O
( O
Figure O
3B O
) O
and O
100 O
% O
express O
TrkC O
( O
Figure O
3C O
) O
at O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
. O

Thus O
, O
all O
developing O
neurons O
express O
TrkC O
in O
combination O
with O
either O
TrkA O
or O
TrkB O
. O

By O
St O
. O

31 O
( O
E7 O
) O
, O
the O
number O
of O
TrkA O
- O
positive O
, O
Islet O
- O
1 O
- O
positive O
cells O
increases O
to O
100 O
% O
( O
Figure O
3D O
) O
and O
immunoreactivities O
for O
both O
TrkB O
and O
TrkC O
appear O
dispersed O
( O
Figure O
3E O
, O
3F O
) O
. O

By O
St O
. O

34 O
( O
E8 O
) O
, O
TrkA O
expression O
is O
well O
- O
sustained O
( O
Figure O
3G O
) O
and O
TrkB O
and O
TrkC O
immunoreactivities O
are O
lost O
( O
Figure O
3H O
, O
3I O
) O
. O

We O
also O
examined O
the O
early O
development O
of O
murine B
sympathetic O
ganglia O
( O
Figure O
4 O
) O
. O

At O
E13 O
, O
the O
newly O
formed O
lumbar O
sympathetic O
ganglia O
can O
be O
observed O
ventral O
to O
the O
spinal O
cord O
and O
notochord O
by O
their O
staining O
for O
Hu O
C O
/ O
D O
and O
TH O
( O
Figure O
4A O
) O
. O

TrkB O
- O
positive O
cells O
can O
be O
seen O
surrounding O
developing O
ganglia O
, O
as O
well O
as O
in O
occasional O
cells O
within O
the O
ganglia O
( O
Figure O
4C O
, O
D O
) O
. O

These O
TrkB O
- O
positive O
cells O
within O
the O
ganglia O
co O
- O
express O
TH O
and O
are O
seen O
at O
a O
frequency O
of O
1 O
- O
2 O
cells O
per O
section O
starting O
at O
E13 O
( O
Figure O
4A O
- O
D O
) O
and O
are O
still O
present O
at O
E15 O
. O
5 O
( O
data O
not O
shown O
) O
. O

In O
neuroblastoma O
cells O
, O
BDNF O
is O
co O
- O
expressed O
with O
TrkB O
, O
suggesting O
that O
autocrine O
stimulation O
is O
a O
means O
by O
which O
proliferation O
is O
sustained O
in O
the O
transformed O
cells O
. O

To O
test O
whether O
BDNF O
, O
the O
ligand O
for O
TrkB O
, O
was O
present O
in O
embryonic O
chick B
sympathetic O
ganglia O
, O
we O
used O
quantitative O
real O
- O
time O
PCR O
with O
TaqMan O
probes O
to O
determine O
the O
relative O
abundance O
of O
BDNF O
transcripts O
in O
total O
RNA O
extracted O
from O
lumbar O
sympathetic O
ganglia O
at O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
, O
St O
. O

31 O
( O
E7 O
) O
, O
St O
. O

34 O
( O
E8 O
) O
, O
and O
E9 O
. O

BDNF O
expression O
within O
the O
ganglia O
parallels O
that O
of O
TrkB O
: O
BDNF O
mRNA O
expression O
levels O
are O
highest O
at O
St O
29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
, O
and O
these O
levels O
decrease O
2 O
- O
fold O
at O
St O
. O

31 O
( O
E7 O
) O
and O
St O
. O

34 O
( O
E8 O
; O
Figure O
5 O
) O
. O

By O
E9 O
, O
BDNF O
levels O
are O
7 O
times O
lower O
than O
at O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
; O
Figure O
5 O
) O
. O

NT3 O
and O
NGF O
promote O
survival O
of O
differentiating O
sympathetic O
neurons O
in O
culture O

To O
determine O
the O
effect O
of O
neurotrophins O
, O
we O
cultured O
cells O
dispersed O
from O
lumbar O
sympathetic O
ganglia O
at O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
because O
, O
at O
this O
stage O
, O
ganglion O
formation O
is O
complete O
, O
the O
number O
of O
TrkA O
- O
, O
TrkB O
- O
, O
and O
TrkC O
- O
positive O
cells O
have O
peaked O
, O
and O
all O
Trk O
- O
expressing O
cells O
have O
initiated O
neural O
differentiation O
. O

First O
, O
we O
identified O
markers O
expressed O
by O
acutely O
isolated O
cells O
. O

As O
shown O
in O
Table O
1 O
, O
80 O
- O
91 O
% O
of O
the O
cells O
are O
p75 O
neurotrophin O
receptor O
( O
NTR O
) O
- O
positive O
, O
indicating O
that O
most O
of O
the O
cells O
are O
neural O
crest O
- O
derived O
and O
little O
mesenchymal O
contamination O
is O
introduced O
by O
the O
isolation O
procedure O
. O

In O
addition O
, O
28 O
- O
33 O
% O
of O
the O
cultured O
cells O
express O
the O
neural O
marker O
Hu O
C O
/ O
D O
. O

Approximately O
half O
of O
these O
Hu O
C O
/ O
D O
- O
positive O
cells O
express O
TrkB O
. O

Conversely O
, O
all O
of O
the O
TrkB O
positive O
cells O
express O
Hu O
C O
/ O
D O
. O

These O
TrkB O
- O
positive O
cells O
comprise O
approximately O
14 O
- O
17 O
% O
of O
the O
total O
cell O
population O
. O

We O
then O
determined O
how O
many O
of O
the O
acutely O
isolated O
cells O
were O
proliferating O
by O
incubating O
them O
for O
12 O
hrs O
in O
BrdU O
- O
containing O
medium O
. O

For O
these O
experiments O
, O
we O
identified O
differentiating O
neurons O
with O
the O
transcription O
factor O
Islet O
- O
1 O
because O
this O
marker O
labels O
nuclei O
, O
thus O
it O
co O
localizes O
with O
any O
BrdU O
that O
has O
been O
incorporated O
into O
the O
DNA O
, O
allowing O
us O
to O
determine O
whether O
the O
cell O
had O
undergone O
S O
- O
phase O
of O
the O
cell O
cycle O
. O

After O
12 O
hrs O
in O
BrdU O
, O
59 O
% O
of O
Islet O
- O
1 O
- O
positive O
nuclei O
stain O
for O
BrdU O
immunoreactivity O
. O

Thus O
, O
cultures O
of O
St O
. O

29 O
/ O
30 O
sympathetic O
ganglia O
contain O
many O
cells O
that O
proliferate O
while O
exhibiting O
markers O
of O
neuronal O
differentiation O
, O
confirming O
previous O
observations O
[ O
2 O
] O
. O

We O
call O
these O
dividing O
neuronal O
precursors O
sympathoblasts O
. O

The O
remaining O
non O
- O
BrdU O
incorporating O
, O
Islet O
- O
1 O
positive O
cells O
are O
likely O
to O
be O
post O
mitotic O
neurons O
. O

Finally O
, O
we O
determined O
the O
trophic O
requirements O
of O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
sympathetic O
neurons O
and O
sympathoblasts O
. O

We O
monitored O
cultures O
over O
a O
three O
day O
period O
after O
plating O
and O
counted O
the O
number O
of O
phase O
bright O
cells O
with O
neurites O
, O
a O
morphological O
feature O
of O
both O
neurons O
and O
sympathoblasts O
. O

In O
the O
absence O
of O
trophic O
factors O
, O
more O
than O
2 O
/ O
3 O
of O
the O
cells O
die O
by O
24 O
hours O
in O
culture O
and O
BDNF O
, O
NT O
- O
3 O
, O
or O
NGF O
alone O
is O
not O
sufficient O
to O
promote O
survival O
( O
Figure O
6 O
) O
. O

However O
, O
NGF O
together O
with O
NT O
- O
3 O
supports O
the O
survival O
of O
a O
significantly O
larger O
number O
of O
cells O
( O
Figure O
6 O
) O
. O

For O
the O
subsequent O
experiments O
, O
all O
neurons O
were O
cultured O
with O
25 O
ng O
/ O
ml O
NT O
- O
3 O
and O
1 O
mu O
g O
/ O
ml O
7S O
NGF O
to O
optimize O
survival O
. O

BDNF O
promotes O
proliferation O
of O
TrkB O
- O
positive O
sympathetic O
neurons O
in O
culture O

To O
determine O
the O
effects O
of O
BDNF O
, O
cultures O
of O
cells O
from O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
sympathetic O
ganglia O
were O
supplemented O
with O
200 O
ng O
/ O
ml O
BDNF O
and O
the O
number O
of O
neurons O
and O
sympathoblasts O
were O
counted O
at O
24 O
, O
48 O
, O
and O
72 O
hours O
using O
phase O
microscopy O
( O
Figure O
7A O
) O
. O

A O
1 O
. O
6 O
- O
fold O
increase O
in O
the O
number O
of O
neurons O
due O
to O
BDNF O
is O
observed O
by O
24 O
hours O
and O
this O
number O
does O
not O
increase O
further O
at O
48 O
or O
72 O
hours O
. O

This O
effect O
of O
BDNF O
is O
concentration O
- O
dependent O
with O
an O
EC50 O
of O
75 O
ng O
/ O
ml O
( O
Figure O
7B O
) O
. O

To O
test O
whether O
the O
increase O
in O
the O
number O
of O
neurons O
and O
sympathoblasts O
caused O
by O
BDNF O
is O
due O
to O
the O
differentiation O
of O
pluripotent O
neural O
crest O
cells O
, O
we O
quantified O
the O
effects O
of O
BDNF O
on O
the O
number O
of O
neurally O
differentiating O
cells O
( O
Hu O
C O
/ O
D O
- O
positive O
) O
versus O
the O
number O
of O
non O
- O
neuronal O
cells O
( O
Hu O
C O
/ O
D O
- O
negative O
) O
after O
identifying O
all O
neural O
crest O
- O
derived O
cells O
by O
staining O
for O
p75NTR O
in O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
cultures O
. O

If O
BDNF O
increases O
the O
number O
of O
neurons O
and O
sympathoblasts O
by O
inducing O
a O
non O
- O
neuronal O
cell O
to O
express O
Hu O
C O
/ O
D O
, O
then O
we O
expected O
that O
the O
total O
cell O
number O
would O
remain O
the O
same O
and O
that O
there O
would O
be O
a O
decrease O
in O
the O
number O
of O
non O
- O
neuronal O
cells O
as O
well O
as O
a O
corresponding O
increase O
in O
the O
number O
of O
neurons O
. O

After O
24 O
hours O
, O
BDNF O
significantly O
increases O
the O
number O
of O
p75NTR O
- O
positive O
cells O
as O
well O
as O
the O
number O
of O
Hu O
C O
/ O
D O
- O
positive O
cells O
( O
Figure O
8A O
) O
. O

However O
, O
there O
was O
no O
statistically O
significant O
change O
in O
the O
number O
of O
non O
- O
neuronal O
cells O
. O

Thus O
, O
it O
is O
unlikely O
that O
BDNF O
increases O
the O
number O
of O
neurons O
and O
sympathoblasts O
by O
inducing O
differentiation O
of O
non O
- O
neuronal O
cells O
. O

To O
determine O
whether O
the O
increase O
in O
the O
total O
number O
of O
neurons O
and O
sympathoblasts O
is O
caused O
by O
BDNF O
- O
induced O
proliferation O
, O
control O
and O
BDNF O
- O
treated O
sympathetic O
cultures O
were O
exposed O
to O
BrdU O
for O
12 O
hours O
after O
plating O
, O
and O
the O
number O
of O
cells O
that O
incorporated O
BrdU O
into O
their O
DNA O
was O
determined O
after O
24 O
hours O
in O
culture O
. O

Even O
in O
the O
control O
condition O
, O
a O
number O
of O
cells O
in O
the O
culture O
are O
dividing O
, O
giving O
a O
high O
baseline O
of O
BrdU O
incorporation O
( O
Figure O
8B O
) O
. O

When O
BDNF O
is O
added O
, O
the O
total O
number O
of O
BrdU O
positive O
cells O
increases O
approximately O
1 O
. O
6 O
- O
fold O
( O
Figure O
8B O
) O
. O

This O
BDNF O
- O
induced O
increase O
in O
the O
total O
number O
of O
BrdU O
positive O
cells O
occurs O
in O
sympathoblasts O
because O
the O
number O
of O
Islet O
- O
1 O
- O
positive O
nuclei O
from O
control O
cultures O
that O
label O
with O
BrdU O
is O
268 O
+ O
/ O
- O
59 O
and O
BDNF O
treatment O
raises O
this O
number O
to O
424 O
+ O
/ O
- O
80 O
, O
which O
corresponds O
to O
an O
increase O
of O
1 O
. O
6 O
- O
fold O
. O

This O
accounts O
for O
the O
1 O
. O
6 O
- O
fold O
increase O
in O
total O
neuron O
number O
and O
total O
BrdU O
- O
positive O
cells O
described O
above O
. O

We O
then O
confirmed O
that O
BDNF O
acts O
on O
TrkB O
- O
positive O
cells O
: O
BDNF O
increases O
the O
number O
of O
TrkB O
- O
expressing O
cells O
that O
incorporate O
BrdU O
2 O
. O
6 O
- O
4 O
- O
fold O
over O
control O
( O
Table O
2 O
) O
and O
it O
also O
increases O
the O
overall O
number O
of O
TrkB O
- O
positive O
cells O
2 O
- O
2 O
. O
5 O
- O
fold O
over O
control O
( O
Table O
2 O
) O
. O

BDNF O
does O
not O
increase O
the O
number O
of O
BrdU O
- O
positive O
, O
TrkB O
- O
negative O
cells O
or O
the O
overall O
number O
of O
TrkB O
- O
negative O
cells O
( O
Table O
2 O
) O
. O

In O
further O
support O
that O
BDNF O
acts O
directly O
on O
TrkB O
- O
expressing O
cells O
, O
an O
antibody O
directed O
against O
the O
extracellular O
domain O
of O
TrkB O
completely O
prevents O
the O
effect O
of O
BDNF O
in O
promoting O
proliferation O
of O
TrkB O
- O
positive O
, O
but O
not O
TrkB O
- O
negative O
cells O
( O
compare O
Figure O
9A O
to O
9B O
) O
. O

Thus O
, O
the O
effect O
of O
BDNF O
is O
restricted O
to O
the O
population O
that O
expresses O
TrkB O
, O
which O
are O
developing O
sympathoblasts O
. O

To O
determine O
whether O
TrkB O
- O
positive O
cells O
are O
actively O
proliferating O
in O
vivo O
, O
embryos O
were O
injected O
with O
BrdU O
at O
St O
. O

27 O
and O
harvested O
at O
St O
. O

29 O
, O
approximately O
24 O
hrs O
later O
. O

The O
majority O
( O
85 O
- O
90 O
% O
) O
of O
TrkB O
- O
positive O
cells O
do O
not O
incorporate O
BrdU O
into O
their O
nuclei O
under O
basal O
conditions O
in O
vivo O
( O
Figure O
10 O
) O
, O
although O
a O
few O
TrkB O
positive O
cells O
with O
labeled O
nuclei O
could O
be O
observed O
( O
arrows O
) O
. O

This O
contrasts O
with O
our O
observation O
that O
71 O
- O
76 O
% O
of O
TrkB O
- O
positive O
cells O
incorporate O
BrdU O
in O
culture O
after O
treatment O
with O
BDNF O
( O
Table O
2 O
) O
, O
suggesting O
that O
endogenous O
BDNF O
does O
not O
achieve O
a O
threshold O
sufficient O
to O
support O
a O
high O
level O
of O
sympathoblast O
proliferation O
in O
vivo O
. O

Discussion O

We O
report O
that O
the O
neurotrophin O
receptor O
TrkB O
is O
expressed O
in O
a O
subset O
of O
embryonic O
sympathoblasts O
during O
the O
early O
development O
of O
lumbar O
paravertebral O
sympathetic O
ganglia O
in O
chicken B
and O
mouse B
embryos O
. O

In O
the O
chicken B
, O
TrkB O
expression O
is O
transient O
, O
and O
completely O
lost O
by O
St O
34 O
( O
E8 O
) O
. O

Since O
BDNF O
induces O
the O
proliferation O
of O
sympathoblasts O
in O
cell O
culture O
, O
yet O
in O
vivo O
there O
is O
little O
proliferation O
observed O
in O
TrkB O
- O
positive O
cells O
in O
nascent O
ganglia O
, O
we O
propose O
that O
if O
TrkB O
activation O
becomes O
unregulated O
by O
excess O
BDNF O
or O
constitutive O
phosphorylation O
of O
TrkB O
[ O
16 O
] O
, O
this O
transient O
population O
of O
TrkB O
- O
positive O
sympathoblasts O
may O
trigger O
the O
genesis O
of O
neuroblastoma O
, O
a O
childhood B
tumor O
found O
in O
the O
paravertebral O
chain O
and O
adrenal O
medulla O
. O

The O
two O
populations O
of O
sympathoblasts O
that O
we O
observe O
support O
previous O
findings O
of O
heterogeneity O
among O
developing O
sympathetic O
neurons O
and O
neural O
crest O
cells O
. O

Early O
sympathetic O
ganglia O
contain O
at O
least O
two O
subpopulations O
: O
early O
differentiating O
neurons O
that O
lack O
TrkB O
expression O
and O
express O
TrkA O
and O
TrkC O
, O
and O
late O
differentiating O
sympathoblasts O
that O
express O
TrkB O
. O

Explant O
cultures O
of O
sympathetic O
ganglia O
from O
E16 O
chick B
embryos O
give O
rise O
to O
two O
neuronal O
populations O
: O
one O
that O
remains O
close O
to O
the O
explant O
, O
and O
one O
that O
migrates O
away O
from O
the O
explant O
[ O
1 O
] O
. O

In O
addition O
, O
early O
neuronal O
subpopulations O
have O
been O
observed O
in O
cultures O
of O
neural O
crest O
cells O
from O
St O
. O

13 O
/ O
14 O
quail B
embryos O
as O
evidenced O
by O
the O
expression O
of O
neuronal O
cell O
type O
- O
specific O
gangliosides O
[ O
17 O
] O
. O

Perhaps O
these O
different O
subpopulations O
will O
ultimately O
give O
rise O
to O
the O
two O
neurochemically O
distinct O
populations O
found O
in O
lumbar O
sympathetic O
ganglia O
: O
the O
noradrenergic O
, O
NPY O
- O
containing O
neurons O
that O
innervate O
internal O
organs O
and O
enteric O
ganglia O
and O
the O
cholinergic O
, O
VIP O
- O
containing O
neurons O
that O
innervate O
vasculature O
in O
the O
hind O
limbs O
. O

The O
effects O
of O
BDNF O
and O
TrkB O
deletion O
and O
over O
expression O
have O
been O
studied O
on O
superior O
cervical O
ganglion O
and O
preganglionic O
neurons O
in O
thoracic O
segments O
of O
the O
spinal O
column O
, O
but O
not O
on O
paravertebral O
sympathetic O
neurons O
. O

In O
the O
superior O
cervical O
ganglion O
, O
an O
increase O
in O
the O
number O
of O
neurons O
of O
BDNF O
null O
mice B
is O
likely O
due O
to O
apoptosis O
induced O
by O
BDNF O
via O
p75NTR O
[ O
18 O
] O
. O

In O
contrast O
, O
the O
responses O
of O
paravertebral O
sympathetic O
neurons O
to O
BDNF O
are O
complex O
and O
subtype O
dependent O
. O

Over O
expression O
of O
BDNF O
leads O
to O
an O
increase O
in O
the O
number O
of O
noradrenergic O
fibers O
innervating O
the O
erector O
pilli O
muscles O
of O
hair O
follicles O
, O
while O
noradrenergic O
fibers O
innervating O
blood O
vessels O
were O
unaffected O
[ O
19 O
] O
. O

If O
our O
results O
indicating O
that O
BDNF O
promotes O
proliferation O
of O
TrkB O
- O
positive O
sympathoblasts O
in O
the O
chicken B
embryo O
can O
be O
extrapolated O
to O
the O
subset O
of O
TrkB O
- O
positive O
sympathoblasts O
in O
murine B
ganglia O
, O
then O
these O
TrkB O
- O
positive O
cells O
may O
be O
neurons O
destined O
to O
innervate O
the O
erector O
pilli O
. O

In O
other O
studies O
, O
TrkB O
null O
mice B
showed O
no O
changes O
in O
morphology O
or O
cell O
number O
in O
superior O
cervical O
ganglia O
[ O
12 O
] O
or O
in O
the O
intermediolateral O
column O
[ O
20 O
] O
; O
but O
this O
may O
not O
be O
predictive O
of O
a O
phenotype O
in O
the O
lumbar O
paravertebral O
chain O
. O

It O
is O
thus O
possible O
that O
BDNF O
/ O
TrkB O
signaling O
could O
play O
a O
specific O
role O
in O
other O
regions O
of O
the O
paravertebral O
sympathetic O
chain O
, O
such O
as O
the O
lumbar O
region O
. O

However O
, O
if O
TrkB O
- O
positive O
cells O
are O
not O
normally O
actively O
proliferating O
in O
vivo O
, O
then O
it O
would O
not O
be O
surprising O
that O
the O
development O
of O
the O
paravertebral O
sympathetic O
chain O
is O
not O
disrupted O
in O
TrkB O
or O
BDNF O
null O
mice B
. O

It O
may O
be O
more O
informative O
to O
examine O
mice B
that O
over O
express O
BDNF O
on O
a O
promoter O
that O
targets O
expression O
to O
embryonic O
lumbar O
ganglia O
. O

Unfortunately O
, O
such O
mice B
do O
not O
exist O
. O

Our O
findings O
that O
the O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
sympathoblasts O
are O
dependent O
on O
both O
NT O
- O
3 O
and O
NGF O
for O
survival O
in O
culture O
are O
consistent O
with O
previous O
work O
on O
mouse B
sympathoblasts O
from O
the O
superior O
cervical O
ganglion O
[ O
11 O
] O
. O

In O
these O
studies O
, O
NT O
- O
3 O
and O
NGF O
deletion O
separately O
led O
to O
a O
decrease O
in O
the O
number O
of O
sympathetic O
neurons O
at O
E17 O
. O
5 O
compared O
to O
control O
. O

Deletion O
of O
both O
NT O
- O
3 O
and O
NGF O
together O
did O
not O
enhance O
cell O
death O
. O

In O
contrast O
, O
cultured O
rat B
superior O
cervical O
ganglion O
sympathetic O
neurons O
respond O
to O
NT O
- O
3 O
at O
E14 O
. O
5 O
and O
then O
to O
NGF O
at O
E19 O
. O
5 O
, O
although O
time O
points O
in O
between O
were O
not O
analyzed O
[ O
6 O
] O
. O

In O
addition O
to O
promoting O
survival O
, O
NT O
- O
3 O
, O
NGF O
, O
and O
BDNF O
also O
induce O
proliferation O
of O
various O
neuronal O
precursors O
at O
different O
stages O
of O
development O
. O

NT O
- O
3 O
can O
promote O
the O
incorporation O
of O
[ O
3H O
] O
- O
thymidine O
into O
cultured O
quail B
neural O
crest O
cells O
from O
the O
trunk O
region O
[ O
21 O
, O
22 O
] O
, O
Later O
in O
rat B
sympathetic O
development O
, O
NT O
- O
3 O
supports O
survival O
of O
neurons O
, O
but O
does O
not O
promote O
proliferation O
[ O
6 O
] O
, O
which O
is O
consistent O
with O
our O
results O
. O

NGF O
promotes O
an O
increase O
in O
BrdU O
incorporation O
from O
25 O
% O
to O
35 O
% O
in O
the O
DRG O
cervical O
segment O
2 O
in O
the O
chick B
embryo O
[ O
23 O
] O
. O

In O
chicken B
embryos O
that O
are O
treated O
with O
NGF O
in O
ovo O
at O
St O
. O

18 O
and O
21 O
, O
there O
is O
an O
increase O
in O
BrdU O
uptake O
after O
formation O
of O
the O
primary O
sympathetic O
chain O
at O
St O
. O

23 O
[ O
24 O
] O
. O

Since O
NGF O
does O
not O
appear O
to O
affect O
proliferation O
of O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
chick B
sympathoblasts O
, O
NGF O
may O
only O
promote O
proliferation O
in O
primary O
, O
but O
not O
secondary O
chain O
sympathoblasts O
. O

Motor O
neuron O
progenitors O
in O
the O
ventral O
neural O
tube O
from O
the O
chick B
embryo I
express O
TrkB O
and O
when O
ventral O
neural O
tube O
explants O
are O
treated O
with O
BDNF O
, O
there O
is O
an O
increase O
in O
the O
number O
of O
motor O
neurons O
produced O
and O
BrdU O
incorporation O
[ O
25 O
] O
. O

BDNF O
also O
promotes O
the O
proliferation O
of O
cultured O
neuroblastoma O
cells O
[ O
13 O
] O
. O

Taken O
together O
, O
these O
results O
are O
consistent O
with O
our O
findings O
that O
NT O
- O
3 O
and O
NGF O
do O
not O
promote O
proliferation O
of O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
sympathoblasts O
, O
and O
support O
the O
assertion O
that O
BDNF O
promotes O
proliferation O
of O
TrkB O
- O
positive O
sympathoblasts O
in O
culture O
. O

Our O
observations O
suggest O
a O
transient O
function O
of O
TrkB O
during O
early O
sympathetic O
development O
in O
supporting O
proliferation O
of O
this O
early O
subpopulation O
of O
sympathoblasts O
. O

However O
, O
the O
in O
vivo O
labeling O
suggests O
that O
only O
a O
minority O
( O
10 O
- O
20 O
% O
) O
of O
this O
population O
is O
dividing O
during O
the O
window O
that O
TrkB O
is O
expressed O
. O

In O
light O
of O
the O
very O
strong O
proliferative O
effect O
produced O
in O
cell O
culture O
, O
these O
TrkB O
expressing O
cells O
could O
respond O
more O
strongly O
if O
endogenous O
BDNF O
rises O
to O
higher O
levels O
, O
or O
if O
the O
mechanism O
that O
down O
regulates O
TrkB O
expression O
becomes O
nonfunctional O
. O

Such O
events O
could O
trigger O
an O
early O
proliferative O
event O
that O
leads O
to O
a O
cascade O
of O
changes O
that O
initiates O
transformation O
of O
cells O
to O
neuroblastoma O
. O

Thus O
, O
these O
early O
TrkB O
expressing O
cells O
help O
solve O
the O
puzzle O
as O
to O
why O
TrkB O
is O
expressed O
in O
aggressive O
and O
invasive O
forms O
of O
neuroblastoma O
, O
particularly O
because O
BDNF O
induces O
cultured O
neuroblastoma O
cells O
to O
become O
more O
proliferative O
, O
invasive O
, O
angiogenic O
, O
and O
resistant O
to O
chemotherapeutic O
reagents O
than O
untreated O
cultures O
[ O
13 O
] O
. O

Future O
studies O
will O
determine O
whether O
constitutive O
expression O
of O
BDNF O
and O
TrkB O
in O
the O
chick B
embryo O
sustains O
proliferation O
of O
differentiating O
sympathoblasts O
. O

Conclusion O

We O
have O
identified O
a O
time O
point O
during O
development O
when O
differentiating O
lumbar O
sympathetic O
neurons O
transiently O
express O
TrkB O
and O
proliferate O
in O
response O
to O
high O
concentrations O
of O
BDNF O
in O
culture O
. O

These O
studies O
suggest O
that O
elevated O
BDNF O
expression O
above O
basal O
levels O
and O
signaling O
through O
TrkB O
may O
be O
a O
mechanism O
that O
contributes O
to O
the O
onset O
of O
neuroblastoma O
. O

A O
further O
understanding O
of O
the O
two O
populations O
of O
sympathetic O
neurons O
and O
the O
fate O
of O
the O
TrkB O
- O
positive O
cells O
will O
provide O
additional O
insight O
into O
the O
development O
of O
paravertebral O
sympathetic O
ganglia O
and O
the O
genesis O
of O
neuroblastoma O
. O

Methods O

Preparation O
of O
tissue O
for O
immunohistochemistry O

The O
lumbar O
spinal O
column O
and O
surrounding O
tissues O
were O
dissected O
from O
chicken B
embryos O
at O
the O
indicated O
stages O
and O
placed O
in O
Zamboni O
' O
s O
fixative O
( O
4 O
% O
( O
w O
/ O
v O
) O
paraformaldehyde O
, O
15 O
% O
( O
v O
/ O
v O
) O
picric O
acid O
in O
0 O
. O
1 O
M O
sodium O
phosphate O
buffer O
, O
pH O
7 O
. O
4 O
) O
for O
two O
hours O
at O
room O
temperature O
. O

Mouse B
embryos O
at O
13 O
- O
15 O
days O
post O
- O
coitus O
were O
collected O
according O
to O
an O
IACUC O
- O
approved O
protocol O
to O
Dr O
. O
L O
. O

Sherman O
at O
the O
Oregon O
Health O
and O
Science O
University O
. O

The O
mouse B
embryos O
were O
immersion O
- O
fixed O
in O
Zamboni O
' O
s O
fixative O
overnight O
at O
4 O
degrees O
C O
then O
washed O
with O
phosphate O
buffered O
saline O
( O
PBS O
; O
130 O
mM O
NaCl O
, O
20 O
mM O
sodium O
phosphate O
buffer O
, O
pH O
7 O
. O
4 O
) O
. O

Fixed O
tissues O
were O
equilibrated O
in O
30 O
% O
sucrose O
in O
1 O
x O
phosphate O
- O
buffered O
saline O
( O
PBS O
) O
. O

Fixed O
mouse B
embryos O
were O
shipped O
to O
Vermont O
in O
sucrose O
. O

Transverse O
30 O
mu O
M O
sections O
of O
the O
spinal O
columns O
were O
cut O
at O
on O
a O
Microm O
HM O
cryostat O
( O
knife O
temperature O
: O
16 O
degrees O
C O
; O
object O
temperature O
: O
23 O
degrees O
C O
) O
and O
collected O
on O
Superfrost O
Plus O
slides O
( O
Fisher O
) O
. O

Sections O
were O
dried O
at O
room O
temperature O
, O
washed O
in O
1 O
x O
PBS O
and O
incubated O
overnight O
in O
blocking O
buffer O
( O
1 O
x O
PBS O
consisting O
of O
10 O
% O
( O
v O
/ O
v O
) O
heat O
- O
inactivated O
horse B
serum O
( O
Invitrogen O
/ O
Gibco O
) O
, O
0 O
. O
5 O
% O
Triton O
X O
- O
100 O
( O
Sigma O
) O
, O
and O
0 O
. O
1 O
% O
sodium O
azide O
( O
Fisher O
) O
) O
. O

Immunohistochemistry O

Sections O
were O
incubated O
with O
primary O
antibodies O
overnight O
at O
4 O
degrees O
C O
, O
followed O
by O
incubation O
with O
secondary O
antibodies O
for O
2 O
hours O
at O
room O
temperature O
. O

Primary O
antibodies O
used O
were O
: O
rabbit B
anti O
- O
p75 O
( O
1 O
: O
1500 O
, O
generous O
gift O
from O
Louis O
Reichardt O
, O
UCSF O
[ O
26 O
] O
) O
, O
mouse B
IgG2b O
anti O
- O
Hu O
C O
/ O
D O
, O
( O
1 O
: O
250 O
, O
Molecular O
Probes O
) O
; O
mouse B
IgG1 O
anti O
- O
Islet O
- O
1 O
, O
( O
1 O
: O
10 O
, O
Developmental O
Studies O
Hybridoma O
Bank O
) O
; O
rabbit B
anti O
- O
chicken B
TrkA O
( O
1 O
: O
500 O
) O
; O
rabbit B
anti O
- O
chicken B
TrkB O
( O
1 O
: O
500 O
) O
; O
rabbit B
anti O
- O
chicken B
TrkC O
( O
1 O
: O
500 O
) O
( O
all O
Trk O
antibodies O
were O
generous O
gifts O
of O
Dr O
. O

Louis O
Reichardt O
, O
UCSF O
[ O
26 O
- O
28 O
] O
) O
; O
mouse B
anti O
- O
HNK O
- O
1 O
( O
1 O
: O
50 O
, O
Developmental O
Studies O
Hybridoma O
Bank O
) O
; O
mouse B
IgG2a O
anti O
- O
tyrosine O
hydroxylase O
( O
1 O
: O
10 O
, O
Developmental O
Studies O
Hybridoma O
Bank O
) O
, O
sheep B
anti O
- O
BrdU O
( O
1 O
: O
100 O
, O
Biodesign O
International O
) O
, O
rabbit B
anti O
- O
tyrosine O
hydroxylase O
( O
1 O
: O
100 O
, O
Chemicon O
) O
, O
and O
goat B
anti O
- O
TrkB O
( O
1 O
: O
1000 O
, O
R O
& O
D O
Systems O
) O
. O

Immunofluorescence O
was O
imaged O
using O
a O
Nikon O
C1 O
confocal O
mounted O
on O
a O
Nikon O
Eclipse O
E800 O
microscope O
with O
a O
10 O
x O
Plan O
Apo O
( O
NA O
0 O
. O
785 O
) O
air O
objective O
or O
a O
60 O
x O
Plan O
Apo O
( O
NA O
1 O
. O
4 O
) O
oil O
objective O
lens O
, O
E7 O
- O
C1 O
software O
, O
and O
UV O
, O
Argon O
, O
and O
He O
/ O
Ne O
lasers O
exciting O
at O
408 O
, O
488 O
, O
and O
543 O
nm O
and O
emitting O
at O
404 O
500 O
- O
530 O
, O
and O
555 O
- O
615 O
nm O
, O
respectively O
. O

A O
Nikon O
Eclipse O
E800 O
microscope O
in O
the O
nearby O
COBRE O
Molecular O
/ O
Cellular O
Core O
Facility O
was O
used O
for O
counting O
immunofluorescent O
cells O
at O
200 O
x O
using O
epifluorescence O
optics O
. O

RNA O
Extraction O
/ O
cDNA O
synthesis O

Sympathetic O
ganglia O
were O
removed O
from O
chick O
embryos O
and O
RNA O
was O
isolated O
using O
TriReagent O
( O
Molecular O
Research O
Center O
) O
, O
an O
acidified O
guanidinium O
with O
phenol O
extraction O
method O
[ O
29 O
] O
. O

RNA O
was O
transcribed O
to O
cDNA O
using O
oligo O
- O
dT O
with O
Superscript O
II O
Reverse O
Transcriptase O
( O
Invitrogen O
) O
at O
42 O
degrees O
C O
for O
1 O
hour O
. O

Real O
- O
time O
PCR O

Relative O
RNA O
levels O
were O
determined O
using O
quantitative O
real O
- O
time O
PCR O
with O
an O
ABI O
7500 O
Fast O
Real O
Time O
PCR O
System O
. O

TaqMan O
probes O
were O
used O
to O
quantify O
the O
progression O
of O
the O
PCR O
reaction O
and O
reactions O
were O
normalized O
using O
the O
constitutively O
expressed O
gene O
chick O
ribosomal O
binding O
protein O
s17 O
( O
CHRPS O
) O
. O

The O
sequences O
were O
used O
for O
primer O
/ O
probes O
sets O
: O
for O
BDNF O
: O
forward O
: O
5 O
' O
- O
AGCCCAGTGAGGAAAACAAG O
- O
3 O
' O
, O
reverse O
: O
5 O
' O
- O
ACTCCTCGAGCAGAAAGAGC O
- O
3 O
' O
, O
probe O
: O
5 O
' O
- O
[ O
6 O
- O
FAM O
] O
- O
TACACATCCCGAGTCATGCT O
- O
[ O
BHQ O
] O
- O
3 O
' O
; O
for O
CHRPS O
( O
chick O
ribosomal O
binding O
protein O
S O
- O
17 O
) O
: O
5 O
' O
AACGACTTCCACACCAACAA O
' O
, O
reverse O
: O
5 O
' O

CTTCATCAGGTGGGTGACAT O
' O
, O
probe O
: O
5 O
' O
- O
[ O
6 O
- O
FAM O
] O
- O
CGCCATCATCCCCAGCAAGA O
[ O
BHQ O
] O
- O
3 O
' O
. O

Primers O
and O
probes O
were O
synthesized O
by O
Operon O
Technologies O
, O
Inc O
( O
Alameda O
, O
CA O
) O
. O

The O
primers O
for O
BDNF O
were O
validated O
against O
primers O
for O
CHRPS O
according O
to O
an O
Applied O
BioSystems O
protocol O
by O
serially O
diluting O
the O
target O
cDNA O
1 O
: O
10 O
, O
determining O
the O
cycle O
threshold O
( O
Ct O
) O
for O
each O
reaction O
, O
and O
plotting O
the O
Ct O
versus O
log O
concentration O
. O

Slopes O
of O
the O
resulting O
lines O
were O
calculated O
and O
primers O
were O
accepted O
if O
their O
Ct O
slopes O
were O
between O
- O
3 O
. O
2 O
and O
- O
3 O
. O
4 O
( O
a O
perfect O
efficiency O
of O
1 O
. O
0 O
yields O
a O
slope O
of O
- O
3 O
. O
3 O
) O
. O

To O
analyze O
the O
data O
, O
the O
delta O
Ct O
method O
of O
relative O
quantification O
was O
used O
, O
where O
the O
Ct O
of O
Chrps O
was O
subtracted O
from O
the O
Ct O
of O
the O
gene O
of O
interest O
( O
Delta O
Ct O
) O
and O
the O
arbitrary O
units O
of O
mRNA O
were O
expressed O
as O
10000 O
/ O
2 O
^ O
( O
Delta O
Ct O
) O
. O

Cell O
culture O

Sympathetic O
neurons O
were O
cultured O
as O
previously O
described O
[ O
30 O
] O
with O
a O
few O
modifications O
. O

Sympathetic O
ganglia O
were O
removed O
from O
the O
lumbar O
region O
of O
the O
paravertebral O
chain O
of O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
chick O
embryos O
and O
placed O
in O
Modified O
Puck O
' O
s O
solution O
with O
glucose O
( O
MPG O
) O
. O

The O
cells O
were O
dissociated O
by O
incubation O
of O
sympathetic O
ganglia O
with O
0 O
. O
1 O
% O
trypsin O
in O
MPG O
at O
37 O
degrees O
C O
for O
10 O
minutes O
followed O
by O
triturating O
with O
a O
fire O
polished O
9 O
" O
Pasteur O
pipette O
. O

Cells O
were O
then O
resuspended O
in O
Dulbecco O
' O
s O
Modified O
Eagle O
Medium O
( O
DMEM O
) O
consisting O
of O
10 O
% O
horse B
serum O
, O
2 O
% O
fetal O
calf B
serum O
, O
and O
10 O
mg O
/ O
ml O
penicillin O
/ O
streptomycin O
. O

For O
neurotrophin O
studies O
, O
the O
culture O
medium O
was O
supplemented O
with O
25 O
ng O
/ O
ml O
NT O
- O
3 O
( O
R O
& O
D O
Systems O
) O
and O
1 O
mu O
g O
/ O
ml O
7S O
NGF O
( O
Alomone O
Labs O
) O
upon O
plating O
, O
and O
50 O
ng O
/ O
ml O
, O
100 O
ng O
/ O
ml O
, O
or O
200 O
ng O
/ O
ml O
BDNF O
( O
R O
& O
D O
Systems O
) O
once O
the O
cells O
adhered O
to O
the O
wells O
. O

Cells O
were O
plated O
on O
poly O
- O
D O
- O
lysine O
/ O
laminin O
coated O
wells O
or O
cover O
slips O
( O
Fisher O
) O
as O
previously O
described O
[ O
30 O
] O
. O

Quantification O
of O
neurons O
and O
sympathoblasts O
using O
phase O
microscopy O

Embryonic O
sympathoblasts O
and O
neurons O
are O
small O
, O
phase O
bright O
cells O
with O
neurites O
. O

The O
total O
number O
of O
cells O
with O
neurites O
the O
length O
of O
two O
cell O
bodies O
were O
counted O
in O
10 O
non O
- O
overlapping O
fields O
of O
view O
evenly O
spaced O
in O
a O
grid O
- O
like O
pattern O
across O
the O
bottom O
of O
a O
well O
from O
a O
24 O
well O
plate O
at O
200 O
x O
using O
a O
Nikon O
Eclipse O
TE200 O
microscope O
. O

BrdU O
labeling O

For O
in O
vitro O
studies O
, O
approximately O
2 O
hours O
after O
plating O
cells O
from O
St O
. O

29 O
/ O
30 O
( O
E6 O
. O
5 O
) O
sympathetic O
ganglia O
, O
cells O
were O
labeled O
with O
10 O
mu O
M O
bromodeoxyuridine O
( O
BrdU O
, O
Sigma O
) O
for O
12 O
hours O
at O
37 O
degrees O
C O
. O

Following O
this O
labeling O
period O
, O
cells O
were O
incubated O
in O
complete O
medium O
without O
BrdU O
for O
an O
additional O
10 O
hrs O
. O

Cells O
were O
then O
fixed O
in O
Zamboni O
' O
s O
fixative O
for O
30 O
min O
at O
room O
temperature O
and O
rinsed O
with O
1 O
x O
PBS O
. O

For O
in O
vivo O
studies O
, O
25 O
mu O
g O
BrdU O
was O
injected O
into O
the O
amnion O
of O
chick O
embryos O
at O
St O
. O

27 O
. O

The O
cells O
and O
sections O
were O
denatured O
with O
2 O
N O
HCl O
at O
37 O
degrees O
C O
for O
1 O
hr O
, O
and O
were O
then O
neutralized O
with O
0 O
. O
1 O
M O
borate O
buffer O
, O
pH O
8 O
. O
5 O
, O
for O
10 O
min O
at O
room O
temperature O
. O

Immunochemistry O
was O
performed O
as O
described O
above O
. O

Abbreviations O

BDNF O
, O
brain O
- O
derived O
neurotrophic O
factor O
; O
BrdU O
, O
Bromodeoxyuridine O
; O
DA O
, O
dorsal O
aorta O
; O
DMEM O
, O
Dulbecco O
' O
s O
Modified O
Eagle O
' O
s O
Medium O
; O
DRG O
, O
dorsal O
root O
ganglion O
; O
E O
, O
embryonic O
day O
; O
HS O
, O
horse B
serum O
; O
MPG O
, O
Modified O
Puck O
' O
s O
solution O
with O
glucose O
; O
NGF O
, O
nerve O
growth O
factor O
; O
NC O
, O
notochord O
; O
NT O
, O
neural O
tube O
; O
NT O
- O
3 O
, O
neurotrophin O
- O
3 O
; O
NTR O
, O
neurotrophin O

receptor O
; O
PBS O
, O
phosphate O
- O
buffered O
saline O
; O
SC O
, O
spinal O
cord O
; O
SCG O
, O
superior O
cervical O
ganglion O
; O
SEM O
, O
standard O
error O
of O
the O
mean O
; O
SG O
, O
sympathetic O
ganglion O
; O
St O
. O
, O
stage O
; O
w O
/ O
v O
, O
weight O
/ O
volume O
; O
v O
/ O
v O
, O
volume O
/ O
volume O
. O

Authors O
' O
contributions O

JAS O
designed O
the O
experiments O
, O
performed O
the O
experiments O
, O
analyzed O
the O
data O
, O
and O
wrote O
the O
manuscript O
. O

GLSS O
contributed O
intellectually O
to O
the O
conception O
and O
design O
of O
this O
study O
, O
and O
assisted O
in O
the O
interpretation O
of O
the O
results O
. O

RN O
supervised O
the O
study O
, O
participated O
in O
the O
design O
of O
experiments O
, O
edited O
the O
manuscript O
, O
and O
obtained O
funding O
for O
the O
project O
. O

All O
authors O
read O
and O
approved O
the O
final O
manuscript O
. O

Video O
analysis O
of O
the O
escape O
flight O
of O
Pileated O
Woodpecker O
Dryocopus O
pileatus I
: O
does O
the O
Ivory O
- O
billed O
Woodpecker O
Campephilus O
principalis O
persist O
in O
continental O
North O
America O
? O

Abstract O

Background O

The O
apparent O
rediscovery O
of O
the O
Ivory O
- O
billed O
Woodpecker O
Campephilus O
principalis I
in O
Arkansas O
, O
USA O
, O
previously O
feared O
extinct O
, O
was O
supported O
by O
video O
evidence O
of O
a O
single O
bird O
in O
flight O
( O
Fitzpatrick O
et O
al O
, O
Science O
2005 O
, O
308 O
: O
1460 O
- O
1462 O
) O
. O

Plumage O
patterns O
and O
wingbeat O
frequency O
of O
the O
putative O
Ivory O
- O
billed O
Woodpecker O
were O
said O
to O
be O
incompatible O
with O
the O
only O
possible O
confusion O
species O
native O
to O
the O
area O
, O
the O
Pileated O
Woodpecker O
Dryocopus O
pileatus I
. O

Results O

New O
video O
analysis O
of O
Pileated O
Woodpeckers O
in O
escape O
flights O
comparable O
to O
that O
of O
the O
putative O
Ivory O
- O
billed O
Woodpecker O
filmed O
in O
Arkansas O
shows O
that O
Pileated O
Woodpeckers O
can O
display O
a O
wingbeat O
frequency O
equivalent O
to O
that O
of O
the O
Arkansas O
bird O
during O
escape O
flight O
. O

The O
critical O
frames O
from O
the O
Arkansas O
video O
that O
were O
used O
to O
identify O
the O
bird O
as O
an O
Ivory O
- O
billed O
Woodpecker O
are O
shown O
to O
be O
equally O
, O
or O
more O
, O
compatible O
with O
the O
Pileated O
Woodpecker O
. O

Conclusion O

The O
identification O
of O
the O
bird O
filmed O
in O
Arkansas O
in O
April O
2004 O
as O
an O
Ivory O
- O
billed O
Woodpecker O
is O
best O
regarded O
as O
unsafe O
. O

The O
similarities O
between O
the O
Arkansas O
bird O
and O
known O
Pileated O
Woodpeckers O
suggest O
that O
it O
was O
most O
likely O
a O
Pileated O
Woodpecker O
. O

Background O

The O
reported O
rediscovery O
of O
the O
Ivory O
- O
billed O
Woodpecker O
in O
2004 O
- O
5 O
in O
the O
Big O
Woods O
of O
Arkansas O
gave O
new O
impetus O
to O
efforts O
to O
conserve O
the O
mature O
bottomland O
woodlands O
of O
the O
south O
- O
eastern O
USA O
. O

Several O
sightings O
have O
been O
reported O
without O
photographic O
evidence O
being O
obtained O
[ O
1 O
] O
. O

Unless O
sightings O
are O
, O
however O
, O
independently O
verifiable O
on O
the O
basis O
of O
photographic O
or O
other O
recorded O
evidence O
, O
the O
possibility O
that O
mistakes O
have O
been O
made O
cannot O
be O
eliminated O
. O

Crucial O
to O
the O
scientific O
case O
for O
the O
persistence O
of O
the O
Ivory O
- O
billed O
Woodpecker O
was O
a O
4 O
s O
video O
of O
a O
large O
woodpecker O
in O
flight O
recorded O
by O
M O
. O
D O
. O

Luneau O
on O
25 O
April O
2004 O
( O
henceforth O
referred O
to O
as O
the O
' O
Luneau O
video O
' O
) O
and O
published O
in O
2005 O
[ O
1 O
] O
, O
which O
was O
claimed O
to O
be O
inconsistent O
with O
the O
plumage O
patterns O
of O
the O
superficially O
similar O
Pileated O
Woodpecker O
( O
a O
common O
resident O
bird O
of O
the O
area O
) O
. O

Both O
species O
are O
large O
, O
black O
- O
and O
- O
white O
woodpeckers O
[ O
2 O
] O
. O

The O
upperwing O
of O
the O
Ivory O
- O
billed O
Woodpecker O
is O
black O
, O
with O
white O
secondary O
feathers O
and O
white O
on O
some O
inner O
primary O
feathers O
. O

Pileated O
Woodpeckers O
have O
a O
largely O
black O
upperwing O
, O
with O
white O
restricted O
to O
the O
' O
wrist O
' O
due O
to O
white O
bases O
to O
the O
primary O
feathers O
. O

The O
underwing O
of O
Pileated O
Woodpecker O
has O
all O
- O
white O
underwing O
coverts O
, O
giving O
an O
appearance O
of O
a O
white O
underwing O
with O
a O
broad O
black O
outline O
( O
the O
black O
flight O
feathers O
) O
. O

These O
plumage O
differences O
result O
in O
the O
Ivory O
- O
billed O
Woodpecker O
having O
a O
white O
trailing O
edge O
to O
the O
wings O
( O
upper O
and O
lower O
sides O
) O
, O
whereas O
the O
Pileated O
Woodpecker O
has O
a O
black O
trailing O
edge O
to O
the O
wings O
. O

Both O
species O
have O
black O
wing O
- O
tips O
. O

These O
and O
other O
plumage O
characteristics O
are O
shown O
in O
[ O
1 O
, O
2 O
] O
. O

The O
wingbeat O
frequency O
of O
the O
bird O
in O
the O
Luneau O
video O
was O
measured O
at O
8 O
. O
6 O
beats O
s O
- O
1 O
, O
similar O
to O
that O
inferred O
from O
archival O
sound O
recording O
of O
a O
single O
Ivory O
- O
billed O
Woodpecker O
, O
but O
claimed O
to O
be O
outside O
the O
range O
of O
Pileated O
Woodpeckers O
( O
which O
generally O
have O
slower O
wingbeats O
) O
[ O
1 O
, O
3 O
] O
. O

Sibley O
et O
al O
[ O
4 O
] O
questioned O
the O
video O
evidence O
, O
in O
particular O
providing O
alternative O
explanations O
for O
the O
plumage O
patterns O
of O
the O
Luneau O
bird O
in O
flight O
and O
at O
rest O
. O

They O
pointed O
out O
individual O
frames O
of O
the O
Luneau O
video O
that O
appear O
to O
show O
three O
features O
that O
are O
each O
inconsistent O
with O
Ivory O
- O
billed O
Woodpecker O
: O
( O
1 O
) O
apparently O
black O
secondary O
feathers O
on O
the O
upper O
surface O
of O
the O
left O
wing O
, O
( O
2 O
) O
particularly O
bright O
white O
primary O
bases O
, O
and O
( O
3 O
) O
a O
black O
band O
curving O
smoothly O
round O
the O
wing O
tip O
( O
see O
Figure O
3 O
in O
[ O
4 O
] O
) O
. O

They O
hypothesized O
that O
flexing O
of O
a O
Pileated O
Woodpecker O
' O
s O
wings O
during O
flight O
could O
produce O
the O
appearance O
of O
white O
trailing O
edges O
on O
both O
wings O
in O
low O
- O
quality O
videos O
[ O
4 O
] O
. O

They O
offered O
, O
however O
, O
no O
direct O
evidence O
to O
show O
that O
this O
could O
cause O
a O
video O
of O
a O
Pileated O
Woodpecker O
to O
look O
like O
the O
bird O
in O
the O
Luneau O
video O
. O

Fitzpatrick O
et O
al O
[ O
5 O
] O
in O
turn O
rebutted O
some O
aspects O
of O
the O
hypothesis O
of O
Sibley O
et O
al O
[ O
4 O
] O
, O
publishing O
video O
stills O
of O
Pileated O
Woodpeckers O
, O
and O
a O
model O
of O
a O
Pileated O
Woodpecker O
, O
that O
appeared O
to O
show O
a O
black O
trailing O
edge O
to O
the O
wings O
inconsistent O
with O
Ivory O
- O
billed O
Woodpecker O
and O
the O
Luneau O
video O
. O

Fitzpatrick O
et O
al O
[ O
5 O
] O
neither O
rebutted O
nor O
discussed O
the O
three O
key O
inconsistencies O
described O
above O
. O

Without O
further O
evidence O
, O
this O
became O
largely O
a O
theoretical O
debate O
over O
interpretation O
of O
field O
characters O
that O
were O
barely O
visible O
in O
the O
very O
small O
images O
originally O
obtained O
. O

On O
one O
hand O
, O
as O
pointed O
out O
in O
Sibley O
et O
al O
[ O
4 O
] O
, O
some O
of O
the O
frames O
of O
the O
bird O
in O
the O
Luneau O
video O
do O
appear O
to O
be O
inconsistent O
with O
Ivory O
- O
billed O
Woodpecker O
. O

On O
the O
other O
hand O
, O
the O
flight O
pattern O
of O
the O
bird O
in O
the O
Luneau O
video O
is O
asserted O
to O
be O
atypical O
for O
Pileated O
Woodpecker O
( O
but O
matching O
anecdotal O
descriptions O
of O
Ivory O
- O
billed O
Woodpecker O
) O
. O

Furthermore O
, O
the O
general O
impression O
of O
the O
bird O
in O
the O
Luneau O
video O
was O
that O
there O
is O
far O
too O
much O
white O
in O
the O
wings O
for O
it O
to O
be O
a O
Pileated O
Woodpecker O
, O
and O
that O
if O
it O
was O
a O
Pileated O
, O
then O
it O
must O
be O
an O
aberrant O
one O
with O
abnormally O
extensive O
white O
plumage O
. O

Such O
birds O
occasionally O
occur O
, O
and O
have O
been O
observed O
in O
the O
Arkansas O
study O
area O
[ O
6 O
] O
. O

This O
study O
was O
undertaken O
to O
determine O
whether O
the O
flight O
and O
plumage O
of O
the O
bird O
in O
the O
Luneau O
video O
really O
was O
inconsistent O
with O
either O
a O
normal O
or O
partial O
albino O
Pileated O
Woodpecker O
. O

Independent O
analyses O
of O
the O
plumage O
patterns O
and O
wingbeat O
frequencies O
observable O
in O
Pileated O
Woodpeckers O
are O
presented O
, O
and O
it O
is O
concluded O
that O
the O
identification O
of O
the O
bird O
in O
the O
Luneau O
video O
as O
definite O
Ivory O
- O
billed O
Woodpecker O
is O
probably O
unsafe O
. O

Results O

On O
January O
28 O
and O
February O
5 O
, O
2006 O
, O
David O
Nolin O
( O
DN O
) O
video O
- O
recorded O
Pileated O
Woodpeckers O
Dryocopus O
pileatus I
at O
a O
bird O
- O
feeder O
in O
Dayton O
, O
Ohio O
, O
USA O
. O

A O
Hi O
- O
8 O
Sony O
Handycam O
was O
used O
, O
hand O
- O
held O
, O
at O
approximately O
5 O
m O
from O
the O
feeder O
. O

Birds O
on O
the O
tree O
trunk O
were O
alarmed O
by O
movement O
, O
and O
their O
escape O
flights O
recorded O
. O

Four O
escape O
flights O
were O
captured O
that O
approximate O
to O
that O
recorded O
for O
the O
putative O
Ivory O
- O
billed O
Woodpecker O
Campephilus B
principalis I
by O
Luneau O
in O
April O
2004 O
and O
published O
in O
Fitzpatrick O
et O
al O
[ O
1 O
] O
. O

The O
videos O
are O
not O
directly O
equivalent O
because O
the O
Pileated O
Woodpeckers O
made O
only O
short O
escape O
flights O
to O
nearby O
trees O
, O
whereas O
the O
putative O
Ivory O
- O
billed O
Woodpecker O
in O
the O
Luneau O
video O
showed O
little O
sign O
of O
coming O
to O
rest O
before O
being O
lost O
from O
view O
. O

Nevertheless O
, O
interesting O
comparisons O
can O
be O
made O
. O

Wingbeat O
frequency O
of O
Pileated O
Woodpecker O

The O
woodpecker O
in O
the O
Luneau O
video O
maintains O
a O
steady O
rapid O
wingbeat O
rate O
of O
8 O
. O
6 O
beats O
s O
- O
1 O
for O
at O
least O
8 O
wingbeats O
[ O
1 O
] O
, O
a O
figure O
that O
was O
confirmed O
by O
independent O
analysis O
during O
preparation O
of O
this O
paper O
. O

The O
Pileated O
woodpeckers O
in O
DN O
' O
s O
video O
do O
not O
do O
this O
- O
after O
initial O
rapid O
flapping O
immediately O
after O
take O
- O
off O
, O
they O
settle O
into O
a O
more O
relaxed O
level O
flight O
. O

As O
shown O
in O
Tables O
1 O
and O
2 O
, O
although O
the O
mean O
wingbeat O
frequencies O
of O
the O
Pileated O
Woodpeckers O
in O
DN O
' O
s O
video O
are O
slower O
than O
the O
8 O
. O
6 O
s O
- O
1 O
recorded O
for O
the O
bird B
in O
the O
Luneau O
video O
[ O
1 O
, O
3 O
, O
5 O
] O
the O
first O
four O
wingbeats O
, O
the O
initial O
escape O
response O
, O
are O
faster O
than O
those O
claimed O
for O
Pileated O
Woodpeckers O
in O
the O
literature O
[ O
1 O
, O
3 O
, O
5 O
] O
. O

For O
the O
four O
escape O
flights O
, O
the O
mean O
frequency O
values O
for O
the O
first O
four O
wingbeats O
are O
7 O
. O
1 O
, O
6 O
. O
7 O
, O
8 O
. O
6 O
, O
and O
8 O
. O
0 O
s O
- O
1 O
, O
respectively O
. O

The O
8 O
. O
6 O
beats O
s O
- O
1 O
of O
the O
bird O
identified O
in O
the O
Luneau O
video O
, O
while O
consistent O
with O
the O
limited O
data O
( O
n O
= O
1 O
; O
see O
Discussion O
) O
for O
Ivory O
- O
billed O
Woodpecker O
, O
is O
equally O
consistent O
with O
Pileated O
Woodpecker O
in O
its O
initial O
escape O
flight O
. O

The O
bird O
in O
the O
Luneau O
video O
maintains O
a O
frequency O
of O
8 O
. O
6 O
s O
- O
1 O
for O
the O
next O
four O
wingbeats O
too O
, O
whereas O
the O
Pileated O
Woodpeckers O
recorded O
here O
all O
slowed O
their O
flight O
as O
they O
prepared O
to O
land O
in O
nearby O
trees O
. O

There O
are O
no O
data O
to O
suggest O
whether O
Pileated O
Woodpeckers O
can O
maintain O
a O
wingbeat O
frequency O
approaching O
8 O
. O
6 O
s O
- O
1 O
for O
eight O
or O
more O
wingbeats O
, O
like O
the O
bird O
in O
the O
Luneau O
video O
. O

It O
remains O
possible O
that O
the O
flight O
pattern O
of O
the O
bird O
in O
the O
Luneau O
video O
is O
unusual O
for O
Pileated O
Woodpecker O
, O
but O
a O
frequency O
of O
8 O
. O
6 O
s O
- O
1 O
is O
consistent O
with O
a O
Pileated O
Woodpecker O
gaining O
initial O
speed O
and O
height O
in O
escape O
flight O
, O
and O
by O
itself O
cannot O
be O
taken O
as O
strong O
evidence O
that O
the O
Luneau O
video O
bird O
was O
an O
Ivory O
- O
billed O
Woodpecker O
. O

This O
is O
discussed O
further O
below O
. O

Plumage O
pattern O
of O
Pileated O
Woodpeckers O
in O
flight O

The O
video O
of O
Pileated O
Woodpeckers O
in O
flight O
was O
obtained O
in O
avi O
format O
, O
decompiled O
and O
examined O
frame O
by O
frame O
. O

Comparisons O
of O
Pileated O
Woodpecker O
images O
with O
key O
images O
of O
Luneau O
video O
are O
shown O
in O
Figures O
1 O
and O
2 O
, O
and O
suggest O
a O
genuine O
resemblance O
between O
the O
bird O
in O
the O
Luneau O
video O
and O
a O
Pileated O
Woodpecker O
. O

Analysis O
is O
complicated O
by O
the O
different O
digital O
processing O
of O
the O
two O
videos O
, O
and O
in O
the O
case O
of O
the O
Nolin O
videos O
it O
is O
important O
to O
concentrate O
only O
on O
those O
frames O
or O
part O
- O
frames O
where O
apparent O
plumage O
features O
are O
not O
an O
artifact O
of O
blurred O
images O
. O

Thirty O
- O
six O
frames O
from O
the O
fourth O
example O
of O
Pileated O
escape O
flight O
, O
which O
most O
resembled O
the O
flight O
path O
of O
the O
Luneau O
video O
bird O
, O
were O
analysed O
systematically O
frame O
by O
frame O
. O

They O
represent O
seven O
complete O
wingbeats O
( O
1 O
. O
20 O
s O
from O
the O
middle O
of O
the O
second O
wingbeat O
to O
middle O
of O
wingbeat O
9 O
) O
and O
were O
directly O
compared O
frame O
- O
by O
- O
frame O
with O
the O
equivalent O
fields O
( O
middle O
wingbeat O
2 O
- O
middle O
wingbeat O
9 O
) O
of O
the O
Luneau O
video O
. O

This O
comparison O
is O
shown O
in O
Figure O
3 O
. O

The O
images O
of O
the O
birds O
are O
not O
identical O
, O
but O
in O
every O
frame O
of O
the O
36 O
frames O
available O
, O
there O
are O
sufficient O
similarities O
to O
suggest O
that O
the O
bird O
in O
the O
Luneau O
video O
is O
consistent O
with O
the O
known O
Pileated O
Woodpecker O
. O

Further O
comparisons O
of O
the O
Luneau O
bird O
with O
the O
other O
three O
Pileated O
escape O
flights O
recorded O
are O
presented O
in O
the O
supplementary O
material O
( O
see O
Additional O
file O
1 O
) O
. O

Key O
findings O
of O
the O
video O
analysis O
are O
: O

1 O
. O

Pileated O
Woodpeckers O
flying O
near O
- O
horizontally O
away O
from O
the O
observer O
show O
much O
more O
white O
in O
poor O
- O
quality O
video O
than O
would O
be O
expected O
from O
their O
general O
plumage O
pattern O
. O

They O
present O
an O
appearance O
of O
a O
black O
- O
bodied O
bird O
with O
largely O
white O
wings O
and O
black O
wingtips O
, O
very O
similar O
to O
the O
bird O
in O
the O
Luneau O
video O
; O
compare O
in O
particular O
Figure O
1B O
, O
frame O
758 O
, O
with O
Figure O
1A O
, O
frame O
283 O
. O
3 O
. O

The O
expected O
appearance O
of O
the O
upperwing O
of O
Pileated O
Woodpecker O
- O
mostly O
black O
with O
a O
small O
white O
patch O
at O
the O
base O
of O
the O
primaries O
- O
is O
often O
not O
seen O
, O
and O
is O
only O
clearly O
resolvable O
when O
birds O
are O
flying O
near O
- O
vertically O
before O
landing O
on O
a O
tree O
trunk O
; O
something O
the O
bird O
in O
the O
Luneau O
video O
did O
not O
do O
. O

2 O
. O

The O
black O
trailing O
edge O
to O
the O
underwing O
of O
Pileated O
Woodpecker O
is O
often O
very O
inconspicuous O
and O
may O
disappear O
completely O
. O

Due O
to O
motion O
and O
flexion O
of O
the O
wing O
, O
the O
black O
trailing O
edge O
is O
much O
more O
obvious O
towards O
the O
wingtips O
. O

This O
produces O
an O
apparent O
plumage O
pattern O
that O
matches O
the O
patterns O
shown O
by O
the O
Luneau O
video O
bird O
( O
compare O
Figure O
1B O
, O
frames O
175 O
and O
457 O
with O
Figure O
1A O
, O
frames O
300 O
and O
416 O
. O
7 O
) O
. O

In O
many O
frames O
of O
Pileated O
Woodpecker O
, O
a O
black O
trailing O
edge O
to O
the O
wing O
is O
discernable O
( O
though O
due O
to O
bleeding O
of O
white O
as O
a O
video O
artifact O
, O
it O
appears O
narrower O
than O
it O
really O
is O
) O
. O

However O
, O
analysis O
of O
the O
bird O
in O
the O
Luneau O
video O
in O
light O
of O
images O
of O
known O
Pileated O
Woodpeckers O
confirms O
that O
a O
similar O
black O
trailing O
edge O
to O
the O
wing O
is O
discernable O
in O
some O
frames O
of O
the O
Luneau O
video O
( O
compare O
Figure O
1B O
, O
frame O
775 O
with O
Figure O
1A O
, O
frame O
366 O
. O
7 O
: O
the O
apparent O
plumage O
patterns O
are O
similar O
, O
and O
inconsistent O
with O
Ivory O
- O
billed O
Woodpecker O
) O
. O

It O
is O
argued O
here O
that O
the O
hypothesis O
put O
forward O
in O
Sibley O
et O
al O
[ O
4 O
] O
is O
correct O
, O
and O
that O
the O
black O
trailing O
edge O
of O
the O
underwing O
of O
Pileated O
Woodpecker O
can O
indeed O
, O
due O
to O
flexion O
of O
the O
wings O
during O
the O
downstroke O
, O
be O
misinterpreted O
as O
the O
black O
leading O
edge O
and O
wingtips O
of O
the O
upperwing O
of O
an O
Ivory O
- O
billed O
Woodpecker O
. O

3 O
. O

Figure O
3 O
shows O
that O
the O
plumage O
patterns O
shown O
by O
the O
Luneau O
bird O
, O
throughout O
several O
wingbeat O
cycles O
, O
are O
compatible O
with O
Pileated O
Woodpecker O
. O

The O
three O
plumage O
features O
described O
in O
Sibley O
et O
al O
[ O
4 O
] O
that O
are O
incompatible O
with O
Ivory O
- O
billed O
Woodpecker O
( O
black O
secondary O
feathers O
on O
upper O
surface O
of O
left O
wing O
, O
brighter O
white O
primary O
bases O
, O
and O
a O
black O
band O
curling O
round O
the O
wing O
tip O
) O
are O
seen O
consistently O
in O
the O
Luneau O
video O
and O
are O
recapitulated O
throughout O
the O
video O
of O
Pileated O
Woodpecker O
. O

Discussion O

Evidence O
is O
presented O
here O
to O
show O
that O
the O
distinctive O
plumage O
features O
of O
Pileated O
Woodpecker O
are O
surprisingly O
difficult O
to O
resolve O
in O
poor O
- O
quality O
video O
of O
birds O
in O
escape O
flight O
away O
from O
the O
camera O
, O
and O
that O
they O
can O
show O
apparent O
plumage O
patterns O
that O
might O
more O
readily O
be O
associated O
with O
Ivory O
- O
billed O
Woodpecker O
. O

Irrespective O
of O
the O
identity O
of O
the O
bird O
in O
the O
Luneau O
video O
, O
this O
knowledge O
will O
be O
critical O
to O
assessment O
of O
further O
claims O
of O
Ivory O
- O
billed O
Woodpeckers O
during O
the O
current O
intensive O
search O
effort O
. O

It O
is O
, O
however O
, O
suggested O
here O
that O
critical O
frames O
used O
for O
identification O
of O
the O
Luneau O
video O
woodpecker O
as O
an O
Ivory O
- O
billed O
Woodpecker O
are O
also O
consistent O
with O
Pileated O
Woodpecker O
. O

The O
wingbeat O
frequency O
of O
the O
bird O
in O
the O
Luneau O
video O
is O
also O
perhaps O
consistent O
with O
Pileated O
Woodpecker O
, O
at O
least O
for O
short O
periods O
of O
flight O
. O

Analysis O
of O
the O
videos O
of O
Pileated O
Woodpecker O
has O
supported O
the O
hypothesised O
interpretations O
of O
key O
frames O
of O
the O
Luneau O
video O
by O
Sibley O
et O
al O
[ O
4 O
] O
. O

Although O
the O
rebuttal O
of O
that O
comment O
in O
Fitzpatrick O
et O
al O
[ O
5 O
] O
asserted O
that O
flexion O
and O
motion O
of O
wings O
of O
Pileated O
Woodpeckers O
could O
not O
produce O
the O
images O
seen O
in O
the O
Luneau O
video O
, O
it O
has O
been O
shown O
here O
that O
they O
can O
. O

The O
Luneau O
video O
as O
presented O
in O
Fitzpatrick O
et O
al O
[ O
1 O
] O
, O
shows O
features O
that O
are O
consistent O
with O
Pileated O
Woodpecker O
, O
and O
inconsistent O
with O
Ivory O
- O
billed O
Woodpecker O
. O

It O
is O
argued O
in O
this O
paper O
that O
, O
in O
fact O
, O
the O
black O
trailing O
edge O
of O
the O
wing O
of O
a O
Pileated O
Woodpecker O
is O
seen O
clearly O
in O
the O
Luneau O
video O
, O
during O
the O
downstroke O
of O
the O
wingbeat O
cycle O
, O
but O
that O
it O
has O
been O
misinterpreted O
as O
black O
wingtips O
( O
Figure O
1 O
, O
2 O
, O
3 O
) O
. O

A O
fuller O
analysis O
of O
the O
Luneau O
video O
by O
the O
Cornell O
University O
team O
is O
presented O
online O
[ O
7 O
] O
. O

Although O
it O
is O
not O
peer O
- O
reviewed O
, O
the O
points O
this O
article O
makes O
should O
be O
taken O
into O
account O
. O

The O
authors O
summarise O
nine O
diagnostic O
traits O
from O
their O
analysis O
of O
the O
Luneau O
video O
that O
identify O
the O
bird O
as O
Ivory O
- O
billed O
Woodpecker O
. O

These O
are O
listed O
and O
discussed O
point O
- O
by O
point O
below O
. O

1 O
. O

' O
The O
underwing O
pattern O
in O
flight O
consistently O
appears O
largely O
white O
, O
giving O
the O
appearance O
of O
having O
black O
wingtips O
but O
lacking O
any O
black O
along O
the O
rear O
, O
or O
trailing O
edge O
. O
' O

Data O
presented O
in O
this O
paper O
show O
that O
this O
statement O
is O
not O
wholly O
supported O
, O
and O
in O
any O
case O
the O
underwing O
of O
Pileated O
Woodpeckers O
can O
present O
the O
same O
appearance O
. O

2 O
. O

' O
The O
upperwing O
pattern O
in O
flight O
consistently O
shows O
a O
broad O
, O
white O
trailing O
edge O
, O
with O
no O
frames O
demonstrating O
the O
conspicuous O
dark O
rear O
border O
to O
be O
expected O
of O
normal O
Pileated O
Woodpeckers O
. O
' O

Notwithstanding O
that O
certain O
frames O
of O
the O
Luneau O
video O
( O
e O
. O
g O
. O
frame O
350 O
) O
do O
appear O
to O
show O
a O
black O
trailing O
edge O
to O
the O
upperwing O
, O
data O
presented O
in O
this O
paper O
shows O
that O
, O
at O
this O
angle O
of O
view O
and O
resolution O
of O
video O
, O
Pileated O
Woodpeckers O
also O
may O
fail O
to O
show O
this O
feature O
. O

This O
analysis O
has O
shown O
that O
the O
hypothesis O
presented O
in O
Sibley O
et O
al O
[ O
4 O
] O
is O
plausible O
, O
i O
. O
e O
. O
that O
some O
of O
the O
frames O
interpreted O
by O
[ O
1 O
] O
to O
show O
the O
upperwing O
of O
an O
Ivory O
- O
billed O
Woodpecker O
may O
in O
fact O
show O
large O
amounts O
of O
white O
and O
the O
black O
trailing O
edge O
from O
the O
underwing O
of O
a O
Pileated O
Woodpecker O
. O

3 O
. O

' O
The O
wings O
are O
longer O
relative O
to O
the O
body O
diameter O
than O
in O
Pileated O
Woodpecker O
and O
consistent O
with O
the O
wing O
shape O
of O
Ivory O
- O
billed O
Woodpecker O
. O
' O

Fitzpatrick O
et O
al O
[ O
5 O
] O
agreed O
that O
accurate O
measurements O
were O
not O
possible O
from O
the O
video O
images O
presented O
in O
their O
original O
paper O
[ O
1 O
] O
, O
and O
it O
seems O
unlikely O
that O
much O
confidence O
can O
be O
placed O
in O
the O
wing O
- O
length O
measurements O
of O
the O
bird O
in O
the O
Luneau O
video O
. O

Comparison O
of O
, O
for O
example O
, O
Figure O
1A O
, O
frame O
283 O
. O
3 O
with O
Figure O
1B O
, O
frame O
578 O
suggests O
that O
any O
differences O
will O
be O
very O
difficult O
to O
prove O
. O

4 O
. O

' O
Reenactment O
of O
the O
scene O
using O
life O
- O
sized O
, O
realistically O
painted O
, O
dynamically O
flapping O
models O
produced O
images O
remarkably O
similar O
to O
those O
of O
the O
Luneau O
video O
using O
the O
Ivory O
- O
billed O
Woodpecker O
model O
, O
and O
images O
clearly O
identifiable O
as O
Pileated O
Woodpecker O
using O
a O
model O
of O
that O
species O
. O
' O

Interpretation O
of O
model O
re O
- O
enactments O
is O
hampered O
by O
the O
fact O
that O
the O
stiff O
, O
flat O
- O
winged O
models O
cannot O
reflect O
the O
wing O
flexion O
and O
curvature O
of O
real O
birds O
. O

Reenactment O
of O
the O
scene O
using O
real O
Pileated O
Woodpeckers O
has O
produced O
images O
remarkably O
similar O
to O
the O
Luneau O
video O
. O

5 O
. O

' O
The O
wingbeat O
frequency O
is O
8 O
. O
6 O
beats O
per O
second O
, O
which O
is O
almost O
identical O
to O
that O
recorded O
for O
Ivory O
- O
billed O
Woodpecker O
( O
as O
documented O
by O
one O
acoustic O
record O
from O
1935 O
) O
. O

The O
wing O
- O
beat O
frequencies O
of O
Pileated O
Woodpecker O
are O
not O
known O
to O
exceed O
7 O
. O
5 O
beats O
per O
second O
, O
and O
more O
typically O
range O
between O
3 O
and O
6 O
beats O
per O
second O
. O
' O

The O
fact O
that O
in O
only O
four O
recorded O
escape O
flights O
of O
Pileated O
Woodpecker O
, O
two O
were O
recorded O
for O
which O
the O
initial O
escape O
flight O
wingbeat O
frequency O
( O
8 O
. O
0 O
s O
- O
1 O
and O
8 O
. O
6 O
s O
- O
1 O
) O
exceeded O
that O
previously O
recorded O
for O
this O
species O
shows O
that O
previous O
datasets O
were O
too O
limited O
to O
make O
this O
conclusion O
. O

Birds O
flap O
more O
rapidly O
at O
take O
off O
to O
gain O
altitude O
and O
speed O
than O
they O
do O
in O
sustained O
level O
flight O
: O
Pileated O
Woodpecker O
flight O
data O
in O
the O
literature O
[ O
1 O
, O
4 O
, O
5 O
] O
was O
derived O
from O
the O
work O
of O
Tobalske O
[ O
8 O
] O
, O
which O
explicitly O
excluded O
the O
initial O
take O
- O
off O
period O
, O
and O
therefore O
cannot O
be O
used O
to O
support O
the O
elimination O
of O
Pileated O
Woodpecker O
in O
the O
Luneau O
video O
. O

Furthermore O
, O
the O
bird O
in O
the O
Luneau O
video O
is O
consistently O
gaining O
height O
from O
a O
low O
position O
above O
water O
and O
, O
whatever O
its O
species O
, O
might O
be O
expected O
to O
flap O
more O
rapidly O
than O
if O
it O
were O
in O
level O
flight O
. O

Tanner O
[ O
9 O
] O
noted O
that O
Pileated O
Woodpeckers O
can O
maintain O
extended O
fast O
direct O
flight O
. O

He O
was O
of O
the O
opinion O
that O
flight O
pattern O
was O
not O
a O
useful O
character O
for O
separating O
the O
two O
species O
in O
the O
field O
, O
and O
that O
Pileated O
Woodpeckers O
frequently O
fly O
in O
a O
manner O
that O
was O
in O
no O
way O
different O
to O
Ivory O
- O
billed O
Woodpeckers O
. O

The O
figure O
of O
8 O
. O
6 O
wingbeats O
per O
second O
for O
the O
Luneau O
bird O
( O
data O
reanalysed O
here O
) O
is O
taken O
as O
consistent O
with O
Ivory O
- O
billed O
Woodpecker O
on O
the O
basis O
of O
analysis O
of O
a O
single O
archival O
audio O
recording O
[ O
3 O
] O
. O

The O
Ivory O
- O
billed O
Woodpecker O
in O
that O
audio O
tape O
is O
clearly O
flapping O
its O
wings O
, O
but O
without O
accompanying O
visual O
confirmation O
it O
is O
not O
clear O
that O
it O
is O
in O
flight O
. O

In O
general O
, O
larger O
birds O
are O
expected O
to O
flap O
their O
wings O
more O
slowly O
than O
smaller O
birds O
of O
comparable O
wing O
morphology O
. O

Tobalske O
[ O
8 O
] O
showed O
that O
, O
across O
species O
, O
smaller O
woodpeckers O
tend O
to O
flap O
more O
quickly O
than O
larger O
ones O
, O
and O
that O
there O
was O
considerable O
intraspecific O
variation O
. O

The O
assertion O
that O
Ivory O
- O
billed O
Woodpeckers O
flap O
their O
wings O
more O
quickly O
than O
Pileated O
Woodpeckers O
is O
therefore O
counterintuitive O
. O

Further O
comment O
is O
conjecture O
: O
while O
the O
flight O
pattern O
and O
wing O
posture O
of O
the O
bird O
in O
the O
Luneau O
video O
may O
be O
unusual O
, O
it O
has O
not O
been O
shown O
that O
it O
is O
outside O
the O
range O
of O
variability O
of O
Pileated O
Woodpecker O
, O
and O
cannot O
therefore O
be O
used O
to O
eliminate O
the O
possibility O
that O
it O
was O
the O
commoner O
species O
. O

6 O
. O

' O
White O
plumage O
on O
the O
back O
is O
visible O
on O
the O
retreating O
bird O
as O
it O
begins O
to O
gain O
altitude O
. O

Ivory O
- O
billed O
Woodpecker O
has O
white O
on O
the O
back O
; O
Pileated O
Woodpecker O
has O
entirely O
black O
back O
. O
' O

This O
was O
discussed O
by O
Sibley O
et O
al O
[ O
4 O
] O
, O
who O
argued O
that O
the O
images O
thought O
to O
show O
white O
on O
the O
dorsum O
were O
too O
small O
to O
be O
accepted O
uncritically O
. O

In O
all O
the O
frames O
of O
the O
Luneau O
video O
that O
appear O
to O
show O
white O
on O
the O
dorsum O
, O
the O
bird B
is O
distant O
( O
dorsal O
white O
is O
not O
visible O
on O
the O
higher O
resolution O
images O
earlier O
in O
the O
video O
) O
and O
partially O
obscured O
, O
making O
it O
difficult O
to O
distinguish O
dorsum O
from O
wing O
. O

Spurious O
areas O
of O
white O
pixels O
appear O
as O
artifacts O
in O
both O
videos O
. O

Nevertheless O
, O
this O
remains O
the O
best O
evidence O
that O
the O
Luneau O
bird O
was O
not O
a O
standard O
Pileated O
Woodpecker O
. O

7 O
. O

' O
The O
dorsal O
view O
of O
the O
right O
wing O
as O
it O
begins O
to O
unfold O
shows O
a O
triangle O
of O
white O
that O
matches O
in O
size O
and O
position O
the O
white O
on O
the O
folded O
wing O
of O
an O
Ivory O
- O
billed O
Woodpecker O
beginning O
to O
launch O
into O
flight O
. O
' O

No O
further O
comment O
is O
provided O
here O
. O

An O
alternative O
explanation O
was O
offered O
by O
Sibley O
et O
al O
[ O
4 O
] O
and O
rebutted O
by O
Fitzpatrick O
et O
al O
[ O
5 O
] O
. O

The O
statement O
requires O
a O
degree O
of O
certainty O
about O
the O
position O
of O
the O
wing O
. O

( O
8 O
) O
' O
The O
distance O
between O
the O
wrist O
area O
and O
the O
tip O
of O
the O
tail O
( O
32 O
- O
36 O
cm O
, O
as O
measured O
when O
the O
bird O
begins O
to O
take O
flight O
) O
is O
comparable O
to O
known O
measurements O
of O
Ivory O
- O
billed O
Woodpecker O
and O
considerably O
larger O
than O
even O
the O
largest O
Pileated O
Woodpecker O
we O
measured O
. O
' O

As O
stated O
under O
( O
3 O
) O
, O
above O
, O
there O
is O
general O
agreement O
that O
accurate O
measurements O
are O
not O
possible O
from O
the O
Luneau O
video O
because O
too O
many O
uncontrolled O
variables O
are O
involved O
[ O
4 O
, O
5 O
] O
. O

9 O
. O

' O
Only O
20 O
seconds O
before O
the O
woodpecker O
flees O
, O
a O
bird O
with O
the O
size O
and O
color O
pattern O
of O
an O
Ivory O
- O
billed O
Woodpecker O
was O
perched O
within O
3 O
m O
of O
the O
site O
from O
which O
the O
woodpecker O
took O
flight O
. O
' O

This O
would O
be O
a O
strong O
argument O
if O
it O
could O
be O
shown O
that O
the O
object O
in O
question O
was O
a O
bird B
and O
not O
, O
as O
is O
now O
apparently O
thought O
likely O
, O
a O
section O
of O
branch O
or O
tree O
stump O
[ O
10 O
] O
. O

The O
Luneau O
video O
reveals O
several O
white O
triangular O
patches O
apparently O
visible O
on O
or O
around O
tree O
trunks O
, O
most O
or O
all O
of O
which O
must O
therefore O
be O
images O
of O
tree O
topography O
or O
video O
artifacts O
. O

This O
was O
discussed O
in O
the O
literature O
( O
see O
[ O
4 O
, O
5 O
] O
) O
. O

Central O
to O
the O
identification O
of O
the O
flying O
bird O
seen O
in O
the O
Luneau O
video O
was O
the O
evidence O
that O
plumage O
and O
flight O
patterns O
were O
inconsistent O
with O
Pileated O
Woodpecker O
. O

A O
very O
basic O
video O
analysis O
presented O
here O
has O
suggested O
that O
this O
may O
not O
be O
the O
case O
, O
and O
that O
further O
research O
is O
needed O
. O

Any O
identification O
of O
the O
bird O
in O
the O
Luneau O
video O
as O
an O
Ivory O
- O
bill O
must O
take O
into O
account O
the O
data O
presented O
here O
and O
in O
Sibley O
et O
al O
[ O
4 O
] O
, O
which O
shows O
it O
is O
largely O
consistent O
with O
Pileated O
Woodpecker O
and O
points O
out O
apparent O
inconsistencies O
with O
Ivory O
- O
billed O
Woodpecker O
. O

This O
does O
not O
of O
course O
necessarily O
imply O
that O
the O
Ivory O
- O
billed O
Woodpecker O
is O
extinct O
, O
nor O
indeed O
entirely O
rule O
out O
the O
possibility O
that O
the O
bird O
in O
the O
Luneau O
video O
was O
one O
. O

There O
appears O
to O
be O
no O
reason O
to O
question O
the O
anecdotal O
sight O
records O
of O
Ivory O
- O
billed O
Woodpecker O
presented O
in O
Fitzpatrick O
et O
al O
[ O
1 O
] O
( O
or O
in O
many O
online O
sources O
) O
, O
because O
some O
of O
them O
appear O
credible O
, O
albeit O
brief O
. O

Audio O
evidence O
has O
since O
been O
published O
[ O
11 O
] O
although O
this O
too O
is O
far O
from O
conclusive O
. O

However O
, O
to O
regard O
the O
Luneau O
video O
by O
itself O
as O
presenting O
anything O
other O
than O
an O
unidentified O
woodpecker O
falls O
below O
the O
standards O
of O
proof O
normally O
required O
for O
scientific O
publication O
: O
the O
images O
are O
not O
good O
enough O
. O

The O
Ivory O
- O
billed O
Woodpecker O
may O
persist O
in O
continental O
North O
America O
, O
and O
there O
is O
enough O
anecdotal O
evidence O
to O
make O
this O
a O
possibility O
, O
but O
the O
Luneau O
video O
does O
not O
support O
the O
case O
. O

The O
balance O
of O
evidence O
would O
suggest O
that O
the O
bird O
in O
the O
Luneau O
video O
is O
more O
likely O
to O
have O
been O
a O
Pileated O
Woodpecker O
, O
but O
the O
search O
for O
Ivory O
- O
billed O
Woodpecker O
should O
continue O
. O

While O
this O
paper O
was O
under O
review O
, O
a O
report O
of O
sight O
records O
and O
sound O
recordings O
of O
Ivory O
- O
billed O
Woodpeckers O
was O
published O
from O
a O
location O
in O
Florida O
[ O
12 O
] O
. O

This O
very O
exciting O
claim O
is O
strengthened O
by O
reports O
of O
sighting O
of O
the O
white O
dorsal O
stripes O
on O
one O
bird O
in O
flight O
. O

Unfortunately O
, O
several O
sightings O
were O
made O
without O
optical O
aids O
and O
cannot O
be O
considered O
proven O
. O

The O
' O
kent O
' O
calls O
recorded O
from O
the O
Florida O
location O
are O
spectrographically O
similar O
to O
the O
' O
bleat O
' O
calls O
of O
young O
White O
- O
tailed O
Deer B
, O
as O
described O
in O
Richardson O
et O
al O
[ O
13 O
] O
. O

A O
clear O
photograph O
will O
be O
required O
from O
this O
location O
too O
before O
the O
presence O
of O
Ivory O
- O
billed O
Woodpeckers O
can O
be O
considered O
confirmed O
. O

It O
is O
hoped O
that O
this O
paper O
will O
help O
with O
assessment O
of O
any O
further O
low O
quality O
photographs O
or O
videos O
. O

Conclusion O

Flight O
and O
plumage O
patterns O
of O
the O
putative O
Ivory O
- O
billed O
Woodpecker O
recorded O
in O
Arkansas O
in O
2005 O
are O
recapitulated O
by O
confirmed O
Pileated O
Woodpeckers O
. O

The O
bird O
in O
the O
Arkansas O
video O
is O
best O
regarded O
as O
not O
fully O
identified O
, O
and O
is O
probably O
a O
Pileated O
Woodpecker O
. O

Methods O

Video O
recording O

Pileated O
Woodpeckers O
were O
attracted O
to O
a O
bird O
feeder O
containing O
suet O
at O
Grants O
Trail O
, O
Dayton O
, O
OH O
45459 O
. O

The O
suet O
feeder O
was O
placed O
approximately O
2 O
. O
1 O
m O
high O
on O
a O
tree O
trunk O
, O
and O
the O
distance O
to O
the O
suet O
feeder O
from O
the O
observation O
point O
was O
approximately O
5 O
m O
. O

Birds O
on O
the O
feeder O
were O
startled O
by O
movement O
of O
window O
blinds O
on O
January O
28 O
and O
February O
5 O
, O
2006 O
, O
and O
their O
escape O
flights O
were O
filmed O
using O
a O
Sony O
Hi O
- O
8 O
SteadyShot O
video O
camera O
at O
29 O
. O
97 O
frames O
s O
- O
1 O
. O

At O
least O
two O
birds O
feature O
in O
the O
videos O
, O
male O
and O
female O
. O

Analogue O
tape O
was O
converted O
to O
digital O
by O
connecting O
the O
Hi O
- O
8 O
camera O
directly O
to O
a O
Sony O
DCR O
- O
HC30 O
digital O
video O
camera O
and O
recording O
onto O
that O
camera O
' O
s O
mini O
dv O
cassette O
. O

The O
resulting O
images O
were O
converted O
to O
an O
avi O
file O
using O
Windows O
Movie O
Maker O
on O
a O
Windows O
XP O
PC O
. O

The O
video O
is O
freely O
available O
in O
wmv O
format O
[ O
14 O
] O
and O
in O
avi O
format O
from O
the O
author O
or O
David O
Nolin O
( O
via O
the O
author O
) O
. O

The O
video O
was O
decompiled O
using O
Blaze O
Media O
Pro O
( O
Mystik O
Media O
, O
Hampstead O
, O
NC O
, O
USA O
) O
for O
a O
detailed O
analysis O
. O

Import O
into O
Avid O
( O
R O
) O
Xpress O
Pro O
HD O
for O
deinterlacing O
did O
not O
reduce O
the O
wing O
flicker O
seen O
in O
the O
images O
, O
and O
further O
professional O
processing O
could O
not O
improve O
the O
resolution O
, O
so O
the O
original O
decompiled O
file O
was O
used O
for O
analysis O
. O

Hence O
some O
frames O
contain O
two O
overlaid O
images O
, O
which O
may O
lower O
the O
resolution O
in O
some O
cases O
. O

The O
decompiled O
file O
was O
examined O
frame O
by O
frame O
and O
compared O
to O
the O
decompiled O
images O
of O
the O
putative O
Ivory O
- O
billed O
Woodpecker O
presented O
in O
Fitzpatrick O
et O
al O
[ O
1 O
] O
. O

Wingbeat O
frequencies O
were O
calculated O
by O
noting O
the O
frame O
number O
of O
the O
midpoint O
of O
the O
downstroke O
of O
each O
wingbeat O
( O
e O
. O
g O
. O
Figure O
1B O
, O
frame O
758 O
) O
and O
calculating O
the O
length O
of O
time O
taken O
per O
wingbeat O
as O
( O
number O
of O
frames O
between O
downstroke O
midpoints O
) O
/ O
29 O
. O
97 O
. O

Authors O
' O
contributions O

JMC O
performed O
the O
data O
analysis O
and O
drafted O
the O
manuscript O
. O

Supplementary O
Material O

A O
global O
gene O
evolution O
analysis O
on O
Vibrionaceae B
family O
using O
phylogenetic O
profile O

Abstract O

Background O

Vibrionaceae B
represent O
a O
significant O
portion O
of O
the O
cultivable O
heterotrophic O
sea O
bacteria O
; O
they O
strongly O
affect O
nutrient O
cycling O
and O
some O
species O
are O
devastating O
pathogens O
. O

In O
this O
work O
we O
propose O
an O
improved O
phylogenetic O
profile O
analysis O
on O
14 O
Vibrionaceae B
genomes O
, O
to O
study O
the O
evolution O
of O
this O
family O
on O
the O
basis O
of O
gene O
content O
. O

The O
phylogenetic O
profile O
is O
based O
on O
the O
observation O
that O
genes O
involved O
in O
the O
same O
process O
( O
e O
. O
g O
. O
metabolic O
pathway O
or O
structural O
complex O
) O
tend O
to O
be O
concurrently O
present O
or O
absent O
within O
different O
genomes O
. O

This O
allows O
the O
prediction O
of O
hypothetical O
functions O
on O
the O
basis O
of O
a O
shared O
phylogenetic O
profiles O
. O

Moreover O
this O
approach O
is O
useful O
to O
identify O
putative O
laterally O
transferred O
elements O
on O
the O
basis O
of O
their O
presence O
on O
distantly O
phylogenetically O
related O
bacteria O
. O

Results O

Vibrionaceae B
ORFs O
were O
aligned O
against O
all O
the O
available O
bacterial O
proteomes O
. O

Phylogenetic O
profile O
is O
defined O
as O
an O
array O
of O
distances O
, O
based O
on O
aminoacid O
substitution O
matrixes O
, O
from O
single O
genes O
to O
all O
their O
orthologues O
. O

Final O
phylogenetic O
profiles O
, O
derived O
from O
non O
- O
redundant O
list O
of O
all O
ORFs O
, O
was O
defined O
as O
the O
median O
of O
all O
the O
profiles O
belonging O
to O
the O
cluster O
. O

The O
resulting O
phylogenetic O
profiles O
matrix O
contains O
gene O
clusters O
on O
the O
rows O
and O
organisms O
on O
the O
columns O
. O

Cluster O
analysis O
identified O
groups O
of O
" O
core O
genes O
" O
with O
a O
widespread O
high O
similarity O
across O
all O
the O
organisms O
and O
several O
clusters O
that O
contain O
genes O
homologous O
only O
to O
a O
limited O
set O
of O
organisms O
. O

On O
each O
of O
these O
clusters O
, O
COG O
class O
enrichment O
has O
been O
calculated O
. O

The O
analysis O
reveals O
that O
clusters O
of O
core O
genes O
have O
the O
highest O
number O
of O
enriched O
classes O
, O
while O
the O
others O
are O
enriched O
just O
for O
few O
of O
them O
like O
DNA O
replication O
, O
recombination O
and O
repair O
. O

Conclusion O

We O
found O
that O
mobile O
elements O
have O
heterogeneous O
profiles O
not O
only O
across O
the O
entire O
set O
of O
organisms O
, O
but O
also O
within O
Vibrionaceae B
; O
this O
confirms O
their O
great O
influence O
on O
bacteria O
evolution O
even O
inside O
the O
same O
family O
. O

Furthermore O
, O
several O
hypothetical O
proteins O
highly O
correlate O
with O
mobile O
elements O
profiles O
suggesting O
a O
possible O
horizontal O
transfer O
mechanism O
for O
the O
evolution O
of O
these O
genes O
. O

Finally O
, O
we O
suggested O
the O
putative O
role O
of O
some O
ORFs O
having O
an O
unknown O
function O
on O
the O
basis O
of O
their O
phylogenetic O
profile O
similarity O
to O
well O
characterized O
genes O
. O

Background O

Over O
the O
past O
ten O
years O
, O
a O
great O
number O
of O
microbial O
genomes O
have O
been O
sequenced O
covering O
a O
wide O
representation O
of O
prokaryots O
as O
well O
as O
multiple O
strains O
of O
some O
species O
. O

The O
study O
of O
these O
genomes O
both O
by O
computational O
and O
experimental O
approaches O
has O
highly O
improved O
our O
understanding O
on O
physiology O
, O
phylogenetic O
relationship O
and O
pathogenicity O
of O
many O
organisms O
. O

Furthermore O
, O
it O
has O
provided O
new O
knowledge O
on O
microbial O
genome O
evolution O
, O
revealing O
a O
gene O
core O
shared O
by O
the O
great O
majority O
of O
bacteria O
, O
genes O
characteristic O
of O
particular O
groups O
and O
" O
novel O
" O
genes O
that O
possibly O
originated O
by O
lateral O
gene O
transfer O
from O
some O
unknown O
source O
. O

Analysis O
performed O
on O
closely O
related O
genomes O
revealed O
that O
a O
substantial O
fraction O
of O
genes O
in O
any O
genome O
seem O
to O
be O
strain O
specific O
. O

These O
genes O
might O
sometime O
arise O
by O
gene O
duplication O
followed O
by O
a O
rapid O
divergence O
, O
or O
by O
lineage O
- O
specific O
loss O
of O
genes O
in O
one O
strain O
, O
resulting O
in O
a O
unique O
gene O
in O
other O
strains O
. O

However O
, O
there O
are O
several O
lines O
of O
evidence O
indicating O
that O
lateral O
gene O
transfer O
may O
be O
the O
main O
mechanism O
to O
acquire O
novel O
genes O
. O

Indeed O
, O
this O
could O
be O
one O
of O
the O
main O
forces O
driving O
bacterial O
adaptation O
and O
evolution O
. O

Phage O
DNA O
is O
thought O
to O
be O
one O
of O
the O
main O
vectors O
for O
lateral O
gene O
transfer O
among O
bacteria O
[ O
1 O
] O
and O
many O
virulence O
factors O
from O
bacterial O
pathogen O
are O
phage O
encoded O
[ O
2 O
] O
. O

For O
example O
, O
the O
genes O
for O
CT O
, O
the O
most O
important O
virulence O
factor O
of O
V B
. I
cholerae I
, O
are O
encoded O
in O
the O
genome O
of O
phage O
CTX O
phi O
, O
integrated O
in O
the O
bacterial O
chromosome O
1 O
. O

Since O
lateral O
gene O
transfer O
plays O
a O
relevant O
role O
in O
bacterial O
evolution O
, O
the O
reconstruction O
of O
phylogeny O
is O
very O
complex O
and O
phylogenetic O
trees O
built O
by O
standard O
sequence O
analysis O
may O
not O
lead O
to O
a O
reliable O
picture O
of O
the O
evolutionary O
history O
. O

In O
fact O
, O
alternative O
trees O
can O
be O
obtained O
when O
different O
proteins O
are O
considered O
. O

For O
many O
aspects O
the O
classification O
of O
bacteria O
on O
the O
basis O
of O
their O
global O
gene O
content O
may O
give O
a O
better O
description O
of O
their O
evolutionary O
history O
. O

This O
may O
be O
particularly O
important O
when O
bacteria O
of O
the O
same O
group O
are O
compared O
, O
since O
newly O
acquired O
genes O
could O
be O
relevant O
to O
confer O
peculiar O
features O
that O
allows O
the O
exploitation O
of O
different O
ecological O
niches O
. O

In O
this O
study O
we O
propose O
a O
bioinformatic O
procedure O
to O
investigate O
bacterial O
genome O
evolution O
, O
taking O
into O
account O
the O
global O
gene O
content O
, O
as O
well O
as O
sequence O
similarity O
. O

We O
based O
our O
analysis O
on O
modified O
phylogenetic O
profiles O
[ O
3 O
] O
; O
however O
, O
we O
do O
not O
consider O
only O
the O
presence O
/ O
absence O
of O
orthologue O
genes O
, O
but O
also O
their O
distance O
, O
based O
on O
a O
substitution O
matrix O
. O

A O
phylogenetic O
profile O
is O
a O
non O
- O
sequence O
- O
homology O
- O
based O
method O
developed O
to O
infer O
a O
possible O
functional O
relationship O
between O
genes O
. O

It O
is O
based O
on O
the O
idea O
that O
proteins O
that O
are O
involved O
in O
the O
same O
metabolic O
pathway O
or O
structural O
complex O
are O
likely O
to O
evolve O
in O
a O
correlate O
fashion O
and O
during O
evolution O
appear O
phylogenetically O
linked O
, O
showing O
a O
tendency O
to O
be O
either O
preserved O
or O
eliminated O
as O
a O
whole O
. O

Therefore O
, O
genes O
showing O
similar O
phylogenetic O
profiles O
are O
likely O
to O
be O
functionally O
related O
. O

We O
extended O
the O
use O
of O
phylogenetic O
profiles O
to O
produce O
an O
evolutionary O
tree O
based O
on O
a O
hierarchical O
clusterization O
of O
organisms O
with O
similar O
phylogenetic O
profiles O
. O

For O
this O
study O
we O
took O
the O
whole O
gene O
dataset O
of O
320 O
prokaryotic O
genomes O
, O
however O
, O
we O
limited O
the O
analysis O
to O
the O
orthologous O
groups O
that O
are O
present O
in O
at O
least O
one O
of O
the O
14 O
considered O
species O
of O
the O
Vibrionaceae B
family O
. O

These O
bacteria O
belong O
to O
the O
Gammaproteobacteria O
group O
and O
are O
highly O
abundant O
in O
aquatic O
environment O
, O
they O
strongly O
influence O
nutrient O
cycling O
and O
various O
species O
are O
also O
devastating O
pathogens O
. O

Since O
we O
focused O
our O
analysis O
on O
this O
particular O
group O
, O
the O
aim O
of O
this O
study O
is O
not O
the O
construction O
of O
a O
global O
evolutionary O
tree O
, O
but O
rather O
a O
Vibrionaceae O
perspective O
of O
bacterial O
diversity O
, O
based O
on O
phylogenetic O
profiles O
. O

Results O
and O
discussion O

Phylogenetic O
matrix O

The O
analysis O
was O
performed O
on O
14 O
bacteria O
belonging O
to O
the O
Vibrionaceae B
family O
( O
Table O
1 O
) O
. O

The O
redundant O
list O
of O
Vibrionaceae B
ORFs O
was O
clustered O
to O
reduce O
the O
number O
of O
proteins O
to O
analyze O
and O
the O
phylogenetic O
profile O
for O
each O
cluster O
was O
calculated O
as O
described O
in O
the O
Method O
section O
. O

Many O
authors O
proposed O
and O
successfully O
applied O
different O
measure O
methods O
to O
calculate O
the O
phylogenetic O
profile O
values O
. O

Pellegrini O
et O
al O
. O

[ O
3 O
] O
firstly O
proposed O
a O
phylogenetic O
profile O
described O
as O
a O
string O
of O
bits O
, O
each O
bit O
representing O
the O
absence O
or O
presence O
of O
an O
homologous O
gene O
in O
a O
given O
genome O
. O

This O
method O
lacks O
a O
weighting O
procedure O
, O
giving O
the O
same O
weight O
( O
value O
1 O
) O
to O
all O
the O
sequences O
that O
are O
considered O
homologous O
given O
a O
similarity O
threshold O
. O

Enault O
and O
colleagues O
proposed O
an O
improved O
phylogenetic O
profile O
based O
on O
a O
normalized O
Blastp O
bit O
score O
[ O
4 O
] O
. O

This O
method O
, O
compared O
to O
the O
approach O
implemented O
by O
Pellegrini O
, O
allows O
weighting O
each O
point O
of O
the O
profile O
proportionally O
to O
the O
length O
and O
the O
quality O
of O
the O
alignment O
. O

Jingchun O
and O
colleagues O
optimized O
the O
phylogenetic O
profiles O
method O
by O
integrating O
phylogenetic O
relationships O
among O
reference O
organisms O
and O
sequence O
homology O
information O
, O
based O
on O
E O
- O
value O
score O
, O
to O
improve O
prediction O
accuracy O
[ O
5 O
] O
. O

The O
measure O
index O
I O
proposed O
in O
this O
work O
is O
similar O
to O
the O
others O
described O
above O
, O
taking O
into O
account O
both O
the O
quality O
and O
the O
length O
of O
the O
alignment O
using O
a O
substitution O
matrix O
. O

Moreover O
our O
approach O
considers O
also O
the O
total O
length O
of O
the O
sequences O
, O
penalizing O
good O
alignments O
occurring O
between O
ORFs O
having O
different O
lengths O
and O
taking O
into O
consideration O
that O
ORFs O
could O
differentiate O
mainly O
for O
the O
presence O
of O
functional O
domains O
. O

The O
final O
phylogenetic O
profile O
for O
each O
cluster O
was O
defined O
as O
the O
median O
of O
all O
the O
profiles O
belonging O
to O
the O
cluster O
, O
named O
" O
meta O
- O
profile O
" O
, O
which O
describes O
the O
profile O
of O
conserved O
ORFs O
belonging O
to O
an O
entire O
family O
. O

Hierarchical O
cluster O
analysis O

A O
hierarchical O
cluster O
analysis O
was O
performed O
on O
the O
entire O
phylogenetic O
profile O
matrix O
and O
it O
was O
calculated O
a O
statistical O
support O
based O
on O
bootstrap O
method O
for O
the O
nodes O
of O
the O
columns O
tree O
( O
Fig O
1 O
) O
. O

The O
branch O
tree O
colors O
represent O
the O
bootstrap O
percentage O
support O
. O

This O
constitutes O
a O
phylogenetic O
tree O
based O
on O
gene O
content O
using O
Vibrionaceae B
ORFs O
as O
a O
reference O
. O

Genomes O
belonging O
to O
the O
same O
taxonomic O
group O
tend O
to O
cluster O
together O
and O
the O
Vibrionaceae B
species O
are O
closely O
related O
. O

As O
expected O
, O
according O
to O
the O
Vibrionaceae B
branch O
lengths O
it O
is O
evident O
that O
variability O
within O
this O
group O
is O
higher O
compared O
to O
the O
other O
groups O
. O

The O
dataset O
used O
for O
phylogenetic O
matrix O
calculation O
is O
indeed O
composed O
by O
Vibrionaceae B
ORFs O
. O

This O
implies O
that O
the O
similarity O
measures O
between O
these O
ORFs O
and O
the O
corresponding O
orthologues O
will O
be O
nearly O
zero O
in O
most O
of O
the O
other O
species O
and O
significantly O
higher O
in O
the O
Vibrionaceae O
family O
, O
increasing O
the O
variability O
into O
this O
group O
. O

Moreover O
the O
average O
percentage O
of O
clusters O
shared O
by O
the O
Vibrionaceae B
members O
is O
only O
47 O
. O
5 O
% O
( O
average O
number O
of O
shared O
clusters O
divided O
by O
the O
total O
number O
of O
clusters O
) O
that O
again O
indicates O
a O
high O
variability O
inside O
this O
family O
. O

It O
is O
also O
interesting O
to O
note O
that O
organisms O
belonging O
to O
the O
same O
or O
closely O
related O
taxa O
split O
into O
different O
subgroups O
. O

This O
highlights O
the O
existence O
of O
a O
high O
variability O
among O
lineages O
, O
due O
to O
genetic O
and O
evolutionary O
processes O
such O
as O
lateral O
gene O
transfer O
, O
concerted O
evolution O
and O
gene O
duplication O
[ O
6 O
] O
. O

In O
terms O
of O
gene O
content O
, O
the O
organisms O
more O
related O
to O
the O
Vibrionaceae B
belong O
to O
the O
gamma O
and O
beta O
proteobacteria O
. O

In O
particular O
Altermonadales O
, O
Enterobacteriales O
and O
Burkholderiales O
are O
closely O
related O
to O
Vibrionaceae B
, O
and O
share O
the O
higher O
number O
of O
cluster O
of O
genes O
( O
average O
percentage O
of O
20 O
% O
) O
. O

As O
expected O
, O
Archea O
are O
the O
most O
distant O
group O
sharing O
just O
3 O
. O
8 O
% O
of O
clusters O
. O

Clusters O
and O
genes O
distribution O
, O
as O
shown O
in O
Fig O
2 O
, O
reveals O
that O
the O
number O
of O
clusters O
and O
genes O
shared O
by O
the O
organisms O
decreases O
as O
the O
number O
of O
organisms O
considered O
increases O
. O

The O
analysis O
was O
performed O
considering O
for O
each O
cluster O
profile O
the O
number O
of O
organisms O
sharing O
the O
same O
numbers O
of O
clusters O
( O
and O
genes O
) O
. O

The O
majority O
of O
gene O
cluster O
groups O
no O
more O
than O
21 O
species O
on O
a O
total O
of O
320 O
. O

The O
highest O
blue O
spike O
corresponds O
to O
the O
higher O
number O
of O
genes O
shared O
by O
105 O
groups O
of O
14 O
organisms O
. O

Among O
these O
groups O
, O
as O
expected O
, O
Vibrionaceae B
are O
highly O
represented O
. O

Other O
species O
represented O
are O
Colwellia O
psychrerythraea O
34H O
and O
Shewanella B
oneidensis I
, O
that O
belong O
to O
the O
Alteromonadales O
family O
. O

The O
cluster O
analysis O
performed O
on O
genes O
is O
shown O
in O
Fig O
. O

3 O
. O

From O
now O
on O
, O
to O
avoid O
confusing O
interpretation O
between O
clusters O
derived O
from O
the O
cluster O
analysis O
and O
cluster O
derived O
from O
the O
ORFs O
clustering O
we O
will O
use O
the O
term O
" O
gene O
" O
in O
place O
of O
cluster O
of O
ORFs O
. O

The O
different O
gradient O
of O
color O
, O
from O
bright O
to O
dark O
red O
, O
represents O
decreasing O
similarity O
values O
. O

The O
cluster O
analysis O
allows O
the O
detection O
of O
three O
main O
groups O
of O
genes O
. O

The O
first O
one O
( O
Fig O
3 O
, O
panel O
B O
) O
contains O
the O
most O
conserved O
and O
established O
genes O
shared O
almost O
by O
all O
the O
organisms O
. O

These O
core O
genes O
can O
be O
defined O
as O
the O
set O
of O
all O
genes O
shared O
as O
orthologous O
by O
all O
members O
of O
an O
evolutionary O
coherent O
group O
. O

In O
our O
analysis O
we O
identify O
four O
clusters O
, O
for O
a O
total O
of O
145 O
genes O
, O
shared O
by O
all O
the O
320 O
organisms O
. O

The O
ORFs O
belonging O
to O
these O
clusters O
are O
predicted O
to O
codify O
for O
the O
ATP O
binding O
subunit O
of O
ABC O
transporters O
( O
annotated O
as O
ABC O
- O
type O
polar O
amino O
acid O
transport O
system O
, O
ABC O
- O
type O
antimicrobial O
peptide O
transport O
system O
, O
ABC O
- O
type O
histidine O
transport O
system O
and O
ABC O
- O
type O
transport O
system O
involved O
in O
lysophospholipase O
L1 O
biosynthesis O
) O
. O

This O
finding O
is O
surprising O
since O
this O
is O
the O
first O
report O
where O
these O
ORFs O
are O
assigned O
to O
the O
core O
genes O
. O

Anyway O
two O
different O
explanations O
can O
be O
traced O
. O

First O
, O
it O
is O
known O
that O
the O
ABC O
transporters O
represent O
an O
essential O
transport O
system O
in O
the O
prokaryotes O
and O
that O
their O
ATP O
binding O
subunits O
are O
apparently O
overrepresented O
compared O
to O
the O
other O
two O
subunits O
( O
ligand O
binding O
and O
permease O
subunit O
) O
in O
all O
genomes O
sequenced O
thus O
far O
[ O
7 O
] O
. O

Second O
, O
one O
organism O
, O
Buchnera B
aphidicola I
, O
presents O
these O
genes O
with O
a O
similarity O
just O
below O
the O
cut O
- O
off O
used O
for O
the O
analysis O
, O
but O
they O
have O
been O
considered O
since O
it O
is O
well O
known O
that O
in O
this O
mutualistic O
endosymbiont O
the O
accelerated O
evolution O
and O
AT O
bias O
affect O
all O
its O
genes O
, O
including O
the O
16S O
rRNA O
[ O
8 O
, O
9 O
] O
. O

The O
dataset O
used O
for O
the O
analysis O
includes O
genomes O
in O
draft O
quality O
( O
Vibrio B
cholerae I
0395 O
, O
Vibrio B
cholerae I
MO10 O
, O
Vibrio B
cholerae I
RC385 O
, O
Vibrio B
cholerae I
V51 O
, O
Vibrio B
cholerae I
V52 O
, O
Photobacterium B
profundum O
3TCK O
, O
Vibrio O
MED222 O
, O
Vibrio B
splendidus12B01 I
) O
. O

Wrong O
ORFs O
prediction O
or O
missing O
genes O
due O
to O
incomplete O
genome O
sequences O
can O
explain O
the O
low O
number O
of O
core O
genes O
identified O
. O

To O
avoid O
such O
problems O
we O
repeated O
the O
analysis O
excluding O
the O
draft O
genomes O
and O
thus O
considering O
312 O
genomes O
. O

The O
results O
, O
reported O
in O
Table O
2 O
, O
show O
an O
increased O
number O
of O
the O
core O
genes O
and O
in O
particular O
ribosomal O
proteins O
and O
tRNA O
synthetase O
, O
as O
reported O
by O
Charlesbois O
and O
Doolittle O
[ O
10 O
] O
. O

This O
could O
be O
considered O
as O
a O
sort O
of O
" O
minimal O
genome O
" O
containing O
the O
group O
of O
genes O
that O
are O
necessary O
to O
maintain O
a O
free O
- O
living O
organism O
. O

The O
low O
number O
of O
genes O
shared O
by O
all O
the O
organisms O
can O
be O
due O
to O
many O
factors O
. O

First O
of O
all O
we O
used O
the O
Vibrionaceae B
ORFs O
as O
a O
reference O
, O
limiting O
the O
number O
of O
genes O
we O
were O
able O
to O
identify O
. O

It O
was O
further O
demonstrated O
that O
the O
core O
gene O
size O
decreases O
as O
more O
genome O
sequences O
are O
analyzed O
[ O
10 O
] O
. O

Genes O
that O
are O
considered O
to O
belong O
to O
the O
core O
set O
when O
close O
organisms O
are O
compared O
, O
are O
classified O
as O
flexible O
genes O
when O
distantly O
related O
genomes O
are O
analyzed O
[ O
6 O
] O
. O

Finally O
, O
genes O
within O
core O
genomes O
might O
be O
transferred O
or O
replaced O
, O
introducing O
new O
versions O
of O
existing O
genes O
into O
genomes O
. O

Such O
transfers O
can O
replace O
even O
highly O
conserved O
genes O
by O
non O
- O
homologous O
counterparts O
but O
the O
advantages O
provided O
are O
difficult O
to O
explain O
. O

It O
is O
also O
to O
take O
into O
consideration O
that O
many O
symbiotic O
and O
parasitic O
bacteria O
undergo O
a O
reduction O
of O
their O
genomes O
, O
loosing O
many O
genes O
required O
by O
free O
- O
living O
cell O
. O

The O
second O
group O
( O
Fig O
3 O
, O
panel O
C O
) O
represents O
genes O
shared O
mainly O
among O
Vibrionaceae B
and O
other O
gamma O
proteobacteria O
( O
as O
Altermonadales O
, O
Burkholderiales O
and O
Enterobactidiales O
) O
. O

Finally O
, O
the O
third O
group O
( O
Fig O
. O
3 O
, O
panel O
D O
) O
is O
composed O
by O
genes O
that O
are O
mainly O
specific O
to O
the O
Vibrionaceae B
. O

k O
- O
mean O
cluster O
analysis O
and O
cluster O
enrichment O

We O
performed O
a O
k O
- O
means O
cluster O
analysis O
, O
setting O
the O
k O
value O
to O
14 O
. O

As O
shown O
in O
Fig O
. O

4 O
, O
the O
clusters O
3 O
, O
4 O
, O
11 O
, O
13 O
and O
14 O
contain O
the O
higher O
percentage O
of O
genes O
, O
accounting O
for O
more O
that O
50 O
% O
of O
the O
total O
genes O
, O
while O
clusters O
9 O
and O
10 O
contain O
the O
lower O
number O
of O
ORFs O
( O
3 O
% O
of O
genes O
) O
. O

The O
variance O
in O
each O
k O
- O
means O
cluster O
is O
very O
low O
( O
Fig O
. O
4 O
) O
, O
meaning O
that O
the O
clusters O
contain O
genes O
with O
compact O
and O
similar O
profiles O
. O

As O
described O
in O
Fig O
. O

4 O
, O
the O
majority O
of O
the O
clusters O
( O
1 O
, O
2 O
, O
3 O
, O
4 O
, O
5 O
, O
6 O
, O
7 O
, O
9 O
, O
12 O
, O
13 O
) O
contains O
genes O
with O
a O
similar O
profile O
, O
with O
the O
average O
values O
( O
red O
line O
) O
near O
zero O
, O
except O
for O
the O
presence O
of O
some O
spikes O
correspondent O
to O
an O
increasing O
similarity O
with O
some O
isolated O
organisms O
. O

As O
shown O
in O
Table O
3 O
, O
clusters O
1 O
, O
3 O
, O
4 O
, O
5 O
, O
and O
9 O
contains O
genes O
that O
have O
a O
high O
similarity O
in O
a O
small O
subset O
of O
organisms O
. O

The O
majority O
of O
these O
ORFs O
are O
annotated O
as O
hypothetical O
proteins O
or O
phage O
related O
proteins O
. O

Clusters O
8 O
, O
10 O
and O
14 O
present O
genes O
shared O
among O
almost O
all O
the O
organisms O
. O

In O
particular O
cluster O
10 O
is O
composed O
by O
the O
core O
genes O
described O
before O
having O
an O
high O
value O
of O
similarity O
widespread O
among O
all O
the O
organisms O
; O
cluster O
8 O
contains O
genes O
shared O
mainly O
by O
gamma O
proteobacteria O
and O
cluster O
14 O
is O
composed O
of O
genes O
in O
common O
between O
Vibrionaceae B
and O
Enterobacteriaceae O
. O

A O
functional O
annotation O
has O
been O
performed O
on O
each O
gene O
cluster O
using O
COG O
( O
Cluster O
of O
Orthologous O
Genes O
) O
, O
KEGG O
pathway O
map O
and O
GO O
databases O
. O

For O
each O
k O
- O
mean O
cluster O
the O
enrichment O
probability O
with O
respect O
to O
the O
total O
number O
of O
clusters O
has O
been O
obtained O
with O
the O
hypergeometric O
distribution O
. O

Fig O
. O

5 O
shows O
COG O
enrichment O
results O
for O
each O
cluster O
. O

As O
expected O
clusters O
represented O
by O
conserved O
genes O
( O
cluster O
8 O
, O
10 O
and O
14 O
) O
have O
the O
higher O
number O
of O
enriched O
COG O
codes O
, O
while O
cluster O
specific O
of O
few O
organisms O
are O
characterized O
by O
a O
small O
number O
of O
enriched O
COGs O
. O

The O
majority O
of O
clusters O
presents O
COG O
codes O
enrichment O
for O
S O
( O
function O
unknown O
) O
, O
R O
( O
poorly O
characterized O
) O
and O
- O
( O
absence O
of O
COG O
code O
) O
categories O
. O

This O
is O
due O
to O
the O
large O
abundance O
of O
unknown O
and O
hypothetical O
proteins O
presents O
in O
the O
Vibrionaceae B
proteomes O
. O

It O
is O
worth O
noting O
that O
cluster O
3 O
, O
mainly O
represented O
by O
Photobacterium B
profundum I
SS9 O
ORFs O
, O
is O
enriched O
only O
by O
C O
( O
Energy O
production O
and O
conversion O
) O
, O
L O
( O
DNA O
replication O
, O
recombination O
and O
repair O
) O
and O
M O
( O
Cell O
envelope O
biogenesis O
, O
outer O
membrane O
) O
. O

Probably O
the O
L O
class O
overrepresentation O
is O
determined O
by O
the O
high O
number O
of O
transposons O
that O
are O
present O
in O
the O
SS9 O
genome O
[ O
11 O
] O
. O

The O
role O
played O
by O
these O
transposable O
elements O
in O
the O
survival O
of O
this O
deep O
- O
sea O
bacterium O
it O
is O
still O
a O
matter O
of O
debate O
[ O
12 O
] O
. O

In O
addition O
V B
. I
vulnificus I
YJ016 O
and O
V B
. I
vulnificus I
CMCP6 O
( O
cluster O
13 O
) O
seem O
to O
share O
genes O
belonging O
to O
the O
enriched O
COG O
classes O
K O
( O
Transcription O
) O
, O
L O
and O
T O
( O
Signal O
transduction O
mechanisms O
) O
. O

It O
was O
previously O
reported O
an O
enrichment O
in O
genes O
belonging O
to O
the O
transcription O
class O
in O
the O
genome O
of O
V B
. I
vulnificus I
respect O
to O
the O
V B
. I
cholerae I
genome O
[ O
13 O
] O
. O

This O
class O
is O
clearly O
related O
to O
the O
T O
class O
and O
seems O
to O
indicate O
that O
this O
organism O
is O
able O
to O
receive O
and O
translate O
in O
a O
transcriptional O
response O
specific O
environmental O
signals O
. O

Despite O
this O
, O
the O
large O
majority O
of O
the O
genes O
in O
clusters O
3 O
and O
13 O
lacks O
COG O
annotation O
. O

Cluster O
7 O
, O
as O
shown O
in O
Table O
3 O
, O
accounts O
organisms O
with O
large O
genome O
size O
( O
see O
Table O
1 O
) O
. O

This O
can O
explain O
the O
fact O
the O
this O
cluster O
contains O
almost O
all O
the O
COG O
class O
enriched O
and O
suggests O
a O
more O
complex O
and O
flexible O
life O
- O
style O
of O
these O
organisms O
compared O
to O
the O
other O
Vibrionaceae B
members O
. O

KEGG O
annotation O
is O
limited O
to O
metabolic O
or O
structural O
complex O
network O
and O
so O
a O
reduced O
number O
of O
genes O
have O
a O
KEGG O
entry O
. O

This O
causes O
the O
presence O
of O
clusters O
without O
enriched O
map O
( O
cluster O
2 O
- O
5 O
, O
7 O
, O
13 O
, O
see O
Fig O
5 O
) O
. O

Also O
in O
this O
case O
, O
the O
clusters O
presenting O
the O
higher O
number O
of O
significant O
KEGG O
map O
are O
those O
containing O
the O
conserved O
genes O
. O

The O
most O
enriched O
KEGG O
clusters O
are O
cluster O
14 O
, O
10 O
and O
8 O
accounting O
for O
the O
majority O
of O
the O
metabolic O
pathways O
. O

Cluster O
1 O
is O
enriched O
for O
map3080 O
( O
type O
IV O
secretion O
system O
) O
. O

In O
fact O
V B
. I
fischeri I
genome O
contains O
10 O
separate O
pilus O
gene O
clusters O
, O
including O
eight O
type O
- O
IV O
pilus O
loci O
. O

The O
presence O
of O
multiple O
pilus O
gene O
clusters O
suggests O
that O
different O
pili O
may O
be O
expressed O
in O
different O
environments O
or O
during O
multiple O
stages O
of O
its O
development O
as O
a O
symbiont O
[ O
14 O
] O
. O

Cluster O
11 O
is O
enriched O
for O
map3090 O
( O
type O
II O
secretion O
system O
) O
. O

The O
type O
II O
pathway O
is O
conserved O
among O
gram O
- O
negative O
bacteria O
, O
including O
many O
pathogens O
, O
and O
secretes O
a O
variety O
of O
virulence O
factors O
and O
degradative O
enzymes O
[ O
15 O
] O
. O

Cluster O
9 O
is O
enriched O
for O
map O
00860 O
( O
Porphyrin O
and O
chlorophyll O
metabolism O
) O
. O

These O
genes O
are O
involved O
in O
the O
cobalamin O
( O
coenzyme O
B12 O
) O
biosynthetic O
pathway O
[ O
16 O
] O
. O

Some O
organisms O
, O
such O
as O
Salmonella B
typhimurium I
and O
Klebsiella B
pneumoniae I
, O
can O
synthesize O
cobalamin O
de O
novo O
[ O
17 O
] O
, O
while O
E B
. I
coli I
and O
large O
part O
of O
the O
Vibrionaceae O
perform O
cobalamin O
biosynthesis O
only O
when O
provided O
with O
cobinamide O
. O

It O
is O
interesting O
to O
observe O
that O
the O
genes O
belonging O
to O
the O
de O
novo O
pathway O
are O
only O
shared O
by O
Archea O
, O
some O
other O
organisms O
like O
Salmonella O
, O
Pseudomonas O
and O
Vibrio O
MED222 O
. O

Finally O
cluster O
6 O
is O
enriched O
by O
map2010 O
( O
ABC O
transporter O
) O
, O
map2020 O
( O
two O
- O
component O
system O
) O
, O
map2030 O
and O
map2031 O
( O
bacterial O
chemotaxis O
) O
, O
map2040 O
( O
Flagellar O
assembly O
) O
and O
map3090 O
( O
type O
II O
secretion O
system O
) O
. O

This O
cluster O
contains O
genes O
shared O
with O
a O
high O
similarity O
by O
all O
Vibrio O
and O
with O
a O
lower O
similarity O
with O
Photobacterium B
profundum I
species O
. O

Among O
the O
Vibrio O
species O
the O
organisms O
showing O
the O
highest O
similarity O
( O
Tab O
. O
3 O
) O
are O
V B
. I
cholerae I
strains O
. O

Vibrionaceae B
specific O
genes O

We O
identify O
1940 O
clusters O
specific O
to O
the O
Vibrionaceae B
. O

All O
the O
Vibrionaceae B
considered O
in O
the O
analysis O
share O
108 O
clusters O
. O

Among O
these O
genes O
we O
identify O
ToxR O
and O
ToxS O
genes O
. O

ToxR O
gene O
encodes O
a O
transmembrane O
regulatory O
protein O
firstly O
identified O
in O
V B
. I
cholerae I
, O
in O
which O
it O
co O
- O
ordinates O
many O
virulence O
factors O
in O
response O
to O
several O
environmental O
parameters O
[ O
18 O
] O
. O

V B
. I
cholerae I
ToxR O
activity O
is O
enhanced O
by O
a O
second O
transmembrane O
protein O
, O
ToxS O
, O
encoded O
downstream O
toxR O
[ O
19 O
] O
. O

This O
family O
of O
proteins O
is O
involved O
in O
response O
to O
temperature O
, O
pH O
, O
osmolarity O
and O
in O
Photobacterium B
profundum I
SS9 O
, O
a O
piezophilic O
bacterium O
, O
to O
hydrostatic O
pressure O
[ O
20 O
] O
. O

The O
widespread O
presence O
of O
these O
genes O
among O
the O
Vibrionaceae B
suggests O
their O
importance O
in O
regulatory O
mechanisms O
. O

We O
identify O
two O
other O
noteworthy O
groups O
of O
genes O
composed O
by O
257 O
and O
160 O
genes O
respectively O
shared O
just O
by O
two O
strains O
, O
mainly O
annotated O
as O
" O
hypothetical O
protein O
" O
. O

The O
first O
group O
of O
genes O
is O
shared O
between O
Photobacterium B
profundum I
SS9 O
and O
Photobacterium B
profundum I
3TCK O
, O
while O
the O
second O
is O
shared O
between O
V B
. I
vulnificus I
CMCP6 O
and O
YJ016 O
. O

These O
strains O
are O
closely O
related O
and O
this O
explains O
the O
high O
number O
of O
shared O
genes O
; O
while O
, O
inside O
the O
Vibrionaceae B
family O
, O
the O
number O
of O
specific O
shared O
genes O
highly O
decreases O
, O
showing O
a O
high O
inter O
- O
species O
variability O
( O
Fig O
. O
6 O
) O

Prophages O
and O
transposases O

Prophages O
recover O
different O
biological O
roles O
both O
as O
quantitatively O
important O
genetic O
elements O
of O
the O
bacterial O
chromosome O
, O
and O
as O
vectors O
of O
lateral O
gene O
transfer O
among O
bacteria O
, O
due O
to O
their O
characters O
of O
mobile O
DNA O
elements O
. O

Indeed O
, O
numerous O
virulence O
factors O
from O
bacterial O
pathogens O
are O
phage O
encoded O
. O

It O
was O
postulated O
that O
this O
role O
of O
prophages O
is O
not O
limited O
to O
pathogenic O
bacteria O
but O
some O
adaptations O
of O
nonpathogenic O
strains O
to O
their O
ecological O
niche O
might O
also O
be O
mediated O
by O
prophages O
acquisition O
[ O
21 O
] O
. O

To O
better O
understand O
the O
importance O
of O
mobile O
elements O
within O
Vibrionaceae B
family O
, O
we O
performed O
a O
hierarchical O
cluster O
analysis O
using O
gene O
profiles O
annotated O
as O
" O
phage O
protein O
" O
and O
" O
transposase O
" O
, O
for O
a O
total O
of O
172 O
clusters O
of O
genes O
( O
Fig O
7 O
) O
. O

We O
found O
that O
a O
high O
inter O
- O
strain O
genetic O
variability O
exists O
and O
phages O
and O
transposases O
are O
both O
shared O
by O
almost O
all O
Vibrionaceae B
, O
and O
specific O
to O
just O
some O
organisms O
. O

We O
identified O
five O
major O
clusters O
of O
mobile O
elements O
that O
are O
specific O
to O
a O
single O
organism O
. O

A O
group O
composed O
by O
26 O
clusters O
containing O
both O
transposase O
and O
phage O
proteins O
seem O
to O
be O
unique O
to O
V B
. I
splendiduds I
12B01 O
( O
Fig O
7 O
) O
. O

Another O
one O
composed O
by O
16 O
clusters O
is O
specific O
of O
V B
. I
vulnificus I
CMCP6 O
( O
Fig O
7 O
) O
while O
V B
. I
parahaemoliticus I
has O
a O
cluster O
of O
11 O
genes O
( O
Fig O
7 O
) O
. O

Moreover O
there O
is O
another O
group O
of O
transposases O
and O
phage O
genes O
shared O
mainly O
by O
V B
. I
cholerae I
0395 I
, O
Shewanella B
oneidensis I
and O
V B
. I
cholerae I
V51 I
( O
Fig O
7 O
) O
. O

Finally O
a O
big O
cluster O
of O
almost O
30 O
genes O
, O
all O
predicted O
to O
codify O
for O
transposases O
, O
was O
found O
in O
P B
. I
profundum I
SS9 O
genome O
( O
Fig O
. O
7 O
) O
. O

The O
high O
presence O
of O
transposases O
in O
this O
bacterium O
seems O
to O
correlate O
with O
its O
deep O
- O
sea O
habitat O
, O
a O
feature O
presumably O
shared O
with O
other O
deep O
- O
sea O
microorganisms O
[ O
12 O
] O
. O

As O
shown O
in O
Fig O
7 O
, O
many O
of O
the O
clusters O
well O
conserved O
in O
an O
organism O
, O
are O
partially O
shared O
with O
a O
low O
similarity O
by O
other O
organisms O
. O

This O
agrees O
with O
the O
idea O
that O
prophages O
are O
not O
maintained O
in O
the O
genome O
over O
a O
long O
period O
of O
time O
and O
part O
of O
their O
genes O
may O
be O
deleted O
from O
the O
chromosome O
. O

Moreover O
, O
microarray O
analysis O
and O
PCR O
scanning O
demonstrated O
that O
prophages O
are O
frequently O
strain O
specific O
within O
a O
given O
bacterial O
species O
[ O
22 O
- O
24 O
] O
. O

According O
to O
the O
modular O
theory O
of O
phage O
evolution O
, O
phage O
genomes O
are O
mosaics O
of O
modules O
, O
groups O
of O
genes O
functionally O
related O
, O
that O
are O
free O
to O
recombine O
in O
genetic O
exchanges O
between O
distinct O
phages O
infecting O
the O
same O
cell O
[ O
21 O
] O
. O

This O
can O
result O
in O
the O
occurrences O
of O
different O
part O
of O
phage O
distributed O
in O
far O
related O
genomes O
. O

Phylogenetic O
profile O
of O
some O
transposases O
is O
similar O
to O
the O
phage O
ones O
, O
suggesting O
a O
possible O
transfer O
mechanism O
phage O
- O
mediated O
for O
such O
mobile O
elements O
. O

Conclusion O

In O
this O
work O
we O
propose O
an O
improved O
phylogenetic O
profile O
analysis O
on O
14 O
Vibrionaceae B
genomes O
, O
to O
study O
this O
family O
on O
the O
basis O
of O
gene O
content O
. O

Using O
a O
phylogenetic O
profile O
for O
each O
cluster O
of O
genes O
defined O
as O
the O
median O
of O
all O
the O
profiles O
belonging O
to O
the O
cluster O
( O
meta O
- O
profile O
) O
we O
investigate O
the O
evolution O
of O
groups O
of O
ORFs O
belonging O
to O
the O
entire O
family O
. O

A O
two O
- O
way O
cluster O
analysis O
allows O
us O
to O
identify O
similarity O
structures O
on O
the O
entire O
phylogenetic O
matrix O
composed O
by O
8 O
, O
239 O
clusters O
of O
genes O
and O
320 O
organisms O
. O

The O
phylogenetic O
tree O
obtained O
with O
the O
cluster O
analysis O
does O
not O
reflect O
the O
global O
evolutionary O
tree O
because O
of O
the O
Vibrionaceae B
ORFs O
dataset O
used O
for O
the O
analysis O
, O
but O
rather O
can O
be O
considered O
as O
the O
Vibrionaceae B
perspective O
of O
bacterial O
diversity O
. O

The O
phylogenetic O
tree O
reflects O
the O
evolutionary O
processes O
that O
shape O
genomes O
, O
as O
lateral O
gene O
transfer O
, O
genes O
genesis O
and O
loss O
. O

In O
this O
context O
, O
the O
tree O
allows O
to O
group O
together O
genomes O
on O
the O
base O
of O
their O
global O
gene O
content O
. O

We O
found O
that O
genomes O
belonging O
to O
the O
same O
taxonomic O
group O
tend O
to O
cluster O
together O
and O
that O
Vibrionaceae B
species O
are O
closely O
related O
. O

Moreover O
organisms O
belonging O
to O
the O
same O
or O
closely O
related O
taxa O
split O
into O
different O
subgroups O
, O
confirming O
the O
existence O
of O
a O
high O
variability O
among O
lineages O
, O
due O
to O
genetic O
and O
evolutionary O
process O
such O
as O
lateral O
gene O
transfer O
, O
concerted O
evolution O
and O
genes O
duplication O
. O

On O
the O
other O
hand O
several O
groups O
of O
genes O
characterised O
by O
different O
homogeneous O
profiles O
have O
been O
identified O
. O

In O
particular O
we O
found O
, O
1 O
) O
a O
set O
of O
conserved O
genes O
( O
with O
a O
high O
similarity O
values O
across O
all O
organisms O
) O
that O
reflects O
the O
" O
minimal O
genome O
" O
composition O
defined O
in O
other O
previous O
works O
; O
2 O
) O
a O
set O
of O
genes O
mainly O
shared O
by O
Vibrionaceae B
and O
other O
Gamma O
proteobacteria O
and O
3 O
) O
genes O
specific O
to O
different O
sets O
of O
Vibrionaceae B
. O

Finally O
a O
further O
analysis O
on O
prophage O
and O
transposase O
has O
confirmed O
the O
high O
inter O
- O
strain O
genetic O
variability O
even O
among O
closely O
related O
species O
. O

The O
increasing O
number O
of O
genomes O
included O
in O
this O
type O
of O
analysis O
surely O
add O
new O
sorces O
of O
variability O
and O
noise O
, O
anyway O
we O
think O
that O
the O
use O
of O
meta O
- O
profiles O
can O
be O
useful O
for O
complexity O
reduction O
and O
data O
analysis O
to O
study O
global O
gene O
evolution O
. O

Methods O

Datasets O

The O
Vibrionaceae B
species O
used O
in O
this O
analysis O
were O
selected O
among O
the O
freely O
available O
complete O
and O
draft O
genome O
sequences O
. O

The O
proteomes O
of O
V B
. I
cholerae I
N16961 O
, O
V B
. I
parahaemolyticus I
, O
V B
. I
vulnificus I
YJ016 O
, O
V B
. I
vulnificus I
CMCP6 O
, O
V B
. I
fischeri I
ES114 O
, O
Photobacterium B
profundum I
SS9 O
were O
downloaded O
from O
the O
NCBI O
ftp O
site O
[ O
25 O
] O
. O

Protein O
sequences O
of O
Vibrio B
cholerae I
MO10 O
, O
Vibrio B
cholerae I
0395 O
, O
Vibrio B
cholerae I
RC385 O
, O
Vibrio B
cholerae I
V51 O
, O
Vibrio B
cholerae I
V52 O
were O
downloaded O
from O
the O
NCBI O
genome O
database O
, O
while O
sequences O
of O
Vibrio B
MED222 O
, O
Vibrio B
splendidus I
12B01 O
, O
Photobacterium B
profundum I
3TCK O
from O
the O
J O
. O

Craig O
Venter O
Institute O
web O
site O
. O

The O
320 O
complete O
genomes O
update O
at O
03 O
/ O
06 O
were O
downloaded O
from O
the O
NCBI O
ftp O
site O
. O

Similarity O
search O
and O
phylogenetic O
profile O
construction O

All O
the O
Vibrionaceae B
ORFs O
were O
merged O
generating O
a O
redundant O
list O
of O
59 O
, O
669 O
proteins O
and O
were O
compared O
to O
all O
open O
reading O
frame O
from O
320 O
bacterial O
and O
archeal O
genomes O
using O
Blastp O
. O

To O
determine O
the O
presence O
of O
an O
orthologous O
we O
used O
a O
combination O
of O
three O
different O
thresholds O
; O
a O
similarity O
value O
equal O
to O
or O
higher O
than O
30 O
% O
, O
an O
alignment O
length O
equal O
or O
higher O
than O
70 O
% O
and O
an O
Evalue O
score O
lower O
than O
or O
equal O
to O
e O
- O
6 O
. O

After O
determining O
the O
presence O
of O
an O
orthologous O
gene O
, O
we O
computed O
a O
similarity O
index O
I O
for O
each O
pair O
of O
orthologous O
( O
a O
point O
of O
the O
phylogenetic O
profile O
) O
as O
follow O
: O

I O
= O
SqsSqq O
. O
min O
( O
lq O
, O
ls O
) O
max O
( O
lq O
, O
ls O
) O

MathType O
@ O
MTEF O
@ O
5 O
@ O
5 O
@ O
+ O
= O
feaafiart1ev1aaatCvA O
= O
wiFfYdH8Gipec8Eeeu0x O
= O
OqFfea0dXdd9vqai O
= O
hGuQ8kuc9pgc9s8qqaq O
= O
dirpe0xb9q8qiLsFr0 O
= O
vr0 O
= O
vr0dc8meaabaqaciaaca O
@ O
532D O
@ O

where O
lq O
and O
ls O
are O
the O
query O
and O
subject O
length O
sequence O
respectively O
and O
Sqs O
is O
the O
similarity O
score O
between O
the O
query O
and O
the O
subject O
sequence O
. O

Sqs O
is O
defined O
as O
follow O
: O

Sqs O
= O
sum O
i O
= O
1M O
alpha O
Aqi O
, O
Asi O
+ O
GP O

MathType O
@ O
MTEF O
@ O
5 O
@ O
5 O
@ O
+ O
= O
feaafiart1ev1aaatCvA O
= O
wiFfYdH8Gipec8Eeeu0x O
= O
OqFfea0dXdd9vqai O
= O
hGuQ8kuc9pgc9s8qqaq O
= O
dirpe0xb9q8qiLsFr0 O
= O
vr0 O
= O
vr0dc8meaabaqaciaaca O
@ O
4620 O
@ O

where O
M O
is O
the O
match O
length O
between O
the O
query O
and O
subject O
sequence O
; O
Aqi O
and O
Asi O
respectively O
the O
query O
and O
subject O
amino O
acid O
in O
position O
i O
; O
alpha O
the O
BLOSUM O
substitution O
matrix O
value O
for O
amino O
acid O
pair O
Aqi O
, O
Asi O
and O
GP O
the O
gap O
penalty O
. O

GP O
is O
defined O
as O
follow O
: O

GP O
= O
GOP O
+ O
GEP O
( O
k O
- O
1 O
) O

where O
GOP O
is O
the O
Gap O
Open O
Penalty O
set O
to O
- O
11 O
, O
GEP O
the O
Gap O
Extension O
Penalty O
set O
to O
- O
1 O
and O
k O
the O
gap O
length O
. O

Sqq O
represents O
the O
score O
of O
the O
self O
- O
aligned O
query O
sequence O
. O

Sqs O
is O
always O
smaller O
than O
Sqq O
and O
the O
score O
S O
range O
between O
0 O
and O
1 O
. O

In O
order O
to O
take O
into O
account O
also O
the O
different O
sequence O
lengths O
, O
we O
multiplied O
the O
score O
S O
by O
the O
ratio O
between O
the O
minimum O
length O
between O
query O
and O
subject O
and O
the O
maximum O
length O
between O
query O
and O
subjects O
. O

In O
this O
way O
the O
total O
score O
is O
weighted O
on O
the O
base O
of O
the O
length O
, O
resulting O
in O
a O
lower O
similarity O
value O
if O
the O
lengths O
of O
the O
sequences O
are O
different O
. O

The O
phylogenetic O
profile O
for O
each O
ORF O
is O
an O
array O
of O
index O
I O
with O
length O
equal O
to O
the O
number O
of O
genomes O
considered O
( O
320 O
) O
. O

ORFs O
clustering O

The O
redundant O
list O
of O
59 O
, O
669 O
Vibrionaceae B
ORFs O
contained O
multiple O
copies O
of O
the O
same O
genes O
due O
to O
the O
presence O
of O
conserved O
genes O
in O
the O
considered O
genomes O
. O

In O
order O
to O
reduce O
the O
redundancy O
, O
we O
clustered O
proteins O
using O
a O
two O
- O
step O
approach O
. O

The O
first O
step O
is O
based O
on O
COG O
( O
Cluster O
of O
Othologous O
Genes O
) O
annotation O
. O

COG O
classifies O
conserved O
genes O
according O
to O
their O
homologous O
relationships O
. O

All O
the O
Vibrionaceae B
ORFs O
were O
annotated O
using O
COG O
clusters O
and O
proteins O
sharing O
the O
same O
COG O
code O
were O
considered O
belonging O
to O
the O
same O
cluster O
. O

In O
particular O
, O
the O
annotation O
process O
consists O
of O
a O
similarity O
search O
of O
all O
the O
ORFs O
against O
the O
COG O
proteins O
using O
blast O
and O
considering O
the O
best O
hit O
for O
each O
protein O
. O

43 O
, O
024 O
ORFs O
presented O
a O
similarity O
with O
a O
COG O
entry O
, O
producing O
2 O
, O
463 O
different O
clusters O
. O

In O
the O
second O
step O
, O
the O
remaining O
16 O
, O
645 O
ORFs O
without O
similarity O
with O
any O
COG O
entry O
were O
clustered O
using O
CD O
- O
HIT O
software O
[ O
26 O
] O
. O

CD O
- O
HIT O
program O
clusters O
protein O
sequence O
database O
at O
high O
sequence O
identity O
threshold O
and O
efficiently O
removes O
high O
sequence O
redundancy O
. O

This O
last O
clustering O
process O
produced O
9 O
, O
613 O
different O
groups O
of O
similar O
proteins O
. O

Finally O
from O
the O
12 O
, O
076 O
total O
clusters O
obtained O
by O
this O
methodology O
, O
those O
composed O
by O
ORFs O
that O
do O
not O
have O
any O
ortologous O
genes O
( O
with O
a O
phylogenetic O
profile O
composed O
by O
an O
array O
with O
all O
zero O
values O
except O
for O
one O
position O
match O
with O
itself O
) O
were O
eliminated O
, O
resulting O
in O
a O
dataset O
composed O
by O
8 O
, O
239 O
distinct O
clusters O
. O

The O
final O
phylogenetic O
profile O
for O
each O
cluster O
( O
meta O
- O
profile O
) O
was O
defined O
as O
the O
median O
of O
all O
the O
profiles O
belonging O
to O
the O
cluster O
. O

At O
the O
end O
of O
these O
procedures O
the O
final O
phylogenetic O
matrix O
was O
composed O
by O
8 O
, O
239 O
rows O
( O
cluster O
of O
genes O
) O
and O
320 O
columns O
( O
organisms O
) O
. O

In O
each O
cell O
the O
median O
of O
the O
index O
in O
the O
cluster O
was O
reported O
. O

Cluster O
analysis O

Several O
clustering O
techniques O
have O
been O
used O
to O
identify O
the O
similarity O
structure O
underneath O
our O
data O
. O

A O
k O
- O
means O
and O
a O
two O
- O
way O
hierarchical O
cluster O
analysis O
with O
Euclidean O
distance O
and O
complete O
linkage O
were O
performed O
on O
the O
phylogenetic O
matrix O
. O

The O
goal O
of O
a O
cluster O
analysis O
is O
to O
partition O
the O
elements O
into O
subsets O
without O
any O
constrains O
or O
a O
priori O
information O
, O
so O
that O
two O
criteria O
are O
satisfied O
: O
homogeneity O
, O
elements O
inside O
a O
cluster O
are O
highly O
similar O
to O
each O
other O
; O
and O
separation O
, O
elements O
from O
different O
clusters O
have O
low O
similarity O
to O
each O
other O
. O

The O
Figure O
of O
Merit O
( O
FOM O
) O
is O
a O
measure O
of O
fit O
of O
the O
expression O
patterns O
for O
the O
clusters O
produced O
by O
a O
particular O
algorithm O
that O
estimates O
the O
predictive O
power O
of O
a O
clustering O
algorithm O
. O

It O
is O
computed O
by O
removing O
each O
sample O
in O
turn O
from O
the O
data O
set O
, O
clustering O
genes O
based O
on O
the O
remaining O
data O
, O
and O
calculating O
the O
fit O
of O
the O
withheld O
sample O
to O
the O
clustering O
pattern O
obtained O
from O
the O
other O
samples O
. O

On O
our O
data O
FOM O
analysis O
identified O
the O
best O
number O
of O
k O
- O
means O
clusters O
between O
10 O
and O
15 O
. O

We O
decided O
to O
set O
k O
( O
in O
the O
k O
- O
means O
analysis O
) O
equal O
to O
14 O
. O

In O
each O
of O
these O
14 O
clusters O
subsequent O
hierarchical O
cluster O
analysis O
was O
performed O
with O
bootstrap O
cluster O
assessment O
. O

All O
the O
previous O
analyses O
were O
performed O
with O
TMEV O
software O
[ O
27 O
] O
, O
freely O
available O
at O
[ O
28 O
] O
. O

Enrichment O
categories O

Each O
cluster O
of O
genes O
has O
been O
annotated O
according O
to O
COG O
code O
, O
GO O
terms O
and O
KEGG O
pathway O
maps O
. O

Class O
enrichment O
( O
with O
respect O
to O
the O
entire O
matrix O
) O
has O
been O
calculated O
according O
to O
the O
hypergeometric O
distribution O
that O
was O
used O
to O
obtain O
the O
chance O
probability O
of O
observing O
the O
number O
of O
genes O
annotated O
with O
a O
particular O
COG O
, O
GO O
and O
KEGG O
category O
among O
the O
selected O
cluster O
. O

The O
probability O
P O
of O
observing O
at O
least O
k O
genes O
of O
a O
functional O
category O
within O
a O
group O
of O
n O
genes O
is O
given O
by O
: O

P O
= O
sum O
i O
= O
kn O
( O
fi O
) O
( O
g O
- O
fn O
- O
i O
) O
( O
gn O
) O

MathType O
@ O
MTEF O
@ O
5 O
@ O
5 O
@ O
+ O
= O
feaafiart1ev1aaatCvA O
= O
wiFfYdH8Gipec8Eeeu0x O
= O
OqFfea0dXdd9vqai O
= O
hGuQ8kuc9pgc9s8qqaq O
= O
dirpe0xb9q8qiLsFr0 O
= O
vr0 O
= O
vr0dc8meaabaqaciaaca O
@ O
47C6 O
@ O

where O
f O
is O
the O
total O
number O
of O
genes O
with O
the O
same O
category O
( O
in O
the O
matrix O
) O
and O
g O
is O
the O
total O
number O
of O
genes O
in O
our O
matrix O
. O

A O
secondary O
structural O
common O
core O
in O
the O
ribosomal O
ITS2 O
( O
internal O
transcribed O
spacer O
) O
of O

Culexspecies B
from O
diverse O
geographical O
locations O

Abstract O

In O
the O
present O
study O
, O
sequence O
and O
structural O
analysis O
of O
ITS2 O
region O
( O
the O
spacer O
segment O
between O
5 O
. O
8S O
and O
28S O
rRNA O
of O

mature O
rRNA O
sequences O
) O
of O
7 O
Culex B
species I
belonging O
to O
5 O
different O
geographical O
locations O
was O
carried O
out O
. O

Alignment O
of O
the O

ITS2 O
sequence O
from O
the O
7 O
species O
revealed O
8 O
homologous O
domains O
. O

Four O
species O
namely O
C O
. O
vishnui O
, O
C O
. O
annulus I
, O
C B
. I
pipiens I
, O

C O
. O
quiquefasciatusshowe O
high O
sequence O
( O
98 O
- O
100 O
% O
) O
and O
RNA O
secondary O
structure O
similarity O
. O

The O
ITS2 O
similarity O
among O
different O

species O
is O
high O
despite O
their O
varying O
geographical O
locations O
. O

Several O
common O
features O
of O
secondary O
structure O
are O
shared O
among O

these O
species O
, O
with O
some O
of O
them O
supported O
by O
compensatory O
changes O
, O
suggesting O
the O
significant O
role O
by O
ITS2 O
as O
an O
RNA O
domain O

during O
ribosome O
biogenesis O
. O

Background O

Culex B
mosquito O
species O
have O
been O
described O
from O
a O
wide O
range O
of O
environments O
and O
are O
involved O
in O
pathogen O
transmission O
from O

human B
to O
reservoirs O
and O
vice O
- O
versa O
. O

[ O
1 O
] O
Sequence O
similarity O
have O
been O

reported O
for O
ITS2 O
( O
the O
spacer O
segments O
between O
5 O
. O
8S O
and O
28S O
rRNA O
of O
mature O
rRNA O
sequences O
) O
from O
5 O
Culex B
species O
of O
diverse O

geographic O
locations O
. O

[ O
3 O
- O
4 O
- O

5 O
- O
9 O
- O

20 O
] O
The O
ITS O
spacers O
are O
versatile O
as O
genetic O
markers O
and O
have O
been O
used O
for O
the O
determination O
of O
taxonomic O

classification O
. O

[ O
17 O
] O
Recent O
functional O
analyses O
performed O
on O
yeast B
ribosomal O

RNA O
genes O
clearly O
show O
that O
the O
structural O
integrity O
of O
the O
transcribed O
spacer O
regions O
is O
an O
essential O
prerequisite O
for O
the O
correct O

processing O
of O
mature O
rRNA O
and O
for O
the O
biogenesis O
of O
active O
ribosomal O
subunits O
. O

[ O
2 O
- O

3 O
] O
The O
derivation O
of O
reliable O
secondary O
structure O
models O
for O
each O
transcribed O
spacer O

region O
would O
represent O
a O
major O
step O
towards O
a O
detailed O
understanding O
of O
their O
biological O
roles O
. O

Comparative O
sequence O
analysis O
provides O
a O
powerful O
tool O
for O
identifying O
biologically O
relevant O
folding O
patterns O
in O
RNA O
molecules O
. O

[ O
4 O
] O
This O
involves O
collection O
of O
sequences O
exhibiting O
substantial O
nucleotide O

differences O
while O
retaining O
unequivocal O
sequence O
similarity O
. O

However O
, O
a O
high O
degree O
of O
sequence O
variation O
in O
the O
transcribed O

spacers O
is O
known O
even O
among O
closely O
related O
species O
. O

[ O
5 O
- O
6 O
- O
7 O
] O
Here O
, O
we O
analyze O
ITS2 O
from O
7 O

Culex B
species O
characteristic O
of O
5 O
different O
geographical O
locations O
to O
assemble O
common O
RNA O
structural O
features O
. O

Methodology O

Dataset O

ITS2 O
nucleotide O
sequences O
from O
7 O
Culex B
mosquitoes I
characteristic O
of O
5 O
geographical O
locations O
( O
Italy O
, O
China O
, O
Africa O
, O

America O
, O
and O
Japan O
) O
were O
downloaded O
from O
GenBank O
( O
www O
. O
ncbi O
. O
nlm O
. O
nih O
. O
gov O
/ O
genbank O

) O
. O
The O
GenBank O
accession O
numbers O
for O
ITS2 O
sequences O

from O
C O
. O
pipens I
( O
North O
America O
) O
, O
C B
. I
tritaeniorhynchus I
( O
China O
) O
, O
C B
. I
quinquefasciatus I

( O
Africa O
) O
, O
C O
. O
tarsalis O
( O
Italy O
) O
, O
C O
. O
annulus O
( O
China O
) O
, O
C O
. O
pseudovishnui O

( O
China O
) O
, O
and O
C O
. O
vishnui O
( O
Japan O
) O
were O
X75817 O
, O
AF305558 O
, O
Z48468 O
, O
U33031 O
, O
AF453488 O
, O
AF453498 O
and O
AF165900 O
, O

respectively O
. O

Sequence O
alignments O

Multiple O
sequence O
alignments O
were O
performed O
using O
CLUSTALW O
with O
a O
gap O
opening O
penalty O
of O
15 O
and O
gap O
extension O
penalty O
of O
6 O
. O
66 O
. O

[ O
8 O
] O

Secondary O
structure O
prediction O

The O
RNA O
secondary O
structures O
for O
ITS2 O
were O
predicted O
using O
RNADRAW O
. O

[ O
21 O
] O

RNADRAW O
predicts O
RNA O
structures O
by O
identifying O
suboptimal O
structures O
using O
the O
free O
energy O
optimization O
methodology O
at O
a O
default O

temperature O
of O
370 O
degrees O
C O
. O

In O
the O
current O
study O
, O
ITS2 O
and O
5 O
. O
8S O
regions O
( O
the O
first O
170 O
nucleotides O
) O
were O
used O
for O
RNA O
structure O

prediction O
. O

The O
minimum O
energy O
structure O
prediction O
algorithm O
in O
RNADRAW O
was O
ported O
from O
the O
RNAFOLD O
program O
included O
in O
the O

Vienna O
RNA O
package O
. O

[ O
24 O
] O
The O
dynamic O
programming O
algorithm O
employed O
in O

RNADRAW O
was O
based O
on O
the O
work O
of O
Zuker O
and O
Stiegler O
[ O
25 O
] O
and O
uses O
energy O

parameters O
taken O
from O
Turner O
[ O
26 O
] O
, O
Freier O
[ O
27 O
] O
and O
Jaeger O
. O

[ O
28 O
] O

Phylogenetic O
analysis O

The O
phylogenetic O
Genebee O
service O
was O
used O
for O
phylogenetic O
tree O
construction O
. O

[ O
22 O

] O

RNA O
fold O

The O
Sribo O
program O
in O
Sfold O
( O
Statistical O
Folding O
and O
Rational O
Design O
of O
Nucleic O
Acids O
) O
was O

used O
to O
predict O
the O
probable O
target O
accessibility O
sites O
( O
loops O
) O
for O
trans O
- O
cleaving O
ribozymes O
in O
ITS2 O
. O

[ O
24 O
] O
The O
prediction O
of O
accessibility O
is O
based O
on O
a O
statistical O
sample O
of O
the O
Boltzmann O

ensemble O
for O
secondary O
structures O
. O

Here O
, O
we O
assessed O
the O
likelihood O
of O
unpaired O
sites O
for O
potential O
ribozyme O
target O
. O

Each O
mRNA O

exists O
as O
a O
population O
of O
different O
structures O
. O

Hence O
, O
stochastic O
approach O
to O
the O
evaluation O
of O
accessible O
sites O
was O
found O

appropriate O
. O

[ O
29 O
] O
The O
probability O
profiling O
approach O
by O
Ding O
and O
Lawrence O

[ O
30 O
] O
reveals O
target O
sites O
that O
are O
commonly O
accessible O
for O

a O
large O
number O
of O
statistically O
representative O
structures O
in O
the O
target O
RNA O
. O

This O
novel O
approach O
bypasses O
the O
long O
- O
standing O

difficulty O
in O
accessibility O
evaluation O
due O
to O
limited O
representation O
of O
probable O
structures O
due O
to O
high O
statistical O
confidence O

in O
predictions O
. O

The O
probability O
profile O
for O
individual O
bases O
( O
W O
= O
1 O
) O
is O
produced O
for O
the O
region O
that O

includes O
a O
triplet O
and O
two O
flanking O
sequences O
of O
15 O
bases O
each O
in O
every O
site O
of O
the O
selected O
cleavage O
triplet O
( O
e O
. O
g O
. O
, O
GUC O
) O
. O

Results O
and O
Discussion O

ITS2 O
sequences O
showed O
a O
taxonomic O
trend O
similar O
to O
that O
in O
phylogeny O
construction O
( O
Figure O
1A O

) O
. O

A O
multiple O
sequence O
alignment O

of O
5 O
. O
8S O
rDNA O
and O
ITS2 O
showed O
in O
Figure O
1B O
indicated O
that O
more O
distantly O
related O
species O
have O

lower O
sequence O
similarity O
in O
the O

ITS2 O
region O
. O

The O
predicted O
features O
of O
ITS2 O
using O
RNADRAW O
are O
given O
in O
Table O
1 O
. O

The O
stems O

( O
double O
stranded O
paired O
regions O
) O

stabilize O
RNA O
secondary O
structures O
and O
the O
number O
of O
stems O
present O
in O
each O
ITS2 O
is O
given O
( O
Table O
1 O
) O
. O

ITS2 O
RNA O
structures O
from O

C B
. I
pipiens I
and O
C B
. I
quinquefasciatus I
have O
the O
highest O
negative O
free O
energy O
( O
- O
149 O
. O
38 O
Kcal O
and O

- O
148 O
. O
23 O
Kcal O
) O
followed O
by O
C O
. I
tarsalis I
( O
- O
129 O
. O
2 O
) O
, O
C O
. O
annulus O
and O
then O
by O
C O
. O
vishnui O

( O
- O
115 O
. O
3 O
) O
, O
C B
. I
pseudovishnui I
( O
- O
105 O
. O
66 O
) O
and O
C B
. I
tritaeniorhyncus I
( O
- O
82 O
. O
57 O
) O
. O

Visual O
comparison O
shows O

that O
this O
is O
related O
to O
the O
trend O
in O
the O
cladogram O
given O
in O
Figure O
1A O
. O

A O
high O
degree O
of O
sequence O
similarity O
is O
observed O
at O
the O

5 O
' O
end O
compared O
to O
the O
3 O
' O
end O
( O
Figure O
1A O
) O
. O

This O
is O
due O
to O
factors O
such O
as O
genetic O
drift O
, O
the O
relative O
number O
and O
size O
of O
repeats O
, O

rates O
of O
unequal O
crossover O
, O
gene O
conversion O
, O
immigration O
and O
the O
number O
of O
the O
loci O
influencing O
the O
length O
. O

[ O
10 O
] O
Furthermore O
, O
a O
high O
level O
of O
sequence O
similarity O
is O
found O
between O
C O
. O

annulus O
and O
C O
. O
vishnui O
as O
well O
as O
C B
. I
quinquefasciatus I
and O
C O
. O

pipiens I
. O

The O
simple O
tandem O
repeats O
shown O
in O
Figure O
1B O
as O
bolded O
regions O
are O
found O
to O
be O
similar O
. O

This O
similarity O
is O
seen O
in O
corresponding O

RNA O
structures O
and O
computed O
energies O
. O

The O
Culex B
species O
considered O
in O
this O
study O
were O
then O
grouped O
into O
three O
classes O
based O
on O
the O
similarity O
of O

RNA O
stems O
and O
loops O
. O

Despite O
their O
different O
geographic O
locations O
with O
varying O
eco O
- O
climatic O
conditions O
, O
class O
I O

( O
C O
. O
annulus O
, O
C O
. I
pseudovishnui I
and O
C O
. O
vishnui O
) O
isolates O
( O
China O
and O
Japan O
) O
showed O
high O
similarity O

in O
secondary O
structural O
features O
. O

Similar O
observations O
were O
seen O
in O
class O
II O
( O
C B
. I
pipiens I
, O
C B
. I
quinquefasciatus I
and O

C B
. I
tarsalis I
) O
isolates O
from O
North O
America O
and O
Africa O
. O

[ O
19 O
] O

A O
high O
degree O
of O
similarity O
in O
the O
5 O
. O
8S O
region O
unlike O
the O
ITS2 O
is O
shown O
for O
different O
isolates O
due O
to O
relative O
evolutionary O

selection O
. O

[ O
9 O
] O
The O
loops O
11 O
and O
12 O
having O
sequences O
UGUCG O
and O
CUUCGGUG O
, O

respectively O
are O
highly O
conserved O
in O
all O
classes O
. O

Figure O
1C O
shows O
the O
distribution O
of O
different O
types O
of O
loops O
( O
hairpin O
, O
bulge O
, O
multi O
branched O
, O
interior O
and O
exterior O
) O
among O

different O
isolates O
. O

The O
segments O
of O
the O
ITS2 O
having O
score O
> O
= O
50 O
are O
further O
probed O
carefully O
for O
target O
site O
to O
assess O
the O

likelihood O
of O
unpaired O
segments O
. O

Interestingly O
, O
the O
observed O
phylogenetic O
trend O
was O
identified O
with O
respect O
to O
the O
target O

accessibility O
sites O
for O
the O
seven O
Culex O
isolates O
. O

The O
order O
of O
preference O
is O
interior O
loop O
, O
bulge O
loop O
, O
multiple O

branched O
loop O
, O
hairpin O
loop O
and O
exterior O
loops O
in O
all O
the O
isolates O
. O

Several O
homologous O
domains O
were O
observed O
in O
the O
ITS O
regions O
of O
Aedes B
mosquito I
species O
by O
Wesson O

et O
al O
[ O
11 O
] O
and O
was O
shown O
that O
these O
domains O
base O
pair O
to O

form O
a O
core O
region O
central O
to O

several O
stem O
features O
. O

This O
suggested O
that O
conserved O
core O
region O
of O
rDNA O
is O
more O
important O
for O
a O
functional O
rRNA O
folding O

pattern O
. O

Barker O
found O
that O
ITS2 O
is O
unique O
in O
the O
16 O
populations O
of O
Boophilus B
microplus I
, O
Boophilus B
decoloratus I
, O

Rhipicephalus B
appendiculatus I
, O
Rhipicephalus B
zambesiensis I
, O
Rhipicephalus B
evertsi I
, O
Rhipicephalus B
sanguineus I
, O
Rhipicephalus B

turanicus O
, O
Rhipicephalus B
pumilio I
and O
Rhipicephalus B
camicasi I
from O
different O
geographical O
locations O
. O

[ O
12 O
] O
These O
results O
suggest O
that O
the O
differences O
and O
similarity O
observed O

in O
the O
ITS2 O
of O
different O
species O
are O
not O
simply O
accumulated O
due O
to O
random O
mutation O
and O
have O
evolved O
for O
functional O
selection O

in O
ribosome O
biogenesis O
. O

However O
, O
it O
was O
shown O
in O
three O
related O
mosquito O
genera O
( O
Aedes O
, O
Psorophora O
and O
Haemogogus O
) O

that O
the O
intra O
- O
spacer O
variable O
regions O
appear O
to O
co O
- O
evolve O
and O
that O
ITS2 O
variation O
is O
constrained O
by O
its O
secondary O
structure O
. O

[ O
11 O
] O
Further O
studies O
have O
demonstrated O
that O
the O
ITS2 O
is O
essential O

for O
the O
correct O
and O
efficient O
processing O
and O
maturation O
of O
26S O
rRNA O
ribosomal O
units O
. O

[ O

13 O
] O
Furthermore O
, O
the O
information O
required O
for O
the O
efficient O
removal O
of O
ITS2 O
from O
its O
RNA O
precursor O
is O
not O

localized O
but O
dispersed O
throughout O
the O
ITS2 O
region O
. O

Thus O
, O
insertions O
and O
deletions O
( O
indels O
) O
that O
affect O
secondary O
structures O

alter O
rRNA O
processing O
. O

Critical O
changes O
in O
the O
rRNA O
folding O
pattern O
due O
to O
evolutionary O
sequence O
variation O
in O
the O
ITS O
spacer O

regions O
may O
thus O
have O
an O
important O
role O
on O
the O
kinetics O
of O
precursor O
rRNA O
formation O
for O
the O
efficient O
functioning O
of O
rDNA O
clusters O
. O

Conclusion O

Comparison O
of O
ITS2 O
sequences O
from O
different O
isolates O
of O
Culex B
show O
similarity O
and O
variations O
. O

Surprisingly O
, O
species O
displaying O

sequence O
similarity O
belong O
to O
different O
geographical O
locations O
with O
diverse O
climatic O
and O
ecological O
conditions O
. O

This O
implies O
that O

the O
ITS2 O
regions O
have O
less O
selective O
pressure O
than O
the O
ribosomal O
regions O
. O

Several O
common O
structural O
folds O
were O
shared O
among O
the O

selected O
mosquitoes O
for O
maintaining O
functional O
equivalents O
. O

Construction O
of O
an O
evolutionary O
tree O
using O
more O
isolates O
of O

Culex O
will O
provide O
an O
understanding O
for O
their O
functional O
selection O
. O

Integration O
in O
primary O
community O
care O
networks O
( O
PCCNs O
) O
: O
examination O
of O
governance O
, O
clinical O
, O
marketing O
, O
financial O
, O
and O
information O
infrastructures O
in O
a O
national O
demonstration O
project O
in O
Taiwan O

Abstract O

Background O

Taiwan O
' O
s O
primary O
community O
care O
network O
( O
PCCN O
) O
demonstration O
project O
, O
funded O
by O
the O
Bureau O
of O
National O
Health O
Insurance O
on O
March O
2003 O
, O
was O
established O
to O
discourage O
hospital O
shopping O
behavior O
of O
people B
and O
drive O
the O
traditional O
fragmented O
health O
care O
providers O
into O
cooperate O
care O
models O
. O

Between O
2003 O
and O
2005 O
, O
268 O
PCCNs O
were O
established O
. O

This O
study O
profiled O
the O
individual O
members O
in O
the O
PCCNs O
to O
study O
the O
nature O
and O
extent O
to O
which O
their O
network O
infrastructures O
have O
been O
integrated O
among O
the O
members O
( O
clinics O
and O
hospitals O
) O
within O
individual O
PCCNs O
. O

Methods O

The O
thorough O
questionnaire O
items O
, O
covering O
the O
network O
working O
infrastructures O
- O
governance O
, O
clinical O
, O
marketing O
, O
financial O
, O
and O
information O
integration O
in O
PCCNs O
, O
were O
developed O
with O
validity O
and O
reliability O
confirmed O
. O

One O
thousand O
five O
hundred O
and O
fifty O
- O
seven O
clinics O
that O
had O
belonged O
to O
PCCNs O
for O
more O
than O
one O
year O
, O
based O
on O
the O
2003 O
- O
2005 O
Taiwan O
Primary O
Community O
Care O
Network O
List O
, O
were O
surveyed O
by O
mail O
. O

Nine O
hundred O
and O
twenty O
- O
eight O
clinic O
members O
responded O
to O
the O
surveys O
giving O
a O
59 O
. O
6 O
% O
response O
rate O
. O

Results O

Overall O
, O
the O
PCCNs O
' O
members O
had O
higher O
involvement O
in O
the O
governance O
infrastructure O
, O
which O
was O
usually O
viewed O
as O
the O
most O
important O
for O
establishment O
of O
core O
values O
in O
PCCNs O
' O
organization O
design O
and O
management O
at O
the O
early O
integration O
stage O
. O

In O
addition O
, O
it O
found O
that O
there O
existed O
a O
higher O
extent O
of O
integration O
of O
clinical O
, O
marketing O
, O
and O
information O
infrastructures O
among O
the O
hospital O
- O
clinic O
member O
relationship O
than O
those O
among O
clinic O
members O
within O
individual O
PCCNs O
. O

The O
financial O
infrastructure O
was O
shown O
the O
least O
integrated O
relative O
to O
other O
functional O
infrastructures O
at O
the O
early O
stage O
of O
PCCN O
formation O
. O

Conclusion O

There O
was O
still O
room O
for O
better O
integrated O
partnerships O
, O
as O
evidenced O
by O
the O
great O
variety O
of O
relationships O
and O
differences O
in O
extent O
of O
integration O
in O
this O
study O
. O

In O
addition O
to O
provide O
how O
the O
network O
members O
have O
done O
for O
their O
initial O
work O
at O
the O
early O
stage O
of O
network O
forming O
in O
this O
study O
, O
the O
detailed O
surveyed O
items O
, O
the O
concepts O
proposed O
by O
the O
managerial O
and O
theoretical O
professionals O
, O
could O
be O
a O
guide O
for O
those O
health O
care O
providers O
who O
have O
willingness O
to O
turn O
their O
business O
into O
multi O
- O
organizations O
. O

Background O

Taiwan O
' O
s O
National O
Health O
Insurance O
( O
NHI O
) O
under O
the O
control O
of O
the O
Bureau O
of O
National O
Health O
Insurance O
( O
BNHI O
) O
, O
was O
launched O
in O
March O
1995 O
to O
replace O
its O
social O
insurance O
system O
that O
was O
covering O
59 O
% O
of O
its O
population O
: O
government O
employees O
, O
labourers O
, O
farmers O
and O
servicemen O
[ O
1 O
] O
. O

By O
June O
2003 O
the O
number O
of O
people B
insured O
had O
reached O
21 O
, O
956 O
, O
729 O
( O
99 O
% O
) O
. O

There O
were O
17 O
, O
259 O
medical O
providers O
( O
92 O
% O
) O
, O
including O
575 O
hospitals O
and O
16 O
, O
684 O
clinics O
contracted O
with O
the O
BNHI O
for O
serving O
the O
enrolled O
population O
. O

The O
unique O
phenomenon O
characterized O
in O
Taiwan O
health O
care O
industry O
different O
from O
those O
in O
the O
western O
countries O
is O
the O
freedom O
of O
patients B
to O
choose O
the O
health O
care O
providers O
they O
want O
, O
no O
matter O
what O
their O
disease O
severity O
is O
. O

Furthermore O
, O
Taiwan O
people B
favor O
the O
larger O
scales O
of O
facilities O
and O
this O
fallacy O
leads O
to O
the O
phenomenon O
of O
big O
- O
hospital O
shopping O
. O

For O
example O
, O
people B
choose O
the O
medical O
centers O
which O
are O
accredited O
as O
the O
highest O
level O
of O
medical O
science O
in O
Taiwan O
when O
they O
only O
suffer O
from O
a O
common O
cold O
. O

In O
the O
spring O
of O
2003 O
, O
the O
SARS O
epidemic O
viciously O
attacked O
the O
health O
of O
Taiwan O
' O
s O
people B
. O

The O
people B
' O
s O
freedom O
to O
choose O
medical O
providers O
caused O
the O
national O
health O
authority O
to O
barely O
control O
and O
traced O
the O
flow O
of O
epidemic O
. O

This O
event O
made O
Taiwan O
national O
health O
authorities O
rethink O
what O
happened O
and O
how O
it O
damaged O
under O
the O
traditional O
fragmented O
health O
care O
providers O
in O
Taiwan O
. O

One O
health O
reform O
launched O
was O
named O
the O
" O
Primary O
Community O
Care O
Network O
( O
PCCN O
) O
demonstration O
project O
" O
, O
a O
nationwide O
health O
care O
financing O
program O
funded O
by O
the O
Bureau O
of O
National O
Health O
Insurance O
( O
BNHI O
) O
in O
March O
2003 O
and O
it O
was O
a O
new O
model O
for O
the O
Taiwan B
government O
to O
redefine O
the O
role O
of O
family O
physicians O
in O
the O
health O
care O
delivery O
system O
. O

A O
PCCN O
in O
Taiwan O
consists O
of O
a O
group O
of O
clinic O
physicians O
whose O
medical O
jobs O
are O
viewed O
as O
family O
care O
and O
at O
least O
one O
hospital O
for O
secondary O
or O
tertiary O
care O
. O

The O
idea O
of O
member O
component O
design O
in O
PCCNs O
was O
aimed O
to O
lead O
the O
Taiwan O
citizens O
to O
choose O
one O
clinic O
physician O
as O
their O
personal O
family O
physician O
for O
health O
maintenance O
and O
this O
family O
physician O
also O
would O
have O
the O
responsibility O
of O
referring O
the O
patients B
to O
specialty O
care O
if O
necessary O
. O

From O
a O
national O
health O
authority O
perspective O
, O
they O
expected O
the O
Taiwan O
people B
to O
put O
an O
end O
to O
their O
fallacy O
that O
" O
bigger O
is O
better O
" O
for O
health O
care O
organizations O
and O
establish O
the O
idea O
of O
" O
human B
health O
" O
, O
starting O
with O
prevention O
and O
primary O
care O
, O
followed O
by O
secondary O
or O
tertiary O
care O
, O
emphasizing O
health O
promotion O
and O
maintenance O
instead O
of O
disease O
curing O
. O

Furthermore O
, O
it O
could O
decrease O
the O
inappropriateness O
of O
medical O
usage O
, O
i O
. O
e O
. O
, O
over O
- O
uses O
of O
secondary O
and O
tertiary O
medical O
services O
in O
the O
high O
- O
tech O
hospitals O
. O

In O
addition O
, O
the O
national O
health O
authority O
was O
expected O
to O
drive O
the O
traditional O
fragmented O
heath O
care O
providers O
into O
coordinated O
medical O
multidisciplinary O
teams O
and O
share O
the O
limited O
medical O
resources O
through O
the O
PCCN O
demonstration O
project O
. O

In O
summary O
, O
the O
PCCN O
demonstration O
project O
was O
aimed O
to O
: O
1 O
) O
change O
the O
traditional O
patients B
' O
customs O
of O
freely O
choosing O
health O
care O
organizations O
and O
establish O
referral O
channels O
along O
the O
continuum O
of O
care O
, O
and O
2 O
) O
establish O
partnerships O
among O
the O
primary O
care O
clinics O
and O
hospitals O
to O
provide O
a O
continuum O
of O
health O
care O
services O
. O

It O
was O
also O
expected O
to O
establish O
the O
primary O
care O
system O
of O
family O
physicians O
to O
provide O
whole O
- O
people B
health O
care O
and O
improve O
care O
quality O
[ O
1 O
] O
. O

Partnership O
structures O
in O
the O
PCCNs O
represent O
the O
virtual O
vertical O
( O
i O
. O
e O
. O
, O
between O
the O
member O
clinics O
and O
hospitals O
) O
and O
virtual O
horizontal O
( O
i O
. O
e O
. O
, O
among O
the O
member O
clinics O
) O
aspects O
of O
organizing O
, O
which O
designate O
the O
formal O
relationships O
between O
individuals O
and O
the O
total O
network O
and O
include O
organizational O
design O
to O
ensure O
effective O
communication O
, O
coordination O
, O
and O
integration O
across O
the O
total O
network O
. O

Each O
PCCN O
consists O
of O
five O
to O
ten O
clinics O
: O
half O
of O
them O
should O
offer O
the O
services O
of O
general O
medicine O
, O
internal O
medicine O
, O
surgery O
, O
obstetrics O
and O
gynecology O
, O
pediatric O
, O
or O
family O
medicine O
. O

And O
each O
PCCN O
has O
a O
central O
headquarters O
, O
usually O
in O
one O
of O
the O
clinic O
facilities O
, O
to O
coordinate O
and O
integrate O
the O
network O
. O

All O
the O
clinic O
physicians O
in O
a O
PCCN O
are O
assigned O
the O
roles O
of O
" O
family O
physicians O
" O
or O
" O
gatekeepers O
" O
who O
recruit O
people B
from O
the O
local O
community O
, O
keep O
background O
and O
medical O
files O
on O
them O
, O
certify O
family O
physician O
education O
training O
programs O
, O
and O
hold O
office O
hours O
in O
the O
member O
hospital O
, O
where O
they O
serve O
as O
joint O
faculty O
members O
for O
further O
medical O
consultations O
or O
medical O
utilizations O
of O
labs O
and O
tests O
, O
if O
necessary O
. O

In O
addition O
, O
the O
hospital O
member O
is O
asked O
to O
help O
clinic O
physicians O
in O
their O
network O
to O
set O
up O
a O
medical O
information O
system O
, O
share O
hospital O
resources O
( O
medical O
equipment O
and O
library O
literature O
) O
with O
the O
clinic O
physicians O
in O
their O
network O
and O
establish O
referral O
channels O
among O
the O
network O
members O
. O

Furthermore O
, O
this O
new O
demonstration O
model O
tries O
to O
minimize O
the O
barriers O
to O
patient B
access O
by O
setting O
up O
24 O
- O
hour O
a O
day O
, O
7 O
- O
day O
a O
week O
medical O
consultation O
telephone O
lines O
for O
providing O
urgent O
services O
onsite O
and O
for O
taking O
care O
of O
the O
patients B
whose O
family O
physicians O
' O
practices O
are O
closed O
to O
assure O
seamless O
care O
channels O
. O

The O
BNHI O
funded O
these O
extra O
demonstration O
actions O
, O
at O
around O
one O
hundred O
thousand O
US O
dollars O
( O
i O
. O
e O
. O
, O
NT O
$ O
3 O
, O
500 O
, O
000 O
) O
for O
each O
PCCN O
under O
the O
current O
fee O
- O
for O
- O
service O
payment O
system O
[ O
1 O
] O
. O

Figure O
1 O
describes O
the O
organizational O
structure O
of O
individual O
PCCNs O
introduced O
in O
the O
demonstration O
project O
in O
Taiwan O
. O

To O
date O
, O
the O
PCCN O
demonstration O
project O
has O
been O
in O
operation O
for O
more O
than O
three O
years O
. O

There O
have O
been O
268 O
PCCNs O
formed O
in O
the O
period O
of O
2003 O
to O
2005 O
around O
Taiwan O
. O

The O
geographical O
distributions O
of O
PCCNs O
and O
their O
members O
were O
described O
in O
Table O
1 O
. O

Analyzing O
all O
1 O
, O
557 O
participating O
clinic O
members O
in O
the O
demonstration O
project O
in O
terms O
of O
medical O
specialties O
, O
they O
cover O
general O
medicine O
, O
internal O
medicine O
, O
surgeries O
, O
obstetrics O
and O
gynecology O
, O
pediatrics O
, O
family O
medicines O
, O
otolaryngology O
, O
ophthalmology O
, O
rehabilitation O
medicine O
, O
dermatology O
, O
and O
psychiatry O
, O
with O
237 O
clinics O
providing O
more O
than O
two O
specialties O
. O

On O
the O
other O
hand O
, O
each O
PCCN O
recruits O
at O
least O
one O
district O
or O
regional O
accredited O
hospital O
for O
acute O
care O
demands O
( O
required O
for O
network O
members O
) O
and O
a O
medical O
center O
for O
tertiary O
care O
support O
( O
not O
required O
for O
network O
members O
) O
. O

There O
are O
6 O
medical O
centers O
, O
52 O
regional O
hospitals O
, O
and O
71 O
district O
hospitals O
joining O
in O
the O
demonstration O
project O
. O

See O
Table O
1 O
for O
more O
detailed O
information O
about O
the O
PCCN O
members O
. O

To O
date O
, O
there O
have O
been O
few O
empirical O
studies O
of O
the O
working O
relationships O
that O
have O
developed O
between O
members O
of O
the O
PCCN O
program O
. O

Partnership O
needs O
a O
method O
to O
determine O
at O
an O
early O
stage O
, O
to O
make O
sure O
whether O
they O
are O
making O
the O
most O
of O
collaboration O
[ O
2 O
] O
and O
the O
acceptance O
of O
the O
contracting O
networks O
in O
Taiwan O
as O
an O
organizational O
innovation O
worthy O
of O
greater O
diffusion O
deserves O
to O
be O
explored O
. O

Therefore O
, O
this O
study O
used O
a O
structured O
questionnaire O
to O
characterize O
the O
relationship O
among O
the O
members O
in O
the O
individual O
PCCNs O
, O
with O
regard O
to O
governance O
, O
clinical O
, O
marketing O
, O
financing O
, O
as O
well O
as O
information O
integration O
infrastructures O
. O

The O
results O
of O
this O
study O
provide O
descriptive O
analyses O
in O
detail O
to O
map O
the O
partnership O
developments O
, O
to O
enrich O
the O
body O
of O
knowledge O
of O
the O
partner O
relationships O
and O
to O
help O
policy O
makers O
understand O
the O
coordinated O
efforts O
of O
these O
health O
care O
providers O
which O
have O
developed O
under O
this O
system O
. O

It O
also O
provides O
the O
recommendations O
for O
heath O
policy O
decision O
- O
making O
and O
management O
of O
networks O
of O
health O
care O
providers O
for O
the O
future O
involvement O
. O

Methods O

This O
study O
was O
aimed O
at O
providing O
descriptive O
analyses O
to O
map O
the O
partnership O
development O
. O

To O
understand O
the O
actual O
integration O
actions O
done O
by O
network O
members O
, O
the O
theoretical O
concept O
employed O
by O
network O
partnerships O
were O
described O
and O
then O
the O
derived O
survey O
instrument O
was O
developed O
. O

Theoretical O
framework O
for O
organization O
design O
of O
network O
integration O

The O
rapid O
organizational O
changes O
in O
the O
health O
care O
industry O
have O
driven O
theorists O
from O
every O
discipline O
and O
across O
the O
world O
to O
seek O
an O
approach O
that O
allows O
organizations O
to O
flourish O
. O

Organization O
theory O
allows O
investigators O
to O
profile O
an O
organization O
from O
the O
aspect O
of O
patterns O
and O
regularities O
in O
organizational O
design O
and O
behavior O
. O

In O
the O
early O
20th O
century O
, O
classical O
management O
theorists O
claimed O
that O
an O
organization O
has O
" O
a O
best O
way O
" O
to O
be O
organized O
and O
managed O
[ O
3 O
] O
. O

That O
implied O
that O
all O
organizations O
would O
own O
the O
" O
same O
" O
organizational O
styles O
or O
structures O
. O

In O
the O
1960s O
, O
several O
theorists O
[ O
4 O
- O
8 O
] O
challenged O
this O
assumption O
by O
applying O
a O
" O
contingency O
approach O
" O
to O
propose O
that O
there O
is O
no O
best O
way O
to O
organize O
an O
organization O
, O
and O
that O
the O
effectiveness O
of O
an O
organizational O
structure O
varies O
with O
the O
situation O
of O
an O
organization O
. O

Furthermore O
, O
it O
is O
proposed O
that O
the O
best O
way O
to O
organize O
an O
organization O
depends O
on O
the O
nature O
of O
the O
environment O
to O
which O
the O
organization O
relates O
. O

Contingency O
theory O
delineates O
the O
concepts O
" O
organization O
' O
s O
internal O
features O
, O
" O
" O
the O
demands O
of O
organizational O
environments O
, O
" O
" O
best O
adaptation O
, O
" O
and O
, O
the O
most O
important O
and O
difficult O
of O
all O
, O
" O
best O
match O
" O
[ O
9 O
] O
. O

Lawrence O
& O
Lorsch O
[ O
7 O
] O
argued O
that O
environments O
characterized O
by O
uncertainty O
and O
rapid O
rates O
of O
change O
in O
market O
conditions O
or O
technology O
impose O
different O
demands O
, O
including O
constraints O
and O
opportunities O
, O
on O
organizations O
than O
do O
placid O
and O
stable O
environments O
. O

Similarly O
to O
Lawrence O
and O
Lorsch O
' O
s O
views O
mentioned O
above O
, O
Galbraith O
[ O
10 O
, O
11 O
] O
stressed O
the O
contingency O
perspective O
on O
information O
processing O
. O

The O
information O
- O
processing O
approach O
emphasizes O
that O
environment O
, O
size O
, O
and O
technology O
impose O
different O
information O
- O
processing O
requirements O
on O
organizations O
, O
and O
thus O
an O
organization O
must O
be O
designed O
to O
encourage O
information O
flow O
in O
both O
vertical O
and O
horizontal O
directions O
to O
achieve O
the O
overall O
tasks O
of O
the O
organization O
and O
, O
finally O
, O
organizational O
effectiveness O
[ O
11 O
- O
14 O
] O
. O

Some O
theorists O
have O
criticized O
conventional O
contingency O
theorists O
who O
presume O
that O
organizational O
structure O
is O
driven O
by O
the O
environment O
. O

Child B
[ O
15 O
] O
, O
Miller O
[ O
16 O
] O
, O
Van O
de O
Ven O
and O
Drazin O
[ O
17 O
] O
, O
and O
Tushman O
and O
Romanelli O
[ O
18 O
] O
raised O
such O
criticisms O
; O
they O
argued O
that O
organizations O
become O
what O
they O
are O
not O
only O
because O
of O
the O
environment O
, O
but O
also O
because O
of O
choices O
made O
by O
members O
, O
especially O
choices O
about O
strategy O
and O
organizational O
design O
. O

As O
Thompson O
' O
s O
words O
in O
the O
book O
Organizations O
in O
Action O
[ O
8 O
] O
put O
it O
, O
" O
organizations O
are O
not O
determined O
simply O
by O
their O
environments O
( O
p O
. O
27 O
) O
. O
" O
He O
also O
pointed O
out O
that O
" O
administration O
may O
innovate O
on O
any O
or O
all O
of O
the O
necessary O
dimensions O
, O
but O
only O
to O
the O
extent O
that O
innovations O
are O
acceptable O
to O
those O
on O
whom O
the O
organization O
can O
and O
must O
depend O
. O
" O
Instead O
of O
assuming O
that O
administrators O
are O
highly O
constrained O
in O
their O
decisions O
, O
strategic O
contingency O
theorists O
emphasized O
" O
the O
importance O
of O
choice O
, O
" O
that O
is O
, O
" O
the O
freedom O
of O
agency O
" O
[ O
15 O
] O
. O

Furthermore O
, O
Pfeffer O
[ O
19 O
] O
explicitly O
pointed O
out O
that O
" O
organizational O
structures O
are O
the O
outcomes O
of O
political O
contests O
within O
organizations O
( O
p O
. O
38 O
) O
. O
" O

Daft O
[ O
14 O
] O
proposed O
a O
top O
management O
model O
to O
delineate O
how O
" O
a O
strategy O
is O
a O
plan O
for O
interacting O
with O
the O
competitive O
environment O
to O
achieve O
organizational O
goals O
. O
" O
He O
stated O
that O
the O
major O
responsibility O
of O
top O
management O
is O
to O
determine O
the O
goals O
, O
strategy O
, O
and O
design O
of O
an O
organization O
to O
adapt O
to O
a O
changing O
environment O
. O

To O
assess O
the O
external O
and O
internal O
environments O
of O
an O
organization O
seems O
to O
be O
the O
first O
task O
for O
top O
managers O
in O
defining O
an O
organization O
' O
s O
goals O
and O
missions O
. O

Then O
, O
guided O
by O
the O
goals O
and O
missions O
of O
the O
organization O
, O
top O
managers O
shape O
the O
design O
of O
the O
organization O
, O
including O
structural O
forms O
, O
information O
system O
, O
technology O
, O
human B
resources O
, O
organizational O
culture O
, O
and O
inter O
- O
organizational O
linkages O
, O
to O
achieve O
the O
final O
organizational O
performance O
. O

Integration O
refers O
to O
the O
mechanisms O
of O
coordination O
, O
the O
ways O
guided O
to O
partnership O
goals O
to O
fit O
internal O
and O
external O
conditions O
[ O
7 O
, O
20 O
, O
21 O
] O
. O

In O
the O
early O
1990s O
, O
proposals O
for O
US O
national O
health O
care O
reform O
recognized O
the O
need O
for O
integrating O
mechanisms O
to O
achieve O
both O
financial O
success O
and O
quality O
of O
care O
of O
a O
well O
- O
organized O
system O
of O
care O
[ O
22 O
, O
23 O
] O
. O

Several O
researchers O
also O
viewed O
inter O
- O
organizational O
cooperation O
as O
resource O
exchanges O
, O
including O
client O
referrals O
, O
money O
, O
and O
staff O
[ O
24 O
- O
27 O
] O
. O

From O
practical O
ways O
of O
viewing O
integration O
, O
the O
success O
of O
integration O
lies O
in O
the O
coordinative O
mechanisms O
and O
partnership O
working O
that O
support O
it O
[ O
28 O
] O
, O
including O
an O
administrative O
organization O
that O
coordinates O
the O
operations O
of O
various O
health O
care O
services O
; O
a O
management O
information O
system O
that O
integrates O
clinical O
, O
utilization O
, O
and O
financial O
data O
and O
follows O
clients B
across O
different O
settings O
; O
a O
care O
coordination O
program O
such O
as O
case O
management O
or O
disease O
management O
that O
works O
with O
clients B
to O
arrange O
health O
care O
services O
; O
and O
a O
financial O
mechanism O
that O
enables O
pooling O
of O
funds O
across O
services O
[ O
29 O
- O
35 O
] O
. O

Fox O
[ O
36 O
] O
suggested O
the O
success O
of O
integrated O
health O
networks O
should O
ensure O
that O
the O
new O
business O
link O
such O
aspects O
as O
technology O
, O
functional O
skills O
, O
customer O
access O
, O
management O
, O
or O
products O
that O
can O
be O
shared O
across O
both O
the O
core O
and O
the O
new O
business O
; O
to O
conduct O
market O
financial O
evaluation O
; O
to O
share O
the O
risk O
of O
vertical O
integration O
with O
outside O
entities O
, O
to O
develop O
the O
management O
structure O
that O
can O
reflect O
the O
degree O
of O
coordination O
necessary O
to O
support O
the O
core O
business O
activities O
; O
to O
ensure O
that O
the O
integration O
strategy O
meets O
the O
needs O
of O
customers O
, O
including O
medical O
treatment O
, O
the O
use O
of O
medical O
technology O
, O
and O
the O
preferred O
methods O
of O
purchase O
; O
and O
to O
measure O
the O
new O
business O
by O
its O
value O
to O
the O
enterprise O
as O
a O

whole O
, O
rather O
than O
by O
its O
profitability O
as O
a O
stand O
- O
alone O
entity O
. O

In O
summary O
, O
the O
effects O
that O
integration O
in O
inter O
- O
organizational O
designs O
has O
on O
network O
management O
were O
substantial O
from O
a O
managerial O
perspective O
. O

Borrowing O
the O
ideas O
of O
strategic O
contingency O
perspective O
[ O
8 O
, O
15 O
, O
19 O
] O
and O
top O
management O
model O
[ O
14 O
] O
, O
it O
could O
be O
imply O
that O
success O
( O
organization O
performance O
) O
in O
reengineering O
a O
network O
lies O
in O
the O
integration O
of O
process O
and O
services O
( O
see O
Figure O
1 O
) O
, O
including O
leadership O
/ O
governing O
structure O
, O
teamwork O
between O
disciplines O
and O
patient B
care O
, O
financial O
planning O
, O
and O
information O
systems O
, O
characterized O
as O
the O
constructs O
of O
governance O
, O
clinical O
, O
financial O
, O
and O
information O
infrastructures O
, O
respectively O
, O
in O
this O
study O
. O

In O
addition O
, O
another O
construct O
, O
marketing O
infrastructure O
, O
was O
especially O
important O
and O
designed O
to O
explore O
for O
PCCNs O
in O
this O
study O
because O
of O
patients B
' O
freedom O
of O
making O
healthcare O
choice O
and O
the O
traditional O
fragmented O
health O
care O
systems O
by O
individual O
health O
care O
organizations O
in O
Taiwan O
. O

One O
major O
reason O
for O
Taiwan O
people B
' O
s O
hospital O
shopping O
preferences O
was O
that O
Taiwan O
people B
usually O
believe O
the O
bigger O
the O
facility O
, O
the O
better O
capacities O
a O
facility O
has O
no O
matter O
on O
any O
aspect O
from O
medical O
professionals O
to O
tangible O
medical O
equipment O
and O
plants O
. O

And O
this O
fallacy O
made O
the O
public O
want O
to O
overuse O
the O
facility O
with O
high O
- O
tech O
medical O
services O
no O
matter O
if O
it O
fits O
their O
needs O
. O

From O
the O
health O
policy O
and O
management O
perspectives O
, O
therefore O
, O
the O
health O
care O
providers O
were O
encouraged O
to O
market O
their O
services O
as O
a O
new O
corporate O
identity O
and O
brand O
strategy O
[ O
37 O
] O
, O
including O
offering O
tangible O
resources O
such O
as O
books O
, O
libraries O
, O
medical O
equipment O
, O
and O
intangible O
resources O
such O
as O
knowledge O
and O
information O
exchanges O
( O
education O
) O
and O
reputation O
sharing O
one O
another O
among O
PCCN O
members O
. O

Furthermore O
, O
through O
the O
process O
of O
marketing O
resource O
exchanges O
, O
therefore O
, O
each O
PCCN O
could O
establish O
the O
images O
of O
" O
one O
system O
, O
one O
brand O
and O
quality O
" O
for O
the O
public O
and O
for O
the O
health O
care O
providers O
. O

It O
also O
makes O
it O
be O
more O
visible O
to O
the O
public O
. O

The O
five O
integration O
infrastructures O
of O
network O
management O
were O
constructed O
as O
a O
conceptual O
framework O
in O
this O
study O
to O
help O
to O
portray O
how O
the O
PCCN O
members O
have O
done O
. O

The O
survey O
instrument O
development O
was O
described O
in O
the O
following O
. O

Survey O
instrument O
development O
: O
integration O
infrastructures O
and O
measurements O
of O
partnerships O

Based O
on O
the O
five O
integration O
infrastructures O
of O
network O
management O
, O
the O
structured O
questionnaire O
were O
derived O
from O
extensive O
literature O
reviews O
. O

Governance O
infrastructure O

Governance O
assumes O
the O
broad O
responsibility O
for O
organizational O
goals O
and O
survival O
and O
involves O
the O
series O
process O
of O
setting O
and O
monitoring O
organizational O
goals O
and O
strategy O
development O
through O
a O
board O
of O
representatives O
[ O
38 O
] O
. O

Governance O
or O
administrative O
integration O
infrastructure O
in O
establishing O
network O
partnerships O
refers O
to O
administrative O
structures O
( O
or O
responsibilities O
) O
created O
to O
facilitate O
communication O
, O
clear O
lines O
of O
authority O
, O
accountability O
, O
and O
responsibility O
for O
patient B
care O
services O
; O
to O
negotiate O
budgets O
and O
financial O
trade O
- O
offs O
; O
and O
to O
present O
a O
cohesive O
, O
consistent O
message O
in O
interactions O
with O
external O
agencies O
and O
the O
community O
[ O
29 O
, O
39 O
- O
41 O
] O
and O
most O
important O
for O
members O
in O
contract O
agreements O
, O
to O
manage O
participation O
[ O
33 O
] O
. O

From O
a O
multidisciplinary O
perspective O
, O
Mitchell O
and O
Shortell O
[ O
42 O
] O
applied O
the O
concepts O
of O
governance O
and O
management O
characteristics O
in O
effective O
community O
health O
partnerships O
. O

The O
construct O
of O
governance O
involved O
several O
tasks O
, O
including O
setting O
priorities O
for O
strategic O
goals O
, O
choosing O
the O
membership O
composition O
, O
obtaining O
the O
necessary O
financial O
resources O
, O
and O
setting O
up O
the O
accountability O
systems O
, O
and O
so O
on O
. O

The O
construct O
of O
the O
management O
refers O
to O
the O
tasks O
of O
engaging O
and O
maintaining O
organizational O
members O
' O
interest O
in O
a O
shared O
vision O
and O
mission O
, O
providing O
appropriate O
structures O
and O
coordination O
mechanisms O
for O
the O
specified O
strategies O
, O
promoting O
constructive O
conflicts O
and O
managing O
destructive O
conflicts O
, O
implementing O
information O
systems O
to O
monitor O
the O
dynamics O
, O
adjusting O
the O
leadership O
in O
the O
overall O
membership O
, O
and O
so O
on O
. O

The O
issues O
of O
governance O
and O
administrative O
integration O
in O
the O
PCCNs O
could O
include O
[ O
2 O
, O
38 O
, O
40 O
, O
41 O
, O
43 O
- O
47 O
] O
: O

* O
planning O
the O
shared O
visions O
and O
missions O

* O
determining O
the O
shared O
service O
strategies O
, O
cooperation O
priorities O
, O
policies O
and O
principles O

* O
identifying O
the O
information O
needed O
and O
how O
to O
get O
it O

* O
organizing O
the O
network O
dynamics O
and O
member O
roles O

* O
leading O
and O
managing O
the O
conflicts O
and O
communication O

* O
designing O
and O
controlling O
the O
shared O
network O
performance O
systems O
, O
including O
indicator O
settings O
, O
feedbacks O
, O
and O
accountability O
. O

Clinical O
infrastructure O

The O
idea O
of O
care O
integration O
begins O
through O
such O
public O
programs O
that O
include O
social O
workers O
in O
public O
welfare O
departments O
, O
caseworkers O
in O
mental O
health O
, O
or O
nurses O
in O
public O
health O
departments O
. O

In O
the O
late O
1980s O
, O
care O
integration O
was O
deemed O
necessary O
for O
the O
streamlining O
of O
care O
and O
negotiating O
the O
maze O
of O
long O
- O
term O
care O
services O
. O

At O
that O
time O
, O
it O
was O
referred O
to O
as O
service O
coordination O
or O
case O
management O
, O
or O
in O
other O
related O
terms O
[ O
29 O
] O
. O

The O
purpose O
of O
care O
integration O
is O
to O
work O
directly O
with O
patients B
and O
their O
families O
over O
time O
to O
help O
them O
arrange O
and O
manage O
the O
complex O
resources O
that O
patients B
may O
need O
to O
maintain O
health O
and O
independent O
functioning O
. O

At O
the O
same O
time O
, O
care O
integration O
is O
used O
to O
achieve O
the O
most O
cost O
- O
effective O
use O
possible O
of O
scarce O
resources O
, O
by O
steering O
patients B
to O
the O
health O
, O
social O
, O
and O
support O
services O
most O
appropriate O
for O
them O
at O
a O
given O
time O
[ O
29 O
] O
. O

Conrad O
and O
Dowling O
[ O
33 O
] O
pointed O
out O
that O
to O
coordinate O
and O
integrate O
patient B
care O
relies O
on O
connecting O
patient B
services O
at O
the O
different O
stages O
of O
the O
patient B
care O
processes O
. O

Care O
coordination O
in O
integrated O
networks O
can O
be O
achieved O
through O
integration O
of O
training O
programs O
and O
some O
clinical O
services O
, O
provision O
of O
complementary O
clinical O
capabilities O
, O
clinical O
geographic O
proximity O
design O
, O
clear O
role O
definition O
of O
each O
institution O
, O
commitment O
and O
flexibility O
of O
leaderships O
and O
medical O
staffs O
, O
and O
the O
support O
of O
a O
large O
referring O
physician O
groups O
embracing O
the O
affiliation O
concepts O
[ O
48 O
] O
. O

The O
issues O
of O
clinical O
integration O
in O
the O
PCCNs O
could O
include O
[ O
48 O
- O
50 O
] O
: O

* O
planning O
and O
differentiating O
target O
markets O
based O
on O
the O
clinical O
services O
of O
the O
network O
members O

* O
uniting O
individual O
clinical O
professionals O
for O
clinical O
project O
planning O

* O
designing O
patient B
- O
centered O
care O
or O
case O
management O
teams O

* O
establishing O
committees O
responsible O
for O
patient B
- O
centered O
case O
report O
meetings O
, O
case O
referral O
, O
transfer O
, O
and O
tracing O
, O
file O
management O
( O
record O
and O
information O
exchanges O
) O
, O
clinical O
quality O
management O
( O
quality O
assurance O
, O
improvement O
, O
risk O
and O
malpractice O
management O
, O
and O
utilization O
review O
) O
, O
and O
medical O
continuing O
education O
and O
on O
- O
job O
education O
. O

Marketing O
infrastructure O

Marketing O
integration O
refers O
to O
how O
to O
work O
together O
as O
a O
whole O
both O
from O
the O
provider O
and O
patient B
perspectives O
. O

One O
of O
the O
case O
reports O
interviewing O
developing O
integrated O
delivery O
system O
or O
networks O
realized O
that O
the O
most O
important O
thing O
is O
how O
an O
integrated O
system O
or O
network O
is O
promoted O
and O
what O
is O
promoted O
for O
the O
consumers O
[ O
51 O
] O
, O
including O
focusing O
on O
product O
development O
, O
making O
sure O
the O
branding O
holds O
together O
, O
marketing O
directly O
to O
consumers B
, O
demonstrating O
values O
to O
consumers B
, O
and O
even O
conducting O
marketing O
research O
to O
make O
efforts O
for O
the O
long O
term O
. O

In O
a O
health O
care O
network O
with O
several O
organizational O
members O
and O
target O
patients B
, O
the O
marketing O
infrastructure O
in O
PCCNs O
here O
refers O
to O
provider O
members O
' O
marketing O
, O
meaning O
the O
resource O
sharing O
and O
market O
development O
in O
a O
PCCN O
as O
a O
whole O
. O

The O
issues O
of O
the O
marketing O
integration O
in O
the O
PCCNs O
could O
include O
[ O
37 O
, O
52 O
- O
54 O
] O
: O

* O
sharing O
the O
literature O
and O
facility O
publications O
among O
the O
network O
members O

* O
uniting O
public O
promotions O
such O
as O
united O
activities O
, O
electronic O
and O
paper O
media O
for O
enhancing O
the O
network O
reputation O
as O
" O
one O
system O
, O
one O
brand O
and O
quality O
" O

* O
differentiating O
target O
markets O
of O
the O
network O
for O
competing O
in O
the O
medical O
industry O
. O

Financial O
infrastructure O

Comprehensive O
, O
flexible O
, O
and O
adequate O
financing O
is O
a O
goal O
of O
the O
ideal O
continuum O
of O
care O
. O

That O
component O
is O
the O
most O
critical O
and O
challenging O
to O
manage O
under O
the O
changes O
in O
the O
health O
care O
delivery O
environment O
. O

Gillies O
et O
al O
. O

[ O
30 O
] O
suggested O
that O
integrating O
financial O
management O
across O
operating O
units O
adds O
the O
greatest O
value O
to O
systems O
or O
organizations O
. O

In O
one O
case O
study O
, O
Bramson O
et O
al O
. O

[ O
55 O
] O
also O
showed O
that O
reducing O
costs O
through O
joint O
purchasing O
by O
the O
radiology O
departments O
of O
a O
vertically O
integrated O
health O
system O
could O
yield O
substantial O
savings O
. O

The O
issues O
of O
the O
financial O
integration O
in O
the O
PCCNs O
could O
include O
: O

* O
budgeting O

* O
uniting O
equipment O
, O
medical O
materials O
, O
and O
drug O
purchasing O
and O
routine O
administrative O
stuff O
management O

* O
pooling O
recruitment O
funds O

* O
designing O
a O
financial O
risk O
and O
sharing O
mechanism O
. O

Information O
infrastructure O

Information O
is O
an O
essential O
component O
of O
an O
organization O
. O

A O
complete O
information O
system O
can O
help O
an O
organization O
to O
integrate O
its O
individual O
units O
and O
efficiently O
manage O
the O
continuum O
. O

The O
ideal O
information O
system O
for O
a O
continuum O
of O
care O
was O
conceived O
of O
and O
formed O
in O
the O
mid O
- O
1980s O
[ O
56 O
] O
. O

During O
the O
late O
1980s O
, O
computer O
technology O
began O
to O
make O
an O
information O
system O
feasible O
and O
affordable O
through O
new O
computer O
chips O
with O
expanded O
capability O
and O
networking O
technology O
. O

In O
the O
1990s O
, O
the O
individual O
services O
of O
the O
continuum O
upgraded O
their O
information O
systems O
to O
combine O
clinical O
, O
financial O
, O
and O
utilization O
data O
[ O
29 O
] O
. O

Some O
studies O
have O
argued O
that O
the O
quality O
of O
information O
systems O
can O
drive O
costs O
down O
, O
because O
a O
good O
information O
system O
can O
give O
physicians O
easy O
electronic O
access O
to O
complete O
the O
documentation O
of O
the O
patients B
' O
clinical O
records O
, O
better O
inform O
them O
about O
reimbursement O
and O
capitation O
issues O
, O
help O
them O
easily O
associate O
and O
manage O
cases O
together O
, O
and O
achieve O
a O
higher O
level O
of O
professional O
satisfaction O
[ O
57 O
, O
58 O
] O
. O

Using O
Inova O
Health O
System O
, O
an O
integrated O
delivery O
system O
in O
northern O
Virginia O
, O
as O
an O
example O
, O
Wager O
, O
Heda O
, O
and O
Austin O
[ O
59 O
] O
showed O
that O
by O
developing O
a O
health O
information O
network O
within O
an O
integrated O
delivery O
system O
, O
Inova O
can O
have O
a O
clinical O
transaction O
system O
for O
hospitals O
and O
other O
entities O
, O
a O
data O
repository O
for O
decision O
support O
and O
outcome O
management O
, O
a O
managed O
care O
information O
system O
to O
support O
managed O
care O
and O
capitation O
contracts O
, O
and O
greater O
capability O
to O
acquire O
physicians O
. O

The O
issues O
of O
information O
coordination O
include O
[ O
60 O
- O
68 O
] O
: O

* O
establishing O
an O
electronic O
medical O
record O
system O
, O
regional O
information O
network O
for O
patient B
clinical O
and O
administrative O
data O
, O
clinical O
service O
arrangements O
and O
administrative O
work O

* O
uniting O
the O
system O
information O
management O
and O
web O
pages O
. O

The O
structured O
questionnaire O
was O
developed O
with O
the O
wording O
of O
practical O
managerial O
actions O
based O
on O
the O
five O
concepts O
just O
mentioned O
. O

There O
were O
19 O
survey O
items O
on O
governance O
infrastructure O
, O
25 O
on O
clinical O
infrastructure O
, O
13 O
on O
marketing O
infrastructure O
, O
20 O
on O
financial O
infrastructure O
, O
and O
7 O
on O
information O
infrastructure O
. O

All O
84 O
items O
were O
, O
simultaneously O
, O
applied O
to O
examine O
the O
relationships O
of O
the O
clinic O
' O
s O
peer O
members O
and O
the O
relationship O
of O
clinic O
and O
hospital O
members O
in O
a O
PCCN O
, O
and O
it O
resulted O
in O
a O
total O
of O
168 O
survey O
questions O
. O

The O
detailed O
information O
of O
the O
item O
questions O
was O
listed O
in O
Table O
2 O
, O
3 O
, O
4 O
, O
5 O
, O
6 O
. O

The O
structured O
questionnaires O
were O
drafted O
from O
previous O
literatures O
and O
then O
examined O
by O
two O
academic O
professors O
for O
theoretical O
accuracy O
. O

Then O
one O
pilot O
study O
was O
pre O
- O
tested O
for O
the O
PCCN O
pioneers O
( O
i O
. O
e O
. O
, O
92 O
network O
clinic O
members O
) O
and O
116 O
hospital O
providers O
which O
have O
partner O
relationships O
with O
other O
health O
care O
organizations O
( O
i O
. O
e O
. O
, O
hospitals O
, O
clinics O
, O
long O
- O
term O
care O
facilities O
) O
. O

The O
wordings O
and O
meanings O
of O
each O
question O
item O
were O
revised O
to O
assure O
content O
validity O
. O

The O
Cronbach O
alpha O
values O
for O
the O
five O
integration O
constructs O
- O
governance O
, O
clinical O
, O
marketing O
, O
finance O
, O
and O
information O
infrastructure O
were O
0 O
. O
946 O
, O
0 O
. O
958 O
, O
0 O
. O
932 O
, O
0 O
. O
944 O
, O
and O
0 O
. O
898 O
for O
the O
measures O
of O
clinic O
- O
clinic O
member O
relationships O
; O
and O
0 O
. O
945 O
, O
0 O
. O
949 O
, O
0 O
. O
916 O
, O
0 O
. O
948 O
, O
and O
0 O
. O
896 O
for O
the O
measures O
of O
clinic O
- O
hospital O
member O
relationships O
. O

Study O
subjects O

To O
find O
the O
member O
partnership O
, O
we O
sent O
questionnaires O
to O
1 O
, O
557 O
individual O
clinics O
which O
had O
belonged O
to O
PCCNs O
for O
at O
least O
one O
year O
, O
based O
on O
information O
contained O
in O
the O
Taiwan O
Primary O
Community O
Care O
Network O
List O
( O
Bureau O
of O
National O
Health O
Insurance O
2003 O
, O
2004 O
and O
2005 O
) O
. O

We O
let O
clinic O
members O
in O
all O
PCCNs O
point O
out O
how O
they O
coordinate O
with O
their O
peer O
clinic O
members O
and O
hospital O
members O
within O
a O
PCCN O
because O
individual O
clinic O
members O
could O
be O
better O
informants O
than O
hospital O
members O
, O
which O
need O
to O
deal O
with O
multiple O
clinic O
relationships O
and O
therefore O
might O
find O
it O
hard O
to O
describe O
the O
coordination O
involvement O
one O
by O
one O
with O
clinic O
members O
. O

Moreover O
, O
networks O
form O
for O
various O
reasons O
and O
it O
might O
lead O
to O
the O
various O
involvements O
by O
individual O
network O
members O
( O
i O
. O
e O
. O
, O
hospital O
and O
clinic O
members O
) O
. O

Therefore O
, O
using O
the O
participating O
clinics O
as O
individual O
survey O
units O
, O
the O
results O
could O
portray O
the O
overall O
dynamics O
and O
processes O
more O
authentically O
and O
detailed O
throughout O
all O
PCCNs O
in O
the O
demonstration O
project O
. O

Nine O
hundred O
and O
twenty O
- O
eight O
clinics O
responded O
( O
59 O
. O
6 O
% O
) O
, O
with O
239 O
clinics O
in O
the O
Taipei O
region O
, O
165 O
in O
the O
northern O
region O
, O
241 O
in O
the O
central O
region O
, O
108 O
in O
the O
southern O
region O
, O
150 O
in O
the O
Kao O
- O
Ping O
region O
, O
and O
15 O
in O
the O
eastern O
region O
of O
Taiwan O
. O

Ten O
clinics O
had O
not O
mentioned O
their O
practicing O
locations O
. O

There O
is O
no O
statistically O
significant O
difference O
in O
geographical O
distribution O
between O
the O
respondents O
and O
the O
study O
population O
( O
chi O
2 O
= O
4 O
. O
208 O
, O
p O
> O
0 O
. O
05 O
) O
. O

Analytical O
techniques O

The O
data O
was O
first O
analyzed O
descriptively O
with O
frequency O
counts O
( O
percentage O
) O
for O
each O
survey O
item O
, O
instead O
of O
using O
mean O
as O
a O
statistical O
method O
, O
because O
the O
variation O
among O
the O
respondents O
may O
not O
represent O
the O
normal O
distribution O
and O
it O
might O
ignore O
the O
extreme O
values O
for O
the O
respondents O
' O
answers O
. O

To O
compare O
how O
the O
respondents O
perceived O
the O
strength O
of O
integration O
existing O
in O
clinic O
- O
clinic O
and O
clinic O
- O
hospital O
relationships O
, O
paired O
t O
- O
tests O
were O
performed O
for O
individual O
survey O
items O
, O
using O
the O
original O
numerical O
scores O
. O

Results O

Profiling O
the O
partnerships O
in O
Taiwan O
PCCNs O
: O
governance O
infrastructure O

With O
regard O
to O
the O
governance O
infrastructures O
, O
the O
frequency O
was O
counted O
for O
each O
survey O
item O
with O
recalculated O
scales O
: O
disagree O
( O
Likert O
scale O
1 O
and O
2 O
) O
, O
fair O
( O
Likert O
scale O
3 O
) O
, O
and O
agree O
( O
Likert O
scale O
4 O
and O
5 O
) O
with O
individual O
items O
. O

In O
clinic O
- O
clinic O
relationship O
( O
Table O
2 O
) O
, O
the O
majority O
of O
clinic O
members O
agree O
that O
the O
determined O
deals O
were O
obeyed O
( O
Table O
2 O
, O
item O
1 O
: O
88 O
. O
69 O
% O
) O
, O
the O
goals O
and O
strategies O
of O
members O
were O
well O
- O
understood O
( O
Table O
2 O
, O
item O
16 O
: O
79 O
. O
74 O
% O
) O
, O
and O
the O
united O
principals O
for O
individual O
members O
were O
developed O
( O
Table O
2 O
, O
item O
15 O
: O
79 O
. O
42 O
% O
) O
. O

The O
higher O
percentages O
were O
also O
found O
in O
clinic O
- O
hospital O
relationship O
in O
the O
same O
items O
( O
Table O
2 O
) O
. O

On O
the O
other O
hand O
, O
establishing O
fair O
coordination O
mechanism O
( O
Table O
2 O
, O
item O
11 O
: O
27 O
. O
91 O
% O
) O
, O
designing O
and O
employing O
the O
network O
performance O
indicators O
( O
Table O
2 O
, O
item O
3 O
: O
21 O
. O
23 O
% O
) O
, O
and O
establishing O
communication O
models O
and O
channels O
( O
Table O
2 O
, O
item O
12 O
: O
19 O
. O
94 O
% O
) O
still O
occupied O
higher O
percentages O
not O
developed O
and O
deserved O
to O
been O
made O
the O
focus O
of O
more O
efforts O
in O
the O
future O
. O

Paired O
t O
- O
test O
analyses O
for O
all O
individual O
survey O
items O
of O
governance O
infrastructure O
showed O
that O
the O
deals O
obeyed O
( O
Table O
2 O
, O
item1 O
) O
and O
plans O
and O
goals O
controlled O
( O
Table O
2 O
, O
item O
2 O
) O
were O
achieved O
more O
in O
clinic O
- O
clinic O
relationships O
than O
those O
in O
clinic O
- O
hospital O
relationships O
; O
however O
, O
the O
design O
of O
network O
performance O
indicators O
( O
Table O
2 O
, O
item O
3 O
) O
, O
development O
of O
disintegration O
policy O
and O
principals O
( O
Table O
2 O
, O
item O
8 O
) O
, O
and O
the O
establishment O
of O
fair O
coordination O
mechanism O
( O
Table O
2 O
, O
item O
11 O
) O
were O
reached O
more O
in O
clinic O
- O
hospital O
relationships O
than O
those O
in O
clinic O
- O
clinic O
relationships O
. O

Profiling O
the O
partnerships O
in O
Taiwan O
PCCNs O
: O
clinical O
infrastructure O

Examining O
the O
extent O
of O
clinical O
infrastructure O
for O
network O
members O
, O
establishing O
two O
- O
directed O
patient B
referral O
systems O
and O
patient B
referral O
information O
files O
( O
Table O
3 O
, O
items O
35 O
& O
37 O
) O
and O
uniting O
medical O
continuing O
education O
and O
on O
- O
job O
education O
( O
Table O
3 O
, O
item O
34 O
) O
were O
shown O
at O
a O
highly O
implemented O
rate O
in O
clinic O
- O
clinic O
( O
more O
than O
70 O
% O
) O
and O
clinic O
- O
hospital O
( O
more O
than O
80 O
% O
) O
relationships O
. O

On O
the O
other O
hand O
, O
network O
members O
had O
higher O
percentages O
( O
more O
than O
40 O
% O
) O
not O
to O
think O
about O
the O
possible O
integration O
mechanisms O
including O
establishing O
committees O
to O
deal O
with O
medical O
malpractice O
( O
Table O
3 O
, O
item O
44 O
) O
, O
planning O
and O
differentiating O
clinical O
market O
areas O
( O
Table O
3 O
, O
item O
20 O
) O
, O
and O
designing O
patient B
- O
centered O
case O
management O
teams O
( O
Table O
3 O
, O
item O
22 O
) O
. O

Overall O
, O
there O
was O
better O
clinical O
integration O
involvement O
for O
all O
the O
described O
items O
in O
clinic O
- O
hospital O
relationships O
than O
those O
in O
clinic O
- O
clinic O
relationships O
within O
a O
network O
in O
this O
study O
( O
see O
Table O
3 O
, O
paired O
t O
- O
tests O
, O
p O
< O
0 O
. O
05 O
) O
. O

Profiling O
the O
partnerships O
in O
Taiwan O
PCCNs O
: O
marketing O
infrastructure O

For O
marketing O
planning O
, O
the O
clinics O
had O
better O
integrated O
marketing O
activities O
with O
their O
respective O
hospitals O
than O
with O
peer O
clinic O
members O
within O
PCCNs O
for O
all O
studied O
items O
( O
Table O
4 O
, O
paired O
t O
- O
test O
, O
p O
< O
0 O
. O
05 O
) O
. O

Examining O
the O
clinic O
- O
clinic O
relationships O
, O
uniting O
social O
activities O
( O
Table O
4 O
, O
item O
53 O
) O
, O
sharing O
the O
individual O
facility O
reports O
for O
updated O
services O
( O
Table O
4 O
, O
item O
46 O
) O
, O
public O
promotion O
( O
Table O
4 O
, O
item O
54 O
) O
, O
and O
uniting O
and O
joining O
the O
facility O
activities O
( O
Table O
4 O
, O
item O
51 O
) O
were O
the O
top O
four O
marketing O
works O
done O
among O
clinic O
members O
( O
more O
than O
60 O
% O
implemented O
rate O
) O
; O
and O
those O
items O
also O
showed O
a O
higher O
implemented O
rate O
( O
more O
than O
70 O
% O
) O
between O
clinic O
and O
hospital O
members O
. O

On O
the O
other O
hand O
, O
facility O
assets O
such O
as O
reports O
( O
Table O
4 O
, O
item O
49 O
) O
, O
and O
professional O
literatures O
and O
books O
( O
Table O
4 O
, O
item O
45 O
) O
were O
not O
well O
- O
shared O
among O
clinic O
members O
( O
" O
never O
- O
thinking O
" O
rate O
: O
42 O
. O
13 O
% O
) O
. O

In O
addition O
, O
uniting O
the O
network O
publication O
could O
make O
more O
efforts O
in O
the O
future O
( O
" O
never O
- O
thinking O
" O
rate O
in O
item O
50 O
: O
39 O
. O
98 O
% O
) O
. O

The O
room O
for O
clinic O
- O
hospital O
partnership O
to O
think O
about O
acting O
was O
kind O
of O
different O
from O
those O
in O
the O
clinic O
- O
clinic O
relationship O
. O

In O
addition O
to O
the O
uniting O
publication O
that O
can O
be O
encouraged O
to O
improve O
the O
clinic O
- O
hospital O
relationship O
( O
Table O
4 O
, O
item O
50 O
: O
27 O
. O
48 O
% O
) O
, O
cooperating O
in O
research O
projects O
( O
Table O
4 O
, O
item O
52 O
: O
25 O
. O
00 O
% O
) O
and O
identifying O
and O
differentiating O
target O
markets O
( O
Table O
4 O
, O
item O
57 O
: O
24 O
. O
57 O
% O
) O
had O
still O
more O
opportunities O
to O
be O
focused O
on O
in O
the O
future O
. O

Profiling O
the O
partnerships O
in O
Taiwan O
PCCNs O
: O
financial O
infrastructure O

The O
PCCN O
members O
were O
found O
to O
have O
a O
lower O
extent O
of O
financial O
integration O
as O
evidenced O
by O
higher O
percentage O
of O
' O
' O
never O
thinking O
' O
' O
scale O
about O
the O
survey O
items O
on O
almost O
all O
items O
( O
see O
Table O
5 O
) O
. O

Slightly O
more O
integration O
( O
that O
is O
, O
' O
' O
acting O
' O
' O
rate O
) O
was O
found O
in O
only O
four O
items O
both O
in O
clinic O
- O
clinic O
relationships O
and O
in O
clinic O
- O
hospital O
relationships O
, O
including O
uniting O
budget O
planning O
( O
Table O
5 O
: O
item O
58 O
) O
, O
sharing O
places O
, O
materials O
, O
and O
equipment O
( O
Table O
5 O
: O
item O
68 O
) O
, O
uniting O
budgeting O
for O
certain O
services O
( O
Table O
5 O
: O
items O
72 O
) O
, O
and O
designing O
the O
resource O
distribution O
principals O
based O
on O
the O
whole O
network O
goals O
( O
Table O
5 O
: O
item O
77 O
) O
. O

Further O
examining O
the O
financial O
infrastructure O
in O
clinic O
- O
clinic O
relationship O
and O
clinic O
- O
hospital O
relationship O
, O
paired O
- O
t O
tests O
revealed O
that O
clinic O
- O
hospital O
partnerships O
were O
involved O
more O
in O
places O
, O
materials O
, O
and O
equipment O
sharing O
and O
maintenance O
( O
Table O
5 O
, O
items O
62 O
and O
68 O
) O
( O
p O
< O
0 O
. O
05 O
) O
and O
higher O
financial O
infrastructure O
coordination O
exists O
in O
clinic O
- O
clinic O
relationships O
( O
Table O
5 O
, O
items O
58 O
, O
59 O
, O
63 O
- O
65 O
, O
72 O
, O
74 O
, O
75 O
, O
and O
77 O
) O
( O
p O
< O
0 O
. O
05 O
) O
. O

Profiling O
the O
partnerships O
in O
Taiwan O
PCCNs O
: O
information O
infrastructure O

There O
was O
significantly O
greater O
integration O
of O
information O
in O
clinic O
- O
hospital O
than O
clinic O
- O
clinic O
relationships O
in O
all O
items O
in O
this O
category O
( O
Table O
6 O
, O
paired O
t O
- O
tests O
, O
p O
< O
0 O
. O
001 O
) O
. O

The O
greatest O
integration O
was O
found O
in O
electronic O
patient B
records O
( O
Table O
6 O
: O
item O
78 O
) O
, O
followed O
by O
information O
integration O
for O
patient B
data O
( O
Table O
6 O
: O
item O
79 O
) O
and O
clinical O
service O
arrangements O
( O
Table O
6 O
: O
item O
81 O
) O
. O

The O
lowest O
level O
of O
integration O
in O
information O
infrastructure O
was O
found O
in O
administrative O
works O
such O
as O
registration O
, O
billing O
and O
so O
on O
( O
Table O
6 O
: O
item O
82 O
, O
' O
' O
never O
- O
thinking O
' O
' O
rate O
more O
than O
50 O
% O
) O
within O
network O
members O
. O

Discussion O

In O
this O
study O
, O
we O
surveyed O
943 O
clinics O
that O
had O
belonged O
to O
Taiwan O
PCCNs O
for O
more O
than O
a O
year O
to O
understand O
the O
nature O
and O
extent O
of O
integration O
to O
which O
they O
and O
their O
associated O
PCCN O
members O
( O
clinics O
and O
hospitals O
) O
had O
in O
governance O
, O
clinical O
, O
marketing O
, O
financial O
, O
and O
information O
infrastructures O
. O

It O
was O
found O
a O
wide O
variance O
in O
the O
kind O
and O
degree O
of O
integration O
among O
them O
and O
a O
lot O
of O
room O
for O
better O
integration O
( O
Table O
2 O
, O
3 O
, O
4 O
, O
5 O
, O
6 O
) O
. O

From O
the O
governance O
perspective O
, O
we O
found O
lower O
integration O
was O
found O
in O
the O
establishment O
of O
fair O
coordination O
mechanism O
( O
Table O
2 O
: O
item O
11 O
) O
among O
member O
clinics O
and O
member O
hospitals O
. O

Coordination O
could O
be O
viewed O
from O
different O
perspectives O
, O
including O
the O
use O
of O
standardized O
languages O
and O
forms O
, O
organizational O
rules O
and O
procedures O
, O
the O
establishment O
of O
common O
rules O
, O
policies O
, O
and O
procedures O
, O
and O
the O
monitoring O
through O
memos O
, O
reports O
, O
and O
a O
computerized O
information O
system O
[ O
69 O
, O
70 O
] O
. O

Facing O
the O
cumbersome O
integration O
processes O
, O
it O
suggests O
that O
each O
PCCN O
' O
s O
headquarters O
should O
become O
actively O
involved O
and O
clarify O
the O
authority O
, O
responsibility O
and O
accountability O
of O
individual O
members O
, O
identify O
the O
potential O
conflict O
sources O
, O
and O
publicize O
the O
rules O
and O
regulation O
of O
network O
integration O
dynamics O
covering O
decision O
making O
processes O
, O
market O
planning O
, O
clinical O
teamwork O
designs O
, O
and O
financial O
reports O
of O
individual O
network O
members O
. O

These O
actions O
could O
enhance O
the O
trust O
and O
respect O
of O
network O
members O
one O
another O
and O
could O
improve O
the O
small O
extent O
of O
integration O
found O
in O
this O
study O
about O
the O
mechanisms O
for O
communication O
models O
and O
channels O
in O
the O
PCCNs O
( O
Table O
2 O
: O
item O
12 O
) O
. O

From O
the O
network O
management O
perspective O
, O
communication O
could O
occur O
between O
the O
various O
entities O
such O
as O
between O
hospital O
and O
clinics O
, O
primary O
care O
physicians O
and O
specialists O
, O
managers O
and O
clinical O
professionals O
, O
and O
even O
among O
the O
clinical O
professionals O
in O
the O
network O
. O

To O
develop O
effective O
and O
timely O
communication O
channels O
was O
the O
key O
for O
the O
management O
of O
integrated O
organizations O
[ O
30 O
- O
32 O
] O
and O
could O
alleviate O
the O
tensions O
that O
sometimes O
occur O
in O
the O
dynamics O
of O
the O
multi O
- O
organizations O
. O

In O
this O
study O
, O
it O
was O
found O
a O
low O
level O
of O
involvement O
of O
medical O
teams O
in O
medical O
projects O
, O
patient B
- O
centered O
case O
management O
, O
and O
case O
report O
meetings O
among O
the O
network O
members O
( O
Table O
3 O
: O
item O
21 O
, O
22 O
, O
and O
23 O
) O
from O
a O
clinical O
integration O
perspective O
. O

Several O
researchers O
have O
addressed O
that O
clinical O
integration O
providing O
a O
process O
of O
medical O
management O
, O
care O
management O
, O
case O
management O
, O
and O
patient B
management O
designed O
to O
transform O
the O
traditionally O
fragmented O
delivery O
system O
into O
a O
more O
cohesive O
system O
[ O
71 O
] O
, O
and O
lead O
to O
higher O
service O
quality O
and O
assure O
financial O
objectives O
[ O
72 O
, O
73 O
] O
. O

More O
attention O
could O
be O
paid O
to O
these O
activities O
in O
the O
future O
. O

In O
addition O
, O
it O
was O
also O
found O
less O
integration O
in O
planning O
and O
differentiating O
clinical O
market O
areas O
( O
Table O
3 O
, O
item O
20 O
) O
among O
the O
network O
members O
in O
the O
category O
of O
clinical O
infrastructure O
. O

This O
may O
result O
from O
the O
existing O
specialty O
diversities O
in O
individual O
PCCNs O
, O
which O
might O
not O
need O
to O
involve O
planning O
and O
differentiating O
market O
area O
based O
on O
the O
members O
' O
clinical O
services O
at O
the O
early O
stage O
of O
network O
development O
. O

There O
was O
more O
involvement O
in O
marketing O
efforts O
in O
clinic O
- O
hospital O
relationships O
than O
in O
clinic O
- O
clinic O
relationships O
. O

Generally O
speaking O
, O
hospitals O
have O
more O
resources O
( O
i O
. O
e O
. O
, O
money O
, O
human B
resources O
, O
materials O
, O
and O
physical O
assets O
) O
than O
clinics O
, O
which O
might O
explain O
the O
stronger O
marketing O
involvements O
between O
the O
clinic O
and O
hospital O
members O
, O
including O
the O
library O
sharing O
( O
books O
and O
literatures O
) O
, O
facility O
brochure O
dissemination O
, O
public O
promoting O
, O
and O
medical O
research O
cooperation O
. O

These O
integration O
efforts O
also O
meet O
the O
expectation O
of O
the O
national O
health O
authority O
for O
resource O
sharing O
and O
medical O
quality O
image O
enhancement O
among O
the O
health O
care O
providers O
. O

Financial O
infrastructure O
was O
found O
to O
be O
the O
least O
integrated O
, O
with O
most O
items O
never O
considered O
. O

Perhaps O
the O
only O
reason O
for O
the O
higher O
score O
of O
budget O
planning O
activities O
( O
Table O
5 O
: O
items O
58 O
and O
77 O
) O
was O
that O
BHNI O
required O
each O
PCCN O
to O
design O
and O
determine O
its O
budgeting O
arrangement O
in O
advance O
before O
joining O
the O
demonstration O
project O
. O

While O
slightly O
more O
financial O
involvement O
was O
made O
among O
network O
members O
( O
Table O
5 O
: O
items O
68 O
, O
72 O
& O
73 O
) O
, O
possibly O
due O
to O
similar O
needs O
, O
there O
remains O
a O
lot O
of O
room O
for O
financial O
integration O
in O
the O
future O
. O

There O
was O
a O
need O
for O
networks O
to O
develop O
electronic O
information O
systems O
, O
though O
creating O
and O
managing O
an O
integrated O
information O
system O
involves O
very O
detailed O
work O
. O

Most O
of O
the O
clinics O
surveyed O
have O
focused O
more O
on O
the O
individual O
public O
members O
' O
administrative O
works O
such O
as O
filing O
patient B
medical O
records O
, O
collecting O
and O
managing O
network O
patient B
clinical O
data O
, O
and O
scheduling O
clinical O
services O
, O
which O
were O
required O
by O
the O
BNHI O
. O

The O
factors O
for O
the O
health O
care O
managers O
to O
adopt O
the O
integrated O
clinical O
information O
systems O
include O
the O
decision O
of O
make O
or O
buy O
, O
adoption O
leadership O
, O
adoption O
objectives O
, O
implementation O
leadership O
, O
phased O
versus O
simultaneous O
implementation O
, O
parallel O
systems O
, O
information O
technology O
implementation O
policies O
and O
practices O
, O
use O
levels O
and O
resistance O
, O
and O
realized O
benefits O
and O
return O
on O
investment O
calculation O
[ O
66 O
] O
, O
which O
might O
be O
very O
cumbersome O
and O
time O
- O
consuming O
. O

It O
suggests O
that O
the O
network O
partners O
might O
be O
engaged O
, O
firstly O
, O
more O
in O
simpler O
network O
cooperation O
such O
as O
the O
administrative O
systems O
for O
patient B
admission O
to O
the O
network O
members O
and O
establishing O
united O
web O
pages O
for O
patients B
to O
access O
their O
family O
physicians O
and O
network O
members O
for O
medical O
and O
public O
promotion O
purposes O
. O

And O
for O
further O
integrated O
information O
investments O
, O
efforts O
must O
be O
redirected O
for O
network O
members O
to O
work O
together O
to O
define O
the O
approach O
to O
specific O
classes O
of O
integration O
for O
the O
long O
term O
[ O
74 O
] O
. O

Conclusion O

This O
study O
tried O
to O
portray O
and O
trace O
how O
the O
facility O
participants B
were O
involved O
in O
the O
Taiwan O
PCCNs O
. O

It O
was O
found O
that O
Taiwan O
PCCNs O
' O
members O
had O
higher O
involvement O
in O
the O
governance O
infrastructure O
, O
which O
was O
usually O
viewed O
as O
the O
most O
important O
for O
establishment O
of O
core O
values O
in O
PCCNs O
' O
organization O
design O
and O
management O
. O

There O
existed O
a O
higher O
extent O
of O
integration O
of O
clinical O
, O
marketing O
, O
and O
information O
infrastructures O
among O
the O
hospital O
- O
clinic O
member O
relationship O
than O
those O
among O
clinic O
members O
within O
individual O
PCCNs O
. O

The O
financial O
infrastructure O
was O
shown O
the O
least O
integrated O
relative O
to O
other O
functional O
infrastructures O
at O
the O
early O
stage O
of O
PCCN O
formation O
. O

Page O
[ O
43 O
] O
argued O
that O
networks O
form O
and O
grow O
for O
various O
reasons O
, O
however O
, O
only O
some O
of O
them O
could O
be O
compatible O
with O
the O
iterative O
processes O
of O
collaboration O
. O

Some O
participants B
in O
the O
PCCNs O
may O
simply O
seek O
short O
- O
term O
economic O
gains O
and O
have O
little O
interest O
in O
joint O
learning O
and O
continuous O
improvement O
. O

From O
an O
organizational O
design O
perspective O
, O
the O
old O
phrase O
proposed O
by O
the O
wisdom O
of O
the O
saying O
about O
developing O
the O
integrated O
organizations O
( O
networks O
) O
should O
be O
- O
" O
coming O
together O
is O
the O
beginning O
, O
and O
working O
together O
is O
the O
success O
. O
" O
Page O
[ O
43 O
] O
examined O
the O
virtual O
provider O
organizations O
such O
as O
physician O
- O
hospital O
organizations O
and O
pointed O
out O
the O
issue O
of O
the O
provider O
attitudes O
and O
behaviors O
as O
the O
critically O
successful O
continuous O
improvements O
in O
the O
health O
care O
environments O
. O

A O
wide O
variance O
of O
degree O
of O
network O
integration O
in O
Taiwan O
PCCNs O
still O
leaves O
room O
to O
improve O
. O

In O
this O
study O
, O
the O
thoroughly O
surveyed O
items O
, O
that O
is O
, O
the O
potential O
network O
design O
content O
, O
were O
employed O
. O

In O
addition O
to O
provide O
how O
the O
network O
members O
have O
done O
their O
initial O
work O
at O
the O
early O
stage O
of O
network O
forming O
in O
this O
study O
, O
the O
detailed O
surveyed O
items O
, O
the O
concepts O
proposed O
by O
the O
managerial O
and O
theoretical O
professionals O
, O
could O
be O
also O
a O
guide O
for O
those O
health O
care O
providers O
who O
have O
a O
willingness O
to O
join O
multi O
- O
organizations O
. O

It O
suggests O
that O
health O
care O
providers O
could O
take O
more O
detailed O
looks O
about O
those O
surveyed O
items O
and O
give O
some O
possible O
opportunities O
to O
create O
the O
potential O
actions O
. O

Further O
research O
could O
be O
empirically O
done O
to O
explore O
the O
relative O
influence O
of O
these O
integration O
mechanisms O
on O
the O
effectiveness O
of O
organizational O
partnerships O
. O

The O
partnerships O
within O
each O
PCCN O
represent O
various O
relationships O
that O
depend O
on O
how O
much O
the O
members O
are O
engaged O
in O
the O
projects O
. O

In O
addition O
to O
the O
macro O
concepts O
including O
governance O
, O
clinical O
, O
marketing O
, O
financial O
, O
and O
information O
infrastructures O
explored O
in O
this O
study O
, O
other O
managerial O
issues O
for O
integrated O
organizations O
were O
also O
suggested O
such O
as O
formation O
of O
an O
integrated O
cultural O
atmosphere O
, O
human B
resources O
management O
, O
physician O
involvement O
, O
mission O
and O
commitment O
establishment O
, O
from O
micro O
organizational O
behavior O
perspective O
[ O
30 O
- O
32 O
, O
34 O
, O
36 O
, O
75 O
, O
76 O
] O
. O

Micro O
managerial O
and O
longitudinal O
research O
designs O
could O
be O
employed O
to O
more O
precisely O
catch O
the O
never O
completing O
integration O
efforts O
in O
the O
future O
. O

Abbreviations O

primary O
community O
care O
network O
( O
PCCN O
) O
; O
Bureau O
of O
National O
Health O
Insurance O
( O
BNHI O
) O

Competing O
interests O

The O
author O
( O
s O
) O
declare O
that O
they O
have O
no O
competing O
interests O
. O

Authors O
' O
contributions O

BYJL O
independently O
designed O
and O
conducted O
this O
study O
. O

Pre O
- O
publication O
history O

The O
pre O
- O
publication O
history O
for O
this O
paper O
can O
be O
accessed O
here O
: O

Recombinant O
activated O
protein O
C O
in O
sepsis O
: O
endothelium O
protection O
or O
endothelium O
therapy O
? O

Abstract O

Endothelium O
dysfunction O
is O
one O
of O
the O
hallmarks O
of O
sepsis O
. O

Looney O
and O
Mattay O
, O
in O
the O
previous O
issue O
of O
Critical O
Care O
, O
highlight O
the O
role O
of O
activated O
protein O
C O
( O
APC O
) O
as O
a O
protective O
endothelial O
drug O
in O
septic O
situations O
. O

Nevertheless O
, O
the O
results O
of O
in O
vivo O
studies O
are O
less O
explicit O
and O
it O
remains O
uncertain O
whether O
these O
properties O
are O
relevant O
in O
human B
septic O
shock O
. O

Before O
considering O
recombinant O
APC O
( O
rAPC O
) O
as O
a O
therapeutic O
drug O
for O
the O
endothelium O
, O
we O
have O
to O
demonstrate O
its O
efficiency O
to O
protect O
or O
to O
reduce O
endothelium O
injury O
when O
infused O
a O
long O
time O
after O
the O
septic O
challenge O
. O

Nevertheless O
, O
if O
rAPC O
is O
efficient O
when O
infused O
in O
the O
early O
phase O
of O
septic O
challenge O
, O
we O
thus O
need O
to O
treat O
our O
patients B
earlier O
. O

At O
the O
least O
, O
genetically O
engineered O
variants O
have O
been O
designed O
with O
greater O
anti O
- O
apoptotic O
activity O
and O
reduced O
anticoagulant O
activity O
relative O
to O
wild O
- O
type O
APC O
. O

Further O
studies O
are O
needed O
to O
demonstrate O
the O
usefulness O
of O
these O
variants O
in O
septic O
shock O
therapy O
. O

The O
use O
of O
recombinant O
activated O
protein O
C O
( O
rAPC O
) O
is O
one O
of O
the O
hottest O
topics O
in O
septic O
shock O
therapy O
. O

The O
pivotal O
phase O
3 O
placebo O
- O
controlled O
Protein O
C O
Worldwide O
Evaluation O
in O
Severe O
Sepsis O
( O
PROWESS O
) O
clinical O
trial O
demonstrated O
a O
19 O
. O
4 O
% O
relative O
risk O
reduction O
in O
28 O
- O
day O
mortality O
( O
6 O
. O
1 O
% O
absolute O
risk O
reduction O
) O
with O
an O
increased O
risk O
( O
3 O
. O
5 O
% O
versus O
2 O
. O
0 O
% O
) O
of O
serious O
bleeding O
events O
compared O
with O
placebo O
. O

Two O
recent O
and O
important O
articles O
have O
highlighted O
the O
role O
of O
APC O
as O
a O
protective O
endothelial O
drug O
[ O
1 O
] O
and O
as O
a O
cyto O
- O
protective O
drug O
[ O
2 O
] O
. O

Beneficial O
effects O
of O
rAPC O
in O
the O
PROWESS O
study O
were O
thought O
to O
be O
related O
to O
a O
reduction O
in O
coagulation O
and O
, O
to O
a O
lesser O
extent O
, O
to O
a O
reduction O
in O
inflammatory O
response O
to O
sepsis O
[ O
3 O
] O
. O

Post O
- O
PROWESS O
investigations O
have O
been O
associated O
with O
a O
myriad O
of O
cellular O
or O
animal O
studies O
demonstrating O
that O
rAPC O
, O
through O
reactions O
mediated O
by O
endothelial O
protein O
C O
receptor O
and O
the O
effector O
receptor O
, O
protease O
activated O
receptor O
- O
1 O
, O
acts O
directly O
on O
cells O
to O
exert O
multiple O
cytoprotective O
effects O
including O
: O
down O
regulation O
of O
pro O
- O
inflammatory O
gene O
expression O
[ O
4 O
] O
; O
anti O
- O
inflammatory O
activities O
[ O
5 O
] O
; O
anti O
- O
apoptotic O
activity O
[ O
6 O
] O
; O
and O
protection O
of O
endothelial O
barrier O
function O
[ O
1 O
, O
2 O
] O
. O

Endothelium O
dysfunction O
is O
one O
of O
the O
hallmarks O
of O
sepsis O
[ O
7 O
] O
. O

Sepsis O
, O
per O
se O
, O
may O
induce O
phenotypic O
modulations O
of O
the O
endothelium O
through O
direct O
or O
indirect O
interaction O
of O
the O
endothelial O
layer O
with O
components O
of O
the O
bacterial O
wall O
, O
inducing O
a O
myriad O
of O
host O
- O
derived O
factors O
from O
endothelial O
cells O
. O

Phenotypic O
modifications O
include O
changes O
in O
pro O
- O
coagulant O
and O
proadhesive O
properties O
, O
increased O
endothelial O
permeability O
, O
endothelial O
cell O
apoptosis O
and O
changes O
in O
vasomotor O
properties O
; O
the O
last O
of O
these O
is O
crucial O
since O
vasoplegia O
is O
directly O
related O
to O
septic O
shock O
mortality O
. O

Recent O
animal O
and O
human B
data O
have O
suggested O
that O
rAPC O
may O
improve O
both O
vascular O
and O
myocardial O
dysfunction O
and O
vascular O
reactivity O
to O
catecholamine O
during O
endotoxin O
and O
/ O
or O
septic O
challenge O
[ O
8 O
, O
9 O
] O
. O

From O
bench O
to O
bedside O

Experimental O
evidence O
supports O
a O
role O
of O
APC O
in O
maintaining O
the O
integrity O
of O
the O
endothelium O
through O
both O
direct O
and O
indirect O
mechanisms O
. O

Nevertheless O
, O
the O
results O
of O
in O
vivo O
studies O
are O
less O
explicit O
. O

In O
a O
retrospective O
study O
of O
septic O
shock O
in O
humans B
, O
Monnet O
and O
colleagues O
[ O
9 O
] O
demonstrated O
that O
APC O
infusion O
was O
associated O
with O
a O
decrease O
in O
the O
amount O
of O
delivered O
norepinephrine O
. O

Wiel O
and O
colleagues O
[ O
10 O
] O
demonstrated O
in O
a O
rabbit B
model O
of O
endotoxin O
induced O
shock O
that O
APC O
decreased O
aorta O
endothelial O
injury O
. O

By O
contrast O
, O
in O
a O
lung O
model O
of O
endotoxin O
induced O
inflammation O
, O
Robriquet O
and O
colleagues O
[ O
11 O
] O
demonstrated O
a O
trend O
to O
an O
increased O
vascular O
permeability O
using O
high O
doses O
of O
human B
APC O
. O

This O
last O
result O
was O
in O
sharp O
contrast O
with O
the O
results O
obtained O
by O
Nick O
and O
colleagues O
[ O
12 O
] O
in O
a O
human B
model O
of O
pulmonary O
endotoxin O
administration O
. O

APC O
appears O
to O
improve O
mortality O
in O
septic O
shock O
with O
a O
high O
APACHE O
2 O
score O
and O
is O
potentially O
detrimental O
in O
severe O
sepsis O
. O

In O
rats B
, O
APC O
markedly O
decreased O
tumour O
necrosis O
factor O
concentrations O
whereas O
they O
remained O
unchanged O
in O
either O
human B
septic O
shock O
or O
endotoxemia O
. O

The O
question O
arises O
, O
therefore O
, O
as O
to O
whether O
it O
is O
truly O
possible O
to O
reconcile O
all O
these O
discrepancies O
? O

Moreover O
, O
can O
these O
stirring O
laboratory O
data O
be O
translated O
into O
clinical O
practice O
? O

Limitations O
of O
experimental O
studies O
: O
endothelium O
protection O
versus O
endothelium O
therapy O

Clearly O
, O
in O
cellular O
and O
animal O
models O
, O
rAPC O
has O
been O
given O
either O
as O
a O
pre O
- O
treatment O
or O
concurrent O
with O
septic O
challenge O
. O

This O
mode O
of O
administration O
favours O
the O
anti O
- O
inflammatory O
effects O
of O
rAPC O
, O
which O
are O
particularly O
efficient O
in O
murine B
models O
in O
protecting O
the O
endothelium O
from O
cytokine O
- O
mediated O
apoptosis O
or O
upregulation O
of O
endothelial O
adhesion O
molecules O
. O

Thus O
, O
studies O
using O
post O
- O
injury O
treatment O
are O
needed O
in O
models O
that O
mimic O
septic O
shock O
, O
such O
as O
experimental O
pneumonia O
or O
peritonitis O
treated O
by O
antibiotics O
and O
volume O
resuscitation O
, O
and O
where O
the O
effects O
of O
rAPC O
would O
be O
investigated O
16 O
to O
24 O
hours O
after O
septic O
challenge O
. O

If O
we O
can O
demonstrate O
the O
efficiency O
of O
rAPC O
to O
protect O
or O
to O
reduce O
endothelium O
injury O
in O
these O
conditions O
, O
we O
can O
ultimately O
postulate O
that O
rAPC O
is O
also O
a O
therapeutic O
drug O
for O
the O
endothelium O
. O

The O
earlier O
the O
better O

If O
rAPC O
is O
efficient O
when O
infused O
in O
the O
early O
phase O
of O
septic O
challenge O
, O
we O
thus O
need O
to O
treat O
our O
patients B
earlier O
. O

At O
least O
two O
studies O
suggest O
that O
treatment O
with O
rAPC O
within O
24 O
hours O
may O
carry O
a O
larger O
survival O
advantage O
for O
patients B
with O
severe O
sepsis O
, O
compared O
with O
those O
treated O
more O
than O
24 O
hours O
after O
organ O
dysfunction O
[ O
13 O
] O
. O

Interventions O
directed O
at O
specific O
endpoints O
, O
when O
initiated O
early O
in O
the O
' O
golden O
hours O
' O
of O
a O
patient B
' O
s O
condition O
, O
seem O
to O
be O
promising O
[ O
14 O
] O
. O

The O
beneficial O
effects O
of O
earlier O
administration O
of O
rAPC O
to O
appropriate O
patients B
may O
fit O
into O
this O
paradigm O
. O

The O
future O

Extensive O
in O
vivo O
and O
in O
vitro O
studies O
have O
focused O
on O
the O
cytoprotective O
effects O
of O
APC O
and O
most O
authors O
agree O
that O
its O
anticoagulant O
and O
cytoprotective O
effects O
are O
mediated O
by O
distinct O
APC O
structural O
features O
. O

Positively O
charged O
residues O
in O
surface O
loops O
in O
the O
APC O
protease O
domain O
have O
been O
identified O
as O
participating O
in O
the O
anticoagulant O
activity O
but O
not O
in O
cellular O
effects O
. O

Hence O
, O
variants O
have O
been O
designed O
with O
greater O
anti O
- O
apoptotic O
activity O
and O
reduced O
anticoagulant O
activity O
relative O
to O
wild O
- O
type O
APC O
[ O
2 O
] O
. O

Whether O
these O
genetically O
engineered O
variants O
actually O
provide O
superior O
pharmacological O
properties O
remains O
to O
be O
elucidated O
in O
vivo O
. O

Such O
investigations O
may O
allow O
the O
design O
of O
therapeutic O
APC O
variants O
with O
decreased O
anticoagulant O
activity O
to O
reduce O
the O
risk O
of O
bleeding O
on O
the O
one O
hand O
, O
but O
also O
with O
normal O
cytoprotective O
properties O
in O
order O
to O
retain O
full O
beneficial O
effects O
on O
sepsis O
outcome O
. O

Abbreviations O

PROWESS O
= O
Protein O
C O
Worldwide O
Evaluation O
in O
Severe O
Sepsis O
; O
rAPC O
= O
recombinant O
activated O
protein O
C O
. O

Competing O
interests O

BL O
has O
received O
reimbursements O
and O
funding O
from O
Eli O
Lilly O
, O
France O
. O

Carbonic O
Anhydrase O
Inhibitors O
. O

Part O
541 O
: O
Metal O
Complexes O
of O
Heterocyclic O
Sulfonamides O
: O
A O
New O
Class O
of O
Antiglaucoma O
Agents O

Abstract O

Metal O
complexes O
of O
heterocyclic O
sulfonamides O
possessing O
carbonic O
anhydrase O
( O
CA O
) O
inhibitory O
properties O
were O
recently O
shown O
to O
be O
useful O
as O
intraocular O
pressure O
( O
IOP O
) O
lowering O
agents O
in O
experimental O
animals O
, O
and O
might O
be O
developed O
as O
a O
novel O
class O
of O
antiglaucoma O
drugs O
. O

Here O
we O
report O
the O
synthesis O
of O
a O
heterocyclic O
sulfonamide O
CA O
inhibitor O
and O
of O
the O
metal O
complexes O
containing O
main O
group O
metal O
ions O
, O
such O
as O
Be O
( O
II O
) O
, O
Mg O
( O
II O
) O
, O
Al O
( O
III O
) O
, O
Zn O
( O
II O
) O
, O
Cd O
( O
II O
) O
and O
Hg O
( O
II O
) O
and O
the O
new O
sulfonamide O
as O
well O
as O
5 O
- O
amino O
- O
1 O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
as O
ligands O
. O

The O
new O
complexes O
were O
characterized O
by O
standard O
physico O
- O
chemical O
procedures O
, O
and O
assayed O
as O
inhibitors O
of O
three O
CA O
isozymes O
, O
CA O
I O
, O
II O
and O
IV O
. O

Some O
of O
them O
( O
but O
not O
the O
parent O
sulfonamides O
) O
strongly O
lowered O
IOP O
in O
rabbits B
when O
administered O
as O
a O
2 O
% O
solution O
into O
the O
eye O
. O

CARBONIC O
ANHYDRASE O
INHIBITORS O
. O

Part O
54 O
METAL O
COMPLEXES O
OF O
HETEROCYCLIC O
SULFONAMIDES O
: O
A O
NEW O
CLASS O
OF O
ANTIGLAUCOMA O
AGENTS O
Claudiu O
T O
. O

Supuran O
* O
, O
Andrea O
Scozzafava O

and O
Andrei O
Jitianu O
2 O

Universit O
& O
degli O
Studi O
, O
Dipartimento O
di O
Chimica O
, O
Laboratorio O
di O
Chimica O
Inorganica O
e O
Bioinorganica O
, O
Via O
Gino O
Capponi O
7 O
, O
1 O
- O
50121 O
, O
Florence O
, O
Italy O
2 O
" O
I O
. O
G O
. O

Murgulescu O
" O
Institute O
of O
Physical O
Chemistry O
, O
Academia O
Romana O
, O
Spl O
. O

Independentei O
202 O
, O
R O
- O
77208 O
Bucharest O
, O
Roumania O
Abstract O
: O
Metal O
complexes O
of O
heterocyclic O
sulfonamides O
possessing O
carbonic O
anhydrase O
( O
CA O
) O
inhibitory O
properties O
were O
recently O
shown O
to O
be O
useful O
as O
intraocular O
pressure O
( O
IOP O
) O
lowering O
agents O
in O
experimental O
animals O
, O
and O
might O
be O
developed O
as O
a O
novel O
class O
of O
antiglaucoma O
drugs O
. O

Here O
we O
report O
the O
synthesis O
of O
a O
heterocyclic O
sulfonamide O
CA O
inhibitor O
and O
of O
the O
metal O
complexes O
containing O
main O
group O
metal O
ions O
, O
such O
as O
Be O
( O
II O
) O
, O
Mg O
( O
II O
) O
, O
AI O
( O
III O
) O
, O
Zn O
( O
II O
) O
, O
Cd O
( O
II O
) O
and O
Hg O
( O
II O
) O
and O
the O
new O
sulfonamide O
as O
well O
as O
5 O
- O
amino O
- O
l O
, O
3 O
, O
4thiadiazole O
- O
2 O
- O
sulfonamide O
as O
ligands O
. O

The O
new O
complexes O
were O
characterized O
by O
standard O
physico O
- O
chemical O
procedures O
, O
and O
assayed O
as O
inhibitors O
of O
three O
CA O
isozymes O
, O
CA O
I O
, O
II O
and O
IV O
. O

Some O
of O
them O
( O
but O
not O
the O
parent O
sulfonamides O
) O
strongly O
lowered O
IOP O
in O
rabbits B
when O
administered O
as O
a O
2 O
% O
solution O
into O
the O
eye O
. O

Introduction O
Sulfonamides O
possessing O
carbonic O
anhydrase O
( O
CA O
, O
EC O
4 O
. O
2 O
. O
1 O
. O
1 O
) O
inhibitory O
properties O
[ O
2 O
] O
such O
as O
acetazolamide O
1 O
, O
methazolamide O
2 O
, O
ethoxzolamide O
3 O
and O
dichlorophenamide O
4 O
have O
been O
used O
for O
more O
than O
40 O
years O
as O
pressure O
lowering O
systemic O
drugs O
in O
the O
treatment O
of O
open O
- O
angle O
glaucoma O
[ O
3 O
, O
4 O
] O
. O

Their O
effect O
is O
due O
to O
inhibition O
of O
at O
least O
two O
CA O
isozymes O
present O
within O
cilliary O
processes O
of O
the O
eye O
, O
ie O
, O
CA O
II O
and O
CA O
IV O
, O
which O
is O
followed O
by O
lowered O
bicarbonate O
formation O
and O
reduction O
of O
aqueous O
humor O
secretion O
[ O
5 O
- O
7 O
] O
. O

Their O
main O
drawback O
is O
constituted O
by O
side O
effects O
such O
as O
fatigue O
, O
augmented O
diuresis O
, O
or O
paresthesias O
, O
due O
to O
CA O
inhibition O
in O
other O
tissues O
/ O
organs O
than O
the O
target O
one O
, O
ie O
, O
the O
eye O
[ O
8 O
] O
. O

N O
- O
- O
N O

XN O
- O
- O
N O
EtO O
S O

NH O
2 O

O O

NH O
O O
: O
S O
- O
- O
- O
- O
O O

NHEt O

The O
above O
- O
mentioned O
side O
effects O
are O
absent O
in O
the O
case O
in O
which O
the O
inhibitor O
has O
topical O
activity O
, O
and O
is O
applied O
directly O
into O
the O
eye O
. O

This O
route O
has O
been O
demonstrated O
only O
in O
1983 O
by O
Maren O
' O
s O
group O
[ O
9 O
] O
and O
was O
followed O
by O
the O
development O
of O
the O
first O
clinical O
agent O
of O
this O
type O
, O
dorzolamide O
5 O
[ O
10 O
, O
11 O
] O
. O

Dorzolamide O
( O
Trusopt O
) O
has O
been O
introduced O
in O
clinical O
use O
in O
1995 O
in O
USA O
and O
Europe O
and O
it O
constituted O
the O
beginning O
of O
a O
radically O
new O
treatment O
of O
glaucoma O
, O
devoid O
of O
the O
severe O
side O
effects O
observed O
with O
the O
systemic O
inhibitors O
[ O
4 O
- O
6 O
] O
. O

The O
success O
of O
topical O
antiglaucoma O
CA O
inhibitors O
fostered O
much O
research O
in O
the O
synthesis O
and O
clinical O
evaluation O
of O
other O
types O
of O
such O
compounds O
[ O
12 O
- O
15 O
] O
. O

307 O

Vol O
. O

4 O
, O
No O
. O
6 O
, O
1997 O

Metal O
Complexes O
of O
Heterocyclic O
Sulfonamides O
: O
A O
New O
Class O
ofAntiglaucoma O
Agents O

On O
the O
other O
hand O
, O
metal O
complexes O
of O
heterocyclic O
sulfonamides O
of O
type O
1 O
- O
5 O
have O
been O
recently O
prepared O
by O
two O
groups O
16 O
- O
20 O
] O
, O
and O
it O
was O
proved O
that O
they O
possess O
much O
stronger O
CA O
inhibitory O
properties O
than O
the O
sulfonamides O
from O
which O
they O
were O
prepared O
18 O
- O
22 O
] O
. O

Although O
the O
mechanism O
of O
CA O
inhibition O
of O
the O
metal O
complexes O
is O
presently O
unknown O
, O
it O
was O
hypothesized O
that O
their O
increased O
inhibitory O
power O
might O
be O
due O
to O
two O
processes O
, O
occurring O
separately O
or O
in O
concert O
, O
ie O
, O
( O
i O
) O
dissociation O
of O
the O
complex O
inhibitor O
in O
sulfonamide O
anions O
and O
metal O
ions O
( O
in O
diluted O
solution O
) O
, O
which O
in O
turn O
both O
interact O
thereafter O
with O
the O
enzyme O
, O
at O
different O
binding O
sites O
, O
and O
( O
ii O
) O
direct O
interaction O
of O
the O
undissociated O
complex O
with O
the O
enzyme O
, O
and O
more O
specifically O
with O
the O
hydrophilic O
patch O
at O
the O
entrance O
of O
CA O
II O
active O
site O
[ O
23 O
] O

, O
this O
being O
the O
isozyme O
most O
susceptible O
to O
inhibition O
with O
this O
class O
of O
compounds O
[ O
2 O
, O
22 O
] O
. O

Whether O
initially O
the O
first O
mechanism O
of O
action O
mentioned O
above O
was O
favored O
by O
us O
[ O
22 O
] O
, O
recent O
evidences O
suggested O
that O
the O
undissociated O
complex O
might O
be O
the O
inhibitory O
species O
, O
at O
least O
for O
some O
isozymes O
[ O
24 O
] O
. O

Since O
metal O
complexes O
are O
much O
more O
inhibitory O
than O
the O
parent O
sulfonamide O
from O
which O
they O
were O
prepared O
, O
it O
appeared O
of O
interest O
to O
test O
whether O
this O
property O
might O
be O
useful O
for O
their O
use O
in O
lowering O
IOP O
in O
experimental O
animals O
and O
whence O
as O
a O
possible O
glaucoma O
therapy O
. O

Recently O
we O
proved O
[ O
25 O
, O
26 O
] O
that O
some O
metal O
complexes O
of O
heterocyclic O
sulfonamides O
( O
which O
themselves O
do O
not O
possess O
IOP O
lowering O
properties O
) O
act O
as O
very O
powerful O
such O
agents O
when O
administered O
as O
diluted O
solutions O
directly O
into O
the O
eye O
of O
experimental O
animals O
, O
and O
would O
thus O
offer O
the O
possibility O
of O
developing O
such O
totally O
novel O
drugs O
. O

Here O
we O
report O
the O
synthesis O
of O
a O
heterocyclic O
sulfonamide O
possessing O
strong O
CA O
inhibitory O
properties O
, O
ie O
, O
5 O
- O
( O
chloroacetamido O
) O
- O
l O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
, O
and O
of O
the O
metal O
complexes O
of O
this O
sulfonamide O
and O
of O
5 O
- O
amino O
- O
1 O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
, O
containing O
some O
main O
group O
metal O
ions O
. O

The O
new O
compounds O
have O
been O
characterized O
by O
standard O
physico O
- O
chemical O
procedures O
, O
and O
were O
assayed O
as O
inhibitors O
of O
three O
CA O
isozymes O
, O
hCA O
I O
, O
hCA O
II O
and O
bCA O
IV O
( O
h O
human B
; O
b O
bovine B
; O
these O
are O
the O
isozymes O
considered O
to O
play O
a O
critical O
role O
in O
aqueous O
humour O
secretion O
within O
the O
eye O
of O
higher O
vertebrates O
[ O
2 O
- O
5 O
] O
) O
. O

Materials O
and O
Methods O

Melting O
points O
were O
recorded O
with O
a O
heating O
plate O
microscope O
and O
are O
not O
corrected O
. O

IR O
spectra O
were O
recorded O
in O
KBr O
pellets O
with O
a O
Carl O
Zeiss O
IR O
- O
80 O
instrument O
. O

1H O
- O
NMR O
spectra O
were O
recorded O
in O
DMSO O
- O
d6 O
as O
solvent O
, O
with O
a O
Bruker O
CPX200 O
instrument O
. O

Chemical O
shifts O
are O
reported O
as O
values O
, O
relative O
to O
Me4Si O
as O
internal O
standard O
. O

Conductimetric O
measurements O
were O
done O
at O
room O
temperature O
( O
1 O
mM O
concentration O
of O
complex O
) O
in O
DMSO O
solution O
with O
a O
Fisher O
conductimeter O
. O

Elemental O
analyses O
were O
done O
by O
combustion O
for O
C O
, O
H O
, O
N O
with O
an O
automated O
Carlo O
Erba O
analyzer O
, O
and O
gravimetrically O
for O
the O
metal O
ions O
, O
and O
were O
0 O
. O
4 O
% O
of O
the O
theoretical O
values O
. O

Thermogravimetric O
measurements O
were O
done O
in O
air O
, O
at O
a O
heating O
rate O
of O
10C O
/ O
min O
. O
, O
with O
a O
Perkin O
Elmer O
3600 O
thermobalance O
. O

Sulfonamides O
used O
as O
standards O
in O
the O
enzymatic O
assay O
( O
except O
for O
5 O
) O
, O
acetazolamide O
, O
pyridine O
, O
and O
chloroacetyl O
chloride O
used O
for O
the O
preparation O
of O
compound O
7 O
, O
solvents O
as O
well O
as O
inorganic O
reagents O
were O
from O
Sigma O
, O
Merck O
and O
Carlo O
Erba O
. O

5 O
- O
Amino O
- O
l O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
6 O
was O
prepared O
from O
acetazolamide O
by O
literature O
procedures O
[ O
27 O
] O
, O
by O
desacetylation O
with O
concentrated O
hydrochloric O
acid O
, O
followed O
by O
neutralization O
with O
sodium O
bicarbonate O
of O
the O
corresponding O
hydrochloride O
( O
Scheme O
1 O
) O
. O

Dorzolamide O
hydrochloride O
5 O
was O
from O
Merck O
, O
Sharp O
and O
Dohme O
or O
was O
prepared O
as O
described O
by O
Ponticello O
et O
al O
10 O
, O
11 O
] O
. O

Human B
CA O
and O
CA O
II O
cDNAs O
were O
expressed O
in O
Escherichia B
coli I
strain I
BL21 I
( I
DE3 I
) O
from O
the O
plasmids O
pACA O
/ O
hCA O
I O
and O
pACAdaCA O
II O
described O
by O
Forsman O
et O
al O
. O

[ O
28 O
] O
( O
the O
two O
plasmids O
were O
a O
gift O
from O
Prof O
. O
Sven O
Lindskog O
, O
Umea O
University O
, O
Sweden O
) O
. O

Cell O
growth O
conditions O
were O
those O
described O
by O
Lindskog O
' O
s O
group O
[ O
29 O
] O
, O
and O
enzymes O
were O
purified O
by O
affinity O
chromatography O
according O
to O
the O
method O
of O
Khalifah O
et O
al O
[ O
30 O
] O
. O

Enzyme O
concentrations O
were O
determined O
spectrophotometrical O
at O
280 O
nm O
, O
utilizing O
a O
molar O
absorptivity O
of O
49 O
mM O
- O
l O
. O
cm O
- O
1 O
for O
hCA O
and O
54 O
mM O
- O
l O
. O
cm O
- O
1 O
for O
hCA O
II O
, O
respectively O
, O
based O
on O
M O
28 O
. O
85 O
kDa O
for O
hCA O
I O
, O
and O
29 O
. O
3 O
kDa O
for O
hCA O
II O
, O
respectively O
[ O
31 O
, O
32 O
] O
. O

bCA O
IV O
was O
isolated O
from O
bovine B
lung O
microsomes O
as O
described O
by O
Maren O
et O
al O
, O
and O
its O
concentration O
has O
been O
determined O
by O
titration O
with O
ethoxzolamide O
[ O
33 O
] O
. O

Synthesis O
of O
5 O
- O
( O
chloroacetamido O
) O
- O
l O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
7 O
An O
amount O
of O
1 O
. O
80 O
g O
( O
10 O
mmol O
) O
of O
5 O
- O
amino O
- O
1 O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
6 O
was O
suspended O
in O
20 O
mL O
of O
anhydrous O
acetonitrile O
and O
0 O
. O
9 O
mL O
( O
0 O
. O
87g O
, O
11 O
mmol O
) O
of O
pyridine O
added O
. O

The O
mixture O
was O
magnetically O
stirred O
at O
4 O
C O
for O
10 O
minutes O
, O
then O
10 O
. O
5 O
mmol O
of O
monochloroacetyl O
chloride O
, O
dissolved O
in O
3 O
mL O
acetonitrile O
, O
were O
added O
dropwise O
for O
5 O
min O
, O
and O
stirring O
was O
continued O
for O
other O
2 O
hours O
at O
room O
temperature O
. O

After O
an O
additional O
30 O
min O
of O
refluxation O
, O
followed O
by O
cooling O
, O
the O
precipitated O
crystals O
were O
filtered O
and O
recrystallized O
from O
ethanol O
. O

Yield O
of O
62 O
% O
white O
crystals O
, O
mp O
246 O
- O
248 O
o O
lit O
[ O
34 O
] O
mp O
IR O
( O
KBr O
) O
, O
cm O
- O
l O
: O
590 O
, O
610 O
, O
660 O
, O
790 O
, O
935 O
, O
1090 O
, O
1115 O
, O
1170 O
, O
1350 O
, O
1400 O
, O
1550 O
, O
1650 O
, O
1720 O
, O
2870 O
, O
3280 O
3370 O
( O
broad O
) O
; O
UV O
spectrum O
, O
, O
max O
, O
nm O
( O
lg O
) O
: O
255 O
( O
3 O
. O
50 O
) O
; O
288 O
( O
4 O
. O
37 O
) O
H O
- O
NMR O
( O
DMSO O
- O
d6 O
) O
, O
i O
, O
ppm O
: O
2 O
. O
96 O
( O
s O

, O
2H O
, O
CH2 O
) O
; O
8 O
. O
20 O
( O
s O
, O
2H O
, O
SOzNH2 O
) O
; O
12 O
. O
22 O
( O
s O
, O
1H O
, O
CONH O
) O
. O

Analysis O
, O
found O
: O
C O
, O
18 O
. O
56 O
; O
H O
, O
1 O
. O
88 O
; O
N O
, O
21 O
. O
76 O
; O
S O
, O
24 O
. O
62 O
% O
; O
C4HsC1N403S2 O
requires O
" O
C O
, O
18 O
. O
72 O
; O
H O
, O
1 O
. O
96 O
; O
N O
, O
21 O
. O
83 O
; O
S O
, O
24 O
. O
98 O
% O
. O

General O
procedure O
for O
the O
preparation O
of O
compounds O
8 O
- O
20 O
An O
amount O
of O
6 O
mmol O
of O
sodium O
salt O
of O
sulfonamides O
6 O
or O
7 O
was O
prepared O
by O
reacting O
the O
corresponding O
sulfonamide O
with O
the O
required O
amount O
of O
an O
alcoholic O
1N O
NaOH O
solution O
, O
in O
ethanol O
as O
solvent O
. O

To O
this O
308 O

Claudiu O
T O
. O
Supuran O
et O
al O
. O

Metal O
- O
Based O
Drugs O

solution O
was O
added O
the O
aqueous O
metal O
salt O
( O
Zn O
( O
II O
) O
, O
Mg O
( O
II O
) O
, O
AI O
( O
III O
) O
, O
Cd O
( O
II O
) O
chlorides O
, O
and O
Be O
( O
II O
) O
, O
Pb O
( O
II O
) O
and O
Hg O
( O
II O
) O
nitrate O
) O
solution O
, O
working O
in O
molar O
ratios O
RSOzNH O
- O
Mn O
+ O
of O
2 O
: O
1 O
for O
the O
divalent O
cations O
and O
3 O
: O
1 O
for O
the O
trivalent O
cation O
, O
respectively O
. O

The O
aqueous O
- O
alcoholic O
reaction O
mixture O
was O
heated O
on O
a O
steam O
bath O
for O
one O
hour O
, O
adjusting O
the O
pH O
at O
7 O
if O
necessary O
, O
and O
after O
being O
cooled O
at O
0 O
C O
the O
precipitated O
complexes O
were O
filtered O
and O
thoroughly O
washed O
with O
alcohol O
- O
water O
1 O
: O
1 O
( O
v O
/ O
v O
) O
and O
air O
dried O
. O

Yields O
were O
in O
the O
range O
of O
85 O
- O
90 O
% O
. O

The O
obtained O
white O
powders O
of O
compounds O
8 O
- O
20 O
melted O
with O
decomposition O
at O
temperatures O
higher O
than O
350 O
C O
, O
and O
were O
poorly O
soluble O
in O
water O
and O
alcohol O
, O
but O
had O
good O
solubilities O
in O
DMSO O
, O
DMF O
as O
well O
as O
mixtures O
of O
DMSO O
- O
water O
, O
DMF O
- O
water O
. O

Pharmacology O
Carbonic O
anhydrase O
inhibition O
Initial O
rates O
of O
4 O
- O
nitrophenyl O
acetate O
hydrolysis O
catalysed O
by O
different O
CA O
isozymes O
were O
monitored O
spectrophotometrical O
, O
at O
400 O
rim O
, O
with O
a O
Cary O
3 O
instrument O
interfaced O
with O
an O
IBM O
compatible O
PC O
[ O
35 O
] O
. O

Solutions O
of O
substrate O
were O
prepared O
in O
anhydrous O
acetonitrile O
; O
the O
substrate O
concentrations O
varied O
between O
2 O
. O
10 O
- O
2 O
and O
1 O
. O
10 O
- O
6 O
M O
, O
working O
at O
25C O
. O

A O
molar O
absorption O
coefficient O
of O
18 O
, O
400 O
M O
- O
l O
. O
cm O
- O
1 O
was O
used O
for O
the O
4 O
- O
nitrophenolate O
formed O
by O
hydrolysis O
, O
in O
the O
conditions O
of O
the O
experiments O
( O
pH O
7 O
. O
40 O
) O
, O
as O
reported O
in O
the O
literature O
[ O
35 O
] O
. O

Non O
- O
enzymatic O
hydrolysis O
rates O
were O
always O
subtracted O
from O
the O
observed O
rates O
. O

Duplicate O
experiments O
were O
done O
for O
each O
inhibitor O
concentration O
, O
and O
the O
values O
reported O
throughout O
the O
paper O
are O
the O
mean O
of O
such O
results O
. O

Stock O
solutions O
of O
inhibitor O
( O
1 O
mM O
) O
were O
prepared O
in O
distilled O
- O
deionized O
water O
with O
1020 O
% O
( O
v O
/ O
v O
) O
DMSO O
( O
which O
is O
not O
inhibitory O
at O
these O
concentrations O
[ O
2 O
] O
) O
and O
dilutions O
up O
to O
0 O
. O
01 O
nM O
were O
done O
thereafter O
with O
distilled O
- O
deionized O
water O
. O

Inhibitor O
and O
enzyme O
solutions O
were O
preincubated O
together O
for O
10 O
min O
at O
room O
temperature O
prior O
to O
assay O
, O
in O
order O
to O
allow O
for O
the O
formation O
of O
the O
E O
- O
I O
complex O
. O

The O
inhibition O
constant O
KI O
was O
determined O
as O
described O
by O
Pocket O
and O
Stone O
[ O
35 O
] O
. O

Enzyme O
concentrations O
were O
3 O
. O
3 O
nM O
for O
hCA O
II O
, O
l0 O
nM O
for O
hCA O
and O
34 O
nM O
for O
bCA O
IV O
( O
this O
isozyme O
has O
a O
decreased O
esterase O
activity O
[ O
36 O
] O
and O
higher O
concentrations O
had O
to O
be O
used O
for O
the O
- O
measurements O
) O
. O

Measurement O
of O
tonometric O
lOP O
Adult O
male O
New O
Zealand I
albino O
rabbits B
weighing O
2 O
- O
3 O
kg O
were O
used O
in O
the O
experiments O
( O
three O
animals O
were O
used O
for O
each O
inhibitor O
studied O
) O
. O

The O
experimental O
procedures O
conform O
to O
the O
Association O
for O
Research O
in O
Vision O
and O
Ophthalmology O
Resolution O
on O
the O
use O
of O
animals O
. O

The O
rabbits B
were O
kept O
in O
individual O
cages O
with O
food O
and O
water O
provided O
ad O
libitum O
. O

The O
animals O
were O
maintained O
on O
a O
12 O
h O
: O
12 O
h O
light O
/ O
dark O
cycle O
in O
a O
temperature O
controlled O
room O
, O
at O
22 O
- O
26 O
C O
. O

Solutions O
of O
inhibitors O
( O
2 O
% O
, O
by O
weight O
) O
were O
obtained O
in O
DMSOwater O
( O
2 O
: O
3 O
, O
v O
/ O
v O
) O
due O
to O
the O
low O
water O
solubility O
of O
some O
of O
these O
derivatives O
. O

Control O
experiments O
with O
DMSO O
( O
at O
the O
same O
concentration O
as O
that O
used O
for O
obtaining O
the O
inhibitors O
solutions O
showed O
that O
it O
does O
not O
possess O
IOP O
lowering O
or O
increasing O
effects O
. O

IOP O
was O
measured O
using O
a O
Digilab O
30R O
pneumatonometer O
( O
BioRad O
, O
Cambridge O
, O
MA O
, O
USA O
) O
as O
described O
by O
Maren O
' O
s O
group O
[ O
37 O
- O
39 O
] O
. O

The O
pressure O
readings O
were O
matched O
with O
two O
- O
point O
standard O
pressure O
measurements O
at O
least O
twice O
each O
day O
using O
a O
Digilab O
Calibration O
verifier O
. O

All O
IOP O
measurements O
were O
done O
by O
the O
same O
investigator O
with O
the O
same O
tonometer O
. O

One O
drop O
of O
0 O
. O
2 O
% O
oxybuprocaine O
hydrochloride O
( O
novesine O
, O
Sandoz O
) O
diluted O
1 O
" O
1 O
with O
saline O
was O
instilled O
in O
each O
eye O
immediately O
before O
each O
set O
of O
pressure O
measurements O
. O

IOP O
was O
measured O
three O
times O
at O
each O
time O
interval O
, O
and O
the O
means O
reported O
. O

IOP O
was O
measured O
first O
immediately O
before O
drug O
administration O
, O
then O
at O
30 O
min O
after O
the O
instillation O
of O
the O
pharmacological O
agent O
, O
and O
then O
each O
30 O
minutes O
for O
a O
period O
of O
several O
hours O
. O

For O
all O
IOP O
experiments O
drug O
was O
administered O
to O
only O
one O
eye O
, O
leaving O
the O
contralateral O
eye O
as O
an O
untreated O
control O
. O

The O
ocular O
hypotensive O
activity O
is O
expressed O
as O
the O
average O
difference O
in O
IOP O
between O
the O
treated O
and O
control O
eye O
, O
in O
this O
way O
minimizing O
the O
diurnal O
, O
seasonal O
and O
interindividual O
variations O
commonly O
observed O
in O
the O
rabbit B
[ O
37 O
- O
39 O
] O
. O

All O
data O
are O
expressed O
as O
mean O
SE O
, O
using O
a O
one O
- O
tailed O
test O
. O

Results O
and O
Discussion O
Reaction O
of O
5 O
- O
amino O
- O
l O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
6 O
[ O
19b O
] O
with O
chloroacteyl O
chloride O
in O
the O
presence O
of O
pyridine O
afforded O
5 O
- O
( O
chloroacetamido O
) O
- O
l O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
7 O
, O
by O
the O
procedure O
already O
reported O
by O
Young O
et O
al O
. O

[ O
34 O
] O
( O
Scheme O
1 O
) O
. O

The O
sulfonamide O
7 O
has O
been O
characterized O
by O
elemental O
analysis O
and O
spectroscopic O
methods O
which O
confirmed O
its O
structure O
( O
only O
its O
m O
. O
p O
. O
has O
been O
reported O
in O
ref O
. O
[ O
34 O
] O
) O
. O

The O
sodium O
salt O
of O
sulfonamides O
6 O
and O
7 O
, O
obtained O
in O
situ O
from O
the O
corresponding O
sulfonamide O
and O
sodium O
hydroxide O
, O
were O
then O
used O
for O
the O
preparation O
of O
coordination O
compounds O
, O
containing O
the O
following O
metal O
ions O
: O
Be O
( O
II O
) O
, O
Mg O
( O
II O
) O
, O
Al O
( O
III O
) O
, O
Zn O
( O
II O
) O
, O
Cd O
( O
II O
) O
and O
Hg O
( O
II O
) O
. O

Mention O
should O
be O
made O
that O
although O
5 O
- O
amino O
- O
l O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
6 O
is O
the O
parent O
compound O
of O
important O
sulfonamide O
CA O
inhibitors O
, O
such O
as O
acetazolamide O
, O
benzolamide O
, O
methazolamide O
, O
etc O
. O
, O
its O
coordination O
chemistry O
has O
been O
scarcely O
investigated O
up O
to O
now O
[ O
22 O
, O
40 O
] O
. O

The O
new O
complexes O
prepared O
in O
this O
work O
are O
shown O
in O
Table O
I O
. O
Both O
compounds O
containing O
the O
sulfonamide O
- O
deprotonated O
species O
of O
sulfonamide O
7 O
( O
LH O
) O
, O
as O
well O
as O
complexes O
in O
which O
the O
anion O
of O
5amino O
- O
1 O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
( O
tda O
) O
act O
as O
ligands O
, O
have O
been O
prepared O
. O

In O
fact O
in O
another O
work O
[ O
40 O
] O
it O
was O
documented O
that O
in O
some O
cases O
, O
sulfonamides O
derived O
from O
this O
ring O
system O
may O
undergo O
hydrolysis O
to O

309 O

Vol O
. O

4 O
, O
No O
. O
6 O
, O
1997 O

Metal O
Complexes O
of O
Heterocyclic O
Sulfonamides O
. O
" O
A O
New O
Class O
ofAntiglaucoma O
Agents O

the O
moiety O
substituting O
the O
5 O
position O
, O
with O
the O
formation O
of O
5 O
- O
amino O
- O
l O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
6 O
, O
which O
thereafter O
coordinates O
metal O
ions O
present O
in O
solution O
. O

N O
CIH O
N O

I O

N O
+ O
NaHCO O
2 O

N O
2 O

S O
' O

- O
NaCl O

I O
S O

N O

II O

SO2NH O

1 O
_ O
12 O
O O
Py O
/ O
MeCN O

6Htda O
+ O
CIOCCH2CI O

0 O
Cl O
CH O
2 O
Scheme O
1 O

HN O

. O
I O

N O

N O

S O
7 O
: O
LH O

II O

SqNH O

Thus O
, O
the O
X O
- O
ray O
crystal O
structure O
of O
the O
complex O
[ O
Zn O
( O
tda O
) O
2 O
( O
NH3 O
) O
] O
. O
H20 O
prepared O
in O
this O
way O
has O
recently O
been O
reported O
by O
this O
group O
[ O
40 O
] O
. O

On O
the O
other O
hand O
, O
when O
the O
ligand O
7 O
has O
not O
been O
hydrolyzed O
( O
during O
the O
preparation O
of O
the O
coordination O
compounds O
) O
in O
the O
presence O
of O
the O
metal O
ion O
to O
5 O
- O
amino O
- O
l O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2sulfonamide O
and O
chloroacetate O
, O
the O
metal O
complexes O
contining O
6 O
as O
ligand O
have O
been O
prepared O
from O
the O
last O
( O
pure O
) O
compound O
( O
as O
sodium O
salt O
) O
and O
the O
corresponding O
metal O
salt O
, O
by O
the O
general O
procedure O
described O
in O
the O
Experimental O
part O
. O

Table O
I O
: O
Prepared O
complexes O
8 O
- O
20 O
, O
containing O
the O
conjugate O
bases O
of O
sulfonamides O
6 O
and O
7 O
as O
ligands O
and O
their O
elemental O
analysis O
data O
. O

L O
stands O
for O
the O
sulfonamide O
deprotonated O
species O
of O
7 O
, O
whereas O
tda O
for O
the O
sulfonamide O
deprotonated O
species O
of O
5 O
- O
amino O
- O
1 O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
6 O
. O

No O
. O
8 O
9 O
10 O
11 O
12 O
13 O
14 O
15 O
16 O
17 O
18 O
19 O
20 O

Complex O

Yield O

( O
% O
) O

% O
Ma O

Analysis O
( O
calculated O
/ O
found O
) O
% O
H O
b O
% O
C O
b O
13 O
. O
0 O
/ O
13 O
. O
1 O
11 O
. O
0 O
/ O
10 O
. O
8 O
11 O
. O
3 O
/ O
11 O
. O
4 O
10 O
. O
2 O
/ O
10 O
. O
1 O
8 O
. O
5 O
/ O
8 O
. O
1 O
7 O
. O
9 O
/ O
7 O
. O
9 O
12 O
. O
7 O
/ O
12 O
. O
8 O
18 O
. O
4 O
/ O
18 O
. O
1 O
18 O
. O
1 O
/ O
17 O
. O
9 O
16 O
. O
6 O
/ O
16 O
. O
2 O
15 O
. O
3 O
/ O
14 O
. O
9 O
13 O
. O
4 O
/ O
13 O
. O
3 O
12 O
. O
7 O
/ O
12 O
. O
5 O
1 O
. O
6 O
/ O
1 O
. O
3 O
2 O
. O
7 O
/ O
2 O
. O
3 O
1 O
. O
4 O
/ O
1 O
. O
1 O
1 O
. O

2 O
/ O
1 O
. O
2 O
1 O
. O
0 O
/ O
1 O
. O
2 O
1 O
. O
6 O
/ O
1 O
. O
3 O
1 O
. O
6 O
/ O
1 O
. O
6 O
1 O
. O
5 O
/ O
1 O
. O
5 O
1 O
. O
5 O
/ O
1 O
. O
3 O
1 O
. O
3 O
/ O
1 O
. O
4 O
1 O
. O
2 O
/ O
1 O
. O
1 O
1 O
. O
1 O
/ O
1 O
. O
2 O
1 O
. O
0 O
/ O
1 O
. O
0 O

% O
N O
b O
30 O
. O
4 O
/ O
30 O
. O
2 O
25 O
. O
6 O
/ O
25 O
. O
5 O
26 O
. O
4 O
/ O
26 O
. O
4 O
13 O
. O
7 O
/ O
23 O
. O
3 O
20 O
. O
0 O
/ O
19 O
. O
8 O
18 O
. O
6 O
/ O
18 O
. O
5 O
29 O
. O
7 O
/ O
29 O
. O
6 O
21 O
. O
5 O
/ O
21 O
. O
3 O
21 O
. O
1 O
/ O
20 O
. O
8 O
19 O
. O
4 O
/ O
19 O
. O
3 O
17 O
. O
9 O
/ O
17 O
. O
8 O
15 O
. O
7 O
/ O
15 O
. O
6 O
14 O
. O
8 O
/ O
14 O
. O
6 O

[ O
Be O
( O
tda O
) O
2 O
] O
[ O
Mg O
( O
tda O
) O
2 O
] O
. O
3 O
H20 O
[ O
Zn O
( O
tda O
) O
2 O
] O
[ O
Cd O
( O
tda O
) O
2 O
] O
[ O
Hg O
( O
tda O
) O
2 O
] O
[ O
Pb O
( O
tda O
) O
z O
( O
OH2 O
) O
2 O
] O
[ O
Al O
( O
tda O
) O
3 O
] O
[ O
BeL2 O
] O
[ O
ALL3 O
] O
[ O
ZnL2 O
] O
[ O
CdL2 O
] O
[ O
HgL2 O
] O
[ O
PbLz O
( O
OH2 O
) O
2 O
] O

78 O
76 O
83 O
90 O
95 O
84 O
72 O
75 O
59 O
87 O
88 O
92 O
95 O

2 O
. O
4 O
/ O
2 O
. O
5 O
5 O
. O
5 O
/ O
5 O
. O
1 O
15 O
. O
4 O
/ O
15 O
. O
0 O
23 O
. O
8 O
/ O
24 O
. O
1 O
35 O
. O
8 O
/ O
35 O
. O
7 O
34 O
. O
4 O
/ O
34 O
. O
7 O
4 O
. O
7 O
/ O
4 O
. O
4 O
1 O
. O
7 O
/ O
1 O
. O
6 O
3 O
. O
4 O
/ O
3 O
. O
5 O
11 O
. O
3 O
/ O
11 O
. O
5 O
18 O
. O
0 O
/ O
18 O
. O
1 O
28 O
. O
1 O
/ O
28 O
. O
3 O
27 O
. O
4 O
/ O
27 O
. O
2 O

aBy O
gravimetry O
; O
bBy O
combustion O
. O

The O
new O
complexes O
have O
also O
been O
characterized O
by O
spectroscopic O
, O
conductimetric O
and O
thermogravimetric O
measurements O
( O
Table O
II O
) O
. O

By O
comparing O
the O
IR O
spectra O
of O
the O
complexes O
and O
the O
corresponding O
ligands O
, O
the O
following O
observations O
should O
be O
made O
: O
( O
i O
) O
the O
shift O
of O
the O
two O
sulfonamido O
vibrations O
( O
both O
the O
symmetric O
as O
well O
as O
the O
the O
assymetric O
one O
) O
, O
towards O
lower O
wavenumbers O
in O
the O
spectra O
of O
the O
complexes O
, O
as O
compared O
to O
the O
spectra O
of O
the O
corresponding O
ligand O
( O
Table O
II O
) O
, O
as O
already O
documented O
previously O
for O
similar O
complexes O
[ O
13 O
- O
22 O
] O
. O

This O
is O
a O
direct O
indication O
that O
the O
deprotonated O
sulfonamido O
310 O

Claudiu O
T O
. O
Supuran O
et O
al O
. O

Metal O
- O
Based O
Drugs O

moieties O
of O
the O
ligands O
interacts O
with O
the O
metal O
ions O
in O
the O
newly O
prepared O
coordination O
compounds O
; O
( O
ii O
) O
the O
amide O
vibrations O
( O
the O
most O
intense O
such O
bands O
at O
1670 O
- O
1680 O
cm O
- O
1 O
) O
of O
ligand O
7 O
appear O
unchanged O
in O
the O
IR O
spectra O
of O
complexes O
15 O
- O
20 O
( O
data O
not O
shown O
) O
, O
suggesting O
that O
these O
moieties O
do O
not O
participate O
in O
coordination O
of O
the O
metal O
ions O
; O
( O
iii O
) O
the O
C O
- O
N O
stretching O
vibration O
in O
the O
spectra O
of O
the O
prepared O
complexes O
is O
shifted O
with O
5 O
- O
20 O
cm O
- O
1 O
towards O
lower O
wavenumbers O
, O
as O
compared O
to O
the O
same O

vibration O
in O
the O
spectra O
of O
sulfonamides O
6 O
and O
7 O
, O
indicating O
that O
one O
of O
the O
endocyclic O
nitrogens O
of O
the O
thiadiazolic O
ring O
( O
presumably O
N3 O
) O
acts O
as O
donor O
atom O
, O
as O
already O
documented O
by O
X O
- O
ray O
crystallographic O
and O
spectroscopic O
determinations O
on O
complexes O
of O
other O
sulfonamides O
( O
such O
as O
1 O
- O
3 O
) O
with O
divalent O
metal O
ions O
[ O
13 O
- O
22 O
] O
( O
Table O
II O
) O
; O
( O
iv O
) O
changes O
in O
the O
region O
3100 O
- O
3160 O
cm O
- O
, O
as O
the O
bands O
present O
in O
the O
spectra O
of O
sulfonamides O
6 O
, O
7 O

are O
present O
in O
the O
spectra O
of O
complexes O
8 O
- O
20 O
too O
, O
but O
they O
are O
not O
well O
resolved O
, O
and O
have O
a O
smaller O
intensity O
. O

This O
is O
probably O
due O
to O
deprotonation O
of O
the O
SO2NH2 O
moiety O
and O
participation O
in O
the O
binding O
of O
cations O
; O
( O
v O
) O
the O
amino O
vibrations O
from O
3320 O
cm O
- O
in O
the O
spectra O
of O
6 O
appear O
unchanged O
in O
the O
spectra O
of O
its O
complexes O
8 O
- O
14 O
( O
data O
not O
s O
. O
hown O
) O
. O

In O
the O
1H O
- O
NMR O
spectra O
of O
compound O
6 O
and O
its O
metal O
complexes O
, O
the O
signal O
of O
the O
amino O
group O
has O
been O
evidenced O
as O
a O
broad O
singlet O
centered O
at O
4 O
. O
54 O
ppm O
( O
Table O
II O
) O
, O
which O
is O
not O
exchangeable O
by O
addition O
of O
D20 O
into O
the O
NMR O
tube O
, O
in O
contrast O
to O
the O
sulfonamido O
NH2 O
protons O
( O
which O
readily O
exchange O
) O
. O

This O
proves O
that O
the O
5 O
- O
amino O
moiety O
is O
not O
involved O
in O
binding O
the O
metal O
ions O
, O
as O
already O
shown O
in O
the O
X O
- O
ray O
crystallographic O
work O
of O
the O
complex O
[ O
Zn O
( O
tda O
) O
z O
( O
NH3 O
) O
] O
. O
H20 O
previously O
reported O
[ O
40 O
] O
. O

For O
sulfonamide O
7 O
the O
CONH O
proton O
resonates O
as O
a O
singlet O
at O
12 O
. O
22 O
ppm O
. O

In O
complexes O
15 O
- O
20 O
only O
very O
minor O
shifts O
of O
this O
signal O
were O
evidenced O
( O
Table O
II O
) O
, O
proving O
basically O
that O
the O
CONH O
moiety O
does O
not O
interact O
with O
the O
metal O
ions O
in O
these O
complexes O
. O

Table O
II O
: O
Spectroscopic O
, O
thermogravimetric O
and O
conductimetric O
data O
for O
compounds O
6 O
- O
20 O
. O

1H O
- O
NMR O
Spectrab O
Comp O
. O

IR O
Spectraa O
, O
cm O
- O
1 O
as O
v O
( O
C O
= O
N O
) O
CONH O
, O
i O
( O
ppm O
) O
v O
( O
SO2 O
) O
S O
; O
v O
( O
SO2 O
) O
6 O
8 O
9 O
10 O
11 O

TG O
analysisc O
calc O
. O
/ O
found O

Conductimetryd O
AM O
( O
- O
1 O
x O
cm2x O
mol O
- O
) O
2 O
7 O
4 O
5 O
3 O
2 O
2 O
6 O
3 O
4 O
2 O
9 O
3 O
2 O
8 O

12 O
13 O
14 O
7 O
15 O
16 O
17 O
18 O
19 O
20 O

1170 O
; O
1150 O
; O
1150 O
; O
1145 O
; O
1145 O
; O
1140 O
; O
1145 O
; O
1150 O
; O
1170 O
; O
1130 O
; O
1140 O
; O
1140 O
; O
1150 O
; O
1140 O
; O
1145 O
; O

1350 O
1300 O
1305 O
1300 O
1305 O
1300 O
1300 O
1300 O
1350 O
1335 O
1330 O
1330 O
1330 O
1335 O
1330 O

1610 O
1600 O
1600 O
1600 O
1600 O
1590 O
1605 O
1605 O
1610 O
1605 O
1610 O
1610 O
1605 O
1600 O
1600 O

A O
A O
A O
A O
A O
A O
A O

e O
e O

12 O
. O
3 O
/ O
12 O
. O
1f O
e O
e O
e O
5 O
. O
9 O
/ O
5 O
. O
7g O

A O
12 O
. O
22 O
( O
1H O
) O
12 O
. O
19 O
( O
2H O
) O
12 O
. O
18 O
( O
3H O
) O
12 O
. O
20 O
( O
2H O
) O
12 O
. O
18 O
( O
2H O
) O
12 O
. O
19 O
( O
2H O
) O
12 O
. O
21 O
( O
2H O
) O

e O
e O
e O
e O
e O
e O
e O
4 O
. O
7 O
/ O
4 O
. O
8g O

a O
In O
KBr O
; O
bin O
DMSO O
- O
d6 O
; O
A O
the O
signal O
of O
the O
5 O
- O
amino O
group O
of O
6 O
( O
appearing O
in O
the O
ligand O
at O
4 O
. O
54 O
ppm O
as O
a O
broad O
singlet O
) O
appears O
at O
the O
same O
chemical O
shift O
( O
4 O
. O
50 O
4 O
. O
55 O
ppm O
) O
in O
complexes O
8 O
- O
14 O
; O
cWeight O
loss O
between O
70 O
- O
250 O
C O
; O
d O
mM O
solution O
, O
in O
DMF O
, O
at O
25C O
; O
e O
No O
weight O
loss O
seen O
under O
250 O
C O
; O
fCorresponding O
to O
three O
lattice O
water O
molecules O
lost O
at O
70 O
- O
110C O
, O
and O
gCorresponding O
to O
two O

coordinated O
water O
molecules O
, O
lost O
at O
160 O
- O
180 O
C O
. O

Thermogravimetric O
analysis O
showed O
the O
presence O
of O
uncoordinated O
water O
molecules O
in O
the O
molecule O
of O
complex O
9 O
( O
the O
three O
waters O
were O
lost O
in O
a O
single O
step O
, O
between O
70 O
- O
110 O
C O
) O
and O
of O
coordinated O
water O
in O
the O
molecules O
of O
the O
lead O
( O
II O
) O
derivatives O
13 O
and O
20 O
. O

All O
these O
compounds O
behaved O
as O
non O
- O
electrolytes O
in O
DMF O
as O
solvent O
( O
Table O
II O
) O
. O

Mention O
should O
be O
made O
that O
the O
Mg O
( O
II O
) O
complex O
of O
sulfonamide O
7 O
could O
not O
be O
isolated O
. O

Instead O
, O
only O
the O
correponding O
complex O
of O
5 O
- O
amino O
- O
l O
, O
3 O
, O
4 O
- O
thiadiazole O
has O
been O
obtained O
from O
reaction O
mixtures O
containing O
magnesium O
salts O
and O
the O
sodium O
salt O
of O
7 O
, O
probably O
due O
to O
a O
metal O
ion O
assisted O
hydrolysis O
of O
7 O
to O
6 O
and O
chloroacetate O
. O

Generally O
such O
hydrolytic O
processes O
involve O
highly O
acidic O
conditions O
and O
prolonged O
heating O
of O
the O
5 O
- O
alkylamido O
- O
1 O
, O
3 O
, O
4 O
- O
thiadiazole O
- O
2 O
- O
sulfonamide O
derivatives O
[ O
41 O
] O
, O
but O
they O
might O
become O
milder O
by O
taking O
into O
account O
the O
putative O
catalytic O
effect O
of O
Mg2 O
+ O
ions O
reported O
here O
. O

The O
data O
shown O
above O
lead O
to O
the O
conclusion O
that O
ligand O
7 O
shares O
a O
common O
coordination O
chemistry O
with O
acetazolamide O
1 O
with O
which O
it O
is O
structurally O
related O
, O
whereas O
6 O
probably O
also O
behaves O
similarly O
to O
acetazolamide O
in O
the O
sense O
that O
the O
5 O
- O
amino O
group O
seems O
not O
to O
be O
involved O
in O
coordinating O
metal O
ions O
, O
at O
311 O

Vol O
. O

4 O
, O
No O
. O
6 O
, O
1997 O

Metal O
Complexes O
of O
Heterocyclic O
Sulfonamides O
: O
A O
New O
Class O
ofAntiglaucoma O
Agents O

least O
in O
the O
complexes O
reported O
by O
us O
here O
( O
and O
also O
in O
the O
compound O
characterized O
by O
X O
- O
ray O
crystallography O
mentioned O
above O
[ O
40 O
] O
) O
. O

Thus O
, O
in O
all O
complexes O
reported O
here O
these O
sulfonamides O
( O
as O
monodeprotonated O
species O
at O
the O
SO2NH O
2 O
moieties O
) O
act O
as O
bidentate O
ligands O
, O
through O
the O
endocyclic O
N O
- O
3 O
and O
the O
NH O
- O
groups O
. O

The O
proposed O
formulae O
of O
the O
new O
complxes O
are O
shown O
below O
. O

Except O
for O
the O
two O
Pb O
( O
II O
) O
complexes O
13 O
and O
20 O
, O
as O
well O
as O
the O
AI O
( O
III O
) O
derivatives O
14 O
and O
16 O
, O
which O
presumably O
are O
pseudo O
- O
octahedral O
, O
the O
other O
derivatives O
are O
supposed O
to O
contain O
tetrahedral O
M O
( O
II O
) O
ions O
. O

R O

R O
13 O
" O
R O
= O
20 O
" O
R O
= O

H2N O

CICH2CONH O

14 O
: O
R O
= O
16 O
: O
R O
= O

H2N O
CICH2CONH O

The O
compounds O
6 O
- O
20 O
together O
with O
the O
standard O
CA O
inhibitors O
1 O
- O
5 O
were O
assayed O
for O
inhibition O
against O
three O
isozymes O
, O
hCA O
I O
, O
hCA O
II O
and O
bCA O
IV O
( O
Table O
III O
) O
. O

As O
seen O
from O
the O
above O
data O
, O
the O
chloracetamido O
derivative O
is O
more O
inhibitory O
than O
acetazolamide O
, O
methazolamide O
and O
dichlorophenamide O
, O
whereas O
the O
unacylated O
compound O
6 O
is O
less O
inhibitory O
than O
the O
above O
sulfonamides O
. O

The O
metal O
complexes O
820 O
are O
much O
more O
inhibitory O
than O
the O
sulfonamides O
from O
which O
they O
derive O
6 O
, O
7 O
and O
than O
all O
other O
simple O
sulfonamides O
assayed O
. O

They O
behave O
similarly O
to O
the O
metal O
complexes O
of O
acetazolamide O
, O
methazolamide O
or O
dorzolamide O
previously O
reported O
by O
this O
group O
, O
which O
were O
all O
more O
inhibitory O
than O
the O
parent O
sulfonamide O
from O
which O
were O
prepared O
[ O
16 O
- O
22 O
, O
40 O
] O
. O

Particularly O
strong O
inhibition O
was O
observed O
for O
the O
Zn O
( O
II O
) O
, O
Hg O
( O
II O
) O
, O
Pb O
( O
II O
) O
and O
Cd O
( O
II O
) O
complexes O
, O
especially O
against O
CA O
II O
and O
CA O
IV O
, O
the O
isozymes O
critical O
for O
aqueous O
humor O
formation O
. O

In O
vivo O
IOP O
lowering O
experiments O
were O
done O
in O
rabbits B
with O
some O
of O
the O
new O
compounds O
prepared O
in O
the O
present O
work O
, O
such O
as O
the O
sulfonamides O
6 O
and O
7 O
, O
and O
their O
Zn O
( O
II O
) O
complexes O
, O
which O
were O
among O
the O
strong O
CA O
II O
and O
CA O
IV O
inhibitors O
in O
the O
obtained O
series O
. O

Some O
of O
the O
IOP O
lowering O
data O
at O
half O
an O
hour O
and O
one O
hour O
after O
the O
instillation O
of O
one O
drop O
of O
2 O
% O
solution O
of O
inhibitor O
within O
the O
rabbit B
eye O
are O
shown O
in O
312 O

Claudiu O
T O
. O
Supuran O
et O
al O
. O

Metal O
- O
Based O
Drugs O

Table O
IV O
, O
with O
dorzolamide O
( O
at O
the O
same O
concentration O
) O
as O
standard O
. O

In O
Fig O
. O
the O
time O
dependence O
of O
IOP O
lowering O
with O
dorzolamide O
5 O
and O
the O
two O
Zn O
( O
II O
) O
complexes O
10 O
and O
17 O
is O
presented O
. O

Table O
III O
. O

CA O
inhibition O
data O
with O
the O
standard O
inhibitors O
1 O
- O
5 O
, O
the O
sulfonamides O
6 O
and O
7 O
, O
and O
their O
metal O
complexes O
8 O
- O
20 O
. O

No O
1 O
2 O
3 O
4 O
5 O
6 O
7 O
8 O
9 O
10 O
11 O
12 O
13 O
14 O
15 O
16 O
17 O
18 O
19 O
20 O
a O

Inhibitor O

KI O
hCA O
Ia O
900 O
780 O
25 O
1200 O
> O
50 O
, O
000 O
1550 O
640 O
1050 O
350 O
50 O
40 O
12 O
80 O
240 O
120 O
80 O
40 O
40 O
9 O
15 O

( O
nM O
) O
bCA O
IVb O
220 O
240 O
13 O
380 O
43 O
780 O
24 O
540 O
220 O
25 O
19 O
10 O
26 O
110 O
12 O
16 O
9 O
10 O
5 O
10 O

hCA O
IIa O
12 O
14 O
8 O
38 O
9 O
230 O
5 O
190 O
110 O
15 O
14 O
7 O
10 O
76 O
5 O
4 O
3 O
3 O
2 O
5 O

Acetazolamide O
Methazolamide O
Ethoxzolamide O

Dichlorophenamide O
Dorzolamide O

Human B
( O
cloned O
) O
isozymes O
; O
b O
From O
bovine B
lung O
microsomes O
. O

Table O
IV O
: O
IOP O
lowering O
following O
topical O
application O
of O
CA O
inhibitors O
, O
half O
an O
hour O
and O
one O
hour O
after O
instillation O
into O
the O
eye O
of O
a O
drop O
( O
50 O
L O
) O
of O
2 O
% O
solution O
of O
inhibitor O
. O

Inhibitor O

lOP O
SE O
1 O
/ O
2 O
h O

a O

( O
mm O
Hg O
) O
lh O
4 O
. O
1 O
0 O
. O
15 O
0 O
0 O
. O
09 O
0 O
0 O
. O
09 O
5 O
. O
0 O
0 O
. O
12 O
8 O
. O
1 O
0 O
. O
21 O
Dorzolamide O
5 O
6 O
7 O
10 O
17 O
a O

2 O
. O
2 O
0 O
. O
10 O
0 O
0 O
. O
10 O
0 O
0 O
. O
10 O
2 O
. O
00 O
. O
09 O
8 O
. O
0 O
0 O
. O
14 O

IOP O

IOP O
control O
eye O
" O
IOP O
treated O
eye O
( O
N O
3 O
) O
. O

As O
seen O
from O
the O
above O
data O
, O
the O
sulfonamides O
6 O
and O
7 O
are O
totally O
ineffective O
as O
lOP O
lowering O
agents O
, O
similarly O
to O
the O
classical O
clinically O
used O
inhibitors O
of O
type O
1 O
- O
5 O
[ O
2 O
, O
3 O
] O
. O

On O
the O
other O
hand O
, O
dorzolamide O
, O
the O
first O
topical O
sulfonamide O
used O
clinically O
in O
the O
treatment O
of O
glaucoma O
is O
an O
effective O
such O
agent O
, O
with O
a O
decrease O
of O
lOP O
of O
around O
4 O
mm O
Hg O
, O
one O
hour O
after O
administration O
directly O
into O
the O
eye O
( O
Table O
IV O
) O
. O

From O
the O
data O
of O
this O
table O
, O
it O
is O
obvious O
that O
the O
metal O
complexes O
of O
heterocyclic O
sulfonamides O
investigated O
by O
us O
behave O
as O
much O
more O
effective O
IOP O
lowering O
agents O
than O
dorzolamide O
, O
and O
their O
effect O
is O
generally O
longerlasting O
( O
Fig O
. O
1 O
) O
. O

A O
last O
remark O
should O
be O
made O
about O
the O
possible O
mechanism O
of O
action O
of O
the O
new O
class O
of O
IOP O
lowering O
agents O
. O

Obviously O
, O
their O
activity O
is O
due O
to O
inhibition O
of O
CA O
isozymes O
present O
in O
the O
cilliary O
processes O
within O
the O
eye O
, O
similarly O
to O
other O
topically O
active O
sulfonamides O
[ O
2 O
- O
6 O
] O
. O

The O
fact O
that O
the O
sulfonamide O
per O
se O
is O
inactive O
via O
the O
topical O
route O
, O
whereas O
the O
metal O
complexes O
result O
much O
better O
than O
the O
drug O

313 O

Vol O
. O

4 O
, O
No O
. O
6 O
, O
1997 O

Metal O
Complexes O
of O
Heterocyclic O
Sulfonamides O
. O
" O
A O
New O
Class O
ofAntiglaucoma O
Agents O

dorzolamide O
, O
indicates O
that O
the O
presence O
of O
metal O
ions O
in O
the O
molecules O
of O
these O
CA O
inhibitors O
is O
essential O
and O
confers O
them O
completely O
new O
properties O
. O

Lowering O
of O
IOP O
( O
ram O
Hg O
) O

" O
| O
| O
, O

/ O

" O
, O
, O

' O
" O
, O
. O
, O

, O
< O

. O
7 O
, O
/ O

/ O

. O
6 O

- O
0 O

- O
10 O
0 O

2 O

Time O
( O
hours O
) O

Fig O
l O
" O
Time O
dependence O
of O
IOP O
lowering O
with O
dorzolamide O
( O
curve O
1 O
) O
; O
the O
zinc O
complex O
10 O
( O
curve O
3 O
) O
and O
the O
zinc O
complex O
17 O
( O
curve O
3 O
) O
, O
after O
topical O
administration O
of O
one O
drop O
of O
2 O
% O
solution O
of O
inhibitor O
in O
rabbit B
. O

Preliminary O
results O
from O
this O
laboratory O
indicate O
that O
the O
metal O
complexes O
of O
topically O
active O
sulfonamides O
show O
also O
increased O
lOP O
lowering O
effects O
with O
respect O
to O
the O
complexes O
prepared O
in O
the O
present O
study O
[ O
42 O
] O
. O

Our O
hypothesis O
is O
that O
the O
presence O
of O
the O
metal O
ion O
in O
the O
molecules O
of O
these O
complex O
inhibitors O
induces O
a O
dramatic O
change O
in O
their O
physico O
- O
chemical O
properties O
as O
compared O
to O
those O
of O
the O
parent O
sulfonamide O
. O

This O
phenomenon O
is O
certainly O
governed O
by O
the O
strong O
polarization O
induced O
by O
the O
metal O
ions O
. O

In O
this O
way O
, O
it O
is O
quite O
probable O
that O
the O
right O
balance O
between O
the O
lipo O
- O
and O
hydrosolubility O
of O
these O
compounds O
is O
achieved O
, O
which O
has O
been O
considered O
to O
be O
the O
critical O
factor O
for O
not O
observing O
topical O
activity O
in O
the O
classical O
CA O
inhibitors O
, O
such O
as O
acetazolamide O
, O
methazolamide O
and O
ethoxzolamide O
, O
which O
were O
either O
too O
lipophilic O
or O
too O
hy O
. O
drosoluble O
[ O
2 O
, O
3 O
] O
. O

So O
, O
by O
choosing O
different O
metal O
ions O
and O
diverse O
sulfonamides O
, O
much O
larger O
possibilities O
arise O
to O
finely O
tune O
the O
pharmacological O
properties O
which O
strongly O
influence O
the O
value O
of O
a O
drug O
. O

In O
conclusion O
we O
describe O
here O
a O
novel O
class O
of O
lOP O
lowering O
agents O
, O
ie O
, O
the O
metal O
complexes O
of O
sulfonamide O
CA O
inhibitors O
. O

These O
derivatives O
appear O
to O
be O
very O
active O
and O
longer O
lasting O
than O
the O
drug O
dorzolamide O
, O
and O
might O
constitute O
the O
premises O
for O
a O
new O
generation O
of O
antiglaucoma O
drugs O
. O

Acknowledgments O
. O

We O
are O
grateful O
to O
Prof O
. O

S B
. I

Lindskog O
( O
Umea O
Univ O
. O
, O
Sweden O
) O
for O
the O
gift O
of O
the O
hCA O
and O
II O
plasmids O
. O

References O
Preceding O
part O
of O
this O
series O
" O
Supuran O
CT O
, O
Scozzafava O
A O
, O
Ilies O
MA O
, O
Iorga O
B O
, O
Cristea O
T O
, O
Chiraleu O
F O
, O
Banciu O
MD O
( O
1997 O
) O
Eur O
J O
Med O
Chem O
, O
submitted O
. O

2 O
Supuran O
CT O
( O
1994 O
) O
" O
Carbonic O
anhydrase O
inhibitors O
" O
In O
Carbonic O
Anhydrase O
and O
Modulation O
of O
Physiologic O
and O
Pathologic O
Processes O
in O
the O
Organism O
, O
( O
Puscas O
I O
, O
Ed O
) O
Helicon O
, O
Timisoara O
, O
pp O
. O

29 O
- O
111 O
. O

3 O
Maren O
TH O
( O
1991 O
) O
" O
The O
links O
among O
biochemistry O
, O
physiology O
and O
pharmacology O
in O
carbonic O
anhydrase O
mediated O
systems O
" O
. O

In O
Carbonic O
Anhydrase O
From O
Biochemistry O
and O
Genetics O
to O
Physiology O
and O
Clinical O
Medicine O
, O
( O
Botr6 O
F O
, O
Gros O
G O
, O
Storey O
BT O
Eds O
) O
VCH O
, O
Weinheim O
, O
pp O
. O

186 O
- O
207 O
. O

4 O
Bayer O
A O
, O
Ferrari O
F O
, O
Maren O
TH O
, O
Erb O
C O
( O
1996 O
) O
dFr O
Ophtalrnol O
19 O
, O
3 O
: O
57 O
- O
362 O
. O

: O
5 O
Maren O
TH O
, O
Conroy O
CW O
, O
Wynns O
GC O
, O
Levy O
NS O
( O
1997 O
) O
d O
Ocul O
Pharrnacol O
Therapeut O
13 O
, O
23 O
- O
30 O
. O

6 O
Sugrue O
MF O
( O
1996 O
) O
d O
Ocul O
Pharmacol O
Ther O
12 O
, O
363 O
- O
376 O
. O

7 O
Bartlett O
JD O
, O
Jaanus O
SD O
( O
1989 O
) O
" O
Carbonic O
anhydrase O
inhibitors O
" O
. O

In O
Clinical O
Ocular O
Pharmacology O
, O
Second O
Edition O
, O
Butterworths O
Publishers O
, O
Boston O
, O
pp O
. O

2 O
: O
54 O
- O
263 O
. O

8 O
Alward O
PD O
, O
Wilensky O
JT O
( O
1981 O
) O
Arch O
Ophthalmo199 O
, O
1973 O
- O
1976 O
. O

314 O

Claudiu O
T O
. O
Supuran O
et O
al O
. O

Metal O
- O
Based O
Drugs O

9 O
Maren O
TH O
, O
Jankowska O
L O
, O
Sanyal O
G O
, O
Edelhauser O
HF O
( O
1983 O
) O
Exp O
Eye O
Res O
36 O
, O
457 O
- O
480 O
. O

10 O
a O
) O
Ponticello O
GS O
, O
Freedman O
MB O
, O
Habecker O
CN O
, O
Lyle O
PA O
, O
Schwam O
H O
, O
Varga O
SL O
, O
Christy O
ME O
, O
Randall O
WC O
, O
Baldwin O
JJ O
( O
1987 O
) O
J O
Med O
Chem O
30 O
, O
591 O
- O
597 O
; O
b O
) O
Greer O
J O
, O
Erickson O
JW O
, O
Baldwin O
JJ O
, O
Varney O
MD O
( O
1994 O
) O
J O
Med O
Chem O
37 O
, O
1035 O
- O
1054 O
. O

11 O
Baldwin O
JJ O
, O
Ponticello O
GS O
, O
Anderson O
GS O
, O
Christy O
ME O
, O
Murcko O
MA O
, O
Randall O
WC O
, O
Schwam O
H O
, O
Sugrue O
MF O
, O
Springer O
JP O
, O
Gautheron O
P O
, O
Grove O
J O
, O
Mallorga O
P O
, O
Viader O
MP O
, O
McKeever O
BM O
, O
Navia O
MA O
( O
1989 O
) O
J O
Med O
Chem O
32 O
, O
2510 O
- O
2513 O
. O

12 O
Chow O
K O
, O
Lai O
R O
, O
Holmes O
JM O
, O
Wijono O
M O
, O
Wheeler O
LA O
, O
Garst O
ME O
( O
1996 O
) O
EurJMed O
Chem O
31 O
, O
175 O
- O
186 O
. O

13 O
Supuran O
CT O
, O
Nicolae O
A O
, O
Popescu O
A O
( O
1996 O
) O
Eur O
JMed O
Chem O
31 O
, O
431 O
- O
438 O
. O

14 O
Supuran O
CT O
, O
Popescu O
A O
, O
Ilisiu O
M O
, O
Costandache O
A O
, O
Banciu O
MD O
( O
1996 O
) O
Eur O
JMed O
Chem O
31 O
, O
439 O
- O
448 O
. O

15 O
Supuran O
CT O
, O
Scozzafava O
A O
, O
Popescu O
A O
, O
Bobes O
- O
Tureac O
R O
, O
Banciu O
A O
, O
Creanga O
A O
, O
Bobes O
- O
Tureac O
G O
, O
Banciu O
MD O
( O
1997 O
) O
Eur O
JMed O
Chem O
32 O
, O
445 O
- O
452 O
. O

16 O
Alzuet O
G O
, O
Casanova O
J O
, O
Ramirez O
JA O
, O
Borras O
J O
, O
Carugo O
O O
( O
1995 O
) O
J O
Inorg O
Bioehem O
57 O
, O
219 O
- O
234 O
. O

17 O
Sumalan O
SL O
, O
Casanova O
J O
, O
Alzuet O
G O
, O
Borras O
J O
, O
Castifieiras O
A O
, O
Supuran O
CT O
( O
1996 O
) O
J O
Inorg O
Biochem O
62 O
, O
3139 O
. O

18 O
a O
) O
Supuran O
CT O
( O
1995 O
) O
Metal O
Based O
Drugs O
2 O
, O
327 O
- O
330 O
; O
b O
) O
Scozzafava O
A O
, O
Supuran O
CT O
( O
1997 O
) O
Metal O
Based O
Drugs O
4 O
, O
19 O
- O
26 O
. O

19 O
a O
) O
Borras O
J O
, O
Cristea O
T O
, O
Supuran O
CT O
( O
1996 O
) O
, O
Main O
Group O
Met O
Chem O
19 O
, O
339 O
- O
346 O
; O
b O
) O
Jitianu O
A O
, O
Ilies O
MA O
, O
Briganti O
F O
, O
Scozzafava O
A O
, O
Supuran O
CT O
( O
1997 O
) O
Metal O
Based O
Drugs O
4 O
, O
1 O
- O
7 O
. O

20 O
a O
) O
Supuran O
CT O
, O
Scozzafava O
A O
( O
1997 O
) O
J O
Enzyme O
Inhibition O
12 O
, O
37 O
- O
51 O
; O
b O
) O
Supuran O
CT O
, O
Briganti O
F O
, O
Scozzafava O
A O
( O
1997 O
) O
J O
Enzyme O
Inhibition O
12 O
, O
175 O
- O
190 O
. O

21 O
Mincione O
G O
, O
Scozzafava O
A O
, O
Supuran O
CT O
( O
1997 O
) O
Metal O
Based O
Drugs O
4 O
, O
27 O
- O
34 O
. O

22 O
Alzuet O
G O
, O
Ferrer O
S O
, O
Borras O
J O
, O
Supuran O
CT O
( O
1994 O
) O
Roum O
Chem O
Quart O
Rev O
2 O
, O
283 O
- O
300 O
. O

23 O
Briganti O
F O
, O
Mangani O
S O
, O
Orioli O
P O
, O
Scozzafava O
A O
, O
Vernaglione O
G O
, O
Supuran O
CT O
( O
1997 O
) O
Biochemistry O
36 O
, O
10384 O
- O
10392 O
. O

24 O
Borras O
J O
, O
Casanova O
J O
, O
Cristea O
T O
, O
Gheorghe O
A O
, O
Scozzafava O
A O
, O
Supuran O
CT O
, O
Tudor O
V O
( O
1996 O
) O
Metal O
Based O
Drugs O
3 O
, O
143 O
- O
148 O
. O

25 O
Supuran O
CT O
, O
Mincione O
F O
, O
Scozzafava O
A O
, O
Briganti O
F O
, O
Mincione O
G O
, O
Ilies O
MA O
( O
1997 O
) O
Eur O
J O
Med O
Chem O
, O
in O

press O
. O

26 O
Supuran O
CT O
, O
Scozzafava O
A O
, O
Saramet O
I O
, O
Banciu O
MD O
( O
1997 O
) O
J O
Enzyme O
Inhibition O
, O
in O
press O
. O

27 O
Jitianu O
A O
, O
Ilies O
MA O
, O
Scozzafava O
A O
, O
Supuran O
CT O
( O
1997 O
) O
Main O
Group O
Met O
Chem O
20 O
, O
151 O
- O
156 O
. O

28 O
Forsman O
C O
, O
Behravan O
G O
, O
Osterman O
A O
, O
Jonsson O
BH O
( O
1988 O
) O
Acta O
Chem O
Scand O
B42 O
, O
314 O
- O
318 O
. O

29 O
Behravan O
G O
, O
Jonasson O
P O
, O
Jonsson O
BH O
, O
Lindskog O
S O
( O
1991 O
) O
Eur O
JBiochem O
198 O
, O
589 O
- O
592 O
. O

30 O
Khalifah O
RG O
, O
Strader O
DJ O
, O
Bryant O
SH O
, O
Gibson O
SM O
( O
1977 O
) O
Biochemistry O
16 O
, O
2241 O
- O
2247 O
. O

31 O
Nyman O
PO O
, O
Lindskog O
S O
( O
1964 O
) O
Biochim O
Biophys O
Acta O
85 O
, O
141 O
- O
151 O
. O

32 O
Henderson O
LE O
, O
Henriksson O
D O
, O
Nyman O
PO O
( O
1976 O
) O
JBiol O
Chem O
251 O
, O
5457 O
- O
5463 O
. O

33 O
Maren O
TH O
, O
Wynns O
GC O
, O
Wistrand O
PJ O
( O
1993 O
) O
Mol O
Pharmaco144 O
, O
901 O
- O
906 O
. O

34 O
Young O
RW O
, O
Wood O
KH O
, O
Vaughan O
JR O
, O
Anderson O
GW O
( O
1956 O
) O
J O
Am O
Chem O
Soc O
78 O
, O
4649 O
- O
4654 O
. O

35 O
Pocker O
Y O
, O
Stone O
JT O
( O
1967 O
) O
Biochemistry O
6 O
, O
668 O
- O
678 O
. O

36 O
Baird O
TT O
, O
Waheed O
A O
, O
Okuyama O
T O
, O
Sly O
WS O
, O
Fierke O
CA O
( O
1997 O
) O
Biochemistry O
36 O
, O
2669 O
- O
2678 O
. O

37 O
Maren O
TH O
, O
Bar O
- O
Ilan O
A O
, O
Conroy O
CW O
, O
Brechue O
WF O
( O
1990 O
) O
Exp O
Eye O
Res O
50 O
, O
27 O
- O
36 O
. O

38 O
Maren O
TH O
, O
Brechue O
WF O
, O
Bar O
- O
Ilan O
A O
( O
1992 O
) O
Exp O
Eye O
Res O
55 O
, O
73 O
- O
79 O
. O

39 O
Brechue O
WF O
, O
Maren O
TH O
( O
1993 O
) O
Invest O
Ophthalmol O
Vis O
Sci O
34 O
, O
2581 O
- O
2587 O
. O

40 O
Borja O
P O
, O
Alzuet O
G O
, O
Server O
- O
Carri6 O
J O
, O
Borras O
J O
, O
Supuran O
CT O
( O
1997 O
) O
J O
Biol O
Inorg O
Chem O
( O
JBIC O
) O
, O
in O
press O
. O

41 O
Supuran O
CT O
, O
Balaban O
AT O
, O
Gheorghiu O
MD O
, O
Schiketanz O
A O
, O
Dinculescu O
A O
, O
Puscas O
I O
( O
1990 O
) O
Rev O
Roum O
Chim O
35 O
, O
399 O
- O
405 O
. O

42 O
Supuran O
CT O
, O
Scozzafava O
A O
( O
1997 O
) O
unpublished O
results O
. O

Received O
: O
September O
24 O
, O
1997 O
Accepted O
: O
October O
17 O
, O
1997 O
Received O
in O
revised O
camera O
- O
ready O
format O
" O
October O
23 O
, O
1997 O

315 O

Synthesis O
, O
Characterization O
and O
Antitumour O
Activity O
of O
Some O
Butyltin O
( O
IV O
) O
Cysteaminates O
and O
N O
, O
N O
- O
Dimethylcysteaminate O

Abstract O

The O
synthesis O
and O
characterization O
of O
four O
di O
- O
and O
tri O
- O
n O
- O
butyltin O
cysteaminates O
and O
N O
, O
N O
- O
dimethylcysteaminate O
and O
three O
protonated O
/ O
quaternized O
derivatives O
are O
reported O
. O

They O
all O
exhibit O
moderate O
or O
high O
in O
vitro O
cytotoxic O
activity O
. O

Six O
of O
seven O
compounds O
presented O
in O
this O
work O
are O
more O
active O
than O
cisplatin O
, O
etoposide O
and O
5 O
- O
fluorouracil O
, O
but O
less O
active O
than O
methotrexate O
and O
doxorubicin O
. O

Metal O
Based O
Drugs O

Vol O
. O

7 O
, O
Nr O
. O

5 O
, O
2000 O

SYNTHESIS O
, O
CHARACTERIZATION O
AND O
ANTITUMOUR O
ACTITY O
OF O
SOME O
BUTYLTIN O
( O
IV O
) O
CYSTEAMINATES O
AND O
N O
, O
N O
- O
DIMETHYLCYSTEAMINATE O
Marcel O
Gielen O
Karel O
Handlir O
. O
2 O
, O
Martin O
Hollein O
and O
Dick O
de O
Vos O
2 O
3 O

Departement O
of O
General O
and O
Organic O
Chemistry O
AOSC O
, O
Faculty O
of O
Applied O
Sciences O
, O
Free O
University O
of O
Brussels O
VUV O
, O
Pleinlaan O
2 O
, O
B O
- O
1050 O
Brussels O
, O
Belgium O
Departement O
of O
General O
and O
Inorganic O
Chemistry O
, O
Faculty O
of O
Chemical O
Technology O
, O
University O
of O
Pafdubice O
, O
Nam O
. O

Cs O
. O
legii O
565 O
, O
53210 O
Pardubice O
, O
Czech O
Republic O
Pharmachemie O
B O
. O

V O
. O
, O
Haarlem O
, O
the O
Netherlands O
Abstract O
. O

The O
synthesis O
and O
characterization O
of O
four O
di O
- O
and O
tri O
- O
n O
- O
butyltin O
cysteaminates O
and O
_ O
N O
, O
N O
- O
dimethylcysteaminate O
and O
three O
protonated O
/ O
quaternized O
derivatives O
are O
reported O
. O

They O
all O
exhibit O
moderate O
or O
high O
in O
vitro O
cytotoxic O
activity O
. O

Six O
of O
seven O
compounds O
presented O
in O
this O
work O
are O
more O
active O
than O
cisplatin O
, O
etoposide O
and O
5 O
- O
fluorouracil O
, O
but O
less O
active O
than O
methotrexate O
and O
doxorubiein O
. O

Introduction O
. O

Several O
organotin O
cysteaminates O
( O
aminoethylthiolates O
) O
have O
been O
studied O
1 O
- O
4 O
as O
compounds O
with O
potentially O
high O
biological O
activity O
, O
but O
little O
is O
known O
about O
their O
cytotoxic O
effect O
. O

The O
present O
paper O
reports O
the O
synthesis O
and O
characterization O
of O
di O
- O
and O
tri O
- O
n O
- O
butyltin O
cysteaminates O
and O
their O
N O
, O
N O
- O
dimethyl O
analogues O
and O
their O
in O
vitro O
cytotoxicity O
. O

It O
is O
shown O
that O
a O
correct O
evaluation O
of O
the O
biological O
activity O
of O
organotin O
compounds O
remains O
5 O
often O
hampered O
by O
their O
low O
water O
solubility O
. O

Therefore O
, O
we O
focused O
on O
the O
synthesis O
of O
derivatives O
with O
potentially O
higher O
water O
solubility O
by O
introducing O
ionic O
groups O
into O
the O
molecule O
by O
protonation O
or O
quatemization O
of O
a O
nitrogen O
atom O
. O

Results O
and O
discussion O
. O

3 O

2 O

4 O

( O
n O
- O
Bu O
) O
n O
Sn O
[ O
SCH2 O
- O
CH2 O
- O
NHm O
( O
CH3 O
) O
2 O
- O
m O
] O
4 O
- O
n O

n O
= O
3 O
, O
n O
= O
3 O
, O
n O
= O
2 O
, O
n O
= O
2 O
, O

m O
= O
2 O
m O
= O
O O
m O
= O
2 O
m O
= O
O O

( O
1 O
) O
( O
2 O
) O
( O
3 O
) O
( O
4 O
) O

3 O

2 O

4 O

( O
n O
- O
Bu O
) O
3 O
Sn O
S O
- O
CH2 O
- O
CH2 O
- O
NHm O
( O
CH3 O
) O
3 O
. O
m O
X O

m O
= O
3 O
, O
x O
= O
cn3so3 O
( O
5 O
) O
m O
1 O
, O
X O
= O
CH3SO3 O
( O
6 O
) O
m O
0 O
, O
X O
I O
( O
7 O
) O

n O
- O
Bu O
CH3 O
- O
CH2 O
- O
CH2 O
- O
CH2 O

( O
2 O
) O
, O
di O
- O
nTri O
- O
n O
- O
butyltin O
cysteaminate O
( O
1 O
) O
, O
tri O
- O
n O
- O
butyltin O
N O
, O
N O
- O
dimethylcysteaminate O
butyltin O
bis O
( O
cysteaminate O
) O
( O
3 O
) O
and O
di O
- O
n O
- O
butyltin O
bis O
( O
N O
, O
N O
- O
dimethylcysteaminate O
) O
( O
4 O
) O
were O
prepared O
by O
the O
reaction O
of O
the O
sodium O
cysteaminate O
( O
prepared O
in O
situ O
by O
the O
reaction O
of O
sodium O
methylate O
with O
the O
hydrochloride O
of O
eysteamine O
) O
with O
the O
suitable O

di O
- O
or O
tri O
- O
n O
- O
butyltin O
chloride O
in O
chloroform O
following O
reference O
by O
the O
reactions O
of O
the O
The O
methanesulphonates O
5 O
and O
6 O
were O
respectively O
synthesized O
equimolar O
amount O
of O
methane O
sulphonic O
acid O
in O
unprotonated O
analogs O
1 O
and O
2 O
with O
an O
chloroform O
. O

After O
purification O
on O
a O
short O
column O
of O
alumina O
and O
evaporation O
of O
the O
solvent O
, O
colourless O
viscous O
oils O
were O
obtained O
. O

The O
quaternized O
methyl O
derivative O
of O
2 O
, O
the O
methiodide O
( O
7 O
) O
, O
was O
prepared O
by O
its O
reaction O
with O
methyl O
iodide O
in O
benzene O
and O
purified O
by O
recrystalization O
from O
chloroform O
. O

233 O

M O
. O

Gielen O
, O
K O
. O

Handlir O
, O
M O
. O

Hollein O
and O
D O
. O

de O
Vos O
Synthesis O
, O
Characterization O
and O
Antitumor O
Activity O
ofSome O
Butyltin O
( O
IV O
) O
Cysteaminates O
and O
N O
, O
N O
- O
Dimethylcysteaminate O

86 O
77 O
85 O
3 O
( O
C4H9 O
) O
2Sn O
( O
SCH2CH2NH2 O
) O
2 O
90 O
4 O
( O
C4Hg O
) O
zSn O
( O
SCH2CHzN O
( O
CH3 O
) O
2 O
) O
2 O
92 O
oil O
5 O
( O
C4H9 O
) O
3 O
SnSCH2CH2NH3 O
CH3SO3 O
94 O
6 O
oil O
( O
C4H9 O
) O
3 O
SnSCH2CH2NH O
( O
CH3 O
) O
2 O
CH3SO3 O
118 O
- O
120 O
74 O
7 O
( O
C4H9 O
) O
. O
SnSCH O
. O
CH O
. O
N O
( O
CH3 O
) O
31 O

The O
elemental O
analyses O
( O
C O
, O
H O
, O
N O
, O
Sn O
) O
agree O
satisfactorily O
with O
the O
proposed O
formul O

Table O
1 O
. O

Melting O
points O
and O
yields O
for O
. O
. O
c0mP0unds O
1 O
( O
) O
rmula O
, O
2 O
comp O
. O

1 O
( O
C4H9 O
) O
3SnSCHzCH2NH2 O
2 O
( O
C4H9 O
) O
3SnSCHzCH2N O
( O
CH3 O
) O
2 O

7 O
. O

m O
. O
p O
. O
, O
. O
. O
b O
. O
p O
. O
. O
, O
. O
C O
120 O
- O
123 O
/ O
40 O
Pa O
129 O
- O
131 O
/ O
35 O
Pa O
167 O
- O
169 O
/ O
35 O
Pa O
172 O
- O
174 O
/ O
40 O
Pa O

' O
o O
' O

yield O
, O
. O
% O

Compounds O
1 O
- O
4 O
, O
and O
7 O
, O
are O
poorly O
soluble O
in O
aqueous O
solutions O
( O
< O
mg O
/ O
mL O
) O
. O

Compounds O
5 O
and O
6 O
exhibit O
higher O
water O
solubilities O
( O
ca O
. O
2 O
mg O
/ O
mL O
) O
. O

Compounds O
1 O
7 O
are O
soluble O
in O
a O
therapeutic O
solution O
( O
0 O
, O
103 O
M O
solution O
of O
NaCl O
in O
water O
/ O
DMSO O
1 O
/ O
9 O
) O
. O

A O
white O
turbidity O
appears O
in O
these O
solutions O
only O
after O
several O
hours O
due O
to O
slow O
hydrolysis O
. O

Compounds O
1 O
7 O
were O
characterized O
by O
multinuclear O
NMR O
spectroscopy O
. O

The O
NMR O
parameters O
are O
given O
in O
Table O
2 O
. O

Table O
2 O
. O

Resonances O
of O
13C O
, O
( O
ca O
20 O
% O
sol O
. O
v O
/ O
v O
) O
1 O
' O
compound O

SN O
and O
ll9Sn O
NMR O
spectra O
of O
2 O

the O
compounds O
1 O
- O
7 O
in O
CDCI3 O
5 O
86 O
. O
3 O
13 O
. O
45 O

6 O
' O
' O
S O
' O
n O
) O
, O
" O
ppm O
6 O
( O
3c O
) O
a O
, O
ppm O

x O

77 O
. O
6 O
12 O
. O
3 O

77 O
. O
5 O
12 O
. O
75 O

3 O
- O
28 O
. O
9 O
22 O
. O
38 O

38 O
. O
' O
18 O
. O
78 O

4 O

6 O
' O
' O
89 O
. O
5 O
13 O
. O
45 O

7 O
93 O
. O
2 O
13 O
. O
60 O

( O
332 O
) O
27 O
. O
93 O

( O
331 O
) O
27 O
. O
97 O

( O
495 O
) O
28 O
. O
27 O

( O
438 O
) O
27 O
. O
51 O

( O
322 O
) O
28 O
. O
30 O

( O
350 O
) O
28 O
. O
34 O

( O
339 O
) O
28 O
. O
47 O

( O
21 O
. O
3 O
) O
26 O
. O
32 O

( O
21 O
. O
1 O
) O
26 O
. O
38 O

( O
28 O
. O
4 O
) O
26 O
. O
42 O

( O
26 O
. O
4 O
) O
25 O
. O
90 O

( O
21 O
. O
4 O
) O
26 O
. O
85 O

( O
21 O
. O
3 O
) O
26 O
. O
81 O

( O
22 O
. O
0 O
) O
26 O
. O
91 O

( O
59 O
. O
6 O
) O
12 O
. O
89 O

( O
59 O
. O
8 O
) O
12 O
. O
95 O

( O
87 O
. O
1 O
) O
13 O
. O
35 O

( O
83 O
. O
8 O
) O
12 O
. O
82 O

( O
62 O
. O
7 O
) O
13 O
. O
45 O

( O
61 O
. O
4 O
) O
13 O
. O
58 O

62 O
. O
2 O
) O
14 O
. O
00 O

( O
- O
) O
30 O
. O
31 O
45 O
. O
02 O

( O
- O
) O
23 O
. O
59 O
63 O
. O
12 O
44 O
. O
71 O

( O
- O
) O
30 O
. O
04 O
44 O
. O
44 O

( O
- O
) O
23 O
. O
84 O
61 O
. O
84 O
44 O
. O
46 O

( O
- O
) O
23 O
. O
92 O
43 O
. O
08 O

( O
- O
; O

( O
- O
) O

20 O
. O
94 O
19 O
. O
70 O
60 O
. O
94 O
69 O
. O
49 O
43 O
. O
42 O
54 O
. O
18 O
39 O
. O
15 O
39 O
. O
16 O
- O
360 O
. O
4 O
- O
354 O
. O
0 O
- O
355 O
. O
3 O
- O
354 O
. O
4 O
- O
349 O
. O
4 O
- O
344 O
. O
2 O
- O
325 O
. O
8 O
1 O
. O
1 O
6 O
. O
2 O
1 O
. O
5 O
1 O
. O
5 O
12 O
. O
7 O
11 O
. O
7 O
30 O
. O
1 O
13 O
a O
' O
values O
of O
nJ O
( O
n O
J O
9 O
Sn O
, O
, O
C O
) O
c0uplihg O
constants O
in O
Hz O
' O
for O
Carbon O
ato O
] O
ns O
in O
parentlee O
' O
s O
difference O
btween O
( O
N O
) O

for O
the O
organo O
. O
i O
compound O
and O
free O
cysteamine O
, O
6 O
( O
N O
) O
361 O
. O
5 O
ppm O
; O
N O
, O
N O
- O
dimethylcysteamine O
, O
5 O
( O
N O
) O
- O
355 O
. O
9 O
ppm O
) O
not O
observed O

C O
resonances O
were O
assigned O
on O
the O
basis O
of O
the O
values O
of O
n O
J O
( O
119 O
Sn O
, O
13 O
C O
) O
coupling O
constants O
and O
standard O
13 O
C O
- O
APT O
techniques O
utilization O
in O
agreement O
with O
ref O
. O
. O

The O
values O
of O
119 O
Sn O
chemical O
shifts O
are O
found O
in O
the O
interval O
characteristic O
for O
fourcoordinated O
tin O
6 O
, O
7 O
. O

The O
values O
of O
the O
coupling O
constants O
j O
( O
1 O
lqSn O
, O
13C O
) O
agree O
with O
the O
structure O
proposed O
. O

Compound O
3 O
is O
characterized O
by O
a O
somewhat O
larger O
upfield O
shift O
, O
close O
to O
the O
upper O
limit O
of O
the O
above O
mentioned O
interval O
, O
together O
with O
a O
high O
value O
of O
1j O
( O
l O
lqSn O
, O
13C O
) O
( O
495 O
Hz O
) O
. O

67 O
The O
C O
- O
Sn O
- O
C O
angle O
, O
est O
' O
mated O
, O
from O
this O
value O
of O
coupling O
constant O
is O
124 O
According O
to O
ref O
. O
this O
behaviour O
can O
be O
due O
to O
intermolecular O
association O
increasing O
the O
coordinatitn O
number O
of O
tin O
in O
concentrated O
solutions O
, O
as O
suggested O
by O
the O
concentration O
dependence O
of O
tS O
( O
Sn O
) O
and O
cryoscopic O
measurements O
in O
benzene O
. O

The O
values O
of O
the O
6 O
( O
SN O
) O
chemical O
shifts O
of O
compounds O
1 O
, O
2 O
and O
4 O
only O
slightly O
differ O
from O
the O
( O
15N O
) O
values O
of O
the O
free O
cysteamine O
. O

The O
significantly O
larger O
i O
( O
15N O
) O
chemical O
shift O
of O
compound O
3 O
is O
however O
much O
lower O
than O
those O
of O
compounds O
5 O
, O
6 O
and O
7 O
containing O
a O
N O
interaction O
in O
compounds O
1 O
tetracoordinated O
nitrogen O
atom O
. O

It O
can O
be O
stated O
that O
the O
Sn O
4 O
is O
negligible O
or O
only O
very O
weak O
in O
chlorofom O
solutions O
. O

This O
conclusion O
is O
not O
in O
contradiction O
with O
the O
proved O
Sn O
N O
interaction O
found O
in O
the O
solid O
state O
8 O
. O

13 O

. O

, O

234 O

Metal O
Based O
Drugs O

Vol O
. O

7 O
, O
Nr O
. O

5 O
, O
2000 O

Antitumour O
activity O
. O

The O
results O
of O
the O
in O
vitro O
antitumour O
tests O
of O
compounds O
1 O
7 O
are O
given O
in O
Table O
3 O
as O
the O
inhibition O
doses O
IDs0 O
observed O
against O
a O
panel O
of O
seven O
human B
tumour O
cell O
lines O
, O
MCF O
- O
7 O
and O
EVSA O
- O
T O
, O
two O
breast O
cancers O
, O
WiDr O
, O
a O
colon O
cancer O
, O
IGROV O
, O
an O
ovarian O
cancer O
, O
M19 O
MEL O
, O
a O
melanoma O
, O
A248 O
, O
a O
renal O
cancer O
, O
and O
H226 O
, O
a O
non O
- O
small O
cell O
lung O
cancer O
. O

The O
antitumour O
tests O
results O
are O
compared O
with O
those O
obtained O
for O
clinically O
used O
reference O
compounds9 O
like O
cisplatin O
, O
doxorubicin O
, O
etoposide O
, O
5 O
- O
fluorouracil O
and O
methotrexate O
. O

Table O
3 O
. O

Inhibition O
doses O

comp O
. O

1 O
2 O
3 O
4 O
5 O
6 O
7 O

MF O
- O
7 O
41 O
41 O
51 O
78 O
30 O
90 O
385 O
699 O
10 O
2 O
594 O
750 O
18 O

EVSA O
- O
T O
33 O
35 O
45 O
65 O
< O
3 O
57 O
367 O

IDs0 O
of O
compounds O
WiDr O
39 O
39 O
211 O
331 O
19 O
40 O
339 O

1 O
- O
7 O
and O
of O
five O
reference O
compounds O
. O

IGROV O
M19MEL O
A498 O
H226 O
67 O
44 O
85 O
39 O
46 O
78 O
88 O
39 O
68 O
70 O
118 O
72 O
110 O
129 O
108 O
112 O
31 O
29 O
36 O
42 O
83 O
133 O
77 O
111 O
327 O
758 O
576 O
411 O
169 O
60 O
580 O
297 O
7 O
558 O
16 O
505 O
442 O
23 O

cisplatin O
doxorubicin O

etoposide O
5 O
- O
fluorouracil O

422 O
8 O
317 O
475 O
5 O

methotrexate O

967 O
11 O
150 O
225 O
< O
3 O

2 O
253 O
90 O
314 O
143 O
37 O

3 O
369 O
199 O
3 O
934 O
340 O
2 O
287 O

It O
is O
evident O
from O
Table O
3 O
that O
all O
studied O
compounds O
exhibit O
excellent O
cytostatic O
activities O
, O
except O
compound O
7 O
showing O
only O
moderate O
activity O
. O

Compounds O
1 O
6 O
are O
more O
active O
than O
cisplatin O
and O
, O
in O
most O
cases O
, O
than O
etoposide O
and O
5 O
- O
fluorouracil O
. O

Their O
activity O
is O
lower O
than O
that O
of O
doxorubicin O
and O
methotrexate O
in O
most O
cases O
for O
the O
studied O
cancer O
cell O
lines O
. O

The O
very O
limited O
set O
of O
acquired O
activity O
values O
show O
that O
the O
activity O
of O
tri O
- O
n O
- O
butyltin O
are O
higher O
than O
those O
of O
di O
- O
n O
- O
butyl O
analogues O
( O
1 O
> O
3 O
, O
2 O
> O
4 O
) O
for O
all O
cell O
lines O
, O
especially O
compounds O
against O
WiDr O
. O

The O
N O
, O
N O
- O
dimethyl O
derivatives O
exhibit O
an O
activity O
comparable O
to O
or O
somewhat O
higher O
than O
that O
of O
unmethylated O
compounds O
. O

Compound O
8 O
, O
with O
a O
quaternized O
nitrogen O
, O
exhibits O
the O
lowest O
cytotoxic O
activities O
, O
approximatively O
9 O
times O
lower O
than O
compound O
2 O
. O

It O
might O
be O
underlined O
that O
the O
ionic O
more O
water O
- O
soluble O
compounds O
5 O
and O
6 O
are O
not O
more O
active O
than O
the O
uncharged O
analogs O
1 O
- O
4 O
, O
whereas O
the O
hydrophilic O
organotin O
polyoxacarboxylates O
exhibit O
much O
higher O
in O
vitro O
activities O
than O
other O
organotin O
carxylates O
51 O
Compounds O
5 O
and O
6 O
( O
with O
a O
protonated O
nitrogen O
) O
exhibit O
activities O
comparable O
or O
even O
considerably O
higher O
( O
compound O
5 O
) O
against O
EVSA O
- O
T O
than O
the O
corresponding O
unprotonated O
compounds O
. O

Compound O
7 O
( O
with O
a O
quaternized O
nitrogen O
) O
exhibits O
the O
lowest O
cytostatic O
activities O
, O
approximately O
9 O
times O
should O
however O
be O
mentioned O
that O
another O
ionic O
organotin O
lower O
than O
compound O
2 O
. O

It O
compound O
, O
bis O
( O
dicyclohexyl O
) O
ammonium O
bis O
( O
2 O
, O
6 O
- O
pyridinecarboxyla O
, O
to O
) O
di O
- O
n O
- O
butylstannate O
, O
where O
the O
organotin O
moiety O
is O
this O
time O
anionic O
, O
is O
also O
as O
active O
as O
the O
uncharged O
analog O
l O
. O

All O
NMR O
spectra O
were O
recorded O
on O
a O
Bruker O
AMX O
360 O
instrument O
using O
a O
5 O
mm O
multinuclear O
tuneable O
probe O
. O

The O
residual O
CHCI3 O
resonance O
at O
7 O
. O
24 O
ppm O
was O
used O
as O
reference O
for O
the O
H O
soectra O
and O
the O
central O
13CDCI3 O
resonance O
at O
77 O
. O
0 O
ppm O
, O
for O
the O
13C O
spectra O
. O

The O
119 O
Sn O
chemical O
shifts O
were O
refered O
to O
the O
external O
tetramethyltin O
[ O
6 O
( O
119 O
Sn O
) O
0 O
. O
0 O
] O
. O

The O
15N O
NMR O
spectra O
were O
measur O
; O
l O
using O
the O
INEPT O
technique O
or O
in O
inverse O
- O
gated O
mode O
. O

( O
reference O
: O
Instrumentation O
. O

external O
nitromethane O
, O
[ O
5 O
( O
' O
N O
) O
0 O
. O
0 O
] O
) O
. O

The O
protocol O
followed O
for O
the O
in O
vitro O
antitumour O
screenings O
has O
been O
already O
reported O
10 O
. O

Acknowledgements O
. O

K O
. O

H O
. O
and O
M O
H O
. O
thank O
the O
Grant O
Agency O
of O
the O
Czech O
Republic O
( O
Grant O
No O
. O
203 O
/ O
00 O
/ O
0920 O
) O
and O
the O
Ministry O
o O
" O
Education O
, O
Youth O
and O
Sport O
of O
the O
Czech O
republic O
, O
associated O
with O
EU O
in O
the O
COST O
8 O
. O
20 O
program O
for O
financial O
support O
of O
this O
work O
. O

We O
are O
grateful O
to O
Mr O
. O
R O
. O

G O
. O

Experimental O
Oostrum O
, O
Dr O
. O
J O
. O
verweij O
, O
Prof O
. O

Dr O
. O
G O
. O

Stoter O
, O
r O
. O
K O
. O

Nooter O
, O
Laboratory O
of O
Chemotherapy O
and O
Pharmacology O
, O
Department O
of O
Medical O
Oncology O
, O
Rotterdam O
Cancer O
235 O

M O
. O

Gielen O
, O
K O
. O

Handlir O
, O
M O
. O

HoHein O
and O
D O
. O

de O
Vos O
Synthesis O
, O
Characterization O
and O
Antitumor O
Activity O
ofSome O
Butyltin O
( O
IV O
) O
Cysteaminates O
and O
N O
, O
N O
- O
Dimethylcysteaminate O

Institute O
, O
NL O
3008 O
AE O
, O
Rotterdam O
, O
The O
Netherlands O
, O
for O
performing O
the O
in O
vitro O
tests O
. O

This O
research O
was O
supported O
by O
the O
Fund O
for O
Scientific O
Research O
Flanders O
( O
Belgium O
) O
, O
grant O
nr O
G O
. O
0074 O
. O
00 O
, O
M O
. O

G O
. O
) O
. O

References O
. O

1 O
. O

B O
. O
S O
. O

Saraswat O
, O
J O
. O

Mason O
, O
Polyhedron O
( O
9 O
) O
, O
5 O
( O
1986 O
) O
, O
1449 O
. O

2 O
. O

G O
. O

Domazetis O
, O
R O
. O

J O
. O

Magee O
, O
B O
. O

D O
. O

James O
, O
J O
. O

Organomet O
. O

Chem O
, O
162 O
( O
1978 O
) O
, O
239 O
. O

3 O
. O

J O
. O
D O
. O

Cashion O
, O
G O
. O

Domazetis O
, O
B O
. O

D O
. O

James O
, O
J O
. O

Organomet O
. O

Chem O
, O
185 O
( O
1980 O
) O
, O
433 O
. O

4 O
. O

G O
. O

Domazetis O
, O
R O
. O

J O
. O

Magee O
, O
B O
. O

D O
. O

James O
, O
J O
. O

Organomet O
. O

Chem O
, O
148 O
( O
1978 O
) O
, O
339 O
. O

5 O
. O

G O
. O

Atassi O
, O
Rev O
. O

Si O
Ge O
Sn O
Pb O
Cpds O
, O
8 O
( O
1985 O
) O
, O
219 O
; O
M O
. O

Kemmer O
, O
M O
. O

Gielen O
, O
M O
. O

Biesemans O
, O
D O
. O

de O
Vos O
, O
R O
. O

Willem O
, O
Metal O
- O
Based O
Drugs O
5 O
( O
1998 O
) O
, O
189 O
; O
M O
. O

Gielen O
, O
M O
. O

Biesemans O
, O
D O
. O

de O
Vos O
, O
R O
. O

Willem O
, O
J O
. O

Inorg O
. O

Biochem O
. O
, O
79 O
( O
2000 O
) O
, O
139 O
. O

6 O
. O

J O
. O

Holecek O
, O
A O
. O

Lycka O
, O
Inorg O
. O

Chim O
. O

Acta O
, O
118 O
( O
1986 O
) O
, O
L O
15 O
. O

7 O
. O

J O
. O

Holecek O
, O
M O
. O

Nadvomik O
, O
K O
. O

Handlir O
, O
A O
. O

Lycka O
, O
J O
. O

Organomet O
. O

Chem O
. O
, O
315 O
( O
1986 O
) O
, O
299 O
. O

8 O
. O

B O
. O
D O
. O

James O
, O
R O
. O

J O
. O

Magee O
, O
W O
. O

C O
. O

Patalinghug O
, O
B O
. O

W O
. O

Skelton O
, O
A O
. O

H O
. O

White O
, O
J O
. O

Organomet O
. O

Chem O
, O
467 O
( O
1994 O
) O
, O
51 O
9 O
. O

P O
. O

Skehan O
, O
R O
. O

Storeng O
, O
D O
. O

Scudiero O
, O
A O
. O

Monks O
, O
J O
. O

McMahon O
, O
D O
. O

Vistica O
, O
J O
. O

T O
. O

Warren O
, O
H O
. O

Bokesh O
, O
S O
. O

Kenney O
, O
M O
. O

R O
. O

Boyd O
, O
J O
. O
. O

Natl O
. O

Cancer O
lnst O
. O
, O
82 O
( O
1990 O
) O
, O
1107 O
. O

10 O
. O

Y O
. O

P O
. O

Kepers O
, O
G O
. O

J O
. O

Peters O
, O
J O
. O

Van O
Ark O
- O
Otte O
, O
B O
. O

Winograd O
, O
H O
. O

M O
. O

Pinedo O
, O
Eur O
. O

J O
. O

Cancer O
, O
27 O
( O
1991 O
) O
, O
897 O
. O

11 O
. O

M O
. O

Kemmer O
, O
L O
. O

Ghys O
, O
M O
. O

Gielen O
, O
M O
. O

Biesemans O
, O
E O
. O

R O
. O

T O
. O

Tiekink O
, O
R O
. O

Willem O
, O
J O
. O

Organomet O
. O

Chem O
. O

582 O
( O
1999 O
) O
, O
195 O
. O

12 O
. O

S B
. I

W O
. O

Ng O
, O
V O
. O

G O
. O

Kumar O
Das O
, O
J O
. O

Holecek O
, O
A O
. O

Lycka O
, O
M O
. O

Gielen O
, O
M O
. O

G O
. O

B O
. O

Drew O
, O
Appl O
. O

Organomet O
. O

Chem O
. O

11 O
( O
1997 O
) O
, O
3 O
9 O
. O

Received O
" O
September O
22 O
, O
2000 O
Accepted O
- O
October O
19 O
, O
2000 O
Received O
in O
revised O
camera O
- O
ready O
format O
- O
October O
20 O
, O
2000 O

236 O

Genomic O
- O
Bioinformatic O
Analysis O
of O
Transcripts O
Enriched O
in O
the O
Third O
- O
Stage O
Larva O
of O
the O
Parasitic O
Nematode O
Ascaris B
suum I

Abstract O

Differential O
transcription O
in O
Ascaris B
suum I
was O
investigated O
using O
a O
genomic O
- O
bioinformatic O
approach O
. O

A O
cDNA O
archive O
enriched O
for O
molecules O
in O
the O
infective O
third O
- O
stage O
larva O
( O
L3 O
) O
of O
A B
. I
suum I
was O
constructed O
by O
suppressive O
- O
subtractive O
hybridization O
( O
SSH O
) O
, O
and O
a O
subset O
of O
cDNAs O
from O
3075 O
clones O
subjected O
to O
microarray O
analysis O
using O
cDNA O
probes O
derived O
from O
RNA O
from O
different O
developmental O
stages O
of O
A B
. I
suum I
. O

The O
cDNAs O
( O
n O
= O
498 O
) O
shown O
by O
microarray O
analysis O
to O
be O
enriched O
in O
the O
L3 O
were O
sequenced O
and O
subjected O
to O
bioinformatic O
analyses O
using O
a O
semi O
- O
automated O
pipeline O
( O
ESTExplorer O
) O
. O

Using O
gene O
ontology O
( O
GO O
) O
, O
235 O
of O
these O
molecules O
were O
assigned O
to O
' O
biological O
process O
' O
( O
n O
= O
68 O
) O
, O
' O
cellular O
component O
' O
( O
n O
= O
50 O
) O
, O
or O
' O
molecular O
function O
' O
( O
n O
= O
117 O
) O
. O

Of O
the O
91 O
clusters O
assembled O
, O
56 O
molecules O
( O
61 O
. O
5 O
% O
) O
had O
homologues O
/ O
orthologues O
in O
the O
free O
- O
living O
nematodes B
Caenorhabditis B
elegans I
and O
C B
. I
briggsae I
and O
/ O
or O
other O
organisms O
, O
whereas O
35 O
( O
38 O
. O
5 O
% O
) O
had O
no O
significant O
similarity O
to O
any O
sequences O
available O
in O
current O
gene O
databases O
. O

Transcripts O
encoding O
protein O
kinases O
, O
protein O
phosphatases O
( O
and O
their O
precursors O
) O
, O
and O
enolases O
were O
abundantly O
represented O
in O
the O
L3 O
of O
A B
. I
suum I
, O
as O
were O
molecules O
involved O
in O
cellular O
processes O
, O
such O
as O
ubiquitination O
and O
proteasome O
function O
, O
gene O
transcription O
, O
protein O
- O
protein O
interactions O
, O
and O
function O
. O

In O
silico O
analyses O
inferred O
the O
C B
. I
elegans I
orthologues O
/ O
homologues O
( O
n O
= O
50 O
) O
to O
be O
involved O
in O
apoptosis O
and O
insulin O
signaling O
( O
2 O
% O
) O
, O
ATP O
synthesis O
( O
2 O
% O
) O
, O
carbon O
metabolism O
( O
6 O
% O
) O
, O
fatty O
acid O
biosynthesis O
( O
2 O
% O
) O
, O
gap O
junction O
( O
2 O
% O
) O
, O
glucose O
metabolism O
( O
6 O
% O
) O
, O
or O
porphyrin O
metabolism O
( O
2 O
% O
) O
, O
although O
34 O
( O
68 O
% O
) O
of O
them O
could O
not O
be O
mapped O
to O
a O
specific O
metabolic O
pathway O
. O

Small O
numbers O
of O
these O
50 O
molecules O
were O
predicted O
to O
be O
secreted O
( O
10 O
% O
) O
, O
anchored O
( O
2 O
% O
) O
, O
and O
/ O
or O
transmembrane O
( O
12 O
% O
) O
proteins O
. O

Functionally O
, O
17 O
( O
34 O
% O
) O
of O
them O
were O
predicted O
to O
be O
associated O
with O
( O
non O
- O
wild O
- O
type O
) O
RNAi O
phenotypes O
in O
C B
. I
elegans I
, O
the O
majority O
being O
embryonic O
lethality O
( O
Emb O
) O
( O
13 O
types O
; O
58 O
. O
8 O
% O
) O
, O
larval O
arrest O
( O
Lva O
) O
( O
23 O
. O
5 O
% O
) O
and O
larval O
lethality O
( O
Lvl O
) O
( O
47 O
% O
) O
. O

A O
genetic O
interaction O
network O
was O
predicted O
for O
these O
17 O
C B
. I
elegans I
orthologues O
, O
revealing O
highly O
significant O
interactions O
for O
nine O
molecules O
associated O
with O
embryonic O
and O
larval O
development O
( O
66 O
. O
9 O
% O
) O
, O
information O
storage O
and O
processing O
( O
5 O
. O
1 O
% O
) O
, O
cellular O
processing O
and O
signaling O
( O
15 O
. O
2 O
% O
) O
, O
metabolism O
( O
6 O
. O
1 O
% O
) O
, O
and O
unknown O
function O
( O
6 O
. O
7 O
% O
) O
. O

The O
potential O
roles O
of O
these O
molecules O
in O
development O
are O
discussed O
in O
relation O
to O
the O
known O
roles O
of O
their O
homologues O
/ O
orthologues O
in O
C B
. I
elegans I
and O
some O
other O
nematodes O
. O

The O
results O
of O
the O
present O
study O
provide O
a O
basis O
for O
future O
functional O
genomic O
studies O
to O
elucidate O
molecular O
aspects O
governing O
larval O
developmental O
processes O
in O
A B
. I
suum I
and O
/ O
or O
the O
transition O
to O
parasitism O
. O

Introduction O

Parasitic O
nematodes O
are O
of O
major O
socio O
- O
economic O
importance O
in O
animals O
. O

For O
example O
, O
hundreds O
of O
millions O
of O
people B
are O
infected O
with O
geohelminths O
( O
soil O
- O
transmitted O
worms O
) O
, O
such O
as O
blood O
- O
feeding O
hookworms O
Ancylostoma B
duodenale I
and O
/ O
or O
Necator B
americanus I
, O
Trichuris B
trichiura I
and O
Ascaris B
spp I
. O

[ O
1 O
] O
, O
causing O
serious O
adverse O
effects O
on O
human B
health O
, O
particularly O
in O
children B
. O

Similarly O
, O
parasitic O
nematodes O
of O
livestock O
, O
such O
as O
pigs B
, O
also O
cause O
substantial O
economic O
losses O
due O
to O
subclinical O
and O
clinical O
diseases O
, O
with O
billions O
of O
dollars O
spent O
annually O
on O
the O
treatment O
and O
control O
of O
gastro O
- O
intestinal O
nematodes O
. O

In O
addition O
to O
the O
socioeconomic O
impact O
that O
these O
parasites O
have O
, O
there O
is O
potential O
for O
the O
emergence O
of O
resistance O
in O
them O
against O
all O
of O
the O
main O
classes O
of O
( O
nematocidal O
) O
compounds O
used O
to O
treat O
the O
diseases O
they O
cause O
[ O
2 O
] O
- O
[ O
5 O
] O
. O

Therefore O
, O
there O
is O
a O
significant O
need O
to O
work O
toward O
discovering O
new O
compounds O
to O
control O
these O
parasites O
. O

Gaining O
an O
improved O
understanding O
of O
the O
molecular O
basis O
of O
parasite O
development O
provides O
such O
an O
avenue O
. O

Compared O
with O
the O
free O
- O
living O
nematode B
Caenorhabditis B
elegans I
, O
there O
is O
very O
little O
information O
on O
fundamental O
molecular O
aspects O
of O
development O
in O
parasitic O
nematodes O
[ O
6 O
] O
- O
[ O
8 O
] O
. O

Since O
the O
genome O
sequence O
of O
C B
. I
elegans I
was O
published O
in O
1998 O
[ O
9 O
] O
, O
many O
aspects O
of O
the O
molecular O
biology O
of O
this O
nematode O
have O
been O
elucidated O
. O

For O
instance O
, O
microarray O
analyses O
have O
been O
used O
to O
examine O
developmental O
and O
gender O
- O
enriched O
gene O
expression O
[ O
10 O
] O
, O
[ O
11 O
] O
, O
and O
the O
functions O
of O
more O
than O
96 O
% O
of O
the O
C B
. I
elegans I
genes O
have O
been O
assessed O
by O
double O
- O
stranded O
RNA O
interference O
( O
RNAi O
, O
or O
gene O
silencing O
; O
[ O
12 O
] O
) O
[ O
13 O
] O
- O
[ O
18 O
] O
. O

Comparative O
analyses O
of O
genetic O
data O
sets O
have O
shown O
that O
parasitic O
nematodes O
usually O
share O
~ O
50 O
- O
70 O
% O
of O
genes O
with O
C B
. I
elegans I
( O
e O
. O
g O
. O
, O
[ O
19 O
] O
, O
[ O
20 O
] O
) O
. O

There O
is O
similarity O
in O
other O
features O
( O
such O
as O
basic O
body O
plan O
and O
moulting O
) O
between O
C B
. I
elegans I
and O
parasitic O
nematodes O
, O
suggesting O
that O
some O
molecular O
pathways O
are O
relatively O
conserved O
[ O
8 O
] O
, O
[ O
21 O
] O
. O

Understanding O
the O
pathways O
linked O
to O
basic O
nematode B
biology O
and O
development O
could O
have O
important O
implications O
for O
finding O
new O
ways O
of O
disrupting O
these O
pathways O
and O
thus O
facilitate O
the O
identification O
of O
new O
drug O
targets O
. O

Despite O
the O
advances O
in O
genomic O
technologies O
[ O
7 O
] O
, O
[ O
22 O
] O
- O
[ O
29 O
] O
and O
the O
study O
of O
C B
. I
elegans I
, O
there O
is O
a O
paucity O
of O
information O
on O
the O
genomics O
of O
parasitic O
nematodes O
of O
animals O
, O
particularly O
in O
relation O
to O
development O
. O

Also O
considering O
the O
major O
socioeconomic O
impact O
of O
Ascaris B
and O
ascariasis O
in O
humans B
and O
pigs B
[ O
30 O
] O
- O
[ O
32 O
] O
, O
several O
characteristics O
, O
including O
the O
large O
size O
of O
the O
adult O
worm O
( O
providing O
the O
opportunity O
of O
investigating O
individual O
organ O
systems O
and O
tissues O
) O
, O
the O
ability O
to O
maintain O
Ascaris B
in O
the O
pig B
, O
store O
eggs O
and O
culture O
larvae O
in O
vitro O
for O
relatively O
long O
periods O
of O
time O
( O
months O
to O
years O
) O
[ O
32 O
] O
as O
well O
as O
the O
discovery O
that O
RNAi O
achieves O
" O
cross O
- O
species O
" O
gene O
silencing O
for O
a O
selected O
number O
of O
genes O
[ O
33 O
] O
, O
[ O
34 O
] O
and O
the O
imminent O
genome O
sequence O
( O
http O
: O

/ O
/ O
www O
. O
sanger O
. O
ac O
. O
uk O
/ O
Projects O
/ O
Helminths O
/ O
) O
all O
indicate O
that O
Ascaris B
could O
serve O
as O
a O
powerful O
model O
system O
for O
investigating O
reproductive O
and O
developmental O
processes O
in O
nematodes O
. O

In O
the O
present O
study O
, O
Ascaris O
from O
pigs B
was O
used O
to O
study O
molecules O
abundantly O
transcribed O
in O
the O
infective O
third O
- O
stage O
larva O
( O
L3 O
) O
. O

Following O
the O
oral O
ingestion O
of O
Ascaris B
eggs O
by O
the O
host O
( O
human B
or O
pig B
) O
, O
L3s O
are O
released O
and O
then O
invade O
/ O
penetrate O
predominantly O
the O
caecal O
wall O
[ O
35 O
] O
to O
then O
undergo O
hepato O
- O
pulmonary O
migration O
, O
after O
which O
ultimately O
the O
adult O
females O
and O
males O
establish O
and O
develop O
in O
the O
small O
intestine O
[ O
36 O
] O
, O
[ O
37 O
] O
. O

The O
molecular O
mechanisms O
linked O
to O
host O
invasion O
and O
parasite O
development O
are O
largely O
unknown O
. O

Here O
, O
we O
constructed O
an O
L3 O
- O
enriched O
cDNA O
library O
using O
the O
method O
of O
suppressive O
- O
subtractive O
hybridization O
( O
SSH O
) O
, O
explored O
transcription O
of O
a O
representative O
subset O
of O
molecules O
by O
microarray O
analysis O
and O
conducted O
bioinformatic O
analyses O
to O
characterize O
these O
molecules O
, O
map O
them O
to O
biochemical O
pathways O
and O
predict O
genetic O
interactions O
based O
on O
comparisons O
with O
C B
. I
elegans I
and O
/ O
or O
other O
organisms O
. O

Materials O
and O
Methods O

Production O
of O
Different O
Developmental O
Stages O
of O
Ascaris B

Experimental O
pigs B
( O
8 O
- O
12 O
weeks O
of O
age O
) O
were O
purchased O
from O
and O
maintained O
in O
the O
Experimental O
Animal O
Center O
of O
South O
China O
Agricultural O
University O
. O

These O
pigs B
were O
treated O
humanely B
, O
according O
to O
the O
Animal O
Ethics O
procedures O
and O
guidelines O
of O
the O
People B
' O
s O
Republic O
of O
China O
. O

Adult O
worms O
( O
males O
and O
females O
) O
of O
A B
. I
suum I
were O
collected O
from O
the O
small O
intestines O
of O
pigs B
from O
an O
abattoir O
in O
Shenzhen O
, O
China O
. O

Infective O
eggs O
and O
infective O
L3s O
of O
A B
. I
suum I
were O
produced O
according O
to O
the O
methods O
described O
previously O
[ O
38 O
] O
. O

In O
brief O
, O
eggs O
from O
the O
uteri O
of O
adult O
females O
of O
A B
. I
suum I
were O
collected O
and O
incubated O
at O
28 O
degrees O
C O
for O
28 O
days O
to O
allow O
them O
to O
develop O
to O
infective O
eggs O
( O
containing O
infective O
L3s O
) O
. O

To O
obtain O
pure O
infective O
L3s O
, O
7 O
. O
5 O
% O
v O
/ O
v O
sodium O
hypochlorite O
was O
used O
to O
treat O
the O
larvated O
eggs O
at O
37 O
degrees O
C O
overnight O
and O
then O
the O
eggs O
were O
shaken O
with O
glass O
- O
beads O
; O
then O
, O
the O
exsheathed O
L3s O
and O
shells O
were O
separated O
by O
density O
gradient O
centrifugation O
using O
lymphocyte O
separating O
medium O
( O
LSM O
) O
[ O
38 O
] O
. O

Following O
the O
experimental O
infection O
of O
helminth O
- O
free O
pigs B
with O
infective O
Ascaris B
eggs O
as O
described O
previously O
[ O
39 O
] O
, O
the O
L3s O
from O
livers O
and O
in O
lungs O
as O
well O
as O
L4s O
in O
intestines O
were O
isolated O
according O
to O
an O
established O
method O
[ O
40 O
] O
. O

All O
parasite O
materials O
were O
snap O
- O
frozen O
in O
liquid O
nitrogen O
prior O
to O
storage O
at O
- O
70 O
degrees O
C O
. O

Construction O
of O
the O
cDNA O
Library O
by O
Subtractive O
- O
Suppressive O
Hybridization O
( O
SSH O
) O

Total O
RNA O
was O
isolated O
from O
adult O
females O
and O
males O
, O
different O
larval O
stages O
or O
eggs O
of O
A B
. I
suum I
using O
TriPure O
reagent O
( O
Roche O
) O
as O
recommended O
by O
the O
manufacturer O
. O

Equal O
amounts O
of O
total O
RNA O
from O
each O
stage O
or O
sex O
were O
pooled O
. O

The O
mRNA O
was O
isolated O
using O
the O
Oligotex O
mRNA O
Kit O
( O
Qiagen O
) O
, O
following O
the O
manufacturer O
' O
s O
protocol O
. O

SSH O
was O
carried O
out O
using O
the O
PCR O
- O
Select O
cDNA O
Subtraction O
kit O
( O
Clontech O
) O
, O
according O
to O
the O
manufacturer O
' O
s O
protocol O
. O

In O
brief O
, O
cDNA O
synthesized O
from O
mRNAs O
from O
infective O
L3s O
was O
subtracted O
against O
cDNA O
synthesized O
from O
the O
pooled O
mRNA O
from O
all O
other O
stages O
included O
herein O
. O

The O
SSH O
library O
was O
constructed O
using O
infective O
L3s O
as O
the O
tester O
and O
pooled O
cDNAs O
from O
all O
other O
stages O
as O
the O
driver O
. O

The O
effectiveness O
of O
this O
subtraction O
process O
has O
already O
been O
demonstrated O
in O
previous O
studies O
[ O
41 O
] O
, O
[ O
42 O
] O
. O

The O
cDNA O
obtained O
following O
SSH O
was O
cloned O
into O
the O
pGEM O
- O
T O
Easy O
plasmid O
vector O
( O
Promega O
) O
and O
competent O
Escherichia B
coli I
( O
JM109 O
) O
transformed O
. O

Positive O
clones O
, O
picked O
randomly O
( O
based O
on O
blue O
/ O
white O
selection O
) O
, O
were O
grown O
overnight O
in O
Luria O
Bertani O
( O
LB O
) O
medium O
( O
shaking O
, O
37 O
degrees O
C O
) O
. O

Individual O
inserts O
were O
PCR O
- O
amplified O
using O
" O
nested O
primers O
" O
1 O
and O
2R O
from O
the O
Subtraction O
kit O
( O
Clontech O
) O
and O
examined O
by O
agarose O
electrophoresis O
. O

Preparation O
of O
Microarray O
Slides O

Clones O
( O
n O
= O
3075 O
) O
from O
the O
subtracted O
library O
were O
picked O
and O
cultured O
overnight O
in O
LB O
containing O
ampicillin O
( O
1000 O
IU O
/ O
ml O
) O
in O
sealed O
96 O
- O
well O
blocks O
. O

Five O
micro O
l O
of O
culture O
suspension O
from O
each O
well O
were O
transferred O
into O
individual O
wells O
thermocycling O
( O
96 O
- O
well O
) O
plates O
and O
the O
inserts O
PCR O
- O
amplified O
using O
primers O
1 O
and O
2R O
. O

Following O
a O
10 O
min O
denaturation O
step O
at O
94 O
degrees O
C O
, O
the O
amplification O
proceeded O
for O
25 O
cycles O
of O
10 O
s O
at O
94 O
degrees O
C O
, O
30 O
s O
at O
68 O
degrees O
C O
and O
1 O
. O
5 O
min O
at O
72 O
degrees O
C O
, O
with O
a O
final O
extension O
for O
5 O
min O
at O
72 O
degrees O
C O
. O

Products O
were O
resolved O
in O
agarose O
gels O
, O
ethanol O
precipitated O
, O
re O
- O
suspended O
in O
16 O
micro O
l O
of O
" O
spotting O
solution O
" O
( O
Shanghai O
BioStar O
Genechip O
, O
Inc O
) O
to O
a O
final O
concentration O
of O
~ O
500 O
ng O
per O
micro O
l O
, O
before O
being O
printed O
on O
to O
glass O
slides O
( O
in O
duplicate O
) O
using O
a O
robotic O
arrayer O
. O

Sixteen O
blanks O
( O
using O
spotting O
solution O
only O
) O
and O
the O
same O
number O
of O
negative O
( O
irrelevant O
cDNAs O
with O
no O
relationship O
to O
Ascaris B
) O
were O
also O
printed O
on O
to O
slides O
and O
served O
as O
negative O
controls O
; O
beta O
- O
actin O
of O
A B
. I
suum I
served O
as O
a O
positive O
control O
to O
assess O
the O
efficiency O
of O
labeling O
and O
hybridization O
. O

The O
slides O
were O
air O
- O
dried O
for O
2 O
h O
, O
and O
cDNA O
in O
the O
spots O
were O
cross O
- O
linked O
at O
254 O
mJ O
. O

The O
printed O
slides O
were O
stored O
at O
4 O
degrees O
C O
. O

Labeling O
of O
cDNA O
Probes O
with O
Fluorescent O
Dyes O
, O
and O
Microarray O
Analysis O

The O
cDNAs O
produced O
from O
total O
RNA O
from O
A B
. I
suum I
eggs O
, O
infective O
L3s O
, O
L3s O
isolated O
from O
pig B
liver O
or O
lung O
, O
fourth O
- O
stage O
larvae O
( O
L4s O
) O
, O
adult O
males O
or O
females O
[ O
as O
described O
in O
the O
section O
' O
Construction O
of O
the O
cDNA O
Library O
by O
Subtractive O
- O
Suppressive O
Hybridization O
( O
SSH O
) O
' O
] O
were O
labeled O
with O
cyanine O
dyes O
. O

Cy3 O
or O
Cy5 O
- O
dCTP O
was O
incorporated O
into O
cDNA O
produced O
from O
30 O
micro O
g O
of O
total O
RNA O
by O
direct O
labeling O
in O
a O
reverse O
transcription O
reaction O
using O
an O
oligo O
( O
dT O
) O
primer O
. O

Labeled O
cDNA O
was O
purified O
using O
DyeEx O
columns O
( O
Qiagen O
) O
. O

Microarray O
slides O
were O
incubated O
with O
a O
pre O
- O
hybridization O
solution O
[ O
5 O
x O
SSC O
, O
1 O
% O
bovine B
serum O
albumin O
( O
BSA O
) O
, O
0 O
. O
1 O
% O
sodium O
dodecyl O
- O
sulphate O
( O
SDS O
) O
] O
for O
6 O
h O
at O
42 O
degrees O
C O
. O

After O
pre O
- O
hybridization O
, O
the O
microarray O
slides O
were O
incubated O
with O
' O
pooled O
' O
Cy3 O
and O
Cy5 O
- O
labeled O
probes O
in O
hybridization O
solution O
( O
5 O
x O
SSC O
, O
1 O
% O
BSA O
, O
0 O
. O
1 O
% O
SDS O
) O
, O
in O
the O
dark O
at O
42 O
degrees O
C O
for O
18 O
h O
, O
and O
then O
washed O
in O
solution O
I O
( O
1 O
x O
SSC O
, O
0 O
. O
2 O
% O
SDS O
) O
for O
10 O
min O
, O
followed O
by O
solution O
II O
( O
0 O
. O
1 O
x O
SSC O
, O
0 O
. O
2 O
% O
SDS O
) O
for O
10 O
min O
at O
60 O
degrees O
C O
, O
according O
to O
the O
protocols O
provided O
by O
Shanghai O
BioStar O

Genechip O
, O
Inc O
. O

A O
" O
dye O
flip O
" O
was O
carried O
out O
to O
control O
for O
any O
bias O
in O
hybridization O
signal O
between O
the O
Cy O
- O
labeled O
cDNA O
probes O
( O
produced O
for O
two O
distinct O
mRNA O
populations O
) O
. O

The O
slides O
were O
dried O
and O
scanned O
( O
ScanArray O
4000 O
scanner O
) O
using O
image O
acquisition O
software O
( O
Shanghai O
BioStar O
Genechip O
Inc O
. O
) O
and O
a O
range O
of O
laser O
power O
and O
photo O
- O
multiplier O
tube O
intensities O
. O

The O
mean O
hybridization O
signal O
( O
derived O
from O
four O
replicates O
of O
the O
same O
array O
) O
were O
corrected O
for O
background O
, O
normalized O
[ O
43 O
] O
, O
log2 O
- O
transformed O
and O
then O
subjected O
to O
statistical O
analysis O
employing O
the O
students O
t O
- O
test O
in O
a O
spreadsheet O
( O
Excel O
, O
Microsoft O
, O
USA O
) O
. O

The O
microarray O
data O
were O
analysed O
for O
differential O
cDNA O
hybridization O
( O
> O
2 O
. O
0 O
- O
fold O
to O
114 O
. O
3 O
- O
fold O
) O
between O
L3 O
and O
each O
of O
the O
other O
stages O
( O
eggs O
, O
lung O
and O
liver O
L3s O
, O
L4 O
, O
adult O
female O
and O
adult O
male O
) O
. O

Verification O
of O
Differential O
Hybridization O
by O
Reverse O
Transcription O
- O
Coupled O
Polymerase O
Chain O
Reaction O
( O
RT O
- O
PCR O
) O
Analysis O

For O
a O
subset O
( O
n O
= O
17 O
) O
of O
representative O
ESTs O
( O
rESTs O
) O
, O
RT O
- O
PCR O
was O
used O
to O
verify O
the O
differential O
transcription O
recorded O
by O
microarray O
analysis O
. O

Double O
- O
stranded O
cDNA O
was O
synthesized O
from O
total O
RNA O
( O
separately O
) O
from O
each O
stage O
or O
sex O
of O
A B
. I
suum I
using O
reverse O
transcriptase O
( O
Superscript O
III O
, O
Invitrogen O
) O
. O

Briefly O
, O
5 O
micro O
g O
of O
total O
RNA O
were O
added O
to O
14 O
micro O
l O
of O
H2O O
and O
1 O
micro O
l O
of O
oligo O
d O
( O
T O
) O
n O
= O
12 O
- O
18 O
primer O
( O
0 O
. O
5 O
micro O
g O
/ O
micro O
l O
) O
, O
heated O
to O
70 O
degrees O
C O
for O
10 O
min O
and O
chilled O
on O
ice O
. O

First O
- O
and O
second O
- O
strand O
cDNAs O
were O
synthesized O
via O
the O
addition O
of O
4 O
micro O
l O
of O
first O
- O
strand O
cDNA O
buffer O
( O
250 O
mM O
Tris O
- O
HCl O
, O
pH O
8 O
. O
3 O
, O
375 O
mM O
KCl O
and O
15 O
mM O
MgCl2 O
) O
, O
2 O
micro O
l O
of O
0 O
. O
1 O
M O
dithiothreitol O
, O
and O
1 O
micro O
l O
of O
10 O
mM O
of O
each O
dNTP O
, O
followed O
by O
an O
incubation O
at O
25 O
degrees O
C O
( O
10 O
min O
) O
, O
42 O
degrees O
C O
( O
50 O
min O
) O
and O
70 O
degrees O
C O
( O
15 O
min O
) O
. O

One O
- O
tenth O
of O
each O
double O
- O
stranded O
cDNA O
produced O
was O
then O
used O
as O
a O
template O
in O
the O
PCR O
. O

The O
transcripts O
were O
amplified O
from O
individual O
cDNAs O
by O
PCR O
using O
oligonucleotide O
primers O
( O
sequences O
available O
upon O
request O
) O
designed O
to O
each O
EST O
. O

The O
PCR O
amplification O
of O
a O
portion O
( O
209 O
bp O
) O
of O
the O
beta O
- O
actin O
gene O
( O
accession O
no O
. O
BI594141 O
) O
using O
forward O
primer O
( O
5 O
' O
- O
CTCGAAACAAGAATACGATG O
- O
3 O
' O
) O
and O
reverse O
primer O
( O
5 O
' O
- O
ACATGTGCCGTTGTATGATG O
- O
3 O
' O
) O
, O
previously O
determined O
to O
be O
present O
in O
all O
developmental O
stages O
and O
both O
sexes O
of O
A B
. I
suum I
[ O
44 O
] O
, O
served O
as O
a O
positive O
control O
. O

Samples O
without O
template O
( O
no O
- O
DNA O
controls O
) O
were O
included O
in O
each O
PCR O
run O
. O

The O
following O
cycling O
conditions O
were O
employed O
: O
one O
cycle O
at O
94 O
degrees O
C O
( O
5 O
min O
) O
, O
94 O
degrees O
C O
( O
30 O
s O
) O
, O
60 O
degrees O
C O
( O
30 O
s O
) O
and O
72 O
degrees O
C O
( O
30 O
s O
) O
for O
30 O
cycles O
, O
followed O
by O
a O
final O
extension O
of O
70 O
degrees O
C O
( O
7 O
min O
) O
. O

Following O
the O
PCR O
, O
5 O
micro O
l O
of O
individual O
amplicons O
were O
resolved O
in O
ethidium O
bromide O
- O
stained O
agarose O
gels O
( O
2 O
% O
) O
and O
then O
photographed O
upon O
transillumination O
. O

The O
relative O
band O
intensities O
were O
analyzed O
using O
UVIsoft O
Image O
Acquisition O
and O
Analysis O
software O
( O
UVITEC O
) O
. O

The O
specificity O
and O
identity O
of O
individual O
amplicons O
were O
confirmed O
by O
direct O
sequencing O
using O
the O
same O
primers O
( O
separately O
) O
as O
employed O
for O
their O
amplification O
. O

Sequencing O
and O
Bioinformatics O
Analyses O

Clones O
from O
the O
SSH O
cDNA O
library O
with O
increased O
hybridization O
in O
microarray O
analysis O
to O
the O
infective O
L3 O
compared O
with O
other O
stages O
were O
sequenced O
using O
standard O
technology O
[ O
45 O
] O
. O

The O
nucleotide O
sequences O
have O
been O
deposited O
in O
the O
GenBank O
database O
under O
accession O
numbers O
ES290984 O
- O
ES291074 O
. O

Following O
the O
processing O
of O
the O
sequences O
( O
i O
. O
e O
. O
, O
removal O
of O
vector O
sequences O
, O
quality O
assurance O
and O
clustering O
) O
, O
contigs O
or O
singletons O
from O
individual O
clusters O
were O
subjected O
to O
BLASTx O
( O
NCBI O
: O
www O
. O
ncbi O
. O
nlm O
. O
nih O
. O
gov I
) O
and O
BLASTn O
( O
EMBL O
- O
EBI O
Parasite O
Genome O
Blast O
Server O
: O
www O
. O
ebi O
. O
ac O
. O
uk O
) O
analysis O
to O
identify O
putative O
homologues O
in O
C B
. I
elegans I
, O
other O
nematodes B
and O
other O
organisms O
( O
e O
- O
value O
of O
< O
= O
1e O
- O
05 O
) O
. O

Peptides O
inferred O
from O
ESTs O
were O
classified O
functionally O
using O
Interproscan O
( O
available O
at O
http O
: O
/ O
/ O
www O
. O
ebi O
. O
ac O
. O
uk O
/ O
InterProScan O
/ O
) O
employing O
the O
default O
search O
parameters O
. O

WormBase O
( O
www O
. O
wormbase O
. O
org O
) O
was O
interrogated O
extensively O
for O
relevant O
information O
on O
C B
. I
elegans I
homologues O
/ O
orthologues O
, O
including O
RNAi O
phenotypic O
, O
transcriptomic O
, O
proteomic O
and O
interactomic O
data O
. O

ESTs O
with O
homologues O
/ O
orthologues O
in O
C B
. I
elegans I
and O
other O
nematodes B
were O
also O
subjected O
to O
analysis O
employing O
the O
KEGG O
Orthology O
- O
Based O
Annotation O
System O
( O
KOBAS O
) O
( O
www O
. O
kobas O
. O
cbi O
. O
pku O
. O
edu O
. O
cn O
) O
, O
which O
predicts O
the O
biochemical O
pathways O
in O
which O
molecules O
are O
involved O
. O

The O
open O
reading O
frames O
( O
ORFs O
) O
inferred O
from O
selected O
ESTs O
with O
orthologues O
in O
C B
. I
elegans I
were O
also O
subjected O
to O
" O
secretome O
analysis O
" O
using O
the O
program O
SignalP O
v O
. O
2 O
. O
0 O
www O
. O
cbs O
. O
dtu O
. O
dk O
/ O
services O
/ O
SignalP O
/ O
) O
, O
employing O
both O
the O
neural O
network O
and O
hidden O
Markov O
models O
to O
predict O
signal O
peptides O
and O
/ O
or O
anchors O
[ O
46 O
] O
- O
[ O
48 O
] O
. O

Also O
, O
transmembrane O
domains O
were O
predicted O
using O
the O
program O
TMHMM O
( O
www O
. O
cbs O
. O
dtu O
. O
dk O
/ O
services O
/ O
TMHMM O
/ O
; O
[ O
49 O
] O
- O
[ O
51 O
] O
) O
, O
and O
subcellular O
localization O
inferred O
employing O
the O
program O
WoLF O
PSORT O
( O
http O
: O
/ O
/ O
wolfpsort O
. O
org O
/ O
; O
[ O
52 O
] O
) O
. O

The O
method O
established O
by O
Zhong O
and O
Sternberg O
[ O
53 O
] O
was O
used O
to O
predict O
the O
interactions O
for O
C B
. I
elegans I
orthologues O
of O
the O
L3 O
- O
enriched O
molecules O
from O
Ascaris B
. O

In O
brief O
, O
interaction O
, O
phenotypic O
, O
expression O
and O
gene O
ontology O
data O
from O
fruitfly O
, O
yeast B
, O
mouse B
and O
human B
were O
integrated O
using O
a O
na O
i O
ve O
Bayesian O
model O
to O
predict O
genetic O
interactions O
among O
C B
. I
elegans I
genes O
( O
[ O
45 O
] O
, O
[ O
53 O
] O
; O
Zhong O
and O
Sternberg O
, O
unpublished O
) O
. O

The O
predicted O
networks O
resulting O
from O
the O
analyses O
were O
saved O
in O
a O
graphic O
display O
file O
( O
gdf O
) O
format O
and O
examined O
using O
the O
graph O
exploration O
system O
available O
at O
http O
: O
/ O
/ O
graphexploration O
. O
cond O
. O
org O
/ O
. O

Images O
were O
labeled O
and O
saved O
in O
the O
joint O
photographic O
experts O
group O
( O
jpeg O
) O
format O
. O

Results O

To O
identify O
molecules O
transcribed O
abundantly O
in O
the O
L3 O
of O
A B
. I
suum I
, O
an O
enriched O
cDNA O
library O
was O
constructed O
by O
SSH O
. O

From O
a O
total O
of O
3075 O
clones O
from O
this O
library O
, O
2921 O
( O
95 O
% O
) O
were O
shown O
to O
contain O
an O
insert O
( O
which O
could O
be O
amplified O
by O
PCR O
) O
. O

From O
2671 O
( O
92 O
% O
) O
of O
these O
clones O
, O
amplicons O
representing O
single O
bands O
of O
~ O
400 O
to O
600 O
bp O
in O
size O
were O
produced O
. O

These O
latter O
amplicons O
were O
arrayed O
( O
in O
duplicate O
) O
on O
to O
slides O
and O
then O
hybridized O
with O
Cy3 O
- O
labeled O
L3 O
- O
cDNA O
or O
with O
Cy5 O
- O
labeled O
cDNA O
from O
eggs O
, O
liver O
/ O
lung O
L3s O
, O
L4s O
, O
adult O
female O
or O
adult O
male O
of O
Ascaris B
. O

Dye O
flip O
was O
conducted O
to O
verify O
the O
hybridization O
data O
. O

Of O
the O
2671 O
( O
duplicate O
) O
spots O
, O
1526 O
had O
a O
significant O
difference O
in O
hybridization O
between O
infective O
L3 O
cDNA O
and O
cDNAs O
from O
all O
other O
stages O
or O
sexes O
of O
A B
. I
suum I
, O
of O
which O
515 O
had O
a O
> O
2 O
. O
0 O
- O
fold O
increased O
hybridization O
for O
the O
L3 O
. O

In O
order O
to O
independently O
verify O
the O
hybridization O
results O
in O
the O
microarray O
, O
a O
PCR O
- O
based O
analysis O
of O
a O
selected O
subset O
( O
n O
= O
17 O
) O
clones O
was O
conducted O
using O
specific O
primer O
pairs O
. O

Having O
verified O
the O
specificity O
and O
identity O
of O
individual O
amplicons O
by O
sequencing O
, O
PCR O
results O
were O
reproducible O
( O
based O
on O
multiple O
runs O
on O
different O
days O
) O
and O
~ O
94 O
% O
( O
16 O
of O
17 O
) O
concordant O
with O
those O
of O
the O
microarray O
analysis O
( O
not O
shown O
) O
. O

There O
was O
complete O
concordance O
for O
representative O
clones O
associated O
with O
a O
differential O
signal O
of O
> O
= O
3 O
. O
0 O
- O
fold O
in O
the O
microarray O
. O

The O
clones O
linked O
to O
the O
515 O
spots O
representing O
increased O
transcription O
( O
> O
2 O
. O
0 O
- O
fold O
) O
in O
infective O
L3 O
compared O
with O
the O
other O
developmental O
stages O
or O
sexes O
included O
were O
subjected O
to O
sequencing O
. O

The O
498 O
sequences O
( O
length O
: O
550 O
+ O
/ O
- O
115 O
bp O
) O
determined O
were O
then O
subjected O
to O
detailed O
bioinformatic O
analyses O
. O

There O
were O
91 O
unique O
clusters O
( O
accession O
numbers O
ES290984 O
- O
ES291074 O
) O
, O
of O
which O
55 O
were O
singletons O
( O
sequences O
determined O
once O
) O
. O

Of O
56 O
molecules O
( O
61 O
. O
5 O
% O
) O
with O
significant O
similarity O
to O
sequences O
other O
than O
A B
. I
suum I
in O
the O
databases O
interrogated O
, O
50 O
( O
54 O
. O
9 O
% O
) O
had O
C B
. I
elegans I
or O
C B
. I
briggsae I
homologues O
, O
and O
six O
had O
similarity O
to O
ESTs O
already O
sequenced O
from O
ascaridoid B
and O
/ O
or O
other O
parasitic O
nematodes B
, O
and O
/ O
or O
other O
organisms O
( O
Table O
1 O
) O
. O

A O
significant O
proportion O
( O
38 O
. O
4 O
% O
) O
did O
not O
have O
any O
similarity O
in O
sequence O
to O
any O
organisms O
for O
which O
data O
are O
presently O
available O
. O

Comparative O
analysis O
specifically O
against O
A B
. I
suum I
EST O
data O
sets O
( O
n O
~ O
42 O
, O
000 O
) O
available O
in O
public O
databases O
confirmed O
independently O
that O
the O
majority O
of O
molecules O
( O
> O
60 O
% O
) O
were O
present O
exclusively O
in O
the O
infective O
L3 O
stage O
or O
were O
orphans O
. O

As O
gene O
ontology O
( O
GO O
) O
provides O
a O
hierarchy O
that O
unifies O
the O
descriptions O
of O
biological O
, O
cellular O
and O
molecular O
functions O
[ O
54 O
] O
, O
this O
approach O
was O
employed O
to O
predict O
the O
classification O
and O
gene O
function O
of O
molecules O
enriched O
in O
infective O
L3 O
of O
A B
. I
suum I
. O

A O
summary O
of O
the O
GO O
categories O
of O
these O
molecules O
is O
displayed O
in O
Fig O
. O

1 O
. O

Of O
the O
91 O
contigs O
, O
32 O
( O
35 O
% O
) O
could O
be O
functionally O
assigned O
to O
' O
biological O
process O
' O
( O
n O
= O
38 O
) O
, O
' O
cellular O
component O
' O
( O
n O
= O
17 O
) O
and O
' O
molecular O
function O
' O
( O
n O
= O
64 O
) O
. O

The O
most O
common O
subcategories O
were O
gluconeogenesis O
( O
13 O
% O
) O
and O
metabolic O
process O
( O
13 O
% O
) O
within O
' O
biological O
process O
' O
, O
extracellular O
region O
( O
24 O
% O
) O
within O
' O
cellular O
component O
' O
, O
and O
catalytic O
activity O
( O
11 O
% O
) O
and O
phosphoenolpyruvate O
carboxykinase O
activity O
( O
8 O
% O
) O
within O
' O
molecular O
function O
' O
( O
Table O
S1 O
) O
. O

A O
focused O
KOBAS O
analysis O
inferred O
the O
50 O
C B
. I
elegans I
orthologues O
/ O
homologues O
to O
be O
involved O
in O
apoptosis O
and O
insulin O
signaling O
( O
2 O
% O
) O
, O
ATP O
synthesis O
( O
2 O
% O
) O
, O
carbon O
metabolism O
( O
6 O
% O
) O
, O
fatty O
acid O
biosynthesis O
( O
2 O
% O
) O
, O
gap O
junction O
( O
2 O
% O
) O
, O
glucose O
metabolism O
( O
6 O
% O
) O
or O
porphyrin O
metabolism O
( O
2 O
% O
) O
, O
although O
34 O
( O
68 O
% O
) O
of O
them O
could O
not O
be O
mapped O
to O
a O
specific O
metabolic O
pathway O
( O
Table O
2 O
) O
. O

Of O
these O
50 O
molecules O
, O
small O
numbers O
were O
predicted O
to O
be O
secreted O
( O
10 O
% O
) O
, O
anchored O
( O
2 O
% O
) O
and O
/ O
or O
transmembrane O
( O
12 O
% O
) O
proteins O
( O
Table O
2 O
) O
. O

Functionally O
, O
17 O
( O
34 O
% O
) O
of O
the O
50 O
molecules O
were O
associated O
with O
( O
non O
- O
wild O
- O
type O
) O
RNAi O
phenotypes O
in O
C B
. I
elegans I
, O
the O
majority O
displaying O
embryonic O
lethality O
( O
Emb O
) O
( O
13 O
types O
; O
58 O
. O
8 O
% O
) O
, O
larval O
arrest O
( O
Lva O
) O
( O
23 O
. O
5 O
% O
) O
and O
larval O
lethality O
( O
Lvl O
) O
( O
47 O
% O
) O
( O
Table O
2 O
) O
. O

Extending O
this O
analysis O
, O
a O
relatively O
complex O
genetic O
interaction O
network O
was O
predicted O
for O
the O
17 O
C B
. I
elegans I
orthologues O
( O
i O
. O
e O
. O
, O
with O
non O
- O
wild O
- O
type O
RNAi O
phenotypes O
) O
( O
see O
Table O
S2 O
) O
. O

Statistically O
highly O
significant O
interactions O
were O
predicted O
for O
nine O
of O
the O
C B
. I
elegans I
genes O
; O
the O
top O
five O
interactors O
are O
displayed O
in O
Fig O
. O

2 O
. O

The O
gene O
ontology O
categories O
for O
eight O
selected O
C B
. I
elegans I
genes O
( O
F33D11 O
. O
10 O
, O
F55A12 O
. O
8 O
, O
kin O
- O
2 O
, O
mec O
- O
12 O
, O
mup O
- O
2 O
, O
pab O
- O
1 O
, O
rpl O
- O
22 O
and O
T21B10 O
. O
2 O
) O
included O
: O
embryonic O
development O
, O
egg O
hatching O
, O
larval O
development O
and O
/ O
or O
growth O
. O

The O
other O
categories O
included O
: O
positive O
regulation O
of O
growth O
rate O
( O
F55A12 O
. O
8 O
, O
kin O
- O
2 O
, O
mup O
- O
2 O
, O
pab O
- O
1 O
, O
rpl O
- O
22 O
and O
T21B10 O
. O
2 O
) O
and O
gamete O
generation O
and O
locomotory O
behaviour O
( O
kin O
- O
2 O
, O
mup O
- O
2 O
, O
pab O
- O
1 O
and O
F55A12 O
. O
8 O
, O
kin O
- O
2 O
, O
mup O
- O
2 O
, O
respectively O
) O
. O

The O
C B
. I
elegans I
homologue O
egl O
- O
3 O
was O
predicted O
to O
be O
involved O
in O
proteolysis O
( O
see O
www O
. O
wormbase O
. O
org O
) O
. O

All O
nine O
C B
. I
elegans I
orthologues O
were O
predicted O
to O
interact O
directly O
with O
a O
total O
of O
296 O
( O
range O
: O
5 O
- O
75 O
) O
other O
genes O
and O
, O
in O
particular O
, O
a O
direct O
genetic O
interaction O
was O
predicted O
between O
pab O
- O
1 O
and O
T21B10 O
. O
2 O
( O
Fig O
. O
2 O
) O
. O

The O
296 O
interactors O
were O
associated O
with O
embryonic O
and O
larval O
development O
( O
n O
= O
198 O
; O
66 O
. O
9 O
% O
) O
, O
information O
storage O
and O
processing O
( O
n O
= O
15 O
; O
5 O
. O
1 O
% O
) O
, O
cellular O
processes O
and O
signalling O
( O
n O
= O
45 O
; O
15 O
. O
2 O
% O
) O
and O
metabolism O
( O
n O
= O
18 O
; O
6 O
. O
1 O
% O
) O
; O
the O
precise O
function O
of O
some O
of O
the O
interactors O
( O
n O
= O
20 O
; O
6 O
. O
7 O
% O
) O
is O
presently O
unknown O
( O
Table O
S2 O
) O
. O

Discussion O

The O
present O
study O
investigated O
transcripts O
in O
infective O
L3s O
of O
A B
. I
suum I
using O
a O
genomic O
- O
bioinformatic O
platform O
. O

The O
focus O
was O
on O
comparisons O
with O
C B
. I
elegans I
homologues O
/ O
orthologues O
, O
because O
the O
entire O
genome O
sequence O
of O
this O
nematode B
is O
known O
[ O
9 O
] O
and O
because O
there O
is O
a O
wealth O
of O
information O
on O
the O
localization O
and O
functionality O
of O
its O
molecules O
( O
www O
. O
wormbase O
. O
org O
; O
http O
: O
/ O
/ O
elegans B
. O
bcgsc O
. O
bc O
. O
ca O
/ O
knockout O
. O
shtml O
) O
. O

The O
functions O
of O
most O
genes O
in O
C B
. I
elegans I
have O
been O
assessed O
using O
RNAi O
( O
e O
. O
g O
. O
, O
[ O
14 O
] O
, O
[ O
15 O
] O
, O
[ O
17 O
] O
, O
[ O
55 O
] O
, O
[ O
56 O
] O
) O
in O
the O
hermaphroditic O
stage O
, O
whereas O
there O
is O
a O
paucity O
of O
functional O
information O
available O
for O
Ascaris B
and O
other O
parasitic O
nematodes O
of O
animals O
[ O
57 O
] O
, O
[ O
58 O
] O
. O

Following O
the O
microarray O
analysis O
of O
> O
2500 O
ESTs O
from O
the O
SSH O
library O
, O
498 O
cDNAs O
inferred O
to O
be O
enriched O
in O
the O
L3 O
, O
based O
on O
hybridization O
signal O
, O
were O
sequenced O
and O
subjected O
to O
comprehensive O
in O
silico O
analyses O
. O

Of O
the O
91 O
clusters O
of O
molecules O
categorized O
, O
50 O
( O
54 O
. O
9 O
% O
) O
had O
C B
. I
elegans I
homologues O
/ O
orthologues O
with O
loss O
- O
of O
- O
function O
phenotypes O
could O
be O
mapped O
to O
key O
pathways O
. O

The O
statistically O
significant O
genetic O
interactions O
predicted O
for O
9 O
of O
the O
50 O
C B
. I
elegans I
orthologues O
[ O
namely O
egl O
- O
3 O
, O
F33D11 O
. O
10 O
, O
F55A12 O
. O
8 O
, O
kin O
- O
2 O
, O
mec O
- O
12 O
, O
mup O
- O
2 O
, O
pab O
- O
1 O
, O
rpl O
- O
22 O
and O
T21B10 O
. O
2 O
( O
= O
enol O
- O
1 O
) O
] O
and O
the O
interaction O
network O
included O
genes O
encoding O
kinases O
, O
alpha O
- O
tubulins O
, O
enolases O
, O
troponin O
and O
other O
named O
and O
unnamed O
proteins O
. O

Eight O
of O
these O
molecules O
( O
enol O
- O
1 O
, O
pab O
- O
1 O
, O
F33D11 O
. O
10 O
, O
rpl O
- O
22 O
, O
F55A12 O
. O
8 O
mec O
- O
12 O
, O
mup O
- O
2 O
and O
kin O
- O
2 O
) O
have O
known O
or O
predicted O
roles O
in O
embryonic O
and O
larval O
growth O
and O
development O
, O
gamete O
generation O
, O
locomotory O
behaviour O
or O
other O
biological O
processes O
in O
C B
. I
elegans I
( O
see O
www O
. O
wormbase O
. O
org O
) O
. O

The O
enolase O
encoded O
by O
enol O
- O
1 O
is O
predicted O
to O
play O
a O
role O
in O
glycolysis O
, O
gluconeogenesis O
, O
phenylalanine O
, O
tyrosine O
and O
tryptophan O
biosynthesis O
( O
cf O
. O
[ O
59 O
] O
) O
. O

Since O
glucose O
is O
the O
main O
source O
for O
ATP O
production O
, O
the O
alteration O
in O
these O
key O
glycolytic O
enzymes O
may O
lead O
to O
cellular O
dysfunction O
, O
such O
as O
impaired O
ion O
- O
motive O
ATPase O
required O
to O
maintain O
potential O
gradients O
, O
operate O
pumps O
and O
maintain O
membrane O
lipid O
asymmetry O
[ O
60 O
] O
. O

Bioinformatic O
analysis O
for O
transmembrane O
helices O
( O
TMHMM O
) O
and O
peptide O
signal O
sequences O
( O
SignalP O
) O
predicted O
ENOL O
- O
1 O
to O
be O
a O
non O
- O
secreted O
protein O
localized O
to O
the O
cytoplasm O
( O
cf O
. O
Table O
2 O
) O
. O

Nonetheless O
, O
enolases O
are O
often O
detected O
in O
the O
excretory O
/ O
secretory O
( O
ES O
) O
products O
of O
parasitic O
helminths O
, O
including O
adult O
A B
. I
suum I
[ O
61 O
] O
, O
and O
appear O
to O
play O
a O
role O
in O
the O
triggering O
of O
nitric O
oxide O
production O
by O
host O
cells O
. O

The O
enol O
- O
1 O
orthologue O
of O
C B
. I
elegans I
has O
been O
predicted O
to O
interact O
specifically O
with O
the O
polyadenylate O
binding O
protein O
gene O
, O
pab O
- O
1 O
, O
inferred O
to O
be O
involved O
in O
coordinated O
gene O
transcription O
and O
expression O
during O
normal O
larval O
development O
[ O
16 O
] O
. O

Poly O
( O
A O
) O
- O
binding O
proteins O
( O
PABPs O
) O
are O
recognized O
to O
be O
central O
to O
the O
regulation O
of O
mRNA O
translation O
and O
stability O
[ O
62 O
] O
. O

Present O
evidence O
suggests O
that O
the O
expression O
of O
PAB O
- O
1 O
is O
regulated O
by O
an O
oligo O
- O
pyrimidine O
tract O
in O
response O
to O
cell O
growth O
and O
relates O
to O
coordinated O
growth O
regulation O
in O
C B
. I
elegans I
[ O
62 O
] O
. O

Furthermore O
, O
gene O
silencing O
of O
pab O
- O
1 O
and O
its O
selected O
interactors O
( O
see O
Fig O
. O
2 O
) O
leads O
to O
embryonic O
lethal O
( O
Emb O
) O
, O
slow O
growth O
( O
Slo O
) O
and O
sterile O
progeny O
( O
Stp O
) O
phenotypes O
( O
see O
www O
. O
wormbase O
. O
org O
) O
. O

Another O
gene O
( O
F33D11 O
. O
10 O
; O
EST O
code O
4F10 O
; O
see O
Table O
2 O
) O
which O
encodes O
an O
ATP O
- O
dependent O
RNA O
helicase O
and O
is O
associated O
with O
embryonic O
lethal O
( O
Emb O
) O
and O
larval O
lethal O
( O
Lvl O
) O
RNAi O
phenotypes O
, O
was O
shown O
to O
be O
highly O
transcribed O
in O
infective O
L3s O
of O
A B
. I
suum I
. O

Helicases O
are O
involved O
in O
a O
variety O
of O
RNA O
metabolic O
processes O
, O
including O
translation O
initiation O
, O
pre O
- O
mRNA O
splicing O
, O
pre O
- O
rRNA O
processing O
, O
rRNA O
maturation O
and O
RNA O
degradation O
[ O
63 O
] O
, O
and O
are O
crucial O
for O
life O
cycle O
progression O
, O
sex O
determination O
and O
early O
embryogenesis O
in O
C B
. I
elegans I
[ O
60 O
] O
. O

The O
high O
transcription O
levels O
of O
a O
homologue O
/ O
orthologue O
in O
the O
L3 O
of O
A B
. I
suum I
might O
suggest O
a O
similar O
role O
in O
this O
ascaridoid O
. O

Similarly O
, O
the O
coordination O
of O
the O
expression O
of O
a O
large O
number O
of O
genes O
is O
required O
for O
normal O
growth O
and O
cell O
proliferation O
during O
larval O
development O
. O

The O
high O
transcription O
level O
for O
the O
ribosomal O
protein O
gene O
homologue O
rpl O
- O
22 O
( O
large O
subunit O
family O
member O
; O
EST O
code O
26G12 O
, O
see O
Table O
2 O
) O
in O
the O
infective O
L3 O
of O
A B
. I
suum I
compared O
with O
other O
developmental O
stages O
is O
likely O
to O
reflect O
the O
substantial O
rate O
of O
cell O
growth O
in O
this O
stage O
[ O
64 O
] O
. O

The O
gene O
( O
F55A12 O
. O
8 O
, O
EST O
code O
4G11 O
; O
see O
Table O
2 O
) O
encoding O
an O
acetyl O
- O
transferase O
with O
a O
putative O
ATPase O
domain O
, O
shown O
to O
be O
enriched O
in O
the O
L3 O
of O
A B
. I
suum I
, O
was O
predicted O
to O
interact O
with O
75 O
other O
genes O
all O
involved O
in O
energy O
production O
and O
/ O
or O
RNA O
processing O
( O
see O
Table O
S2 O
) O
. O

Several O
molecules O
involved O
in O
ATP O
synthesis O
and O
mitochondrial O
pathways O
( O
e O
. O
g O
. O
, O
cytochrome O
oxidase O
c O
subunits O
1 O
, O
2 O
and O
3 O
, O
ADP O
/ O
ATP O
translocases O
, O
NADH O
dehydrogenases O
, O
ATPases O
and O
ATP O
synthetases O
) O
have O
been O
reported O
previously O
to O
be O
highly O
represented O
in O
the O
L3 O
stage O
of O
Anisakis B
simplex I
[ O
65 O
] O
, O
thus O
supporting O
the O
proposal O
that O
substantial O
energy O
is O
required O
for O
larval O
development O
as O
well O
as O
the O
transition O
from O
the O
free O
- O
living O
to O
the O
parasitic O
stage O
and O
the O
invasion O
of O
the O
host O
. O

There O
is O
also O
likely O
to O
be O
a O
substantial O
energy O
requirement O
for O
muscle O
contraction O
linked O
to O
larval O
motility O
in O
A B
. I
suum I
, O
as O
the O
L3s O
penetrate O
the O
caecal O
wall O
in O
the O
porcine B
host O
, O
before O
undergoing O
hepato O
- O
pulmonary O
migration O
[ O
35 O
] O
. O

Accordingly O
, O
genes O
encoding O
a O
specialized O
tubulin O
expressed O
in O
mechanoreceptors O
( O
mec O
- O
12 O
, O
EST O
code O
13E09 O
) O
and O
a O
troponin O
( O
mup O
- O
2 O
, O
EST O
code O
01G03 O
; O
see O
Table O
2 O
) O
, O
both O
predicted O
to O
interact O
with O
a O
total O
number O
of O
32 O
tubulin O
- O
and O
myosin O
- O
encoding O
genes O
, O
also O
supported O
a O
link O
to O
extensive O
muscle O
contraction O
and O
motility O
in O
A B
. I
suum I
L3s O
. O

Also O
, O
neuroactive O
peptides O
are O
required O
to O
regulate O
the O
responsiveness O
of O
nematode O
larvae O
to O
mechanical O
stimuli O
[ O
66 O
] O
. O

A O
homologue O
encoded O
by O
egl O
- O
3 O
was O
shown O
to O
be O
highly O
transcribed O
in O
the O
L3 O
of O
A B
. I
suum I
; O
EGL O
- O
3 O
is O
predicted O
to O
be O
a O
pro O
- O
hormone O
convertase O
involved O
in O
the O
maturation O
of O
neuropeptides O
and O
could O
be O
associated O
with O
mechano O
- O
sensory O
responses O
and O
touch O
sensitivity O
linked O
to O
the O
host O
invasion O
. O

A O
regulatory O
subunit O
of O
a O
cAMP O
- O
dependent O
protein O
kinase O
( O
kin O
- O
2 O
, O
EST O
code O
22H01 O
; O
see O
Table O
2 O
) O
was O
predicted O
to O
interact O
with O
72 O
other O
genes O
all O
involved O
in O
diverse O
cellular O
processes O
, O
such O
as O
nuclear O
trafficking O
, O
and O
DNA O
replication O
and O
repair O
( O
see O
Table O
S2 O
) O
. O

Based O
on O
gene O
ontology O
terms O
, O
kin O
- O
2 O
is O
implicated O
in O
gamete O
generation O
, O
growth O
, O
larval O
development O
, O
post O
- O
embryonic O
body O
morphogenesis O
, O
signal O
transduction O
and O
/ O
or O
protein O
amino O
acid O
phosphorylation O
( O
see O
Table O
S2 O
) O
. O

Gene O
silencing O
of O
kin O
- O
2 O
in O
C B
. I
elegans I
leads O
to O
phenotypes O
, O
such O
as O
larval O
lethal O
( O
Lvl O
) O
, O
larval O
arrest O
( O
Lva O
) O
, O
body O
morphology O
defect O
( O
Bmd O
) O
, O
dumpy O
( O
Dpy O
) O
, O
uncoordinated O
( O
Unc O
) O
and O
sterile O
progeny O
( O
Stp O
) O
( O
www O
. O
wormbase O
. O
org O
) O
, O
suggesting O
that O
its O
homologue O
in O
A B
. I
suum I
is O
central O
to O
larval O
maturation O
. O

The O
KOBAS O
analysis O
predicted O
the O
protein O
KIN O
- O
2 O
to O
be O
involved O
in O
the O
insulin O
- O
signaling O
pathway O
, O
previously O
implicated O
in O
controlling O
the O
exit O
from O
dauer O
in O
C B
. I
elegans I
and O
the O
activation O
of O
L3s O
of O
the O
canine B
hookworm O
, O
Ancylostoma B
caninum I
, O
following O
exsheathment O
[ O
67 O
] O
. O

In O
a O
recent O
study O
, O
Brand O
and O
Hawdon O
[ O
68 O
] O
were O
able O
to O
inhibit O
( O
with O
a O
phosphoinositide O
- O
3 O
- O
OH O
- O
kinase O
inhibitor O
) O
the O
activation O
of O
infective O
L3s O
of O
both O
of O
the O
hookworms B
Ancylostoma B
caninum I
and O
Ancylostoma B
ceylanicum I
via O
the O
insulin O
signaling O
pathway O
, O
thus O
lending O
some O
credence O
to O
the O
hypothesis O
that O
this O
pathway O
plays O
an O
critical O
role O
in O
regulating O
the O
transition O
from O
the O
free O
- O
living O
to O
the O
parasitic O
stage O
[ O
68 O
] O
. O

Recently O
, O
it O
has O
been O
proposed O
that O
transcriptional O
and O
feeding O
responses O
to O
serum O
- O
stimulation O
in O
Ancylostoma B
caninum I
are O
regulated O
by O
parallel O
systems O
, O
with O
the O
insulin O
signaling O
pathway O
playing O
a O
significant O
role O
in O
the O
' O
resumption O
of O
feeding O
' O
in O
activated O
larvae O
[ O
69 O
] O
. O

Protein O
kinases O
are O
also O
likely O
to O
be O
involved O
in O
pathways O
linked O
to O
sexual O
maturation O
in O
developing O
larvae O
. O

As O
already O
proposed O
for O
adult O
stages O
of O
H B
. I
contortus I
[ O
45 O
] O
, O
the O
protein O
kinase O
gene O
cdk O
- O
1 O
is O
predicted O
to O
play O
a O
pivotal O
role O
in O
the O
germline O
, O
oogenesis O
and O
spermiogenesis O
pathways O
of O
this O
parasitic O
nematode O
. O

Other O
protein O
kinases O
, O
such O
as O
PEPCK O
, O
and O
phosphatases O
, O
were O
shown O
herein O
to O
be O
transcribed O
at O
high O
levels O
in O
the O
L3 O
stage O
compared O
with O
other O
developmental O
stages O
of O
A B
. I
suum I
( O
see O
Table O
2 O
) O
, O
which O
is O
in O
accordance O
to O
findings O
reported O
recently O
for O
Anisakis B
simplex I
[ O
65 O
] O
. O

Due O
to O
their O
major O
regulatory O
effects O
in O
eukaryotic O
signaling O
events O
and O
regulatory O
and O
sensory O
functions O
, O
protein O
kinases O
have O
been O
considered O
interesting O
targets O
for O
anti O
- O
parasitic O
drugs O
[ O
70 O
] O
. O

In O
conclusion O
, O
this O
study O
has O
given O
some O
interesting O
insights O
into O
early O
molecular O
processes O
in O
the O
L3 O
of O
A B
. I
suum I
. O

Approximately O
60 O
% O
of O
the O
transcripts O
enriched O
in O
the O
L3 O
stage O
of O
A B
. I
suum I
have O
homologues O
/ O
orthologues O
in O
C B
. I
elegans I
. O

The O
bioinformatic O
analyses O
of O
selected O
molecules O
suggest O
that O
a O
complex O
genetic O
network O
regulates O
or O
controls O
larval O
growth O
and O
development O
in O
A B
. I
suum I
L3s O
, O
and O
some O
of O
these O
might O
be O
involved O
in O
or O
regulate O
the O
switch O
from O
the O
free O
- O
living O
to O
the O
parasitic O
stage O
. O

Some O
caution O
is O
warranted O
in O
drawing O
conclusions O
regarding O
molecular O
mechanisms O
regulating O
the O
transition O
to O
parasitism O
in O
parasitic O
nematodes O
from O
information O
on O
C B
. I
elegans I
, O
as O
latter O
is O
a O
free O
- O
living O
nematode O
. O

Also O
, O
while O
the O
method O
of O
data O
integration O
is O
essential O
for O
the O
reliable O
prediction O
of O
genetic O
interactions O
, O
it O
might O
limit O
the O
capacity O
of O
the O
approach O
somewhat O
to O
infer O
nematode O
- O
specific O
interactions O
. O

As O
additional O
datasets O
of O
genes O
and O
gene O
functions O
become O
available O
for O
various O
parasitic O
nematodes O
, O
more O
informed O
inferences O
can O
be O
made O
regarding O
the O
functions O
of O
nematode O
- O
specific O
genes O
, O
particularly O
those O
involved O
in O
the O
transition O
to O
parasitism O
. O

The O
imminent O
genome O
sequence O
of O
A B
. I
suum I
( O
http O
: O
/ O
/ O
www O
. O
sanger O
. O
ac O
. O
uk O
/ O
Projects O
/ O
Helminths O
/ O
) O
should O
all O
assist O
in O
this O
endeavour O
. O

Also O
, O
functional O
analysis O
of O
selected O
molecules O
representing O
selected O
ESTs O
identified O
herein O
, O
utilizing O
gene O
silencing O
approaches O
established O
recently O
[ O
33 O
] O
, O
[ O
34 O
] O
, O
could O
provide O
some O
insights O
into O
developmental O
processes O
in O
Ascaris B
and O
related O
ascaridoid B
nematodes O
and O
provide O
avenues O
for O
the O
development O
of O
novel O
approaches O
for O
their O
control O
. O

Supporting O
Information O

Minocycline O
attenuates O
lipopolysaccharide O
( O
LPS O
) O
- O
induced O
neuroinflammation O
, O
sickness O
behavior O
, O
and O
anhedonia O

Abstract O

Background O

Activation O
of O
the O
peripheral O
innate O
immune O
system O
stimulates O
the O
secretion O
of O
CNS O
cytokines O
that O
modulate O
the O
behavioral O
symptoms O
of O
sickness O
. O

Excessive O
production O
of O
cytokines O
by O
microglia O
, O
however O
, O
may O
cause O
long O
- O
lasting O
behavioral O
and O
cognitive O
complications O
. O

The O
purpose O
of O
this O
study O
was O
to O
determine O
if O
minocycline O
, O
an O
anti O
- O
inflammatory O
agent O
and O
purported O
microglial O
inhibitor O
, O
attenuates O
lipopolysaccharide O
( O
LPS O
) O
- O
induced O
neuroinflammation O
, O
sickness O
behavior O
, O
and O
anhedonia O
. O

Methods O

In O
the O
first O
set O
of O
experiments O
the O
effect O
of O
minocycline O
pretreatment O
on O
LPS O
- O
induced O
microglia O
activation O
was O
assessed O
in O
BV O
- O
2 O
microglia O
cell O
cultures O
. O

In O
the O
second O
study O
, O
adult O
( O
3 O
- O
6 O
m O
) O
BALB O
/ O
c O
mice B
received O
an O
intraperitoneal O
( O
i O
. O
p O
. O
) O
injection O
of O
vehicle O
or O
minocycline O
( O
50 O
mg O
/ O
kg O
) O
for O
three O
consecutive O
days O
. O

On O
the O
third O
day O
, O
mice B
were O
also O
injected O
( O
i O
. O
p O
. O
) O
with O
saline O
or O
Escherichia B
coli I
LPS O
( O
0 O
. O
33 O
mg O
/ O
kg O
) O
and O
behavior O
( O
i O
. O
e O
. O
, O
sickness O
and O
anhedonia O
) O
and O
markers O
of O
neuroinflammation O
( O
i O
. O
e O
. O
, O
microglia O
activation O
and O
inflammatory O
cytokines O
) O
were O
determined O
. O

In O
the O
final O
study O
, O
adult O
and O
aged O
BALB O
/ O
c O
mice B
were O
treated O
with O
the O
same O
minocycline O
and O
LPS O
injection O
regimen O
and O
markers O
of O
neuroinflammation O
were O
determined O
. O

All O
data O
were O
analyzed O
using O
Statistical O
Analysis O
Systems O
General O
Linear O
Model O
procedures O
and O
were O
subjected O
to O
one O
- O
, O
two O
- O
, O
or O
three O
- O
way O
ANOVA O
to O
determine O
significant O
main O
effects O
and O
interactions O
. O

Results O

Minocycline O
blocked O
LPS O
- O
stimulated O
inflammatory O
cytokine O
secretion O
in O
the O
BV O
- O
2 O
microglia O
- O
derived O
cell O
line O
and O
reduced O
LPS O
- O
induced O
Toll O
- O
like O
- O
receptor O
- O
2 O
( O
TLR2 O
) O
surface O
expression O
on O
brain O
microglia O
. O

Moreover O
, O
minocycline O
facilitated O
the O
recovery O
from O
sickness O
behavior O
( O
i O
. O
e O
. O
, O
anorexia O
, O
weight O
loss O
, O
and O
social O
withdrawal O
) O
and O
prevented O
anhedonia O
in O
adult O
mice B
challenged O
with O
LPS O
. O

Furthermore O
, O
the O
minocycline O
associated O
recovery O
from O
LPS O
- O
induced O
sickness O
behavior O
was O
paralleled O
by O
reduced O
mRNA O
levels O
of O
Interleukin O
( O
IL O
) O
- O
1 O
beta O
, O
IL O
- O
6 O
, O
and O
indoleamine O
2 O
, O
3 O
dioxygenase O
( O
IDO O
) O
in O
the O
cortex O
and O
hippocampus O
. O

Finally O
, O
in O
aged O
mice B
, O
where O
exaggerated O
neuroinflammation O
was O
elicited O
by O
LPS O
, O
minocycline O
pretreatment O
was O
still O
effective O
in O
markedly O
reducing O
mRNA O
levels O
of O
IL O
- O
1 O
beta O
, O
TLR2 O
and O
IDO O
in O
the O
hippocampus O
. O

Conclusion O

These O
data O
indicate O
that O
minocycline O
mitigates O
neuroinflammation O
in O
the O
adult O
and O
aged O
brain O
and O
modulates O
the O
cytokine O
- O
associated O
changes O
in O
motivation O
and O
behavior O
. O

Background O

The O
bi O
- O
directional O
communication O
between O
the O
immune O
system O
and O
the O
central O
nervous O
system O
( O
CNS O
) O
is O
necessary O
for O
mounting O
the O
appropriate O
immunological O
, O
physiological O
, O
and O
behavioral O
responses O
to O
immune O
stimulation O
[ O
1 O
] O
. O

CNS O
innate O
immune O
cells O
including O
microglia O
and O
macrophages O
play O
integral O
roles O
in O
receiving O
and O
propagating O
inflammatory O
signals O
that O
are O
initiated O
at O
the O
periphery O
. O

Activation O
of O
peripheral O
innate O
immune O
cells O
elicits O
the O
secretion O
of O
inflammatory O
cytokines O
, O
including O
interleukin O
( O
IL O
) O
- O
1 O
, O
IL O
- O
6 O
, O
and O
tumor O
necrosis O
factor O
- O
alpha O
( O
TNF O
alpha O
. O
) O
, O
that O
use O
neural O
[ O
2 O
, O
3 O
] O
, O
humoral O
[ O
4 O
] O
and O
blood O
brain O
barrier O
pathways O
[ O
5 O
] O
to O
relay O
this O
signal O
to O
the O
CNS O
. O

This O
inflammatory O
signal O
, O
in O
turn O
, O
induces O
CNS O
macrophages O
and O
microglia O
to O
produce O
the O
same O
cytokines O
[ O
6 O
] O
, O
which O
target O
neuronal O
substrates O
and O
elicit O
a O
sickness O
behavior O
syndrome O
that O
is O
normally O
adaptive O
and O
beneficial O
to O
the O
host O
[ O
1 O
] O
. O

An O
amplified O
or O
excessive O
inflammatory O
cytokine O
response O
in O
the O
brain O
, O
however O
, O
is O
associated O
with O
a O
myriad O
of O
complications O
including O
cognitive O
dysfunction O
[ O
7 O
- O
10 O
] O
, O
prolonged O
sickness O
behavior O
[ O
11 O
- O
14 O
] O
, O
and O
depressive O
- O
like O
behavior O
[ O
15 O
] O
. O

Microglia O
are O
primarily O
involved O
in O
immune O
surveillance O
[ O
16 O
, O
17 O
] O
, O
but O
when O
activated O
have O
macrophage O
- O
like O
capabilities O
including O
phagocytosis O
, O
inflammatory O
cytokine O
production O
, O
and O
antigen O
presentation O
[ O
18 O
] O
. O

Normally O
these O
neuroinflammatory O
changes O
are O
transient O
with O
microglia O
returning O
to O
a O
resting O
state O
as O
the O
immune O
stimulus O
is O
resolved O
. O

Aging O
or O
neurological O
disease O
, O
however O
, O
may O
provide O
a O
brain O
environment O
where O
microglia O
are O
more O
" O
reactive O
or O
primed O
" O
to O
a O
peripheral O
immune O
challenge O
[ O
19 O
] O
. O

Recent O
findings O
indicate O
that O
several O
markers O
of O
glial O
activation O
such O
as O
major O
histocompatibility O
complex O
( O
MHC O
) O
class O
II O
, O
complement O
receptors O
, O
and O
scavenger O
receptors O
are O
increased O
in O
brain O
during O
normal O
aging O
[ O
13 O
, O
20 O
- O
26 O
] O
. O

Furthermore O
, O
we O
and O
others O
have O
reported O
that O
a O
biological O
consequence O
of O
this O
reactive O
glial O
profile O
is O
an O
exaggerated O
neuroinflammatory O
response O
to O
innate O
immune O
challenge O
[ O
9 O
, O
10 O
, O
12 O
- O
14 O
, O
27 O
, O
28 O
] O
. O

Active O
microglia O
and O
CNS O
macrophages O
also O
contribute O
to O
the O
production O
of O
oxidative O
and O
neuroactive O
mediators O
that O
may O
influence O
behavior O
. O

For O
instance O
, O
inflammatory O
cytokines O
in O
the O
CNS O
upregulate O
the O
enzyme O
IDO O
[ O
29 O
, O
30 O
] O
, O
which O
metabolizes O
tryptophan O
( O
TRP O
) O
into O
L O
- O
kynurenine O
( O
KYN O
) O
[ O
31 O
] O
. O

TRP O
degradation O
to O
KYN O
can O
reduce O
TRP O
levels O
that O
are O
required O
for O
serotonin O
synthesis O
[ O
32 O
] O
and O
can O
lead O
to O
the O
production O
of O
neuroactive O
mediators O
including O
3 O
- O
hydroxykynurenine O
( O
3HK O
) O
and O
quinolinic O
acid O
( O
QUIN O
) O
[ O
31 O
] O
. O

High O
levels O
of O
3HK O
and O
QUIN O
induce O
neuronal O
damage O
through O
oxidative O
stress O
[ O
33 O
] O
and O
over O
stimulation O
of O
N O
- O
methyl O
- O
D O
- O
aspartate O
( O
NMDA O
) O
receptors O
[ O
34 O
, O
35 O
] O
. O

A O
recent O
study O
indicates O
that O
while O
several O
cell O
types O
in O
the O
CNS O
express O
IDO O
, O
only O
microglia O
maintain O
all O
the O
enzymes O
required O
to O
produce O
3HK O
and O
QUIN O
[ O
36 O
] O
. O

Because O
IDO O
mediated O
TRP O
degradation O
impacts O
both O
serotonergic O
and O
glutamatergic O
pathways O
, O
this O
may O
be O
an O
important O
mechanism O
underlying O
mood O
and O
behavior O
complications O
concomitant O
with O
inflammation O
[ O
37 O
- O
39 O
] O
. O

Because O
activated O
microglia O
are O
suspected O
to O
cause O
or O
exacerbate O
several O
neurodegenerative O
diseases O
, O
pharmacological O
strategies O
to O
suppress O
microglial O
activity O
are O
being O
explored O
as O
therapies O
. O

Minocycline O
is O
a O
tetracycline O
derived O
antibiotic O
that O
has O
anti O
- O
inflammatory O
properties O
in O
the O
CNS O
that O
are O
separate O
from O
its O
antimicrobial O
action O
[ O
40 O
] O
. O

Minocycline O
readily O
crosses O
the O
blood O
brain O
barrier O
and O
attenuates O
inflammation O
associated O
with O
microglial O
activation O
. O

For O
example O
, O
minocycline O
blocks O
the O
deleterious O
effects O
of O
neuroinflammation O
on O
neurogenesis O
, O
long O
- O
term O
potentiation O
, O
and O
neuronal O
survival O
[ O
41 O
- O
43 O
] O
. O

The O
mechanism O
of O
action O
is O
unclear O
, O
but O
recent O
studies O
indicate O
that O
minocycline O
abrogates O
MAPkinase O
and O
NF O
kappa O
B O
dependent O
signaling O
pathways O
in O
primary O
microglia O
and O
microglia O
cell O
cultures O
[ O
44 O
] O
. O

Moreover O
, O
in O
the O
brain O
of O
rats B
, O
minocycline O
abrogates O
microglial O
expression O
of O
CD11b O
and O
MHC O
II O
through O
a O
protein O
kinase O
- O
c O
dependent O
mechanism O
[ O
45 O
] O
. O

This O
is O
relevant O
because O
minocycline O
attenuates O
neuroinflammation O
in O
several O
rodent B
models O
of O
disease O
including O
Amyotrophic O
Lateral O
Sclerosis O
[ O
46 O
] O
, O
Experimental O
Autoimmune O
Encephalomyelitis O
( O
EAE O
) O
[ O
45 O
] O
and O
MPTP O
- O
induced O
Parkinson O
' O
s O
disease O
[ O
47 O
] O
. O

However O
, O
the O
extent O
to O
which O
minocycline O
facilitates O
the O
recovery O
from O
cytokine O
- O
mediated O
sickness O
behavior O
is O
unknown O
. O

The O
present O
study O
investigated O
the O
degree O
to O
which O
minocycline O
- O
an O
anti O
- O
inflammatory O
agent O
and O
purported O
microglial O
inhibitor O
- O
reduced O
LPS O
- O
induced O
neuroinflammation O
and O
sickness O
behavior O
. O

We O
show O
that O
minocycline O
blocked O
LPS O
- O
stimulated O
inflammatory O
cytokine O
secretion O
in O
the O
BV O
- O
2 O
microglia O
- O
derived O
cell O
line O
and O
reduced O
LPS O
- O
induced O
Toll O
- O
like O
- O
receptor O
- O
2 O
( O
TLR2 O
) O
surface O
expression O
on O
brain O
microglia O
. O

Moreover O
, O
our O
data O
show O
that O
minocycline O
pretreatment O
attenuated O
LPS O
- O
induced O
weight O
loss O
, O
social O
withdrawal O
, O
and O
anhedonia O
in O
adult O
mice B
. O

The O
attenuation O
of O
sickness O
behavior O
was O
paralleled O
with O
minocycline O
dependent O
decrease O
in O
markers O
of O
neuroinflammation O
( O
IL O
- O
1 O
beta O
, O
TLR2 O
, O
and O
IDO O
) O
in O
adult O
and O
aged O
mice B
. O

These O
findings O
support O
our O
hypothesis O
that O
the O
ability O
to O
mitigate O
cytokine O
expression O
in O
the O
brain O
during O
systemic O
inflammatory O
events O
may O
be O
useful O
in O
preventing O
cognitive O
and O
behavioral O
deficits O
. O

Methods O

Animals O

Male O
BALB O
/ O
c O
mice B
, O
adults O
( O
3 O
month O
old O
) O
and O
juvenile O
( O
3 O
- O
4 O
week O
old O
) O
were O
purchased O
from O
Harlan O
( O
Indianapolis O
, O
IN O
) O
. O

For O
age O
comparisons O
, O
male O
BALB O
/ O
c O
mice B
( O
3 O
- O
4 O
and O
20 O
- O
22 O
month O
old O
) O
were O
purchased O
from O
the O
National O
Institute O
on O
Aging O
specific O
pathogen O
free O
colony O
. O

Upon O
arrival O
, O
mice B
were O
individually O
housed O
in O
polypropylene O
cages O
and O
maintained O
at O
21 O
degrees O
C O
under O
a O
12 O
h O
light O
: O
12 O
h O
dark O
cycle O
with O
ad O
libitum O
access O
to O
water O
and O
rodent B
chow O
. O

At O
the O
end O
of O
each O
study O
, O
mice B
were O
examined O
postmortem O
for O
gross O
signs O
of O
disease O
( O
e O
. O
g O
. O
, O
splenomeglia O
or O
tumors O
) O
. O

Data O
from O
mice B
determined O
to O
be O
unhealthy O
were O
excluded O
from O
analysis O
( O
< O
5 O
% O
) O
. O

All O
procedures O
were O
in O
accordance O
with O
the O
National O
Institute O
of O
Health O
Guidelines O
for O
the O
Care O
and O
Use O
of O
Laboratory O
Animals O
and O
were O
approved O
by O
The O
Ohio O
State O
University O
Institutional O
Laboratory O
Animal O
Care O
and O
Use O
Committee O
. O

Cell O
culture O

BV O
- O
2 O
microglia O
cell O
lines O
were O
cultured O
in O
growth O
medium O
( O
DMEM O
supplemented O
with O
10 O
% O
FBS O
, O
sodium O
bicarbonate O
3 O
. O
7 O
g O
/ O
l O
, O
200 O
mM O
glutamine O
, O
100 O
U O
/ O
ml O
penicillin O
G O
, O
100 O
mu O
g O
/ O
ml O
streptomycin O
, O
0 O
. O
25 O
mu O
g O
/ O
ml O
fungizone O
) O
as O
previously O
described O
[ O
12 O
] O
. O

Cultures O
were O
maintained O
at O
37 O
degrees O
C O
with O
95 O
% O
humidity O
and O
5 O
% O
CO2 O
and O
growth O
medium O
was O
replenished O
every O
third O
day O
until O
confluence O
. O

Cultures O
were O
washed O
twice O
and O
supplemented O
with O
warm O
growth O
medium O
containing O
experimental O
treatments O
. O

Cell O
viability O
was O
measured O
by O
the O
MTS O
cell O
proliferation O
assay O
according O
to O
the O
manufacturer O
' O
s O
instructions O
( O
Promega O
, O
Madison O
, O
WI O
) O
. O

CNS O
macrophage O
/ O
microglia O
isolation O

CNS O
macrophages O
and O
microglia O
were O
collected O
from O
whole O
brain O
homogenates O
as O
described O
previously O
[ O
48 O
] O
, O
but O
with O
several O
modifications O
. O

Mice B
were O
euthanized O
by O
CO2 O
asphyxiation O
and O
whole O
brains O
were O
collected O
. O

Brains O
were O
homogenized O
in O
Hank O
' O
s O
balanced O
salt O
solution O
( O
HBSS O
) O
pH O
7 O
. O
4 O
. O

Brain O
homogenates O
were O
passed O
through O
a O
70 O
mu O
m O
nylon O
cell O
strainer O
and O
centrifuged O
at O
400 O
x O
g O
for O
10 O
min O
. O

Supernatants O
were O
removed O
and O
cell O
pellets O
were O
re O
- O
suspended O
in O
70 O
% O
isotonic O
Percoll O
( O
GE O
- O
healthcare O
, O
Uppsala O
, O
Sweden O
) O
at O
room O
temperature O
. O

A O
discontinuous O
Percoll O
density O
gradient O
was O
set O
up O
as O
follows O
: O
70 O
% O
, O
35 O
% O
, O
and O
0 O
% O
isotonic O
Percoll O
. O

This O
suspension O
was O
centrifuged O
for O
30 O
minutes O
at O
400 O
x O
g O
. O

A O
mixed O
population O
of O
CNS O
macrophages O
and O
microglia O
was O
collected O
from O
the O
interphase O
between O
the O
70 O
% O
and O
35 O
% O
Percoll O
layers O
. O

Cells O
were O
washed O
and O
then O
re O
- O
suspended O
in O
sterile O
HBSS O
. O

The O
number O
of O
viable O
cells O
was O
determined O
using O
a O
hemacytometer O
and O
0 O
. O
2 O
% O
trypan O
blue O
staining O
. O

Flow O
cytometry O

Flow O
cytometric O
analysis O
of O
microglial O
cell O
surface O
markers O
was O
performed O
as O
described O
previously O
, O
but O
with O
a O
few O
modifications O
[ O
48 O
] O
. O

In O
brief O
, O
Fc O
receptors O
on O
macrophages O
and O
microglia O
were O
blocked O
with O
anti O
- O
CD16 O
/ O
CD32 O
antibody O
( O
eBiosciences O
, O
CA O
) O
. O

Next O
, O
cells O
were O
incubated O
with O
either O
Panel O
- O
1 O
( O
anti O
- O
CD11b O
APC O
, O
anti O
- O
CD45 O
FITC O
, O
and O
anti O
- O
MHC O
II O
PE O
from O
eBiosciences O
, O
CA O
) O
or O
Panel O
- O
2 O
antibodies O
( O
anti O
- O
CD11b O
APC O
, O
anti O
- O
CD45 O
FITC O
, O
and O
anti O
- O
TLR2 O
PE O
from O
eBiosciences O
, O
CA O
) O
. O

Expression O
of O
these O
surface O
receptors O
was O
determined O
by O
flow O
cytometry O
using O
a O
Becton O
- O
Dickinson O
FACSCaliber O
four O
color O
Cytometer O
. O

Thirty O
thousand O
events O
were O
collected O
and O
microglia O
were O
differentiated O
from O
macrophages O
based O
on O
the O
levels O
of O
CD11b O
and O
CD45 O
surface O
expression O
. O

Microglia O
stain O
CD11b O
+ O
/ O
CD45low O
and O
macrophages O
stain O
CD11b O
+ O
/ O
CD45high O
[ O
48 O
, O
49 O
] O
. O

Flow O
data O
were O
analyzed O
using O
FlowJo O
software O
( O
Tree O
Star O
, O
San O
Carlos O
, O
CA O
) O
. O

Behavior O
tests O

Social O
exploratory O
behavior O

To O
assess O
the O
motivation O
to O
engage O
in O
social O
exploratory O
behavior O
, O
a O
novel O
juvenile O
conspecific O
was O
introduced O
into O
the O
test O
subject O
' O
s O
home O
cage O
for O
a O
10 O
- O
min O
period O
. O

Behavior O
was O
video O
taped O
and O
the O
cumulative O
amount O
of O
time O
the O
subject O
engaged O
in O
social O
investigation O
was O
determined O
from O
the O
video O
records O
by O
a O
trained O
observer O
who O
was O
blind O
to O
the O
experimental O
treatments O
. O

Baseline O
social O
behavior O
was O
measured O
at O
time O
0 O
for O
all O
experimental O
treatments O
. O

Social O
behavior O
was O
determined O
as O
the O
amount O
of O
time O
that O
the O
experimental O
subject O
spent O
investigating O
( O
e O
. O
g O
. O
, O
anogenital O
sniffing O
, O
trailing O
) O
the O
juvenile O
. O

Results O
are O
expressed O
as O
percent O
decrease O
in O
time O
engaged O
in O
social O
behavior O
compared O
to O
respective O
baseline O
measures O
. O

Sucrose O
preference O

To O
assess O
sucrose O
preference O
, O
mice B
were O
provided O
two O
solutions O
, O
water O
or O
water O
supplemented O
with O
2 O
% O
sucrose O
, O
in O
50 O
ml O
conical O
tubes O
with O
stoppers O
fitted O
with O
ball O
- O
type O
sipper O
tubes O
. O

Prior O
to O
testing O
conditions O
, O
all O
mice B
were O
acclimated O
to O
the O
two O
bottle O
test O
choice O
. O

All O
mice B
drank O
both O
the O
water O
and O
the O
2 O
% O
sucrose O
solution O
, O
but O
preferred O
drinking O
the O
sucrose O
over O
the O
water O
( O
data O
not O
shown O
) O
. O

On O
the O
day O
of O
testing O
, O
mice B
were O
fluid O
and O
food O
deprived O
for O
2 O
h O
prior O
to O
testing O
[ O
50 O
] O
. O

At O
the O
start O
of O
the O
dark O
phase O
of O
the O
photoperiod O
, O
drinking O
water O
and O
the O
2 O
% O
sucrose O
solution O
were O
placed O
in O
the O
home O
cage O
overnight O
( O
15 O
h O
) O
. O

At O
the O
end O
of O
each O
testing O
period O
the O
fluid O
content O
of O
the O
conical O
tubes O
was O
measured O
and O
sucrose O
preference O
was O
determined O
using O
the O
equation O
: O
Sucrose O
intake O
/ O
Total O
fluid O
intake O
( O
water O
+ O
sucrose O
intake O
) O
x O
100 O
[ O
51 O
] O
. O

Plasma O
cytokine O
measurement O

IL O
- O
6 O
and O
IL O
- O
1 O
beta O
were O
measured O
in O
the O
plasma O
as O
previously O
described O
[ O
52 O
] O
. O

In O
brief O
, O
mice B
were O
euthanized O
by O
CO2 O
asphyxiation O
and O
blood O
was O
collected O
by O
cardiac O
puncture O
into O
EDTA O
coated O
syringes O
. O

Samples O
were O
centrifuged O
( O
6000 O
x O
g O
for O
15 O
min O
at O
4 O
degrees O
C O
) O
and O
plasma O
was O
collected O
and O
stored O
frozen O
( O
- O
80 O
degrees O
C O
) O
until O
assaying O
. O

Plasma O
samples O
were O
assayed O
for O
IL O
- O
6 O
using O
a O
customized O
ELISA O
that O
we O
have O
described O
in O
detail O
[ O
52 O
] O
and O
for O
IL O
- O
1 O
beta O
using O
a O
commercial O
ELISA O
kit O
( O
R O
& O
D O
Systems O
, O
Minneapolis O
, O
MN O
) O
. O

Assays O
were O
sensitive O
to O
8 O
pg O
/ O
ml O
of O
IL O
- O
6 O
and O
1 O
. O
5 O
pg O
/ O
ml O
of O
IL O
- O
1 O
beta O
, O
and O
inter O
- O
and O
intra O
- O
assay O
coefficients O
of O
variation O
were O
less O
than O
10 O
% O
. O

Real O
time O
PCR O

Total O
RNA O
was O
isolated O
from O
brain O
using O
the O
Tri O
Reagent O
protocol O
( O
Sigma O
, O
St O
. O
Louis O
, O
MO O
) O
. O

RNA O
samples O
were O
subjected O
to O
a O
DNase O
I O
digestion O
procedure O
and O
then O
reverse O
transcribed O
to O
cDNA O
using O
a O
RT O
RETROscript O
kit O
( O
Ambion O
, O
Austin O
, O
TX O
) O
. O

Quantitative O
real O
time O
PCR O
was O
performed O
using O
the O
Applied O
Biosystems O
( O
Foster O
, O
CA O
) O
Assay O
- O
on O
Demand O
Gene O
Expression O
protocol O
as O
previously O
described O
[ O
13 O
] O
. O

In O
brief O
, O
cDNA O
was O
amplified O
by O
real O
time O
PCR O
where O
a O
target O
cDNA O
( O
IL O
- O
1 O
beta O
, O
IL O
- O
6 O
, O
MHC O
II O
, O
TLR2 O
, O
or O
IDO O
) O
and O
a O
reference O
cDNA O
( O
glyceraldehyde O
- O
3 O
- O
phosphate O
dehydrogenase O
) O
were O
amplified O
simultaneously O
using O
an O
oligonucleotide O
probe O
with O
a O
5 O
' O
fluorescent O
reporter O
dye O
( O
6 O
- O
FAM O
) O
and O
a O
3 O
' O
quencher O
dye O
( O
NFQ O
) O
. O

Fluorescence O
was O
determined O
on O
an O
ABI O
PRISM O
7300 O
- O
sequence O
detection O
system O
( O
Applied O
Biosystems O
, O
CA O
) O
. O

Data O
were O
analyzed O
using O
the O
comparative O
threshold O
cycle O
( O
Ct O
) O
method O
and O
results O
are O
expressed O
as O
fold O
difference O
. O

Experimental O
protocols O

For O
the O
cell O
culture O
studies O
, O
minocycline O
was O
prepared O
in O
dimethyl O
sulfoxide O
( O
DMSO O
) O
and O
BV O
- O
2 O
cells O
were O
washed O
and O
replenished O
with O
growth O
mediumsupplemented O
with O
0 O
, O
25 O
, O
50 O
, O
100 O
, O
200 O
, O
or O
400 O
mu O
g O
/ O
ml O
minocycline O
. O

After O
30 O
min O
, O
LPS O
at O
10 O
ng O
/ O
ml O
was O
added O
to O
the O
culture O
medium O
. O

Supernatants O
were O
collected O
4 O
h O
later O
and O
IL O
- O
6 O
and O
IL O
- O
1 O
beta O
concentrations O
were O
determined O
by O
ELISA O
. O

Total O
proteins O
were O
determined O
from O
cell O
culture O
homogenates O
by O
the O
Bio O
- O
Rad O
Dc O
protein O
assay O
according O
to O
the O
manufacturer O
' O
s O
instructions O
( O
Bio O
- O
Rad O
Lboratories O
, O
Hercules O
, O
CA O
) O
. O

Each O
treatment O
was O
replicated O
a O
minimum O
of O
four O
times O
. O

Cell O
viability O
was O
confirmed O
by O
the O
MTS O
cell O
proliferation O
assay O
according O
to O
the O
manufacturer O
' O
s O
instructions O
( O
Promega O
, O
Madison O
, O
WI O
) O
. O

For O
all O
mouse B
studies O
, O
minocycline O
( O
Sigma O
, O
St O
. O
Louis O
, O
MO O
) O
was O
dissolved O
in O
sterile O
water O
and O
sonicated O
to O
ensure O
complete O
solubilization O
. O

In O
the O
first O
mouse B
study O
, O
adult O
male O
BALB O
/ O
c O
mice B
received O
an O
intraperitoneal O
( O
i O
. O
p O
. O
) O
injection O
of O
vehicle O
or O
minocycline O
( O
50 O
mg O
/ O
kg O
) O
for O
three O
consecutive O
days O
. O

On O
the O
3rd O
day O
, O
mice B
were O
also O
injected O
i O
. O
p O
. O
with O
saline O
or O
Escherichia B
coli I
LPS O
( O
0 O
. O
33 O
mg O
/ O
kg O
; O
serotype O
0127 O
: O
B8 O
, O
Sigma O
, O
St O
. O
Louis O
, O
MO O
) O
and O
were O
euthanized O
by O
CO2 O
asphyxiation O
24 O
h O
later O
( O
n O
= O
6 O
) O
. O

The O
LPS O
dosage O
was O
selected O
because O
it O
elicits O
a O
proinflammatory O
cytokine O
response O
in O
the O
brain O
resulting O
in O
mild O
transient O
sickness O
behavior O
in O
adult O
mice B
[ O
13 O
, O
53 O
] O
. O

Macrophage O
/ O
microglial O
cells O
were O
isolated O
from O
whole O
brain O
homogenates O
and O
TLR2 O
and O
MHC O
II O
surface O
expression O
were O
determined O
by O
flow O
cytometry O
. O

The O
minocycline O
injection O
regimen O
and O
dosage O
was O
selected O
because O
a O
repeated O
pretreatment O
course O
with O
minocycline O
is O
necessary O
to O
attenuate O
neuroinflammation O
[ O
41 O
- O
43 O
, O
45 O
] O
. O

In O
the O
second O
study O
, O
adult O
male O
BALB O
/ O
c O
mice B
received O
an O
i O
. O
p O
. O
injection O
with O
vehicle O
or O
minocycline O
for O
three O
consecutive O
days O
. O

On O
the O
third O
day O
, O
motivation O
to O
engage O
in O
social O
behavior O
was O
determined O
immediately O
before O
i O
. O
p O
. O
injection O
of O
saline O
or O
LPS O
( O
0 O
. O
33 O
mg O
/ O
kg O
) O
and O
again O
2 O
, O
4 O
, O
8 O
, O
12 O
, O
and O
24 O
h O
later O
( O
n O
= O
8 O
) O
. O

Body O
weight O
and O
food O
intake O
were O
measured O
at O
each O
time O
point O
over O
the O
24 O
h O
period O
. O

In O
a O
related O
, O
but O
separate O
study O
, O
adult O
mice B
were O
treated O
with O
minocycline O
and O
LPS O
as O
described O
and O
anhedonia O
was O
assessed O
24 O
- O
39 O
h O
following O
i O
. O
p O
. O
injection O
of O
saline O
or O
LPS O
( O
0 O
. O
33 O
mg O
/ O
kg O
) O
( O
n O
= O
15 O
) O
. O

Body O
weight O
, O
food O
intake O
, O
water O
intake O
, O
and O
sucrose O
intake O
were O
determined O
over O
the O
testing O
period O
. O

In O
the O
third O
study O
, O
adult O
BALB O
/ O
c O
mice B
were O
treated O
with O
minocycline O
and O
then O
LPS O
as O
described O
. O

Mice B
were O
euthanized O
by O
CO2 O
asphyxiation O
4 O
later O
. O

Brains O
were O
removed O
and O
dissected O
to O
collect O
different O
brain O
regions O
. O

Brain O
regions O
were O
stored O
at O
- O
20 O
degrees O
C O
in O
RNAlater O
( O
Qiagen O
, O
CA O
) O
. O

Total O
RNA O
was O
isolated O
from O
brain O
samples O
and O
assayed O
using O
quantitative O
PCR O
( O
n O
= O
8 O
) O
. O

Plasma O
was O
also O
collected O
and O
stored O
( O
- O
80 O
degrees O
C O
) O
until O
assaying O
. O

In O
a O
final O
study O
, O
adult O
( O
3 O
- O
4 O
month O
old O
) O
or O
aged O
( O
20 O
- O
22 O
month O
old O
) O
male O
BALB O
/ O
c O
mice B
were O
treated O
with O
minocycline O
and O
LPS O
as O
described O
and O
euthanized O
4 O
h O
later O
. O

Brains O
were O
dissected O
to O
collect O
different O
brain O
regions O
and O
were O
stored O
at O
- O
20 O
degrees O
C O
in O
RNAlater O
( O
Qiagen O
, O
CA O
) O
. O

Total O
RNA O
was O
isolated O
from O
the O
hippocampus O
and O
assayed O
using O
quantitative O
PCR O
( O
n O
= O
8 O
) O
. O

Statistical O
analysis O

All O
data O
were O
analyzed O
using O
Statistical O
Analysis O
Systems O
( O
SAS O
) O
General O
Linear O
Model O
procedures O
. O

Data O
were O
subjected O
to O
one O
, O
two O
- O
( O
Mino O
x O
LPS O
, O
Age O
x O
LPS O
, O
Mino O
x O
Age O
) O
or O
three O
- O
way O
( O
Mino O
x O
LPS O
x O
Time O
, O
Mino O
x O
LPS O
x O
Age O
) O
ANOVA O
to O
determine O
significant O
main O
effects O
and O
interactions O
between O
main O
factors O
. O

When O
appropriate O
, O
differences O
between O
treatment O
means O
were O
evaluated O
by O
an O
F O
- O
protected O
t O
- O
test O
using O
the O
Least O
- O
Significant O
Difference O
procedure O
of O
SAS O
. O

All O
data O
are O
expressed O
as O
treatment O
means O
+ O
/ O
- O
standard O
error O
of O
the O
mean O
( O
SEM O
) O
. O

Results O

Minocycline O
attenuates O
LPS O
- O
induced O
cytokine O
production O
in O
BV O
- O
2 O
microglia O

Minocycline O
is O
a O
tetracycline O
- O
type O
antibiotic O
that O
has O
anti O
- O
inflammatory O
properties O
in O
the O
CNS O
[ O
41 O
- O
43 O
, O
45 O
] O
. O

To O
determine O
the O
degree O
to O
which O
minocycline O
suppresses O
microglia O
activation O
, O
BV O
- O
2 O
microglia O
- O
derived O
cell O
lines O
were O
used O
. O

In O
the O
first O
experiment O
, O
BV O
- O
2 O
cells O
were O
treated O
with O
LPS O
and O
IL O
- O
6 O
production O
was O
determined O
4 O
h O
later O
. O

Fig O
. O

1A O
shows O
that O
LPS O
increased O
IL O
- O
6 O
production O
in O
a O
dose O
dependent O
manner O
F O
( O
5 O
, O
23 O
) O
= O
101 O
, O
P O
< O
0 O
. O
001 O
) O
. O

In O
the O
second O
experiment O
, O
BV O
- O
2 O
cells O
were O
incubated O
with O
DMSO O
or O
minocycline O
and O
then O
stimulated O
with O
LPS O
. O

Minocycline O
reduced O
LPS O
- O
induced O
IL O
- O
6 O
secretion O
in O
a O
dose O
dependent O
manner O
( O
Mino O
x O
LPS O
interaction O
, O
F O
( O
4 O
, O
23 O
) O
= O
16 O
. O
87 O
, O
P O
< O
0 O
. O
001 O
, O
Fig O
. O
1B O
) O
. O

Minocycline O
pretreatment O
had O
a O
similar O
anti O
- O
inflammatory O
effect O
on O
LPS O
- O
stimulated O
IL O
- O
1 O
beta O
secretion O
( O
Fig O
. O
1C O
) O
. O

In O
a O
third O
experiment O
, O
minocycline O
suppressed O
LPS O
- O
induced O
MHC O
II O
, O
TLR2 O
, O
IL O
- O
1 O
beta O
, O
and O
IL O
- O
6 O
mRNA O
levels O
( O
P O
< O
0 O
. O
05 O
, O
for O
each O
, O
Fig O
. O
1D O
) O
. O

The O
MTS O
assay O
verified O
that O
neither O
cell O
survival O
nor O
proliferation O
was O
affected O
by O
the O
experimental O
treatments O
( O
data O
not O
shown O
) O
. O

LPS O
- O
induced O
TLR2 O
surface O
expression O
on O
microglia O
is O
reduced O
by O
minocycline O

Because O
minocycline O
attenuated O
LPS O
- O
induced O
cytokine O
secretion O
and O
TLR2 O
mRNA O
expression O
in O
BV O
- O
2 O
cells O
we O
next O
sought O
to O
determine O
if O
minocycline O
suppresses O
markers O
of O
microglial O
activation O
in O
the O
brain O
of O
mice B
. O

Mice B
were O
injected O
i O
. O
p O
. O
with O
vehicle O
or O
minocycline O
for O
3 O
consecutive O
days O
then O
challenged O
with O
saline O
or O
LPS O
i O
. O
p O
. O

Markers O
of O
activation O
, O
TLR2 O
and O
MHC O
II O
, O
were O
determined O
on O
microglia O
collected O
24 O
h O
later O
. O

The O
representative O
bivariate O
density O
plot O
in O
Fig O
. O

2A O
shows O
that O
there O
were O
two O
populations O
of O
CD11b O
/ O
CD45 O
positive O
cells O
and O
that O
more O
cells O
stained O
CD11b O
+ O
/ O
CD45low O
( O
microglia O
) O
than O
CD11b O
+ O
/ O
CD45high O
( O
CNS O
macrophages O
) O
. O

ANOVA O
revealed O
that O
LPS O
injection O
increased O
TLR2 O
surface O
expression O
on O
microglia O
( O
F O
( O
1 O
, O
20 O
) O
= O
17 O
. O
6 O
, O
P O
< O
0 O
. O
004 O
, O
Fig O
. O
2B O
& O
D O
) O
, O
but O
this O
induction O
was O
abrogated O
by O
minocycline O
pretreatment O
( O
Tendency O
for O
Mino O
x O
LPS O
interaction O
, O
F O
( O
1 O
, O
20 O
) O
= O
2 O
. O
66 O
, O
P O
= O
0 O
. O
10 O
, O
Fig O
. O
2C O
& O
D O
) O
. O

It O
is O
important O
to O
note O
that O
because O
minocycline O
and O
saline O
controls O
did O
not O
differ O
in O
their O
TLR2 O
expression O
, O
these O
data O
were O
grouped O
together O
as O
the O
Control O
group O
( O
Fig O
. O
2B O
& O
C O
) O
. O

In O
addition O
, O
neither O
minocycline O
nor O
LPS O
treatment O
had O
a O
significant O
main O
effect O
on O
MHC O
class O
II O
surface O
expression O
on O
microglia O
( O
data O
not O
shown O
) O
. O

These O
data O
indicate O
that O
minocycline O
attenuated O
LPS O
- O
induced O
TLR2 O
expression O
on O
microglia O
. O

Minocycline O
facilitates O
the O
recovery O
from O
LPS O
- O
induced O
sickness O
behavior O

CNS O
macrophages O
and O
microglia O
produce O
inflammatory O
cytokines O
and O
secondary O
messengers O
that O
modulate O
behavioral O
responses O
. O

Therefore O
, O
we O
next O
investigated O
if O
minocycline O
reduced O
the O
sickness O
response O
associated O
with O
peripheral O
LPS O
injection O
. O

In O
this O
experiment O
, O
adult O
mice B
were O
treated O
with O
minocycline O
and O
LPS O
as O
described O
. O

Social O
exploratory O
behavior O
was O
measured O
before O
i O
. O
p O
. O

LPS O
injection O
and O
again O
2 O
, O
4 O
, O
8 O
, O
and O
24 O
h O
later O
. O

Fig O
. O

3A O
shows O
that O
LPS O
injection O
caused O
a O
reduction O
in O
social O
exploratory O
behavior O
( O
F O
( O
1 O
, O
57 O
) O
= O
218 O
, O
P O
< O
0 O
. O
001 O
) O
that O
was O
time O
dependent O
( O
F O
( O
4 O
, O
57 O
) O
= O
66 O
. O
5 O
, O
P O
< O
0 O
. O
001 O
) O
. O

Moreover O
, O
the O
LPS O
- O
associated O
reduction O
in O
social O
exploration O
was O
attenuated O
by O
minocycline O
( O
Mino O
x O
LPS O
interaction O
, O
F O
( O
1 O
, O
57 O
) O
= O
7 O
. O
5 O
, O
P O
< O
0 O
. O
007 O
) O
. O

For O
example O
, O
at O
8 O
h O
post O
LPS O
, O
social O
exploration O
was O
reduced O
by O
35 O
% O
in O
minocycline O
pretreated O
mice B
given O
LPS O
compared O
to O
a O
67 O
% O
reduction O
in O
vehicle O
pretreated O
mice B
given O
LPS O
( O
P O
< O
0 O
. O
001 O
) O
. O

While O
minocycline O
administration O
alone O
reduced O
food O
intake O
and O
body O
weight O
in O
control O
mice B
( O
P O
< O
0 O
. O
05 O
, O
for O
each O
) O
, O
it O
also O
protected O
against O
LPS O
- O
associated O
anorexia O
( O
Mino O
x O
LPS O
interaction O
, O
F O
( O
1 O
, O
60 O
) O
= O
70 O
. O
0 O
, O
P O
< O
0 O
. O
001 O
, O
Fig O
. O
3B O
) O
and O
weight O
loss O
( O
Mino O
x O
LPS O
interaction O
, O
F O
( O
1 O
, O
60 O
) O
= O
29 O
. O
7 O
, O
P O
< O
0 O
. O
001 O
, O
Fig O
. O
3C O
) O
. O

Because O
sickness O
can O
also O
be O
associated O
with O
longer O
lasting O
changes O
in O
motivation O
[ O
38 O
] O
, O
we O
next O
sought O
to O
determine O
if O
minocycline O
abrogated O
LPS O
- O
induced O
anhedonia O
[ O
54 O
, O
55 O
] O
. O

In O
this O
experiment O
, O
mice B
were O
subjected O
to O
the O
same O
minocycline O
injection O
regimen O
and O
LPS O
challenge O
as O
above O
and O
sucrose O
preference O
was O
assessed O
24 O
- O
39 O
h O
post O
LPS O
injection O
. O

By O
24 O
h O
post O
LPS O
injection O
, O
food O
and O
water O
intake O
returned O
to O
baseline O
and O
LPS O
treated O
mice B
still O
exhibited O
a O
marked O
reduction O
in O
sucrose O
preference O
from O
24 O
- O
39 O
h O
( O
F O
( O
1 O
, O
59 O
) O
= O
14 O
. O
3 O
, O
P O
< O
0 O
. O
003 O
) O
. O

Moreover O
, O
this O
LPS O
- O
dependent O
reduction O
in O
sucrose O
preference O
was O
prevented O
by O
minocycline O
pretreatment O
( O
Mino O
x O
LPS O
interaction O
, O
F O
( O
1 O
, O
59 O
) O
= O
9 O
. O
9 O
, O
P O
< O
0 O
. O
004 O
, O
Fig O
. O
4 O
) O
. O

For O
example O
, O
minocycline O
pretreated O
mice B
injected O
with O
LPS O
maintained O
the O
same O
strong O
preference O
for O
sucrose O
as O
saline O
and O
minocycline O
controls O
( O
i O
. O
e O
. O
, O
approximately O
85 O
% O
preference O
) O
. O

These O
data O
can O
be O
interpreted O
to O
indicate O
that O
minocycline O
blocks O
anhedonia O
associated O
with O
peripheral O
LPS O
challenge O
. O

Minocycline O
reduces O
LPS O
- O
induced O
neuroinflammation O

Pro O
- O
inflammatory O
cytokines O
in O
the O
CNS O
are O
partially O
responsible O
for O
the O
behavioral O
symptoms O
of O
sickness O
( O
e O
. O
g O
. O
, O
anorexia O
, O
social O
withdrawal O
, O
and O
anhedonia O
) O
[ O
1 O
] O
. O

Therefore O
, O
we O
investigated O
the O
degree O
to O
which O
minocycline O
reduces O
neuroinflammation O
( O
IL O
- O
1 O
beta O
, O
IL O
- O
6 O
, O
and O
IDO O
) O
after O
peripheral O
injection O
of O
LPS O
. O

In O
this O
experiment O
, O
mice B
were O
subjected O
to O
the O
minocycline O
injection O
regimen O
and O
LPS O
challenge O
as O
above O
and O
cytokine O
mRNA O
levels O
were O
determined O
in O
the O
cortex O
and O
hippocampus O
4 O
h O
after O
LPS O
injection O
. O

In O
mice B
pretreated O
with O
vehicle O
, O
LPS O
markedly O
increased O
IL O
- O
1 O
beta O
mRNA O
levels O
in O
the O
hippocampus O
( O
F O
( O
1 O
, O
31 O
) O
= O
62 O
, O
P O
< O
0 O
. O
0001 O
) O
and O
cortex O
( O
F O
( O
1 O
, O
31 O
) O
= O
17 O
. O
25 O
, O
P O
< O
0 O
. O
0003 O
) O
. O

The O
LPS O
- O
induced O
IL O
- O
1 O
beta O
mRNA O
expression O
was O
reduced O
in O
both O
brain O
regions O
in O
mice B
receiving O
minocycline O
prior O
to O
LPS O
injection O
: O
( O
hippocampus O
, O
F O
( O
1 O
, O
31 O
) O
= O
9 O
. O
63 O
, O
P O
< O
0 O
. O
01 O
) O
and O
cortex O
, O
F O
( O
1 O
, O
31 O
) O
= O
7 O
. O
23 O
, O
P O
= O
0 O
. O
08 O
, O
Fig O
. O

5A O
) O
. O

LPS O
caused O
a O
similar O
induction O
of O
IL O
- O
6 O
mRNA O
levels O
in O
the O
hippocampus O
( O
F O
( O
1 O
, O
31 O
) O
= O
37 O
. O
2 O
, O
P O
< O
0 O
. O
001 O
) O
and O
cortex O
( O
F O
( O
1 O
, O
31 O
) O
= O
22 O
. O
5 O
, O
P O
< O
0 O
. O
001 O
) O
, O
but O
minocycline O
pretreatment O
only O
significantly O
attenuated O
LPS O
- O
induced O
IL O
- O
6 O
mRNA O
levels O
in O
the O
hippocampus O
( O
F O
( O
1 O
, O
31 O
) O
= O
10 O
. O
27 O
, O
P O
< O
0 O
. O
004 O
, O
Fig O
. O
5B O
) O
. O

IDO O
mRNA O
levels O
were O
determined O
from O
the O
same O
RNA O
pool O
. O

Fig O
. O

6D O
shows O
that O
LPS O
injection O
increased O
IDO O
mRNA O
expression O
in O
the O
hippocampus O
( O
F O
( O
1 O
, O
31 O
) O
= O
11 O
. O
69 O
, O
P O
< O
0 O
. O
002 O
) O
and O
cortex O
( O
F O
( O
1 O
, O
31 O
) O
= O
5 O
. O
26 O
, O
P O
< O
0 O
. O
03 O
) O
. O

This O
LPS O
- O
induced O
IDO O
mRNA O
expression O
was O
attenuated O
by O
minocycline O
in O
the O
hippocampus O
( O
F O
( O
1 O
, O
31 O
) O
= O
11 O
. O
69 O
, O
P O
< O
0 O
. O
002 O
) O
and O
cortex O
( O
F O
( O
1 O
, O
31 O
) O
= O
5 O
. O
26 O
, O
P O
< O
0 O
. O
03 O
) O
. O

It O
is O
important O
to O
note O
that O
IDO O
mRNA O
was O
undetected O
in O
saline O
treated O
mice B
. O

Therefore O
, O
the O
fold O
IDO O
change O
was O
relative O
to O
the O
IDO O
mRNA O
levels O
in O
mice B
receiving O
minocycline O
prior O
to O
LPS O
. O

Minocycline O
reduces O
LPS O
- O
induced O
IL O
- O
6 O
, O
but O
not O
IL O
- O
1 O
beta O
, O
in O
the O
plasma O

Because O
cytokine O
signals O
can O
be O
relayed O
from O
the O
periphery O
to O
the O
brain O
by O
humoral O
pathways O
[ O
56 O
] O
, O
plasma O
cytokine O
levels O
of O
IL O
- O
6 O
and O
IL O
- O
1 O
beta O
were O
determined O
4 O
h O
post O
LPS O
injection O
. O

As O
expected O
, O
LPS O
injection O
caused O
a O
marked O
increase O
in O
plasma O
IL O
- O
1 O
beta O
( O
F O
( O
1 O
, O
36 O
) O
= O
52 O
. O
5 O
, O
P O
< O
0 O
. O
001 O
) O
and O
IL O
- O
6 O
levels O
( O
F O
( O
1 O
, O
36 O
) O
34 O
. O
01 O
, O
P O
< O
0 O
. O
01 O
) O
. O

Minocycline O
pretreatment O
reduced O
LPS O
- O
induced O
IL O
- O
6 O
levels O
in O
the O
plasma O
( O
F O
( O
1 O
, O
36 O
) O
6 O
. O
68 O
, O
P O
< O
0 O
. O
01 O
) O
but O
had O
no O
significant O
main O
effect O
on O
LPS O
- O
induced O
IL O
- O
1 O
beta O
levels O
( O
Fig O
. O
6 O
) O
. O

Minocycline O
attenuates O
LPS O
- O
induced O
exaggerated O
neuroinflammation O
in O
aged O
mice B

Aged O
BALB O
/ O
c O
mice B
( O
22 O
- O
24 O
m O
) O
have O
an O
exaggerated O
neuroinflammatory O
response O
to O
LPS O
injection O
[ O
10 O
, O
13 O
, O
14 O
] O
. O

Therefore O
, O
we O
next O
sought O
to O
determine O
if O
the O
heightened O
inflammatory O
response O
in O
the O
brain O
of O
aged O
mice B
was O
reduced O
by O
minocycline O
. O

In O
this O
experiment O
, O
adult O
and O
aged O
mice B
were O
subjected O
to O
the O
minocycline O
injection O
regimen O
and O
LPS O
challenge O
as O
above O
. O

As O
we O
have O
reported O
previously O
, O
MHC O
II O
mRNA O
expression O
was O
increased O
by O
age O
( O
P O
< O
0 O
. O
03 O
, O
Fig O
. O
7A O
) O
[ O
13 O
, O
14 O
] O
, O
but O
MHC O
II O
levels O
were O
unaffected O
by O
either O
LPS O
or O
minocycline O
treatment O
( O
not O
shown O
) O
. O

Consistent O
with O
the O
data O
presented O
in O
Fig O
. O

2 O
, O
ANOVA O
revealed O
a O
significant O
main O
effect O
of O
LPS O
injection O
on O
TLR2 O
mRNA O
expression O
in O
the O
hippocampus O
( O
F O
( O
1 O
, O
63 O
) O
= O
85 O
. O
5 O
, O
P O
< O
0 O
. O
001 O
) O
. O

Moreover O
, O
LPS O
caused O
a O
greater O
increase O
in O
TLR2 O
mRNA O
in O
the O
hippocampus O
of O
aged O
mice B
compared O
to O
adults O
( O
LPS O
x O
Age O
interaction O
, O
F O
( O
1 O
, O
63 O
) O
= O
12 O
. O
70 O
, O
P O
< O
0 O
. O
01 O
) O
. O

Furthermore O
, O
minocycline O
pretreatment O
attenuated O
LPS O
- O
induced O
TLR2 O
mRNA O
levels O
in O
both O
adult O
and O
aged O
mice B
( O
Mino O
x O
LPS O
interaction O
, O
F O
( O
1 O
, O
63 O
) O
= O
9 O
. O
02 O
, O
P O
< O
0 O
. O
004 O
) O
. O

Parallel O
to O
the O
results O
for O
TLR2 O
, O
LPS O
caused O
a O
greater O
increase O
in O
IL O
- O
1 O
beta O
and O
IDO O
mRNA O
levels O
in O
hippocampus O
of O
aged O
mice B
compared O
to O
adults O
( O
Age O
x O
LPS O
, O
F O
( O
1 O
, O
60 O
) O
= O
8 O
. O
64 O
, O
P O
< O
0 O
. O
01 O
for O
IL O
- O
1 O
beta O
and O
F O
( O
1 O
, O
60 O
) O
= O
4 O
. O
0 O
, O
P O
< O
0 O
. O
05 O
for O
IDO O
) O
. O

Minocycline O
pretreatment O
attenuated O
LPS O
- O
induced O
mRNA O
levels O
of O
IL O
- O
1 O
beta O
( O
Mino O
x O
LPS O
, O
F O
( O
1 O
, O
60 O
) O
= O
8 O
. O
76 O
, O
P O
< O
0 O
. O
01 O
, O
Fig O
. O
7C O
) O
and O
IDO O
( O
Mino O
x O
LPS O
, O
F O
( O
1 O
, O
60 O
) O
= O
9 O
. O
7 O
, O
P O
< O
0 O
. O
003 O
, O
Fig O
. O
7D O
) O
. O

While O
LPS O
induced O
higher O
IL O
- O
6 O
mRNA O
levels O
in O
the O
hippocampus O
of O
both O
adult O
and O
aged O
mice B
( O
F O
( O
1 O
, O
59 O
) O
= O
44 O
. O
5 O
, O
P O
< O
0 O
. O
001 O
) O
, O
there O
was O
not O
an O
Age O
x O
LPS O
interaction O
. O

Minocycline O
pretreatment O
attenuated O
the O
LPS O
- O
induced O
increase O
in O
hippocampal O
IL O
- O
6 O
mRNA O
( O
Mino O
x O
LPS O
, O
F O
( O
1 O
, O
59 O
) O
= O
5 O
. O
4 O
, O
P O
< O
0 O
. O
02 O
, O
Fig O
. O
7E O
) O
. O

Taken O
together O
these O
data O
indicate O
that O
minocycline O
pretreatment O
was O
effective O
in O
attenuating O
the O
exaggerated O
neuroinflammation O
in O
aged O
mice B
. O

Discussion O

In O
the O
elderly O
, O
systemic O
infection O
is O
associated O
with O
an O
increased O
frequency O
of O
behavioral O
and O
cognitive O
complications O
[ O
57 O
, O
58 O
] O
. O

We O
have O
reported O
that O
stimulation O
of O
the O
peripheral O
immune O
system O
in O
older O
( O
20 O
- O
24 O
m O
) O
BALB O
/ O
c O
mice B
causes O
exaggerated O
neuroinflammation O
that O
is O
paralleled O
by O
prolonged O
sickness O
[ O
13 O
] O
, O
impaired O
working O
memory O
[ O
10 O
] O
, O
and O
depressive O
- O
like O
behaviors O
[ O
15 O
] O
. O

Therefore O
, O
it O
is O
important O
to O
understand O
the O
mechanisms O
that O
can O
modulate O
cytokine O
- O
mediated O
pathways O
in O
the O
brain O
. O

Here O
we O
show O
that O
minocycline O
treatment O
reduced O
LPS O
- O
induced O
TLR2 O
expression O
in O
BV O
- O
2 O
cells O
and O
on O
microglia O
isolated O
from O
adult O
mice B
. O

Moreover O
, O
we O
demonstrate O
that O
minocycline O
was O
effective O
in O
facilitating O
the O
recovery O
from O
LPS O
- O
induced O
sickness O
and O
preventing O
anhedonia O
in O
adult O
mice B
. O

Furthermore O
, O
we O
show O
that O
minocycline O
attenuated O
LPS O
- O
induced O
neuroinflammation O
in O
adults O
and O
normalized O
the O
exaggerated O
neuroinflammation O
in O
aged O
mice B
. O

Our O
findings O
, O
using O
cell O
culture O
and O
animal O
experiments O
, O
support O
the O
notion O
that O
minocycline O
attenuates O
microglial O
activation O
and O
limits O
production O
of O
inflammatory O
mediators O
. O

For O
instance O
, O
minocycline O
pretreatment O
of O
BV O
- O
2 O
cultures O
decreased O
LPS O
- O
stimulated O
cytokine O
production O
in O
a O
dose O
dependent O
manner O
( O
Fig O
. O
1A O
) O
. O

In O
BV O
- O
2 O
cells O
, O
minocycline O
also O
attenuated O
mRNA O
expression O
of O
inflammatory O
genes O
including O
IL O
- O
6 O
, O
IL O
- O
1 O
beta O
, O
MHC O
II O
, O
and O
TLR2 O
( O
Fig O
. O
1D O
) O
. O

These O
data O
are O
consistent O
with O
other O
studies O
using O
minocycline O
and O
LPS O
in O
BV O
- O
2 O
cells O
[ O
44 O
, O
59 O
] O
. O

Based O
on O
these O
data O
we O
next O
investigated O
if O
microglial O
activation O
could O
be O
attenuated O
in O
the O
brain O
. O

Because O
LPS O
increases O
brain O
cytokine O
production O
we O
expected O
that O
MHC O
II O
expression O
would O
also O
be O
increased O
. O

Contrary O
to O
our O
predictions O
, O
neither O
MHC O
II O
mRNA O
levels O
( O
Fig O
. O
7 O
) O
in O
the O
brain O
nor O
MHC O
II O
surface O
expression O
on O
microglia O
( O
CD11b O
+ O
/ O
CD45low O
) O
( O
data O
not O
shown O
) O
were O
increased O
by O
LPS O
injection O
. O

In O
an O
EAE O
model O
, O
minocycline O
reduced O
microglial O
expression O
of O
MHC O
II O
[ O
45 O
] O
, O
but O
one O
key O
difference O
from O
our O
study O
is O
that O
the O
induction O
of O
EAE O
pathology O
requires O
functional O
antigen O
presentation O
on O
MHC O
II O
[ O
60 O
] O
. O

It O
is O
postulated O
that O
microglia O
have O
several O
activation O
states O
that O
depend O
on O
the O
specific O
inflammatory O
stimulus O
[ O
61 O
] O
. O

Thus O
, O
in O
situations O
of O
transient O
peripheral O
innate O
immune O
stimulation O
, O
markers O
in O
the O
CNS O
such O
as O
Toll O
- O
Like O
receptors O
[ O
6 O
] O
may O
be O
indicative O
of O
microglia O
activation O
. O

In O
support O
of O
this O
premise O
, O
our O
data O
show O
that O
LPS O
injection O
increases O
TLR2 O
surface O
expression O
on O
microglia O
( O
CD11b O
+ O
/ O
CD45low O
) O
, O
which O
is O
inhibited O
by O
minocycline O
pretreatment O
( O
Fig O
. O
2 O
) O
. O

These O
data O
are O
consistent O
with O
other O
studies O
showing O
that O
central O
or O
peripheral O
LPS O
challenge O
increases O
TLR2 O
mRNA O
in O
the O
brain O
[ O
6 O
, O
14 O
] O
. O

Taken O
together O
our O
findings O
can O
be O
interpreted O
to O
suggest O
that O
minocycline O
attenuates O
pathways O
associated O
with O
microglia O
activation O
following O
peripheral O
LPS O
challenge O
. O

One O
of O
the O
important O
findings O
of O
this O
study O
was O
that O
reduction O
of O
neuroinflammation O
by O
minocycline O
was O
associated O
with O
facilitated O
recovery O
from O
LPS O
- O
induced O
sickness O
behavior O
. O

These O
results O
are O
akin O
to O
our O
previous O
work O
with O
the O
anti O
- O
oxidant O
, O
alpha O
- O
tocopherol O
[ O
52 O
] O
, O
and O
an O
NFKB O
decoy O
inhibitor O
[ O
62 O
] O
. O

Consistent O
with O
our O
previous O
studies O
[ O
52 O
, O
53 O
, O
62 O
, O
63 O
] O
, O
reductions O
in O
neuroinflammatory O
cytokines O
( O
Fig O
. O
5 O
) O
did O
not O
prevent O
the O
induction O
of O
the O
LPS O
- O
induced O
sickness O
response O
( O
2 O
- O
4 O
h O
) O
, O
but O
rather O
facilitated O
the O
recovery O
from O
sickness O
( O
8 O
- O
24 O
h O
) O
( O
Fig O
. O
3A O
) O
. O

Recovery O
may O
be O
a O
critical O
issue O
because O
brain O
cytokines O
and O
the O
corresponding O
physiological O
and O
behavioral O
responses O
are O
beneficial O
to O
the O
host O
[ O
1 O
] O
. O

The O
potential O
risk O
for O
a O
maladaptive O
response O
occurs O
when O
the O
normally O
transient O
neuroinflammatory O
response O
is O
amplified O
or O
protracted O
[ O
64 O
] O
. O

Therefore O
pharmacological O
agents O
, O
similar O
to O
minocycline O
, O
that O
attenuate O
neuroinflammatory O
responses O
, O
but O
do O
not O
completely O
inhibit O
them O
, O
may O
be O
important O
in O
preventing O
the O
development O
of O
more O
severe O
and O
long O
- O
lasting O
cognitive O
and O
behavioral O
complications O
. O

The O
results O
of O
the O
sucrose O
preference O
experiments O
support O
the O
idea O
that O
limiting O
exposure O
to O
neuroinflammation O
decreases O
the O
duration O
of O
behavioral O
responses O
. O

For O
example O
, O
while O
minocycline O
did O
not O
inhibit O
cytokine O
expression O
( O
Fig O
. O
5 O
) O
or O
the O
induction O
of O
sickness O
( O
Fig O
. O
3A O
) O
, O
minocycline O
pretreatment O
completely O
reversed O
the O
reduction O
in O
sucrose O
preference O
( O
i O
. O
e O
. O
, O
anhedonia O
) O
associated O
with O
LPS O
injection O
( O
Fig O
. O
4 O
) O
. O

It O
is O
also O
important O
to O
mention O
that O
while O
LPS O
- O
associated O
sickness O
and O
anhedonia O
are O
interrelated O
, O
these O
behaviors O
can O
be O
differentiated O
from O
one O
another O
. O

For O
instance O
, O
reduced O
social O
exploration O
was O
evident O
2 O
- O
24 O
h O
post O
injection O
( O
Fig O
. O
3A O
) O
, O
but O
only O
decreased O
sucrose O
preference O
was O
exhibited O
24 O
to O
39 O
h O
later O
( O
Fig O
. O
4 O
) O
. O

This O
separation O
between O
behaviors O
is O
consistent O
with O
other O
studies O
investigating O
sickness O
and O
longer O
- O
lasting O
changes O
in O
motivation O
[ O
15 O
, O
65 O
, O
66 O
] O
. O

IDO O
mediated O
TRP O
metabolism O
represents O
a O
potential O
connection O
between O
activation O
of O
CNS O
innate O
immune O
cells O
and O
longer O
lasting O
behavioral O
responses O
. O

IDO O
mediated O
TRP O
metabolism O
in O
the O
brain O
may O
affect O
behavior O
by O
impacting O
both O
serotonin O
and O
glutamate O
pathways O
[ O
39 O
] O
. O

We O
have O
reported O
that O
IDO O
induction O
and O
activity O
is O
amplified O
in O
the O
brain O
of O
aged O
mice B
and O
is O
associated O
with O
prolonged O
depressive O
- O
like O
behavior O
[ O
15 O
] O
. O

Here O
we O
show O
that O
IDO O
mRNA O
induction O
is O
blocked O
by O
minocycline O
in O
the O
brain O
of O
both O
adult O
and O
aged O
mice B
( O
Figs O
. O
5 O
& O
7 O
) O
. O

These O
data O
are O
consistent O
with O
a O
recent O
report O
showing O
a O
causal O
relationship O
between O
IDO O
activity O
and O
acute O
depressive O
effects O
in O
adult O
CD O
- O
1 O
mice B
. O

In O
this O
study O
, O
O O
' O
Connor O
et O
al O
. O
report O
that O
both O
1 O
- O
methyl O
tryptophan O
( O
a O
competitive O
inhibitor O
of O
IDO O
) O
and O
minocycline O
blocked O
IDO O
induction O
and O
prevented O
depressive O
- O
like O
immobility O
in O
the O
tail O
suspension O
and O
forced O
swimming O
tests O
[ O
66 O
] O
. O

Thus O
, O
in O
the O
present O
study O
, O
the O
minocycline O
blockade O
of O
IDO O
induction O
may O
explain O
the O
abrogation O
of O
LPS O
- O
induced O
anhedonia O
. O

Another O
interesting O
finding O
was O
that O
while O
minocycline O
pretreatment O
in O
adult O
mice B
attenuated O
LPS O
- O
induced O
brain O
IL O
- O
1 O
beta O
at O
4 O
h O
( O
Fig O
. O
5 O
) O
, O
it O
had O
no O
effect O
on O
plasma O
IL O
- O
1 O
beta O
levels O
( O
Fig O
. O
6 O
) O
. O

Because O
IL O
- O
1 O
beta O
signals O
can O
be O
relayed O
from O
the O
periphery O
to O
the O
brain O
by O
humoral O
pathways O
[ O
5 O
] O
, O
these O
findings O
suggest O
that O
minocycline O
has O
anti O
- O
inflammatory O
properties O
within O
the O
brain O
. O

These O
data O
are O
consistent O
with O
related O
findings O
that O
minocycline O
readily O
crosses O
the O
blood O
brain O
barrier O
to O
elicit O
an O
anti O
- O
inflammatory O
effect O
[ O
41 O
- O
43 O
] O
. O

With O
regard O
to O
IL O
- O
6 O
, O
minocycline O
pretreatment O
attenuated O
both O
brain O
and O
plasma O
levels O
at O
4 O
h O
post O
LPS O
injection O
. O

Because O
circulating O
IL O
- O
6 O
levels O
can O
be O
increased O
by O
CNS O
mediated O
pathways O
including O
activation O
of O
the O
hypothalamus O
- O
pituitary O
- O
adrenal O
( O
HPA O
) O
axis O
[ O
67 O
] O
and O
the O
sympathetic O
nervous O
system O
[ O
68 O
] O
, O
the O
specific O
reduction O
in O
plasma O
IL O
- O
6 O
by O
minocycline O
may O
reflect O
the O
reduction O
in O
brain O
inflammation O
at O
4 O
h O
( O
Fig O
. O
5 O
) O
. O

In O
support O
of O
this O
notion O
, O
we O
and O
others O
have O
reported O
that O
i O
. O
c O
. O
v O
. O
injection O
of O
LPS O
or O
IL O
- O
1 O
beta O
increase O
plasma O
IL O
- O
6 O
levels O
, O
but O
not O
IL O
- O
1 O
beta O
levels O
[ O
14 O
, O
68 O
, O
69 O
] O
. O

The O
final O
critical O
finding O
of O
this O
study O
was O
that O
minocycline O
was O
effective O
in O
attenuating O
neuroinflammation O
independent O
of O
age O
. O

Consistent O
with O
other O
aging O
and O
neuroinflammation O
studies O
, O
our O
data O
show O
that O
LPS O
caused O
exaggerated O
neuroinflammation O
in O
aged O
mice B
compared O
to O
adults O
[ O
10 O
, O
13 O
- O
15 O
] O
. O

It O
is O
important O
to O
mention O
that O
while O
there O
was O
an O
age O
- O
related O
difference O
in O
MHC O
II O
expression O
in O
the O
hippocampus O
of O
saline O
treated O
mice B
( O
Fig O
. O
7A O
) O
there O
was O
not O
an O
age O
- O
related O
difference O
in O
IL O
- O
1 O
beta O
and O
IL O
- O
6 O
mRNA O
levels O
. O

These O
data O
differ O
from O
a O
previous O
report O
using O
BALB O
/ O
c O
mice B
showing O
an O
increase O
in O
IL O
- O
6 O
in O
older O
mice B
[ O
70 O
] O
. O

This O
may O
be O
because O
the O
mice B
used O
in O
the O
present O
study O
were O
approximately O
4 O
months O
younger O
than O
the O
mice B
used O
previously O
. O

Nonetheless O
, O
microglia O
can O
be O
primed O
or O
reactive O
with O
increased O
MHC O
II O
expression O
, O
but O
do O
not O
necessarily O
produce O
inflammatory O
cytokines O
in O
this O
state O
[ O
19 O
] O
. O

The O
key O
results O
are O
that O
peripheral O
LPS O
injection O
causes O
a O
greater O
induction O
of O
TLR2 O
, O
IL O
- O
1 O
beta O
, O
and O
IDO O
mRNA O
in O
the O
aged O
brain O
than O
in O
the O
adult O
brain O
and O
that O
minocycline O
pretreatment O
normalizes O
this O
age O
- O
related O
exaggerated O
neuroinflammation O
( O
Fig O
. O
7 O
) O
. O

These O
findings O
are O
also O
important O
because O
an O
amplified O
neuroinflammatory O
response O
in O
the O
aged O
brain O
is O
a O
precursor O
to O
complications O
including O
deficits O
in O
working O
memory O
, O
memory O
consolidation O
, O
and O
depressive O
- O
like O
behavior O
[ O
9 O
, O
10 O
, O
15 O
] O
. O

Based O
on O
the O
biochemical O
and O
behavioral O
data O
obtained O
from O
this O
study O
, O
we O
predict O
that O
minocycline O
will O
abrogate O
the O
prolonged O
LPS O
- O
induced O
sickness O
[ O
13 O
] O
and O
depressive O
- O
like O
behavior O
exhibited O
by O
aged O
BALB O
/ O
c O
mice B
[ O
15 O
] O
. O

We O
acknowledge O
, O
however O
, O
that O
future O
studies O
are O
necessary O
to O
test O
these O
predictions O
. O

Conclusion O

The O
present O
study O
demonstrates O
that O
minocycline O
reduces O
LPS O
- O
induced O
microglial O
activation O
, O
CNS O
cytokine O
production O
, O
and O
behavioral O
symptoms O
of O
sickness O
( O
e O
. O
g O
. O
, O
social O
withdrawal O
and O
anhedonia O
) O
. O

These O
findings O
are O
potentially O
important O
because O
they O
indicate O
that O
minocycline O
can O
be O
used O
to O
mitigate O
cytokine O
expression O
in O
the O
brain O
and O
have O
a O
beneficial O
affect O
on O
behavioral O
responses O
. O

Taken O
together O
, O
these O
data O
support O
the O
idea O
that O
pharmacological O
strategies O
aimed O
at O
decreasing O
neuroinflammation O
associated O
with O
microglial O
activation O
are O
important O
for O
improving O
recovery O
from O
sickness O
and O
reducing O
the O
frequency O
of O
neurobehavioral O
complications O
. O

List O
of O
abbreviations O

3 O
- O
Hydroxy O
- O
L O
- O
Kynuriene O
( O
3HK O
) O
, O
Allophycocyanin O
( O
APC O
) O
, O
Analysis O
of O
variance O
( O
ANOVA O
) O
, O
Central O
Nervous O
System O
( O
CNS O
) O
, O
Dulbecco O
' O
s O
Modified O
Eagle O
' O
s O
Medium O
( O
DMEM O
) O
, O
Dimethyl O
Sulfoxide O
( O
DMSO O
) O
, O
Experimental O
Autoimmune O
Encephalomyelitis O
( O
EAE O
) O
, O
Enzyme O
Linked O
Immmunosorbent O
Assay O
( O
ELISA O
) O
, O
Fluorescein O

Isothiocyanate O
( O
FITC O
) O
, O
Fetal O
Bovine B
Serum O
( O
FBS O
) O
, O
Hank O
' O
s O
Balanced O
Salt O
Solution O
( O
HBSS O
) O
, O
Indoleamine O
2 O
, O
3 O
dioxygenase O
( O
IDO O
) O
, O
Intraperitoneal O
( O
i O
. O
p O
. O
) O
, O
Intracerebroventricu O
( O
i O
. O
c O
. O
v O
. O
) O
, O
Interleukin O
( O
IL O
) O
, O
Kynurenine O
( O
KYN O
) O
, O
Lipopolysaccharide O
( O
LPS O
) O
, O
Major O
Histocompatibility O
Complex O
class O
II O
( O

MHC O
II O
) O
, O
Mitogen O
Activated O
Protein O
Kinase O
( O
MAP O
- O
kinase O
) O
, O
Nuclear O
factor O
kappa O
B O
( O
NF O
kappa O
B O
) O
, O
N O
- O
methyl O
- O
D O
- O
aspartate O
( O
NMDA O
) O
, O
R O
- O
Phycoerthrin O
( O
PE O
) O
, O
Quinolinic O
acid O
( O
QUIN O
) O
, O
Statistical O
Analysis O
Systems O
( O
SAS O
) O
, O
Standard O
Error O
of O
the O
Mean O
( O
SEM O
) O
, O
Toll O
- O
like O
Receptor O
- O
2 O
( O
TLR2 O
) O
, O
and O
Tryptophan O
( O
TRP O
) O
. O

Competing O
interests O

The O
authors O
of O
this O
manuscript O
declare O
that O
there O
are O
no O
actual O
or O
potential O
conflicts O
of O
interest O
. O

The O
authors O
affirm O
that O
there O
are O
no O
financial O
, O
personal O
or O
other O
relationships O
with O
other O
people B
or O
organizations O
that O
have O
inappropriately O
influenced O
or O
biased O
their O
research O
. O

Authors O
' O
contributions O

CJH O
was O
involved O
in O
research O
experimentation O
, O
completion O
of O
statistical O
analysis O
, O
and O
writing O
of O
the O
manuscript O
. O

YH O
, O
AW O
, O
MH O
and O
JH O
assisted O
with O
experimentation O
and O
data O
analysis O
. O

MB O
and O
JFS O
contributed O
to O
the O
design O
of O
the O
experiments O
and O
assisted O
in O
editing O
the O
manuscript O
. O

JPG O
directed O
all O
aspects O
of O
this O
research O
project O
including O
the O
experimental O
design O
, O
research O
experimentation O
, O
completion O
of O
statistical O
analysis O
, O
and O
writing O
of O
the O
manuscript O
. O

A O
case O
of O
demand O
ischemia O
from O
phendimetrazine O

Abstract O

Introduction O

Phendimetrazine O
is O
a O
medication O
currently O
being O
used O
to O
help O
patients B
with O
weight O
loss O
. O

It O
shares O
a O
chemical O
structure O
with O
amphetamines O
. O

As O
such O
, O
it O
shares O
some O
of O
the O
same O
toxicities O
, O
which O
can O
include O
cardiac O
toxicity O
. O

This O
case O
highlights O
this O
principle O
. O

Case O
presentation O

a O
54 O
year O
old O
Caucasian O
female O
presented O
to O
our O
urgent O
care O
facility O
with O
complaints O
of O
chest O
pains O
and O
other O
symptoms O
suggestive O
of O
acute O
coronary O
syndrome O
. O

Ultimately O
, O
she O
was O
transferred O
to O
the O
emergency O
room O
. O

After O
evaluation O
there O
, O
it O
appeared O
she O
was O
having O
demand O
ischemia O
from O
prescription O
diet O
pills O

Conclusion O

This O
case O
report O
demonstrates O
the O
potential O
dangers O
of O
amphetamine O
based O
diet O
pills O
. O

There O
have O
been O
other O
cases O
of O
cardiomyopathies O
related O
to O
phendimetrazine O
, O
but O
it O
is O
something O
that O
is O
rarely O
recognized O
in O
an O
outpatient O
setting O
. O

A O
case O
such O
as O
this O
demonstrates O
the O
importance O
of O
obtaining O
a O
careful O
medication O
history O
in O
all O
patients B
and O
in O
recognizing O
diet O
pills O
with O
an O
amphetamine O
base O
can O
cause O
cardiac O
toxicity O
. O

Case O
presentation O

A O
54 O
year O
- O
old O
Caucasian O
female O
presented O
to O
our O
urgent O
care O
facility O
complaining O
of O
nausea O
and O
vomiting O
, O
sense O
of O
impending O
doom O
and O
vague O
chest O
pain O
radiating O
toward O
her O
left O
side O
for O
about O
five O
hours O
. O

She O
never O
had O
similar O
symptoms O
in O
the O
past O
. O

She O
also O
denied O
anything O
that O
could O
have O
precipitated O
these O
symptoms O
. O

Her O
only O
past O
medical O
history O
was O
significant O
for O
spina O
bifida I
. O

Her O
medications O
included O
occasional O
Fiorinal O
( O
unknown O
dose O
) O
, O
Xanax O
0 O
. O
5 O
mg O
as O
needed O
, O
and O
Phendimetrazine O
( O
unclear O
dose O
) O
. O

Her O
social O
history O
was O
significant O
for O
smoking O
1 O
/ O
2 O
pack O
per O
day O
cigarette O
use O
. O

She O
denied O
alcohol O
use O
. O

Family O
history O
was O
non O
contributory O
. O

She O
worked O
from O
home O
. O

Her O
physical O
exam O
showed O
a O
tachycardia O
of O
around O
100 O
beats O
per O
minute O
, O
respiratory O
rate O
of O
16 O
, O
temperature O
of O
98 O
. O
1 O
, O
and O
O2 O
saturation O
of O
100 O
% O
on O
room O
air O
. O

She O
was O
approximately O
5 O
' O
7 O
" O
and O
145 O
pounds O
. O

In O
general O
, O
she O
was O
an O
anxious O
appearing O
, O
diaphoretic O
woman B
in O
moderate O
distress O
, O
she O
had O
no O
elevated O
JVD O
at O
30 O
degrees O
, O
her O
heart O
was O
tachycardic O
, O
but O
otherwise O
without O
murmur O
, O
gallops O
, O
or O
rubs O
, O
her O
lungs O
were O
clear O
, O
abdomen O
soft O
, O
and O
she B
had O
no O
peripheral O
edema O
. O

An O
EKG O
was O
checked O
which O
appears O
below O
( O
figure O
1 O
) O
. O

After O
examination O
, O
there O
was O
concern O
for O
acute O
coronary O
syndrome O
( O
ACS O
) O
. O

She O
was O
given O
nitroglycerin O
with O
relief O
of O
her O
chest O
discomfort O
. O

She O
was O
also O
given O
aspirin O
to O
chew O
. O

EMS O
was O
called O
and O
she O
was O
transferred O
to O
a O
local O
emergency O
room O
. O

She O
was O
hospitalized O
there O
for O
three O
days O
and O
after O
her O
discharge O
, O
we O
got O
permission O
from O
her O
to O
request O
records O
. O

While O
hospitalized O
, O
she O
was O
ruled O
out O
for O
ACS O
with O
negative O
troponins O
. O

She O
was O
also O
given O
beta O
blockade O
which O
resolved O
her O
tachycardia O
and O
her O
T O
wave O
changes O
on O
EKG O
. O

The O
next O
morning O
, O
she O
had O
an O
adenosine O
stress O
test O
which O
revealed O
normal O
uptake O
with O
no O
areas O
of O
ischemia O
and O
an O
ejection O
fraction O
of O
55 O
% O
. O

She O
was O
monitored O
for O
one O
more O
day O
and O
then O
discharged O
with O
instructions O
to O
discontinue O
her O
diet O
pills O
. O

Discussion O

Phendimetrazine O
is O
a O
medication O
currently O
being O
used O
for O
weight O
loss O
, O
with O
potential O
for O
illicit O
use O
. O

It O
has O
a O
similar O
chemical O
composition O
of O
amphetamines O
, O
which O
is O
thought O
to O
account O
for O
its O
clinical O
actions O
[ O
1 O
] O
. O

Amphetamines O
are O
well O
recognized O
as O
an O
etiology O
of O
cardiac O
ischemia O
, O
however O
phendimetrazine O
is O
more O
rarely O
described O
in O
the O
literature O
as O
causing O
cardiac O
events O
. O

[ O
2 O
, O
3 O
] O
. O

Acute O
effects O
include O
hyperpyrexia O
, O
mydriasis O
, O
chest O
pain O
, O
arrhytmias O
, O
delirium O
, O
and O
, O
rhabdomylosis O
, O
among O
others O
[ O
2 O
] O
. O

Long O
term O
use O
has O
been O
associated O
with O
dilated O
cardiomyopathies O
, O
some O
of O
which O
have O
resolved O
with O
discontinuation O
of O
the O
medication O
[ O
3 O
] O
. O

In O
this O
particular O
case O
, O
it O
appears O
she O
may O
have O
developed O
a O
demand O
ischemia O
from O
the O
medication O
. O

It O
is O
not O
known O
how O
much O
of O
the O
drug O
she O
was O
taking O
. O

Initially O
, O
she O
was O
resistant O
to O
accepting O
that O
phendimetrazine O
could O
induce O
side O
effects O
, O
and O
there O
was O
suspicion O
that O
she O
could O
have O
been O
taking O
more O
of O
the O
drug O
that O
recommended O
. O

In O
addition O
, O
she O
was O
not O
prescribed O
the O
medication O
and O
would O
not O
admit O
to O
where O
she O
obtained O
it O
. O

As O
the O
public O
seems O
to O
have O
more O
focus O
on O
using O
medications O
to O
induce O
weight O
loss O
, O
this O
may O
be O
a O
more O
recognized O
complication O
and O
heart O
conditions O
should O
likely O
be O
monitored O
prior O
to O
starting O
amphetamine O
based O
weight O
loss O
pills O
. O

Conclusion O

Due O
to O
potentially O
detrimental O
effects O
of O
this O
medication O
, O
phendimetrazine O
should O
be O
used O
cautiously O
in O
many O
situations O
. O

As O
it O
shares O
its O
chemical O
structure O
with O
amphetamines O
, O
it O
also O
shares O
many O
of O
the O
side O
effects O
and O
the O
potential O
for O
abuse O
/ O
addiction O
. O

There O
have O
been O
other O
reports O
in O
literature O
describing O
adverse O
outcomes O
from O
phendimetrazine O
as O
well O
as O
other O
weight O
loss O
medications O
. O

Therefore O
, O
cautious O
use O
is O
warranted O
. O

Abbreviations O

ACS O
: O
Acute O
Coronary O
Syndrome O
. O

Competing O
interests O

The O
authors O
declare O
that O
they O
have O
no O
competing O
interests O
. O

Authors O
' O
contributions O

DL O
, O
JJ O
, O
GG O
have O
all O
been O
involved O
in O
and O
approve O
of O
the O
writing O
of O
this O
case O
presentation O
. O

Consent O

Written O
informed O
consent O
was O
obtained O
from O
the O
patient B
for O
publication O
purposes O
. O

A O
copy O
can O
be O
obtained O
if O
requested O
by O
the O
Editor O
in O
Chief O
of O
this O
journal O
. O

Advanced O
software O
concepts O
for O
employing O
microcomputers O
in O
the O

laboratory O

Abstract O

IIII O

II O

, O
Advanced O
software O
concepts O
for O

employing O
microcomputers O
in O
the O
laboratory O
Scott O
B O
. O
Tilden O
and O
M O
. O
Bonner O
Denton O
, O
Department O
of O
Chemistry O
, O
University O
of O
Arizona O
, O
Tucson O
, O
Arizona O
85721 O
, O
USA O
. O

Introduction O
While O
a O
proliferation O
of O
commercial O
chemical O
instrumentation O
is O
appearing O
today O
employing O
microprocessors O
for O
a O
variety O
of O
control O
and O
data O
reduction O
applications O
, O
the O
great O
potential O
of O
microprocessors O
has O
not O
been O
exploited O
extensively O
for O
individual O
custom O
applications O
. O

The O
primary O
reason O
for O
this O
phenomenon O
is O
altogether O
too O
clear O
microprocessor O
software O
is O
either O
difficult O
to O
develop O
or O
inefficient O
in O
memory O
requirements O
and O
speed O
. O

This O
problem O
is O
even O
more O
important O
in O
situations O
requiring O
constant O
software O

modification O
. O

Initially O
, O
most O
instrument O
manufacturers O
utilized O
cross O
assemblers O
supported O
on O
large O
" O
number O
cruncher O
" O
computers O
to O
generate O
the O
required O
machine O
code O
binary O
program O
. O

More O
recently O
, O
the O
trend O
has O
been O
toward O
the O
use O
of O
a O
" O
developmental O
system O
" O
( O
at O
a O
cost O
comparable O
to O
a O
moderate O
minicomputer O
- O
the O
authors O
use O
the O
term O
" O
mini O
" O
in O
contrast O
to O
" O
micro O
" O
reluctantly O
because O
of O
the O
ever O
increasing O
overlap O
in O
computing O
capability O
) O
to O
write O
and O
debug O
assembly O
level O
porgrams O
which O
are O
subsequently O
converted O
to O
binary O
and O
incorporated O
into O
an O
instrument O
in O
the O
form O
of O
" O
read O
only O
memory O
" O
( O
ROM O
) O
. O

While O
this O
approach O

Interpretative O
Machine O
Code O

Source O
Code O
File O

to O
Execute O
Command O
A O
Hachine O
Code O
Various O

to O
Execute O
Comma O
nd O
B O
ne O
Code O

' O
commands O
making O
up O

, O
lachi O

to O
Execute O
Command O
C O

users O
source O
program O

i O

Machine O
Code O

to O
Execute O
Command O
D O

etc O
. O

Figure O
1 O
. O

The O
interpretative O
cycle O
of O
common O
types O
of O
languages O
such O
as O
BASIC O
. O

After O
examining O
each O
command O
in O
the O
source O
file O
, O
the O
interpreter O
searches O
for O
and O
branches O
to O
the O
corresponding O
block O
of O
machine O
code O
; O
thus O
, O
program O
execution O
always O
remains O
within O
the O
interpreter O
. O

128 O

The O
Journal O
of O
Automatic O
Chemistry O

Tilden O
& O
Denton O
IIII O

Advanced O
software O
concepts O

III O

Users O
Source O
File O

The O
Compiler O

Command O
A O

Comma O
nd O
B O
Command O
C O
Command O
D O
etc O
. O

/ O
z O
/ O
li O
i O

II O

. O
Machine O
Code O

etc O
. O

. O
lachi O
ne O
Code O

to O
Execute O
Command O
A O

etc O
. O
to O
Execute O

Machine O
Code O

to O
Execute O
Command O
C O

Machine O
Code O

etc O
. O

Figure O
2 O
. O

Note O
that O
the O
compiler O
transforms O
each O
source O
" O
command O
" O
into O
executable O
machine O
code O
. O

This O
code O
will O
, O
subsequently O
, O
be O
loaded O
and O
executed O
independently O
of O
the O
compiler O
. O

has O
proven O
cost O
effective O
for O
high O
volume O
mass O
produced O
applications O
, O
it O
possesses O
serious O
limitations O
for O
system O
updates O
and O
custom O
applications O
. O

Additionally O
, O
the O
ability O
to O
program O
efficiently O
at O
the O
assembly O
level O
is O
a O
talent O
requiring O
a O
significant O
expenditure O
of O
time O
to O
develop O
. O

During O
the O
past O
several O
years O
, O
a O
virtual O
deluge O
of O
sophisticated O
, O
flexible O
, O
high O
performance O
computer O
hardware O
has O
been O
introduced O
primarily O
aimed O
at O
a O
rapidly O
growing O
" O
hobbyist O
" O
market O
. O

Manufacturers O
quickly O
realilsed O
that O
to O
sell O
the O
public O
hardware O
, O
some O
form O
of O
reasonably O
high O
level O
software O
must O
be O
made O
available O
. O

A O
variety O
of O
BASIC O
interpreters O
, O
ranging O
from O
rather O
" O
dumb O
" O
to O
" O
quite O
intelligent O
" O
have O
since O
evolved O
. O

The O
more O
intelligent O
BASIC O
interpreters O
have O
several O
highly O
attractive O
attributes O
for O
" O
hobbyist O
" O
applications O
. O

The O
language O
is O
both O
easy O
to O
master O
and O
conversational O
. O

Error O
and O
caution O
messages O
are O
provided O
as O
aids O
during O
programming O
. O

Why O
not O
apply O
the O
" O
hobbyist O
" O
technology O
toward O
the O
implementation O
of O
custom O
laboratory O
systems O
? O

Many O
investigators O
have O
and O
, O
no O
doubt O
, O
many O
more O
will O
take O
this O
approach O
. O

However O
, O
BASIC O
interpreters O
possess O
serious O
limitations O
in O
terms O
of O
system O
speed O
, O
flexibility O
and O
input O
/ O
output O
( O
I O
/ O
O O
) O
capabilities O
. O

In O
BASIC O
, O
each O
command O
must O
first O
be O
interpreted O
and O
then O
executed O
( O
see O
Figure O
1 O
) O
. O

In O
many O
cases O
, O
the O
interpretation O
process O
takes O
much O
more O
time O
than O
the O
actual O
execution O
. O

This O
problem O
is O
compounded O
by O
the O
fact O
that O
commands O
interpreted O
in O
the O
past O
must O
be O
re O
- O
interpreted O
each O
time O
they O
are O
used O
causing O
iterative O
programs O
to O
be O
very O
slow O
. O

While O
speed O
is O
often O
not O
a O
serious O
limitation O
in O
playing O
computer O
games O
, O
laboratory O
application O
requiring O
high O
speed O
data O
acquisition O
and O
/ O
or O
data O
manipulation O
are O
common O
. O

Additionally O
, O
the O
more O
intelligent O
BASICs O
make O
very O
inefficient O
use O
of O
memory O
often O
requiring O
minimum O
of O
12 O
or O
16 O
K O
bytes O
( O
twelve O
or O
sixteen O
thousand O
eight O
bit O
words O
) O
' O
Volume O

cS O

H O

A O

D O

H O
Figure O
3 O
. O

The O
' O
threaded O
' O
code O
approach O
used O
in O
CONVERS O
. O

In O
' O
threaded O
' O
code O
programming O
modules O
for O
sub O
- O
routines O
are O
used O
repetitively O
in O
a O
variety O
of O
combinations O
to O
allow O
implementation O
of O
an O
infinite O
number O
of O
functions O
. O

Note O
that O
the O
flow O
of O
logic O
threads O
its O
way O
in O
a O
very O
non O
. O
linear O
fashion O
through O
these O
modules O
. O

No O
. O
3 O
April O
1979 O

129 O

Tilden O

& O
Denton O

Advanced O
software O
concepts O
| O
I O

Stack O

Upper O
Memory O
Bound O

User O
' O
s O
Application O
Dictionaries O

4 O
. O
5k O
Octal O
CONVERS O
- O
Di O
sk O
Operating O
System O

3 O
. O
7k O
Octal O
Standard O
High O

Level O
Dictionary O
1 O
. O
2k O
Octal O
Initial O
Machine O

Code O
Dictionary O

I O
@ O
@ O
Octal O

Figure O
4 O
. O

Memory O
map O
of O
the O
CON O
VERS O
dictionary O
. O

The O
stack O
which O
is O
composed O
of O
data O
parameters O
etc O
. O
acts O
as O
a O
constantly O
expanding O
or O
contracting O
memory O
buffer O
which O
allows O
one O
subroutine O
to O
communicate O
with O
another O
without O
either O
needing O
to O
know O
the O
other O
' O
s O
location O
. O

In O
contrast O
to O
interpreters O
, O
high O
level O
compilers O
, O
such O
as O
FORTRAN O
, O
offer O
a O
much O
faster O
" O
run O
time O
" O
execution O
speed O
. O

This O
is O
accomplished O
through O
generation O
of O
the O
required O
machine O
code O
during O
a O
series O
of O
programming O
operations O
. O

Compilers O
using O
FORTRAN O
, O
which O
are O
designed O
to O
run O
on O
many O
minicomputers O
and O
some O
micros O
, O
often O
first O
transform O
user O
symbolic O
source O
code O
into O
assembly O
code O
. O

An O
assembler O
program O
, O
subsequently O
, O
transforms O
this O
into O
the O
required O
machine O
code O
. O

This O
ready O
- O
to O
- O
run O
machine O
code O
is O
often O
loaded O
along O
with O
a O
run O
time O
package O
which O
executes O
in O
the O
manner O
shown O
in O
Figure O
2 O
. O

While O
this O
approach O
greatly O
improves O
execution O
speed O
, O
the O
need O
for O
loading O
several O
different O
soft O
- O
ware O
routines O
increases O
the O
" O
hassle O
" O
associated O
with O
editing O
and O
debugging O
. O

Thus O
, O
this O
makes O
some O
form O
of O
mass O
memory O
, O
such O
as O
a O
disk O
or O
magnetic O
tape O
, O
almost O
mandatory O
. O

Additionally O
, O
I O
/ O
O O
algorithms O
generally O
must O
be O
implemented O
in O
assembly O
level O
code O
! O

One O
obvious O
question O
immediately O
arises O
- O
why O
not O
incorporate O
the O
most O
desirable O
characteristics O
of O
both O
interpreters O
and O
compilers O
into O
a O
single O
language O
? O

Additionally O
, O
due O
to O
the O
unique O
requirements O
found O
in O
many O
applications O
, O
why O
not O
allow O
the O
programmer O
additional O
flexibility O
by O
providing O
him O
with O
the O
ability O
to O
actually O
develop O
his O
own O
individual O
modifications O
and O
additions O
to O
the O
language O
itself O
? O

Other O
desirable O
features O
would O
include O
high O
memory O
efficiency O
, O
high O
level O
I O
] O
O O
programming O
, O
ease O
of O
understanding O
the O
language O
' O
s O
" O
inter O
- O
working O
" O
and O
the O
ability O
to O
be O
transferred O
from O
one O
CPU O
to O
another O
with O
minimum O
effort O
. O

Type O
" O
ACQUIRE O
" O

ACQUIRE O

# O
of O
characters O

A O
G O

FORMAT O
T O
PLOT O

START O
SCAN O
GALC O
% O
T O

DISPLAY O
RETURN O

EXECUTIVE O

Figure O
5 O
. O

If O
a O
previously O
defined O
program O
name O
( O
A O
CO O
U O
R O
tQ O
is O
entered O
when O
in O
EXECUTE O
mode O
, O
a O
dictionary O
search O
takes O
place O
locating O
the O
ACQUIRE O
entry O
. O

Once O
found O
, O
this O
entry O
contains O
all O
the O
required O
machine O
code O
and O
/ O
or O
calls O
to O
addresses O
of O
other O
previously O
compiled O
machine O
code O
modules O
to O
completely O
execute O
the O
desired O
function O
. O

130 O

The O
Journal O
of O
Automatic O
Chemistry O

Tilden O
& O
Denton O

Advanced O
software O
concepts O

2000 O

VARIABLE O
VARIABLE O

START O
STOP O

5000 O
20 O

VARIABLE O
VARIABLE O
7 O

INCREMENT O

0 O

LOCATION O

A O
/ O
D7 O

INDEVICE O
START O

INITIALIZE O
STEP O

@ O

LOCATION O

LOCATION O

@ O

INCREMENT O
5 O
INDEVICE O

@ O

+ O
10 O

LOCATION O

DELAY O
MOVE O
SCAN O

BEGIN O
- O
HERE O
LOCATION O

AND O

IF O
DELAY O

A O
/ O
D7 O

ELSE O

BEGIN O

THEN O

@ O

5 O

OUTDEVICE O

STEP O

INITIALIZE O

BEGIN O
- O
HERE O

MOVE O

LOCATION O

@ O

STOP O

@ O

> O

IF O

END O

ELSE O

BEGIN O

THEN O

3000 O
6500 O
10 O

START O
START O

INCREMENT O

SCAN O

Figure O
6 O
. O

Ten O
lines O
of O
typical O
CONVERS O
code O
to O
scan O
wavelengths O
between O
two O
easily O
changed O
limits O
and O
acquire O
data O
. O

By O
reference O
to O
notes O
in O
the O
text O
it O
can O
be O
easily O
understood O
. O

NOTE O
: O
a O
number O
or O
name O
pushes O
the O
number O
of O
address O
occupied O
by O
the O
name O
on O
the O
stack O
. O

The O
symbol O
@ O
, O
pips O
the O
top O
number O
from O
the O
stack O
uses O
it O
as O
the O
address O
from O
which O
to O
obtain O
a O
number O
and O
pushes O
that O
number O
on O
the O
stack O
. O

Development O
of O
CONVERS O
During O
the O
past O
two O
years O
, O
a O
different O
approach O
to O
software O
has O
been O
taking O
place O
at O
the O
University O
of O
Arizona O
referred O
to O
as O
an O
" O
Interpretive O
Compiler O
" O
called O
CONVERS O
. O

This O
package O
, O
which O
is O
conceptually O
similar O
to O
the O
FORTH O
language O
currently O
being O
used O
in O
several O
minicomputerastronom O
applications O
] O
, O
is O
able O
to O
provide O
many O
of O
the O
desirable O
features O
found O
in O
both O
interpreters O
and O
compilers O
by O
separating O
the O
compile O
and O
execute O
states O
( O
as O
a O
compiler O
does O
) O
while O
maintaining O
a O
resident O
user O
interactive O
and O
conversational O
executive O
which O
oversees O
system O
operation O
. O

The O
ability O
to O
realise O
such O
advanced O
software O
capabilities O
in O
a O
very O
modest O
amount O
of O
memory O
( O
less O
than O
4 O
K O
bytes O
on O
an O
8080 O
based O
micro O
) O
is O
the O
direct O
result O
of O
exploiting O
threaded O
code O
programming O
techniques O
( O
see O
Figure O
3 O
) O
. O

The O
approach O
involves O
highly O
efficient O
use O
of O
simple O
macroinstructions O
to O
build O
more O
complex O
subroutines O
which O
are O
recombined O
with O
additional O
macroinstructions O
to O
form O
super O
subroutines O
. O

This O
process O
of O
combining O
previously O
defined O
modules O
to O
form O
ever O
increasingly O
sophisticated O
routines O
for O
performing O
the O
task O
at O
hand O
is O
the O
essence O
of O
threaded O
code O
programming O
. O

When O
initially O
loaded O
and O
running O
, O
CONVERS O
acts O
much O
like O
an O
interpreter O
, O
i O
. O
e O
. O
it O
is O
conversational O
, O
ready O
to O
either O
execute O
a O
previously O
programmed O
algorithm O
or O
accept O
a O
new O
one O
. O

However O
, O
in O
contrast O
to O
BASIC O
, O
when O
a O
new O
program O
is O
being O
entered O
under O
CONVERS O
, O
it O
is O
immediately O
transformed O
into O
binary O
machine O
code O
or O
to O
the O
binary O
starting O
addresses O
of O
other O
previously O
entered O
and O
compiled O
machine O
code O
programs O
. O

During O
this O
process O
, O
the O
operator O
is O
kept O
informed O
of O
the O
status O
of O
the O
program O
by O
a O
series O
of O
error O
and O
diagnostic O
Volume O

messages O
. O

When O
the O
new O
program O
has O
been O
completed O
, O
it O
is O
entered O
in O
a O
program O
library O
or O
dictionary O
, O
which O
is O
constantly O
building O
up O
from O
low O
memory O
( O
see O
Figure O
4 O
) O
. O

If O
the O
operator O
now O
wants O
to O
execute O
this O
program O
, O
he O
can O
request O
it O
from O
his O
terminal O
. O

A O
dictionary O
search O
will O
begin O
at O
the O
last O
entry O
and O
progress O
until O
the O
requested O
program O
is O
located O
. O

Once O
located O
, O
the O
requested O
program O
will O
run O
in O
its O
entirety O
without O
need O
for O
any O
additional O
dictionary O
searches O
. O

For O
example O
, O
let O
us O
assume O
an O
algorithm O
, O
called O
ACQUIRE O
, O
has O
been O
programmed O
to O
take O
data O
from O
some O
hypothetical O
experimental O
system O
. O

When O
ACQUIRE O
is O
requested O
from O
the O
terminal O
, O
a O
dictionary O
search O
is O
initiated O
. O

The O
program O
names O
ACQUIRE O
( O
see O
Figure O
5 O
) O
, O
once O
located O
, O
contains O
the O
starting O
addresses O
of O
a O
series O
of O
previously O
defined O
modules O
which O
implement O
the O
various O
steps O
necessary O
to O
perform O
the O
desired O
experiment O
. O

For O
example O
, O
the O
module O
SCAN O
which O
might O
be O
intended O
to O
scan O
a O
monochromator O
' O
s O
wavelength O
in O
some O
desired O
manner O
has O
been O
previously O
defined O
and O
tested O
. O

This O
ability O
to O
easily O
test O
each O
module O
separately O
and O
then O
efficiently O
combine O
a O
series O
of O
modules O
to O
perform O
a O
more O
complex O
function O
, O
test O
this O
function O
, O
and O
then O
employ O
. O
it O
in O
a O
vastly O
more O
complex O
function O
, O
etc O
. O
, O
i O
. O
e O
. O
testing O
each O
step O
as O
the O
threaded O
code O
is O
made O
increasingly O
complex O
, O
is O
a O
major O
factor O
contributing O
to O
the O
speed O
with O
which O
software O
can O
be O
developed O
using O
CONVERS O
. O

Use O
of O
a O
software O
stack O
also O
contributes O
toward O
improved O
memory O
efficiency O
and O
simplified O
programming O
. O

The O
stack O
is O
an O
area O
of O
memory O
set O
aside O
to O
handle O
parameters O
, O
data O
numbers O
, O
etc O
. O

One O
of O
the O
primary O
advantages O
of O
the O
stack O
is O
that O
entries O
can O
leave O
temporary O
parameters O
on O
the O
stack O
without O
having O
to O
assign O
specific O
131 O

No O
. O
3 O
April O
1979 O

Tilden O
& O
Denton O

Advanced O
software O
concepts O

DUAL O
REGULATED O
POWER O
SUPPLY O
I0 O
KV O
I00 O

GRATING O

PIEZOELECTRIC O
TRANSLATOR O

OP O
TOACOUS O
TIC O
CELL O

, O
. O
. O

C02He O
, O
N2 O

MIRROR O
TRANSLATOR O

CHOPPER O

POWER O
DE O
TECTOR O

STEPPER O
MOTOR O

DRIVER O

ADC O

LOCK O
- O
IN O
AMP O

CONTROLLER O

LOCK O
- O
IN O

AMP O

MICROFLOPPY O

DISK O
24K O
RAM O

1 O

Figure O
7 O
. O

The O
optoacoustic O
experiment O
in O
which O
a O
microcomputer O
is O
used O
to O
control O
laser O
wavelength O
and O
to O
monitor O
laser O
power O
and O
optoacoustic O
signal O
. O

memory O
locations O
to O
store O
them O
. O

This O
not O
only O
can O
save O
considerable O
memory O
, O
but O
also O
allows O
programs O
to O
be O
easily O
relocatable O
since O
one O
algorithm O
need O
only O
know O
that O
a O
previous O
routine O
left O
so O
many O
words O
of O
data O
, O
etc O
. O
on O
the O
stack O
. O

It O
need O
not O
know O
where O
the O
previous O
routine O
is O
nor O
even O
where O
the O
stack O
is O
located O
. O

A O
series O
of O
stack O
handling O
routines O
, O
which O
should O
appear O
quite O
familiar O
to O
many O
small O
calculator O
users O
, O
provide O
an O
array O
of O
capabilities O
, O
including O
the O
ability O
to O
PUSH O
a O
number O
on O
the O
stack O
, O
POP O
, O
it O
off O
, O
duplicate O
it O
, O
SWAP O
the O
top O
two O
numbers O
, O
locate O
a O
number O
some O
distance O
into O
the O
stack O
, O
and O
copy O
it O
on O
top O
of O
the O
stack O
, O
etc O
. O

Additionally O
, O
a O
variety O
of O
logic O
functions O
familiar O
to O
the O
minicomputer O
user O
are O
provided O
including O
OR O
, O
AND O
, O
shift O
left O
, O
shift O
right O
, O
greater O
than O
, O
less O
than O
, O
etc O
. O

Input O
/ O
output O
( O
I O
/ O
O O
) O
is O
normally O
accomplished O
using O
the O
stack O
in O
conjunction O
with O
the O
INDEVICE O
or O
OUTDEVICE O
commands O
. O

For O
example O
, O
to O
take O
data O
from O
a O
device O
located O
at O
I O
/ O
O O
, O
port O
7 O
, O
the O
number O
seven O
is O
" O
pushed O
" O
onto O
the O
stack O
( O
7 O
) O
, O
goes O
to O
this O
I O
/ O
O O
port O
, O
takes O
in O
a O
number O
and O
" O
pushes O
" O
the O
number O
on O
the O
stack O
. O

OUTDEVICE O
functions O
in O
a O
similar O
manner O
, O
requiring O
the O
number O
to O
be O
sent O
to O
the O
desired O
device O
to O
be O
" O
pushed O
" O
onto O
the O
stack O
followed O
by O
the O
device O
' O
s O
I O
/ O
O O
port O
address O
. O

Hence O
, O
to O
send O
the O
number O
131 O
to O
device O
11 O
, O
the O
number O
131 O
is O
pushed O
on O
the O
stack O
followed O
by O
11 O
and O
then O
OUTDEVICE O
. O

This O
" O
pops O
" O
the O
top O
number O
( O
11 O
) O
from O
the O
stack O
, O
uses O
it O
as O
the O
output O
port O
and O
then O
sends O
the O
number O
131 O
to O
that O
location O
. O

SOUND O

BELL O

BELL O

BELL O

The O
colon O
denotes O
changing O
from O
EXECUTE O
to O
COMPILE O
mode O
. O

After O
typing O
the O
name O
of O
the O
new O
routine O
, O
in O
this O
case O
to O
be O
called O
SOUND O
, O
typing O
the O
name O
of O
the O
earlier O
defined O
routine O
( O
BELL O
a O
previously O
defined O
simple O
program O
to O
rng O
the O
terminal O
bell O
) O
initiates O
a O
dictionary O
search O
to O
locate O
this O
routine O
' O
s O
starting O
address O
which O
subsequentially O
is O
entered O
three O
times O
. O

The O
resulting O
SOUND O
routine O
contains O
machine O
code O
calls O
to O
the O
BE O
LL O
routine O
which O
, O
itself O
, O
is O
composed O
of O
machine O
code O
. O

Of O
course O
, O
SOUND O
could O
also O
have O
been O
defined O
using O
a O
DO O
- O
LOOP O
, O
i O
. O
e O
. O
SOUND O
3 O

DO O

BELL O

LOOP O

where O
the O
numbers O
three O
and O
one O
set O
the O
upper O
and O
lower O
indices O
. O

If O
it O
were O
desirable O
to O
change O
the O
actual O
number O
of O
bell O
rings O
from O
some O
other O
program O
, O
this O
value O
could O
be O
defined O
as O
a O
VARIABLE O
let O
' O
s O
call O
it O
NOISE O
. O

3 O

VARIABLE O
NOISE O

In O
this O
case O
, O
the O
number O
three O
is O
first O
pushed O
on O
the O
stack O
, O
VARIABLE O
transfers O
the O
top O
number O
on O
the O
stack O
( O
the O
three O
) O
to O
a O
dictionary O
location O
named O
NOISE O
. O

If O
SOUND O
were O
now O
defined O
as O
: O
SOUND O

NOISE O

@ O

DO O

BELL O

LOOP O

Applications O
of O
CONVERS O
To O
appreciate O
the O
ease O
with O
which O
real O
programs O
can O
be O
written O
, O
a O
few O
examples O
will O
be O
considered O
. O

A O
trivial O
program O
, O
called O
SOUND O
, O
which O
rings O
the O
terminal O
bell O
three O
times O
, O
might O
be O
written O
: O
132 O

the O
bell O
would O
again O
ring O
three O
times O
. O

In O
this O
case O
, O
when O
the O
word O
NOISE O
iS O
encountered O
, O
its O
address O
is O
pushed O
on O
the O
stack O
, O
the O
@ O
is O
a O
simple O
program O
which O
goes O
to O
the O
address O
indicated O
on O
the O
top O
of O
the O
stack O
( O
that O
of O
NOISE O
) O
and O
replaces O
it O
with O
the O
actual O
value O
located O
at O
that O
address O
( O
the O
number O
three O
) O
. O

At O
any O
future O
time O
, O
the O
value O
of O
VARIABLE O
can O
be O
changed O
by O
" O
pushing O
" O
the O
new O
value O
onto O
the O
stack O
, O
followed O
by O
the O
address O
of O
the O
variable O
to O
be O
changed O
, O
generated O
by O
its O
name O
and O
an O
exclamation O
mark O
. O

To O
change O
N O
O O
IS O
E O
to O
5 O
, O
The O
Journal O
of O
Automatic O
Chemistry O

Tilden O
& O
Denton O

Advanced O
software O
concepts O

5 O
NOISE O
a O
number O
five O
is O
pushed O
onto O
the O
stack O
, O
NOISE O
pushes O
its O
address O
on O
the O
stack O
, O
and O
goes O
to O
the O
address O
indicated O
by O
the O
" O
top O
" O
number O
on O
the O
stack O
and O
deposits O
the O
next O
number O
. O

Now O
sound O
would O
ring O
the O
terminal O
bell O
five O
times O
. O

A O
much O
less O
trivial O
program O
which O
could O
be O
written O
to O
scan O
and O
take O
data O
from O
a O
monochromator O
equipped O
with O
a O

DENCO O
SM2A O
stepper O
motor O
controller O
[ O
2 O
] O
( O
the O
SM2A O
takes O
a O
parallel O
number O
as O
an O
address O
, O
sends O
one O
of O
two O
stepper O
motors O
to O
this O
location O
and O
outputs O
an O
arrival O
flag O
when O
the O
address O
is O
reached O
) O
is O
given O
in O
Figure O
6 O
. O

Assume O
that O
the O
experimental O
system O
is O
configured O
so O
the O
SM2A O
is O
at O
I O
/ O
O O
, O
port O
5 O
and O
an O
analog O
to O
digital O
converter O
to O
acquire O
data O
is O
at O
I O
/ O
O O
, O
port O
7 O
. O

Let O
us O
assume O
that O
, O
initially O
, O
a O
scan O
is O
designed O
from O
a O
starting O
stepper O
motor O
location O
of O
2000 O
to O
a O
final O
location O
of O
5000 O
, O
taking O
data O
every O
20 O
steps O
. O

The O
program O
illustrated O
in O
figure O
6 O
acts O
in O
the O
following O
manner O
. O

Line O
defines O
a O
variable O
called O
START O
to O
be O
2000 O
, O
which O
is O
the O
starting O
location O
of O
fhe O
scan O
. O

The O
end O
of O
the O
scan O
is O
defined O
as O
5000 O
in O
the O
next O
line O
. O

Line O
3 O
defines O
the O
increment O
between O
data O
points O
. O

A O
variable O
called O
LOCATION O
where O
the O
. O
next O
address O
is O
stored O
is O
defined O
in O
line O
4 O
. O
. O

Colon O
, O
in O
line O
5 O
, O
puts O
the O
system O
in O
the O
compile O
mode O
, O
A O
/ O
D7 O
will O
be O
the O
name O
of O
the O
module O
which O
when O
called O
will O
cause O
a O
7 O
to O
be O
pushed O
onto O
the O
stack O
. O

INDEVICE O
will O
' O
pop O
' O
it O
back O
off O
and O
use O
it O
as O
a O
device O
address O
to O
go O
and O
take O
a O
data O
point O
and O
push O
the O
data O
onto O
the O
stack O
. O

Line O
6 O
defines O
a O
module O
INITIALIZE O
, O
START O
puts O
its O
address O
on O
the O
stack O
, O
@ O
replaces O
the O
address O
with O
the O
value O
at O
that O
address O
, O
LOCATION O
puts O
its O
address O
on O
the O
stack O
, O
' O
! O
' O
goes O
to O
the O
address O
specified O
by O
the O
number O
on O
the O
stack O
and O
deposits O
the O
second O
number O
and O
the O
net O
result O
value O
at O
START O
is O
put O
into O
LOCATION O
. O

Line O
7 O
defines O
STEP O
to O
take O
values O
from O

LOCATION O
and O
INCREMENT O
adds O
them O
together O
and O
puts O
the O
result O
into O
LOCATION O
, O
i O
. O
e O
. O
LOCATION O
puts O
its O
address O
on O
the O
stack O
, O
@ O
replaces O
the O
top O
value O
on O
the O
stack O
with O
the O
number O
stored O
at O
the O
address O
, O
INCREMENT O
@ O
gets O
the O
value O
at O
INCREMENT O
and O
puts O
it O
in O
to O
the O
stack O
, O
and O
adds O
the O
top O
two O
stack O
numbers O
and O
pushes O
the O
result O
on O
the O
stack O
. O

LOCATION O
puts O
its O
address O
on O
the O
stack O
and O
goes O
to O
the O
address O
specified O
by O
the O
top O
number O
on O
the O
stack O
and O
deposits O
the O
second O
number O
. O

Themodule O
defined O
in O
line O
8 O
takes O
a O
number O
from O
the O
stepper O
motor O
controller O
( O
assume O
device O
numbered O
5 O
) O
pushes O
on O
the O
stack O
, O
and O
pushes O
the O
value O
10 O
on O
the O
stack O
and O
does O
a O
logical O
AND O
to O
see O
if O
the O
controllers O
flag O
is O
set O
, O
if O
this O
is O
true O
the O
A O
/ O
D7 O
module O
will O
be O
called O
to O
input O
data O
, O
if O
the O
flag O
is O
not O
set O
, O
the O
program O
is O
returned O
to O
BEGIN O
- O
HERE O
. O

Therefore O
the O
DELAY O
module O
is O
a O
loop O
waiting O
for O
the O
stepper O
motor O
to O
arrive O
at O
its O
new O
location O
followed O
by O
a O
call O
to O
A O
/ O
D7 O
data O
acquisition O
module O
. O

MOVE O
defined O
in O
line O
9 O
gets O
the O
value O
stored O
at O
LOCATION O
pushes O
the O
device O
code O
onto O
the O
stack O
performs O
an O
outdevice O
( O
which O
uses O
the O
top O
stack O
number O
as O
a O
1 O
/ O
0 O
port O
address O
to O
send O
the O
next O
value O
to O
call O
the O
STEP O
module O
( O
ie O
the O
value O
at O
LOCATION O
) O
, O
this O
increments O
LOCATION O
by O
the O
INCREM O
E O
N O
T O
value O
and O
finally O
calls O
D O
E O
L O
A O
Y O
and O
waits O
for O
a O
flag O
from O
the O
stepper O
motor O
controller O
to O
signal O
its O
arrival O
at O
the O
desired O
address O
and O
then O
takes O
a O
data O
point O
. O

The O
final O
line O
shown O
in O
the O
example O
Called O
SCAN O
itself O
calls O
the O
INITIALIZE O
module O
( O
which O
sets O
LOCATION O
to O
the O
START O
location O
for O
the O
Scan O
) O
it O
also O
calls O
MOVE O
( O
which O
sends O
this O
value O
to O
the O
motor O
LOCATION O
controller O
, O
increments O
the O
value O
stored O
in O
LOCATION O
by O
INCREMENT O
and O
calls O
DELAY O
which O
waits O
for O
the O
motor O
to O
arrive O
and O
then O
takes O
a O
data O
point O
) O
. O

Next O
the O
incremented O
value O
from O
LOCATION O
is O
placed O
on O
the O
stack O
( O
LOCATION O
) O
followed O
by O
the O
STOP O

I0 O
KW O
AMPLIFIER O
MATCHING O

, O
NETWORKIS O
PREAMP O

MOTOR O
CON O
TROL O
ER O

MICRODISK O
90K O
byte O

INTEL O
8080 O
based O
MICROCOMPUTER O
16K O

RAM O

Figure O
8 O
. O

A O
schematic O
of O
the O
inductively O
coupled O
plasma O
emission O
spectrometer O
in O
which O
the O
microcomputer O
is O
used O
to O
control O
radio O
frequency O
power O
and O
' O
flame O
' O
positioning O
as O
well O
as O
to O
monitor O
light O
intensity O
. O

Volume O

No O
. O
3 O
April O
1979 O

133 O

Tilden O
& O
Denton O

Advanced O
software O
concepts O

value O
( O
STOP O
@ O
) O
, O
the O
two O
are O
compared O
to O
to O
see O
if O
the O
incremented O
value O
at O
LOCATION O
is O
larger O
then O
the O
STOP O
value O
, O
if O
it O
is O
the O
program O
ends O
, O
if O
not O
, O
it O
repeats O
the O
process O
starting O
at O
BEGIN O
- O
- O
HERE O
. O

It O
should O
be O
noted O
that O
whilst O
many O
variables O
have O
been O
pushed O
on O
the O
stack O
, O
only O
the O
data O
will O
remain O
, O
since O
each O
time O
a O
value O
is O
used O
it O
is O
' O
popped O
' O
( O
removed O
) O
from O
the O
stack O
. O

If O
a O
different O
spectra O
region O
is O
to O
be O
scanned O
i O
. O
e O
. O
from O
3000 O
to O
6500 O
with O
10 O
increments O
the O
variables O
need O
only O
be O

> O

data O
acquisition O
programs O
have O
been O
easily O
implemented O
. O

Memory O
requirements O
and O
operating O
speed O
have O
been O
found O
to O
be O
far O
superior O
to O
conventional O
approaches O
. O

Additionally O
, O
new O
system O
users O
have O
encountered O
a O
difficulty O
in O
utilising O
previously O
developed O
custom O
software O
for O
a O
particular O
experiment O
even O
when O
documentation O
was O
vague O
. O

Discussion O
The O
authors O
hope O
that O
this O
short O
introduction O
to O
only O
a O
few O
of O
the O
concepts O
employed O
in O
CONVERS O
will O
generate O
interest O
in O
its O
capabilities O
. O

A O
much O
more O
complete O
discussion O
is O
available O
in O
the O
form O
of O
a O
user O
' O
s O
manual O
[ O
3 O
] O
available O
from O
the O
authors O
. O

changed O
thus O
3000 O
START O
! O

6500 O
STOP O
10 O
INCREMENT O
and O
type O
SCAN O
, O
system O
will O
now O
scan O
from O
3000 O
to O
6500 O
taking O
data O
every O
10 O
steps O
. O

While O
the O
code O
might O
look O
a O
little O
strange O
at O
first O
, O
it O
quickly O
becomes O
very O
easy O
to O
work O
with O
. O

The O
SCAN O
program O
of O
Figure O
6 O
could O
be O
combined O
with O
other O
modules O
as O
shown O
in O
Figure O
5 O
to O
perform O
some O
more O
complex O
experimental O
function O
. O

Each O
module O
of O
the O
program O
can O
be O
easily O
tried O
out O
to O
ensure O
that O
it O
is O
operational O
before O
proceeding O
with O
the O
next O
. O

Presently O
, O
CONVERS O
is O
being O
used O
in O
the O
authors O
' O
laboratories O
for O
a O
variety O
of O
spectrochemical O
investigations O
, O
including O
laser O
excited O
optoacoustic O
spectroscopy O
( O
Figure O
7 O
) O
and O
inductively O
coupled O
plasma O
optical O
emission O
spectroscopy O
( O
Figure O
8 O
) O
. O

Rather O
complex O
interactive O
control O
and O

ACKNOWLEDGEMENTS O
The O
development O
of O
the O
CONVERS O
system O
was O
partially O
supported O
by O
the O
Office O
of O
Naval O
Research O
and O
a O
Alfred O
P O
. O
Cloan O
Foundation O
Research O
Fellowship O
to O
M O
. O

Bonnet O
Denton O
. O

REFERENCES O
C O
. O

Moore O
, O
Astronomical O
Astrophysics O
Supplemental O
, O
15 O
, O
( O
1974 O
) O
497 O
. O

[ O
2 O
] O
M O
. O
B O
. O

Denton O
, O
J O
. O
D O
. O

Mack O
, O
M O
. O
W O
. O

Routh O
and O
D O
. O
B O
. O

SwartzClmerican O

[ O
3 O
] O

Laboratory O
, O
8 O
, O
69 O
( O
1976 O
) O
. O

CONVERS O
An O
Interpretive O
Compiler O
, O
developed O
by O
Scott O
B O
. O
Tilden O
and O
M O
. O

Bonner O
Denton O
, O
Department O
of O
Chemistry O
, O
University O
of O
Arizona O
, O
Tucson O
, O
Arizona O
85721 O
, O
USA O
. O

The O
use O
of O
a O
microcomputer O
for O
flexible O
automation O
of O
a O
liquid O

chromatograph O
A O
. O
D O
. O

Mills O
, O
i O
. O

Mackenzie O
and O
R O
. O
J O
. O

Dolphin O
* O
Philips O
Research O
Laboratories O
, O
Redhill O
, O
Surrey O
, O
RH1 O
5HA O
, O
U O
. O
K O
. O

Introduction O
Microprocessors O
are O
being O
used O
to O
add O
inexpensive O
automatic O
control O
and O
data O
handling O
facilities O
to O
a O
variety O
of O
chemical O
instruments O
. O

With O
a O
microcomputer O
it O
is O
now O
possible O
to O
realise O
the O
flexibility O
formerly O
available O
only O
with O
a O
relatively O
large O
and O
expensive O
minicomputer O
in O
an O
instrument O
little O
different O
in O
size O
and O
cost O
from O
one O
controlled O
by O
inflexible O
hardware O
. O

In O
many O
ways O
chromatography O
is O
an O
ideal O
process O
for O
such O
automation O
. O

Most O
instruments O
are O
given O
a O
high O
workload O
and O
, O
although O
many O
applications O
may O
be O
routine O
and O
repetitive O
, O
the O
versatility O
of O
the O
technique O
requires O
an O
instrument O
which O
can O
easily O
be O
used O
in O
a O
variety O
of O
modes O
. O

In O
addition O
to O
improving O
the O
convenience O
to O
the O
user O
, O
automation O
of O
a O
liquid O
chromatrograph O
should O
enhance O
the O
performance O
of O
the O
instrument O
. O

Some O
aspects O
of O
high O
performance O
liquid O
chromatography O
( O
HPLC O
) O
which O
can O
benefit O
in O
this O
way O
are O
as O
follows O
: O
( O
1 O
) O
Accurate O
control O
of O
solvent O
flow O
rate O
will O
compensate O
for O
changes O
in O
pressure O
drop O
and O
lead O
to O
more O
reliable O
retention O
times O
. O

( O
2 O
) O
The O
composition O
of O
the O
mobile O
phase O
can O
be O
accurately O
controlled O
in O
either O
isocratic O
or O
gradient O
elution O
chromatography O
using O
, O
for O
example O
, O
a O
proportioning O
valve O
on O
the O
low O
pressure O
side O
of O
the O
pump O
. O

( O
3 O
) O
Automatic O
sampling O
can O
be O
operated O
in O
a O
variety O
of O
modes O
to O
process O
a O
number O
of O
samples O
without O
supervision O
. O

It O
is O
also O
more O
precise O
than O
manual O
injection O
. O

( O
4 O
) O
A O
built O
- O
in O
data O
handling O
facility O
can O
present O
the O
analyst O
with O
an O
easily O
read O
post O
- O
run O
report O
of O
the O
analytical O
results O
with O
accurate O
peak O
area O
measurements O
even O
for O
peaks O
which O
are O
poorly O
resolved O
. O

Although O
liquid O
chromatographs O
incorporating O
microprocessors O
for O
control O
and O
data O
handling O
purposes O
are O
commercially O
available O
, O
these O
instruments O
are O
, O
so O
far O
, O
This O
paper O
describes O
, O
in O
detail O
, O
the O
relatively O
inflexible O
. O
automation O
of O
a O
liquid O
chromatograph O
using O
an O
inexpensive O
general O
purpose O
microcomputer O
, O
which O
has O
previously O
been O
applied O
in O
atomic O
absorption O
spectrophotometry O
[ O
1 O
] O
and O
for O
column O
switching O
in O
HPLC O
2 O
] O
. O

Figure O
illustrates O
the O
interconnection O
of O
the O
chromatograph O
and O
the O
microcomputer O
which O
controls O
the O
mobile O
phase O
flow O
rate O
, O
operates O
an O
automatic O
sampler O
and O
analyses O
data O
from O
the O
detector O
. O

The O
control O
and O
data O
handling O
functions O
are O
integrated O
in O
a O
program O
which O
enables O
the O
user O
to O
communicate O
with O
the O
instrument O
, O
in O
plain O
English O
, O
via O
a O
visual O
display O
unit O
( O
VDU O
) O
or O
teletypewriter O
keyboard O
. O

A O
variety O
of O
operational O
modes O
is O
offered O
, O
giving O
the O
analyst O
an O
opportunity O
to O
establish O
the O
best O
conditions O
for O
a O
particular O
separation O
before O
leaving O
the O
instrument O
to O
perform O
its O
given O
tasks O
without O
further O
interaction O
. O

The O
microcomputer O
Hardware O
The O
computer O
is O
a O
general O
purpose O
instrument O
constructed O
using O
a O
set O
of O
ready O
made O
circuit O
cards O
( O
Philips O
, O
Science O
and O
The O
Journal O
of O
Automatic O
Chemistry O

* O
Present O
address O
: O
lye O
Unicam O
Ltd O
. O
, O
York O
Street O
, O
Cambridge O
, O
CB1 O
2PX O
, O
U O
. O
K O
. O

134 O

TOPS O
+ O
+ O
FATCAT O
: O
Fast O
flexible O
structural O
alignment O
using O
constraints O
derived O
from O
TOPS O
+ O
Strings O
Model O

Abstract O

Background O

Protein O
structure O
analysis O
and O
comparison O
are O
major O
challenges O
in O
structural O
bioinformatics O
. O

Despite O
the O
existence O
of O
many O
tools O
and O
algorithms O
, O
very O
few O
of O
them O
have O
managed O
to O
capture O
the O
intuitive O
understanding O
of O
protein O
structures O
developed O
in O
structural O
biology O
, O
especially O
in O
the O
context O
of O
rapid O
database O
searches O
. O

Such O
intuitions O
could O
help O
speed O
up O
similarity O
searches O
and O
make O
it O
easier O
to O
understand O
the O
results O
of O
such O
analyses O
. O

Results O

We O
developed O
a O
TOPS O
+ O
+ O
FATCAT O
algorithm O
that O
uses O
an O
intuitive O
description O
of O
the O
proteins O
' O
structures O
as O
captured O
in O
the O
popular O
TOPS O
diagrams O
to O
limit O
the O
search O
space O
of O
the O
aligned O
fragment O
pairs O
( O
AFPs O
) O
in O
the O
flexible O
alignment O
of O
protein O
structures O
performed O
by O
the O
FATCAT O
algorithm O
. O

The O
TOPS O
+ O
+ O
FATCAT O
algorithm O
is O
faster O
than O
FATCAT O
by O
more O
than O
an O
order O
of O
magnitude O
with O
a O
minimal O
cost O
in O
classification O
and O
alignment O
accuracy O
. O

For O
beta O
- O
rich O
proteins O
its O
accuracy O
is O
better O
than O
FATCAT O
, O
because O
the O
TOPS O
+ O
strings O
models O
contains O
important O
information O
of O
the O
parallel O
and O
anti O
- O
parallel O
hydrogen O
- O
bond O
patterns O
between O
the O
beta O
- O
strand O
SSEs O
( O
Secondary O
Structural O
Elements O
) O
. O

We O
show O
that O
the O
TOPS O
+ O
+ O
FATCAT O
errors O
, O
rare O
as O
they O
are O
, O
can O
be O
clearly O
linked O
to O
oversimplifications O
of O
the O
TOPS O
diagrams O
and O
can O
be O
corrected O
by O
the O
development O
of O
more O
precise O
secondary O
structure O
element O
definitions O
. O

Software O
Availability O

The O
benchmark O
analysis O
results O
and O
the O
compressed O
archive O
of O
the O
TOPS O
+ O
+ O
FATCAT O
program O
for O
Linux O
platform O
can O
be O
downloaded O
from O
the O
following O
web O
site O
: O

Conclusion O

TOPS O
+ O
+ O
FATCAT O
provides O
FATCAT O
accuracy O
and O
insights O
into O
protein O
structural O
changes O
at O
a O
speed O
comparable O
to O
sequence O
alignments O
, O
opening O
up O
a O
possibility O
of O
interactive O
protein O
structure O
similarity O
searches O
. O

Background O

Structural O
biology O
is O
one O
of O
the O
most O
successful O
fields O
of O
modern O
biology O
. O

Over O
50 O
, O
000 O
solved O
protein O
structures O
illustrate O
details O
of O
many O
specific O
biological O
processes O
. O

The O
same O
data O
also O
provide O
us O
with O
information O
about O
the O
global O
features O
of O
protein O
structure O
space O
and O
can O
be O
studied O
to O
discover O
the O
evolutionary O
, O
physical O
, O
and O
mathematical O
rules O
governing O
them O
. O

How O
many O
fundamentally O
different O
protein O
shapes O
( O
folds O
) O
are O
there O
? O

How O
do O
protein O
structures O
evolve O
? O

How O
do O
new O
structural O
features O
appear O
, O
and O
if O
they O
are O
coupled O
with O
changes O
in O
function O
, O
how O
does O
this O
process O
occur O
? O

Such O
questions O
can O
be O
studied O
by O
classifying O
, O
comparing O
and O
analyzing O
known O
protein O
structures O
. O

Two O
different O
, O
but O
synergistic O
strategies O
are O
typically O
used O
for O
this O
purpose O
. O

In O
classification O
systems O
such O
as O
SCOP O
[ O
1 O
] O
or O
CATH O
[ O
2 O
] O
, O
human B
intuition O
is O
used O
to O
simplify O
the O
description O
of O
protein O
structures O
to O
a O
manageable O
size O
, O
and O
a O
human B
eye O
, O
sometimes O
supported O
by O
automated O
analysis O
, O
can O
recognize O
patterns O
and O
types O
of O
structures O
. O

In O
the O
second O
approach O
, O
specialized O
comparison O
algorithms O
, O
such O
as O
DALI O
[ O
3 O
] O
, O
CE O
[ O
4 O
] O
, O
or O
FATCAT O
[ O
5 O
] O
can O
be O
used O
to O
calculate O
a O
distance O
- O
like O
metric O
in O
the O
protein O
structure O
space O
. O

This O
in O
turn O
can O
be O
used O
to O
cluster O
proteins O
into O
groups O
. O

Many O
such O
algorithms O
have O
been O
developed O
over O
the O
past O
few O
decades O
and O
have O
been O
mostly O
used O
for O
the O
classification O
of O
protein O
structures O
into O
families O
. O

An O
exact O
solution O
of O
an O
alignment O
between O
two O
structures O
is O
formally O
equivalent O
to O
a O
threading O
problem O
and O
is O
therefore O
NP O
- O
complete O
[ O
6 O
] O
. O

However O
, O
a O
practical O
solution O
can O
be O
obtained O
by O
heuristics O
reducing O
the O
problem O
to O
a O
manageable O
size O
[ O
7 O
] O
. O

In O
human B
classification O
systems O
, O
the O
protein O
is O
usually O
reduced O
to O
a O
set O
of O
several O
structural O
elements O
, O
which O
obviously O
involve O
many O
arbitrary O
thresholds O
. O

Automated O
algorithms O
have O
the O
same O
problem O
and O
also O
suffer O
from O
inconsistencies O
between O
different O
numerical O
measures O
of O
protein O
structure O
similarity O
[ O
8 O
] O
. O

Interestingly O
, O
despite O
these O
problems O
, O
results O
of O
different O
approaches O
are O
broadly O
similar O
. O

They O
all O
identify O
approximately O
a O
few O
hundred O
general O
classes O
of O
protein O
structures O
, O
usually O
called O
folds O
[ O
1 O
] O
or O
topologies O
[ O
2 O
] O
, O
distinguished O
by O
how O
the O
main O
chain O
of O
the O
protein O
folds O
around O
itself O
in O
the O
three O
- O
dimensional O
space O
. O

At O
the O
same O
time O
, O
the O
comparison O
of O
different O
approaches O
, O
both O
between O
and O
within O
the O
two O
classes O
, O
shows O
that O
fold O
/ O
topologies O
( O
or O
cluster O
) O
definitions O
are O
somewhat O
fuzzy O
, O
with O
some O
proteins O
being O
occasionally O
difficult O
to O
classify O
and O
joining O
different O
groups O
depending O
on O
various O
assumptions O
. O

This O
lead O
some O
to O
question O
the O
concept O
of O
the O
fold O
[ O
9 O
] O
, O
but O
practical O
application O
of O
protein O
structure O
comparison O
leaves O
little O
doubt O
that O
protein O
structure O
space O
has O
some O
natural O
granularity O
that O
overlaps O
well O
with O
the O
traditional O
fold O
classification O
. O

Comparison O
and O
classification O
of O
protein O
structures O
is O
significantly O
simplified O
by O
the O
fact O
that O
proteins O
have O
naturally O
modular O
structures O
, O
being O
mostly O
composed O
of O
locally O
regular O
structures O
: O
alpha O
helices O
and O
beta O
strands O
. O

These O
two O
types O
of O
secondary O
structures O
constitute O
a O
little O
over O
50 O
% O
of O
an O
average O
protein O
' O
s O
length O
. O

With O
the O
average O
length O
of O
a O
secondary O
structure O
being O
around O
10 O
amino O
acids O
, O
this O
makes O
it O
possible O
to O
describe O
protein O
structure O
as O
an O
arrangement O
of O
a O
much O
smaller O
number O
of O
elements O
. O

Protein O
structures O
are O
often O
visualized O
in O
a O
simplified O
form O
, O
with O
the O
so O
- O
called O
ribbon O
diagram O
with O
secondary O
structures O
shown O
as O
helices O
and O
arrows O
being O
the O
most O
popular O
( O
see O
Figure O
1 O
) O
. O

This O
picture O
can O
be O
simplified O
further O
by O
showing O
individual O
secondary O
structure O
elements O
as O
simple O
symbols O
( O
circles O
or O
boxes O
/ O
triangles O
) O
. O

These O
depictions O
, O
called O
fold O
diagrams O
, O
originally O
proposed O
in O
the O
70s O
[ O
10 O
- O
12 O
] O
are O
best O
captured O
by O
a O
TOPS O
( O
Topology O
of O
Protein O
Structures O
) O
algorithm O
, O
which O
attempts O
to O
automate O
the O
process O
of O
creation O
of O
the O
topology O
cartoon O
[ O
13 O
] O
. O

While O
useful O
in O
protein O
classification O
, O
such O
simplified O
descriptions O
are O
not O
used O
in O
the O
most O
popular O
automated O
protein O
structure O
comparison O
algorithms O
such O
as O
DALI O
[ O
3 O
] O
or O
CE O
[ O
4 O
] O
. O

Kleywegt O
and O
Jones O
developed O
a O
method O
for O
finding O
similar O
motifs O
based O
on O
comparing O
distance O
matrices O
that O
are O
constructed O
by O
representing O
protein O
as O
a O
set O
of O
SSEs O
with O
their O
directional O
vectors O
and O
angle O
between O
those O
vectors O
[ O
14 O
] O
. O

Programs O
that O
used O
SSEs O
either O
for O
structure O
comparison O
based O
on O
hierarchical O
superposition O
of O
both O
SSEs O
and O
atomic O
representation O
[ O
15 O
] O
or O
for O
finding O
common O
substructures O
in O
the O
comparison O
process O
based O
on O
subgraph O
isomorphism O
, O
such O
as O
[ O
16 O
, O
17 O
] O
and O
recent O
applications O
of O
the O
TOPS O
diagram O
[ O
18 O
, O
19 O
] O
, O
usually O
struggle O
with O
translating O
the O
comparison O
results O
from O
the O
secondary O
structure O
to O
the O
individual O
residue O
level O
. O

Although O
the O
SSM O
method O
uses O
graph O
- O
matching O
procedures O
at O
the O
SSE O
level O
followed O
by O
an O
interactive O
3D O
alignment O
of O
the O
protein O
C O
- O
alpha O
atom O
[ O
20 O
] O
, O
it O
lacks O
the O
topological O
relationships O
between O
the O
SSEs O
, O
which O
are O
essential O
features O
in O
identifying O
common O
scaffolds O
in O
distantly O
related O
proteins O
. O

A O
TOPS O
pattern O
was O
used O
to O
guide O
the O
sequence O
alignment O
, O
for O
instance O
, O
to O
build O
multiple O
structural O
alignments O
of O
the O
distantly O
related O
family O
of O
beta O
- O
rich O
protein O
domains O
[ O
21 O
] O
. O

The O
Multiple O
Sequence O
Alignment O
Tool O
( O
MSAT O
) O
automates O
this O
approach O
, O
merging O
it O
with O
a O
popular O
ClustalW O
program O
[ O
22 O
] O
. O

DALI O
[ O
3 O
] O
, O
CE O
[ O
4 O
] O
or O
FATCAT O
[ O
5 O
] O
introduce O
their O
own O
methods O
of O
decomposing O
the O
protein O
structure O
into O
smaller O
units O
, O
such O
as O
7 O
x O
7 O
dense O
distance O
map O
fragments O
( O
DALIs O
) O
or O
aligned O
fragment O
pairs O
( O
AFPs O
) O
( O
CE O
and O
FATCAT O
) O
. O

The O
large O
number O
of O
such O
fragments O
and O
the O
combinations O
of O
the O
fragments O
that O
need O
to O
be O
evaluated O
by O
structure O
comparison O
programs O
is O
the O
main O
reason O
for O
the O
significant O
computational O
requirements O
of O
such O
algorithms O
. O

However O
, O
more O
importantly O
, O
TOPS O
+ O
method O
is O
used O
here O
to O
enable O
a O
structural O
comparison O
that O
takes O
into O
account O
flexibility O
in O
protein O
structures O
and O
not O
only O
classifies O
the O
differences O
, O
but O
also O
can O
recognize O
such O
rearrangements O
- O
which O
is O
a O
first O
such O
application O
using O
the O
SSEs O
language O
. O

In O
this O
contribution O
, O
we O
explore O
the O
question O
of O
whether O
it O
would O
be O
possible O
to O
combine O
insights O
provided O
by O
topology O
diagrams O
into O
automated O
protein O
structure O
alignment O
algorithms O
, O
focusing O
on O
the O
FATCAT O
program O
developed O
previously O
in O
our O
group O
. O

Methods O

Flexible O
structure O
alignment O
method O
FATCAT O

FATCAT O
[ O
5 O
] O
is O
a O
unique O
structure O
alignment O
method O
that O
allows O
for O
flexibility O
in O
the O
structures O
being O
compared O
. O

It O
builds O
the O
alignment O
by O
chaining O
aligned O
fragment O
pairs O
( O
AFPs O
) O
[ O
23 O
] O
together O
using O
a O
unified O
scoring O
function O
where O
AFP O
extensions O
, O
gaps O
, O
and O
twists O
each O
have O
their O
specific O
scores O
( O
Figure O
2 O
) O
. O

Introducing O
a O
twist O
into O
the O
alignment O
is O
penalized O
, O
but O
this O
penalty O
may O
be O
compensated O
for O
by O
the O
gain O
in O
the O
score O
of O
the O
resulting O
alignment O
( O
i O
. O
e O
. O
, O
longer O
alignment O
and O
/ O
or O
better O
RMSD O
) O
. O

Rigid O
alignment O
can O
be O
treated O
as O
a O
special O
case O
, O
in O
which O
no O
twist O
is O
allowed O
in O
chaining O
AFPs O
. O

FATCAT O
program O
provides O
alignment O
in O
both O
, O
" O
rigid O
" O
mode O
and O
" O
flexible O
" O
( O
default O
) O
mode O
. O

FATCAT O
, O
as O
well O
as O
most O
other O
protein O
structure O
comparison O
programs O
, O
is O
very O
slow O
when O
compared O
to O
sequence O
alignments O
. O

The O
computing O
time O
of O
FATCAT O
is O
determined O
by O
the O
size O
of O
the O
collection O
of O
AFPs O
detected O
between O
the O
two O
structures O
being O
compared O
. O

FATCAT O
is O
available O
from O
a O
server O
with O
an O
option O
to O
search O
in O
SCOP O
or O
PDB O
databases O
for O
similar O
structures O
. O

This O
search O
typically O
takes O
between O
8 O
to O
16 O
hours O
of O
CPU O
time O
, O
and O
this O
is O
the O
main O
obstacle O
to O
broader O
use O
of O
this O
option O
. O

FATCAT O
has O
been O
used O
to O
construct O
a O
Flexible O
Structure O
Neighborhood O
( O
FSN O
) O
database O
that O
contains O
pre O
- O
computed O
results O
of O
structure O
similarity O
searches O
and O
it O
takes O
several O
weeks O
of O
CPU O
time O
to O
update O
the O
FSN O
database O
. O

Other O
protein O
structure O
comparison O
resources O
, O
such O
as O
DALI O
or O
CE O
have O
very O
similar O
problems O
. O

TOPS O
cartoons O
and O
TOPS O
graph O
models O

As O
discussed O
in O
the O
Background O
, O
TOPS O
cartoons O
capture O
the O
simplified O
, O
fold O
- O
level O
description O
of O
protein O
structure O
and O
at O
the O
same O
time O
can O
be O
automated O
[ O
24 O
] O
. O

The O
TOPS O
algorithm O
uses O
structural O
features O
such O
as O
hydrogen O
bonds O
and O
chirality O
of O
the O
beta O
strands O
to O
provide O
a O
scoring O
function O
to O
optimize O
the O
cartoon O
( O
see O
Figure O
1 O
( O
b O
) O
) O
. O

In O
TOPS O
, O
the O
secondary O
structural O
elements O
( O
SSEs O
) O
are O
derived O
from O
the O
DSSP O
program O
[ O
25 O
] O
. O

Based O
on O
TOPS O
cartoons O
, O
a O
formal O
graph O
model O
and O
graph O
- O
based O
definitions O
of O
protein O
topology O
and O
pattern O
discovery O
and O
comparison O
methods O
were O
developed O
[ O
26 O
, O
27 O
] O
. O

The O
TOPS O
database O
and O
comparison O
, O
pattern O
discovery O
and O
matching O
programs O
are O
accessible O
from O
. O

Novel O
TOPS O
+ O
and O
TOPS O
+ O
strings O
models O

The O
TOPS O
model O
was O
further O
enhanced O
to O
incorporate O
features O
such O
as O
protein O
- O
ligand O
interaction O
information O
and O
more O
detailed O
secondary O
structural O
segment O
information O
. O

This O
enhanced O
model O
is O
called O
TOPS O
+ O
model O
( O
see O
Figure O
3a O
) O
. O

This O
TOPS O
+ O
model O
can O
be O
described O
formally O
in O
a O
TOPS O
+ O
strings O
language O
( O
Figure O
3b O
) O
at O
a O
reduced O
linear O
level O
. O

The O
enhanced O
TOPS O
+ O
strings O
models O
can O
be O
used O
in O
fast O
string O
- O
based O
structure O
matching O
and O
comparison O
, O
at O
the O
same O
time O
avoiding O
issues O
of O
NP O
- O
completeness O
associated O
with O
graph O
alignments O
. O

In O
detail O
, O
each O
node O
( O
SSE O
segment O
) O
of O
the O
TOPS O
+ O
strings O
is O
described O
by O
its O
type O
, O
orientation O
, O
PDB O
start O
number O
, O
segment O
length O
, O
total O
number O
of O
incoming O
( O
InArc O
) O
and O
outgoing O
( O
OutArc O
) O
arcs O
( O
edges O
) O
, O
total O
number O
of O
ArcTypes O
, O
and O
total O
number O
of O
ligand O
arcs O
( O
LigArc O
) O
. O

The O
type O
of O
the O
segment O
( O
SSEType O
) O
could O
be O
one O
of O
[ O
E O
, O
e O
, O
H O
, O
h O
, O
U O
, O
u O
] O
, O
where O
, O
" O
E O
" O
and O
" O
e O
" O
represent O
the O
" O
up O
" O
- O
and O
" O
down O
" O
- O
oriented O
beta O
strands O
; O
" O
H O
" O
and O
" O
h O
" O
indicate O
the O
" O
up O
" O
- O
and O
" O
down O
" O
- O
oriented O
alpha O
helices O
; O
and O
" O
U O
" O
and O
" O
u O
" O
represent O
ligand O
- O
bound O
and O
ligand O
- O
free O
loops O
. O

The O
InArcType O
can O
be O
classified O
as O
an O
/ O
a O
[ O
R O
, O
L O
, O
P O
, O
A O
] O
, O
where O
" O
R O
" O
and O
" O
L O
" O
represent O
right O
and O
left O
chiralities O
; O
and O
" O
P O
" O
and O
" O
A O
" O
represent O
parallel O
and O
anti O
- O
parallel O
hydrogen O
bonds O
, O
respectively O
. O

The O
OutArcType O
is O
represented O
in O
a O
similar O
manner O
by O
[ O
R O
' O
, O
L O
' O
, O
P O
' O
, O
A O
' O
] O
. O

Ligand O
arcs O
are O
indicated O
by O
LT O
= O
AA O
, O
where O
LT O
is O
the O
ligand O
type O
and O
AA O
is O
the O
PDB O
number O
. O

For O
example O
, O
Figure O
3 O
( O
a O
) O
and O
3 O
( O
b O
) O
contain O
visual O
representations O
of O
TOPS O
+ O
and O
TOPS O
+ O
strings O
models O
, O
respectively O
, O
for O
the O
protein O
domain O
d1fnb O
_ O
1 O
. O

Here O
the O
triangles O
represent O
the O
beta O
strands O
; O
the O
red O
curve O
represents O
the O
alpha O
helix O
; O
gray O
ellipsoids O
indicate O
loops O
; O
and O
green O
arcs O
indicate O
hydrogen O
bonds O
between O
two O
beta O
strands O
, O
called O
anti O
- O
parallel O
beta O
sheets O
. O

The O
length O
of O
a O
TOPS O
+ O
strings O
model O
is O
defined O
by O
number O
of O
SSEs O
; O
thus O
, O
the O
length O
of O
d1fnb O
_ O
1 O
is O
19 O
. O

For O
further O
details O
, O
see O
[ O
28 O
] O
. O

TOPS O
+ O
strings O
comparison O
method O

TOPS O
+ O
is O
a O
comparison O
method O
that O
computes O
a O
distance O
between O
TOPS O
+ O
strings O
models O
of O
two O
proteins O
based O
on O
a O
dynamic O
programming O
approach O
and O
identifies O
the O
longest O
common O
subsequence O
( O
LCS O
) O
, O
consisting O
of O
the O
list O
of O
the O
topologically O
equivalent O
SSEs O
between O
two O
proteins O
. O

For O
example O
, O
Figure O
3 O
( O
c O
) O
shows O
the O
TOPS O
+ O
strings O
alignment O
between O
Dihydropteridine O
reductase O
proteins O
from O
rat B
( O
1dhr O
) O
and O
human B
( O
1hdr O
) O
. O

The O
TOPS O
+ O
strings O
models O
for O
1dhr O
and O
1hdr O
are O
represented O
by O
a O
linear O
string O
- O
model O
, O
where O
a O
yellow O
triangle O
and O
red O
curves O
indicate O
the O
beta O
strands O
and O
alpha O
helices O
in O
their O
" O
up O
" O
or O
" O
down O
" O
orientations O
, O
respectively O
. O

The O
grey O
line O
and O
purple O
stubs O
represent O
the O
loop O
regions O
and O
the O
NAD O
ligand O
interactions O
, O
respectively O
. O

Note O
that O
the O
ligand O
- O
interaction O
information O
is O
optional O
and O
in O
this O
work O
we O
have O
not O
used O
it O
. O

The O
incoming O
and O
outgoing O
arcs O
are O
depicted O
in O
the O
SSEs O
( O
top O
and O
bottom O
of O
the O
beta O
strands O
) O
, O
where O
red O
and O
green O
arcs O
represent O
the O
parallel O
and O
anti O
- O
parallel O
hydrogen O
- O
bond O
interactions O
that O
show O
beta O
- O
sheet O
information O
, O
while O
yellow O
and O
blue O
arcs O
indicate O
the O
right O
and O
left O
chirality O
relationships O
between O
the O
SSEs O
. O

A O
pink O
arrow O
between O
the O
TOPS O
+ O
strings O
elements O
indicates O
the O
conserved O
SSE O
. O

The O
dotted O
arrows O
indicate O
the O
conserved O
alpha O
helices O
and O
beta O
strands O
, O
while O
the O
plain O
arrows O
indicate O
the O
conserved O
loop O
regions O
. O

TOPS O
+ O
+ O
FATCAT O
method O

In O
this O
work O
, O
we O
want O
to O
test O
the O
general O
idea O
of O
pruning O
the O
search O
space O
of O
the O
FATCAT O
comparison O
process O
using O
topological O
constraints O
derived O
from O
the O
TOPS O
+ O
strings O
alignment O
. O

Many O
of O
the O
AFPs O
considered O
in O
the O
FATCAT O
alignment O
could O
be O
easily O
eliminated O
from O
the O
comparison O
by O
constraining O
the O
alignment O
region O
. O

Here O
we O
explore O
constraints O
obtained O
from O
the O
TOPS O
+ O
strings O
alignment O
, O
which O
identifies O
topologically O
equivalent O
secondary O
structure O
elements O
( O
alpha O
helices O
, O
beta O
strands O
, O
and O
loops O
) O
for O
this O
purpose O
. O

Such O
equivalences O
define O
blocks O
that O
restrict O
the O
alignment O
region O
; O
AFPs O
that O
fall O
outside O
these O
regions O
are O
simply O
not O
considered O
( O
see O
Figure O
4 O
( O
b O
) O
) O
. O

We O
introduce O
a O
parameter O
r O
to O
control O
the O
strictness O
of O
constraints O
by O
TOPS O
+ O
strings O
alignments O
; O
r O
equals O
0 O
if O
the O
alignment O
region O
is O
strictly O
restrained O
by O
TOPS O
+ O
strings O
alignment O
, O
and O
r O
is O
set O
to O
1 O
by O
default O
in O
our O
program O
to O
allow O
certain O
flexibility O
to O
the O
constrained O
alignment O
region O
( O
Figure O
4 O
( O
c O
) O
) O
. O

We O
then O
can O
speed O
up O
the O
FATCAT O
alignment O
by O
considering O
only O
the O
AFPs O
within O
the O
constrained O
alignment O
area O
( O
Figure O
4 O
( O
d O
) O
) O
. O

The O
rigid O
structural O
alignment O
can O
be O
treated O
as O
a O
special O
case O
of O
TOPS O
+ O
+ O
FATCAT O
, O
in O
which O
no O
twist O
is O
allowed O
in O
chaining O
AFPs O
. O

However O
, O
the O
TOPS O
+ O
+ O
FATCAT O
program O
provides O
alignment O
in O
both O
, O
" O
rigid O
" O
mode O
and O
" O
flexible O
" O
mode O
( O
default O
) O
. O

Benchmarking O

For O
benchmarking O
and O
comparison O
, O
we O
have O
used O
the O
PDB40 O
dataset O
of O
1 O
, O
901 O
protein O
domain O
pairs O
( O
DP O
) O
corresponding O
to O
SCOP O
version O
1 O
. O
61 O
from O
the O
ASTRAL O
database O
[ O
29 O
] O
. O

Table O
1 O
provides O
the O
SCOP O
superfamily O
level O
homolog O
versus O
non O
- O
homolog O
statistics O
for O
the O
four O
main O
SCOP O
classes O
i O
. O
e O
. O
, O
all O
- O
alpha O
, O
all O
- O
beta O
, O
alpha O
/ O
beta O
, O
alpha O
+ O
beta O
, O
and O
all O
proteins O
regardless O
of O
their O
structural O
classes O
. O

Evaluation O
Analyses O

We O
performed O
the O
Receiver O
Operating O
Characteristics O
( O
ROC O
) O
curve O
and O
the O
AUC O
( O
Area O
Under O
the O
ROC O
Curve O
) O
analyses O
to O
compare O
the O
performance O
of O
the O
TOPS O
+ O
+ O
FATCAT O
method O
with O
the O
original O
FATCAT O
method O
, O
using O
SCOP O
classification O
at O
the O
superfamily O
level O
as O
a O
standard O
of O
comparison O
[ O
30 O
] O
. O

Results O

ROC O
and O
AUC O
Analyses O

We O
have O
compared O
the O
performance O
of O
the O
TOPS O
+ O
+ O
FATCAT O
method O
against O
the O
original O
FATCAT O
method O
using O
the O
SCOP O
classification O
information O
at O
the O
superfamily O
level O
. O

We O
have O
plotted O
the O
ROC O
curves O
based O
on O
P O
- O
values O
obtained O
from O
the O
FATCAT O
and O
the O
TOPS O
+ O
+ O
FATCAT O
methods O
. O

We O
have O
plotted O
the O
ROC O
curves O
separately O
for O
the O
main O
SCOP O
classes O
, O
i O
. O
e O
. O
, O
all O
- O
alpha O
, O
all O
- O
beta O
, O
alpha O
/ O
beta O
, O
alpha O
+ O
beta O
, O
and O
all O
proteins O
regardless O
of O
the O
class O
they O
belong O
to O
( O
see O
Figure O
5 O
( O
a O
) O
to O
5 O
( O
e O
) O
) O
. O

In O
the O
graph O
, O
the O
x O
- O
and O
y O
- O
axes O
represent O
the O
false O
positive O
and O
true O
positive O
rates O
of O
the O
performance O
of O
the O
comparison O
methods O
respectively O
. O

In O
the O
legend O
, O
rF O
- O
pvalue O
and O
fF O
- O
pvalue O
indicate O
results O
from O
the O
rigid O
and O
flexible O
FATCAT O
methods O
, O
respectively O
; O
similarly O
, O
rT2F O
- O
pvalue O
and O
fT2F O
- O
pvalue O
represent O
the O
rigid O
and O
flexible O
TOPS O
+ O
+ O
FATCAT O
methods O
, O
respectively O
. O

We O
have O
calculated O
the O
AUC O
values O
for O
all O
the O
SCOP O
classes O
based O
on O
ROC O
curves O
obtained O
from O
the O
FATCAT O
and O
TOPS O
+ O
+ O
FATCAT O
methods O
with O
the O
flexible O
/ O
rigid O
options O
( O
see O
Table O
2 O
) O
. O

For O
all O
protein O
classes O
, O
the O
rigid O
FATCAT O
performs O
best O
, O
usually O
followed O
by O
the O
flexible O
FATCAT O
, O
the O
rigid O
TOPS O
+ O
+ O
FATCAT O
, O
and O
the O
flexible O
TOPS O
+ O
+ O
FATCAT O
. O

The O
performance O
of O
all O
four O
methods O
is O
best O
for O
all O
alpha O
and O
all O
beta O
proteins O
, O
and O
all O
four O
perform O
markedly O
worse O
( O
but O
similar O
to O
each O
other O
) O
for O
alpha O
/ O
beta O
proteins O
. O

Only O
alpha O
+ O
beta O
proteins O
show O
a O
clear O
difference O
between O
the O
FATCAT O
and O
TOPS O
+ O
+ O
FATCAT O
methods O
. O

It O
is O
important O
to O
note O
that O
the O
TOPS O
+ O
strings O
models O
consider O
the O
parallel O
and O
anti O
- O
parallel O
properties O
of O
the O
beta O
- O
sheet O
information O
in O
the O
form O
of O
total O
number O
of O
incoming O
and O
outgoing O
arcs O
with O
their O
ArcTypes O
. O

Thus O
, O
the O
TOPS O
+ O
+ O
FATCAT O
method O
discriminates O
the O
protein O
domain O
pairs O
more O
efficiently O
compared O
to O
the O
original O
FATCAT O
method O
. O

For O
example O
, O
in O
the O
all O
- O
beta O
protein O
domain O
pairs O
, O
both O
the O
flexible O
and O
the O
rigid O
TOPS O
+ O
+ O
FATCAT O
methods O
perform O
well O
. O

The O
flexible O
TOPS O
+ O
+ O
FATCAT O
method O
covers O
nearly O
84 O
% O
of O
protein O
domains O
with O
0 O
% O
false O
positives O
, O
but O
the O
flexible O
and O
rigid O
FATCAT O
methods O
cover O
only O
76 O
% O
and O
49 O
% O
of O
the O
true O
positives O
, O
respectively O
, O
with O
0 O
% O
false O
positives O
. O

The O
zoomed O
- O
in O
version O
of O
the O
ROC O
curves O
with O
up O
to O
10 O
% O
false O
positives O
for O
all O
- O
beta O
rich O
protein O
families O
is O
shown O
in O
Figure O
5 O
( O
f O
) O
; O
where O
both O
the O
rigid O
TOPS O
+ O
+ O
FATCAT O
( O
green O
) O
and O
flexible O
( O
red O
) O
TOPS O
+ O
+ O
FATCAT O
methods O
have O
coverage O
rates O
of O
82 O
% O
and O
84 O
% O
true O
positives O
respectively O
with O
0 O
% O
false O
positives O
. O

The O
overall O
results O
for O
all O
protein O
classes O
show O
that O
TOPS O
+ O
+ O
FATCAT O
performance O
is O
only O
slightly O
lower O
( O
3 O
% O
- O
7 O
% O
AUC O
value O
difference O
( O
see O
Table O
2 O
) O
) O
as O
compared O
to O
FATCAT O
while O
providing O
a O
significant O
, O
more O
than O
10 O
- O
fold O
speedup O
( O
see O
next O
section O
) O
. O

AFP O
and O
Runtime O
Analyses O

We O
tested O
both O
the O
FATCAT O
and O
TOPS O
+ O
+ O
FATCAT O
methods O
using O
the O
Mac O
OS O
X O
version O
10 O
. O
4 O
. O
10 O
computer O
system O
with O
a O
2 O
x O
2 O
. O
66 O
- O
GHz O
Dual O
- O
Core O
Intel O
Xeon O
processor O
and O
1 O
- O
GB O
667 O
MHz O
memory O
. O

We O
have O
performed O
runtime O
analysis O
on O
1 O
, O
901 O
protein O
domain O
pairs O
and O
counted O
the O
total O
number O
of O
AFPs O
and O
the O
corresponding O
runtime O
from O
both O
the O
FATCAT O
and O
the O
TOPS O
+ O
+ O
FATCAT O
methods O
. O

The O
results O
show O
an O
exponential O
increase O
in O
AFPs O
( O
Figure O
6 O
( O
b O
) O
) O
and O
corresponding O
runtime O
( O
Figure O
6 O
( O
a O
) O
) O
for O
the O
FATCAT O
method O
as O
compared O
to O
the O
TOPS O
+ O
+ O
FATCAT O
method O
( O
see O
Table O
3 O
) O
For O
example O
, O
the O
average O
number O
of O
AFPs O
for O
the O
TOPS O
+ O
+ O
FATCAT O
method O
is O
530 O
, O
but O
the O
average O
number O
of O
AFPs O
for O
the O
FATCAT O
method O
is O
15 O
, O
019 O
. O

This O
represents O
the O
number O
of O
average O
AFPs O
used O
by O
the O
FATCAT O
method O
is O
increased O
by O
a O
factor O
of O
28 O
( O
see O
Table O
3 O
) O
. O

This O
result O
leads O
to O
the O
conclusion O
that O
TOPS O
+ O
+ O
FATCAT O
is O
22 O
times O
faster O
compared O
to O
the O
FATCAT O
because O
this O
method O
must O
take O
into O
account O
more O
number O
of O
AFPs O
in O
the O
comparison O
process O
( O
see O
Table O
3 O
) O
. O

Case O
Studies O

While O
the O
overall O
accuracy O
of O
both O
rigid O
and O
flexible O
FATCAT O
methods O
is O
better O
than O
their O
TOPS O
+ O
+ O
FATCAT O
equivalents O
, O
an O
interesting O
example O
where O
the O
opposite O
is O
true O
lies O
in O
the O
comparison O
of O
two O
proteins O
, O
d2trxa O
_ O
( O
108 O
aa O
) O
from O
Escherichia B
coli I
and O
d1kte O
_ O
_ O
( O
105 O
aa O
) O
from O
Sus B
scrofa I
( O
pig B
) O
from O
the O
thioredoxin O
- O
like O
superfamily O
. O

For O
this O
pair O
, O
the O
flexible O
_ O
TOPS O
+ O
+ O
FATCAT O
method O
provides O
an O
alignment O
with O
88 O
equivalent O
positions O
with O
1 O
. O
67 O
A O
chain O
RMSD O
and O
3 O
. O
06 O
A O
of O
optimal O
RMSD O
without O
any O
twist O
, O
giving O
the O
alignment O
with O
10 O
% O
sequence O
identity O
( O
see O
Table O
4 O
) O
. O

On O
the O
other O
hand O
, O
the O
flexible O
_ O
FATCAT O
method O
provides O
an O
alignment O
with O
86 O
aligned O
positions O
using O
a O
twist O
in O
the O
C O
- O
terminal O
region O
; O
it O
has O
a O
higher O
chain O
RMSD O
of O
5 O
. O
14 O
A O
, O
and O
its O
optimal O
RMSD O
is O
3 O
. O
48 O
A O
. O

For O
more O
information O
regarding O
the O
chain O
and O
optimal O
RMSDs O
refer O
[ O
5 O
] O
. O

The O
flexible O
_ O
FATCAT O
method O
uses O
the O
twist O
to O
align O
a O
helix O
in O
the O
C O
- O
terminal O
region O
, O
which O
is O
positioned O
incorrectly O
with O
a O
beta O
- O
sheet O
core O
( O
see O
Table O
4 O
) O
. O

Figure O
7 O
( O
a O
) O
shows O
the O
superposition O
of O
d2trxa O
_ O
( O
gray O
) O
and O
d1kte O
_ O
_ O
( O
orange O
) O
domains O
from O
the O
flexible O
_ O
FATCAT O
method O
, O
where O
the O
blue O
color O
indicates O
the O
d1kte O
_ O
_ O
protein O
domain O
from O
the O
flexible O
_ O
TOPS O
+ O
+ O
FATCAT O
method O
. O

The O
incorrect O
alignment O
of O
the O
C O
- O
terminal O
domain O
alpha O
helix O
of O
the O
d1kte O
_ O
_ O
domain O
( O
orange O
) O
is O
visible O
in O
the O
core O
of O
the O
beta O
- O
sheet O
region O
. O

Figure O
7 O
( O
b O
) O
and O
7 O
( O
c O
) O
shows O
the O
AFPs O
from O
the O
flexible O
_ O
FATCAT O
and O
flexible O
_ O
TOPS O
+ O
+ O
FATCAT O
methods O
, O
respectively O
. O

The O
hinge O
region O
provides O
a O
twist O
in O
the O
flexible O
_ O
FATCAT O
method O
indicated O
by O
an O
arrow O
and O
the O
AFPs O
represented O
by O
a O
different O
color O
( O
see O
Figure O
7 O
( O
b O
) O
) O
. O

In O
this O
case O
, O
the O
alignment O
constraints O
from O
the O
TOPS O
+ O
strings O
alignment O
allow O
the O
TOPS O
+ O
+ O
FATCAT O
method O
to O
avoid O
a O
spurious O
alignment O
. O

The O
Erythrocruorin O
protein O
domain O
d1eca O
_ O
_ O
( O
136 O
aa O
) O
from O
Chironomus B
thummi I
and O
the O
Phycocyanin O
alpha O
subunit O
protein O
domain O
d1cpca O
_ O
( O
162 O
aa O
) O
from O
Fremyella B
diplosiphon I
( O
Cyanobacterium B
) O
belong O
to O
the O
Globin O
- O
like O
superfamily O
. O

For O
these O
protein O
domain O
pairs O
, O
the O
FATCAT O
method O
provides O
a O
better O
alignment O
with O
120 O
and O
118 O
aligned O
positions O
with O
the O
chain O
RMSD O
of O
4 O
. O
02 O
A O
based O
on O
the O
flexible O
and O
rigid O
options O
, O
respectively O
. O

The O
flexible O
_ O
TOPS O
+ O
+ O
FATCAT O
method O
gives O
an O
alignment O
of O
63 O
aligned O
positions O
with O
the O
3 O
. O
23 O
A O
optimal O
RMSD O
and O
the O
6 O
. O
28 O
A O
chain O
RMSD O
. O

In O
this O
case O
, O
the O
flexible O
_ O
TOPS O
+ O
+ O
FATCAT O
method O
misses O
the O
N O
- O
terminal O
region O
helix O
and O
misaligns O
some O
helices O
. O

For O
example O
, O
Figure O
8 O
( O
a O
) O
shows O
the O
superposition O
of O
d1eca O
_ O
_ O
( O
gray O
) O
and O
d1cpca O
_ O
( O
orange O
) O
domains O
from O
the O
flexible O
_ O
FATCAT O
method O
, O
while O
d1cpca O
_ O
( O
blue O
) O
domain O
is O
from O
the O
flexible O
_ O
TOPS O
+ O
+ O
FATCAT O
method O
. O

The O
AFP O
chaining O
alignment O
and O
the O
actual O
alignment O
from O
FATCAT O
are O
shown O
in O
Figure O
8 O
( O
b O
) O
and O
8 O
( O
e O
) O
, O
respectively O
. O

Figure O
8 O
( O
c O
) O
shows O
the O
AFP O
alignment O
from O
TOPS O
+ O
+ O
FATCAT O
, O
in O
which O
this O
method O
misses O
the O
N O
- O
terminal O
region O
and O
incorrectly O
aligns O
some O
of O
the O
C O
- O
terminal O
regions O
( O
see O
Figure O
8 O
( O
d O
) O
) O
. O

However O
, O
the O
rigid O
_ O
TOPS O
+ O
+ O
FATCAT O
method O
produces O
an O
alignment O
of O
108 O
aligned O
positions O
with O
optimal O
and O
chain O
RMSDs O
of O
3 O
. O
22 O
A O
and O
6 O
. O
28 O
A O
respectively O
. O

In O
general O
, O
TOPS O
comparison O
does O
not O
work O
well O
for O
alpha O
- O
rich O
proteins O
due O
to O
the O
lack O
of O
hydrogen O
bonds O
between O
SSEs O
[ O
26 O
] O
. O

The O
same O
is O
true O
for O
TOPS O
+ O
strings O
comparison O
to O
some O
extent O
; O
however O
, O
this O
method O
takes O
advantage O
of O
ligand O
- O
interaction O
information O
to O
compare O
protein O
domains O
more O
efficiently O
; O
for O
example O
the O
DNA O
binding O
motifs O
such O
as O
helix O
- O
turn O
- O
helix O
and O
helix O
- O
loop O
- O
helix O
can O
be O
easily O
recognized O
[ O
28 O
] O
. O

However O
, O
we O
have O
not O
explored O
that O
ligand O
pattern O
discovery O
option O
within O
the O
TOPS O
+ O
strings O
comparison O
in O
this O
paper O
. O

In O
addition O
, O
the O
TOPS O
+ O
strings O
alignment O
provides O
only O
a O
basic O
alignment O
; O
the O
scoring O
function O
to O
find O
the O
best O
alignment O
has O
not O
been O
optimized O
. O

These O
problems O
can O
be O
addressed O
in O
future O
development O
by O
considering O
the O
advanced O
TOPS O
+ O
and O
TOPS O
+ O
strings O
models O
based O
on O
helix O
- O
helix O
packing O
relationships O
and O
SSE O
- O
ligand O
interaction O
properties O
together O
with O
the O
right O
and O
left O
chiralities O
. O

Furthermore O
, O
the O
TOPS O
+ O
strings O
comparison O
can O
be O
optimized O
in O
both O
the O
comparison O
process O
as O
well O
as O
in O
the O
alignment O
process O
in O
order O
to O
take O
into O
account O
indels O
( O
insertion O
/ O
deletion O
) O
of O
SSEs O
which O
exist O
in O
nature O
across O
the O
different O
members O
of O
the O
protein O
superfamilies O
[ O
31 O
] O
. O

Discussion O
and O
conclusion O

The O
overall O
results O
for O
all O
protein O
classes O
show O
that O
TOPS O
+ O
+ O
FATCAT O
performance O
is O
only O
slightly O
lower O
( O
3 O
% O
- O
7 O
% O
AUC O
value O
difference O
) O
as O
compared O
to O
FATCAT O
while O
providing O
a O
significant O
, O
more O
than O
10 O
- O
fold O
speedup O
. O

The O
main O
reason O
for O
the O
discrepancies O
is O
that O
TOPS O
+ O
strings O
alignments O
occasionally O
misalign O
the O
secondary O
structure O
elements O
and O
subsequent O
FATCAT O
alignment O
, O
constrained O
by O
the O
TOPS O
+ O
strings O
alignment O
, O
cannot O
overcome O
the O
earlier O
errors O
. O

There O
is O
a O
clear O
trade O
- O
off O
between O
the O
runtime O
and O
the O
accuracy O
; O
limiting O
the O
pool O
of O
fragments O
being O
compared O
speeds O
up O
the O
algorithm O
but O
results O
in O
( O
slightly O
) O
lower O
accuracy O
. O

At O
the O
same O
time O
, O
these O
results O
offer O
clear O
suggestions O
for O
future O
development O
. O

Using O
a O
more O
advanced O
version O
of O
the O
TOPS O
+ O
strings O
comparison O
method O
would O
remove O
some O
of O
the O
false O
positives O
might O
be O
at O
a O
cost O
of O
significantly O
slowing O
the O
total O
performance O
of O
the O
TOPS O
+ O
+ O
FATCAT O
method O
. O

Authors O
' O
contributions O

MV O
developed O
the O
TOPS O
+ O
+ O
FATCAT O
algorithm O
, O
performed O
the O
calculations O
and O
prepared O
the O
figures O
, O
YY O
provided O
advice O
and O
oversight O
in O
the O
project O
, O
verified O
the O
code O
and O
provided O
FATCAT O
results O
for O
comparison O
, O
AG O
contributed O
to O
the O
original O
idea O
and O
to O
writing O
of O
the O
manuscript O
. O

Are O
there O
sensitive O
subgroups O
for O
the O
effects O
of O
airborne O
particles O
? O

Abstract O

Recent O
studies O
have O
shown O
that O
particulate O
air O
pollution O
is O
a O
risk O
factor O
for O
hospitalization O
for O
heart O
and O
lung O
disease O
; O
however O
, O
little O
is O
known O
about O
what O
subpopulations O
are O
most O
sensitive O
to O
this O
pollutant O
. O

We O
analyzed O
Medicare O
hospital O
admissions O
for O
heart O
disease O
, O
chronic O
obstructive O
pulmonary O
disorders O
( O
COPD O
) O
and O
pneumonia O
in O
Chicago O
, O
Cook O
County O
, O
Illinois O
, O
between O
1985 O
and O
1994 O
. O

We O
examined O
whether O
previous O
admissions O
or O
secondary O
diagnoses O
for O
selected O
conditions O
predisposed O
persons B
to O
having O
a O
greater O
risk O
from O
air O
pollution O
. O

We O
also O
considered O
effect O
modification O
by O
age O
, O
sex O
, O
and O
race O
. O

We O
found O
that O
the O
air O
- O
pollution O
- O
associated O
increase O
in O
hospital O
admissions O
for O
cardiovascular O
diseases O
was O
almost O
doubled O
in O
subjects O
with O
concurrent O
respiratory O
infections O
. O

The O
risk O
was O
also O
increased O
by O
a O
previous O
admission O
for O
conduction O
disorders O
. O

For O
COPD O
and O
pneumonia O
admissions O
, O
diagnosis O
of O
conduction O
disorders O
or O
dysrhythmias O
increased O
the O
risk O
of O
particulate O
matter O
< O
10 O
microm O
in O
aerodynamic O
diameter O
( O
PM O
( O
10 O
) O
) O
- O
associated O
admissions O
. O

Persons B
with O
asthma O
had O
twice O
the O
risk O
of O
a O
PM O
( O
10 O
) O
- O
associated O
pneumonia O
admission O
and O
persons B
with O
heart O
failure O
had O
twice O
the O
risk O
of O
PM O
( O
10 O
) O
- O
induced O
COPD O
admissions O
. O

The O
PM O
( O
10 O
) O
effect O
did O
not O
vary O
by O
sex O
, O
age O
, O
and O
race O
. O

These O
results O
suggest O
that O
patients B
with O
acute O
respiratory O
infections O
or O
defects O
in O
the O
electrical O
control O
of O
the O
heart O
are O
a O
risk O
group O
for O
particulate O
matter O
effects O
. O

Articles O

Are O
There O
Sensitive O
Subgroups O
for O
the O
Effects O
of O
Airborne O
Particles O
? O

Antonella O
Zanobetti O
, O
1 O
Joel O
Schwartz O
, O
1 O
, O
2 O
and O
Diane O
Gold1 O
, O
2 O
1Environmental O

Epidemiology O
Program O
, O
Department O
of O
Environmental O
Health O
, O
Harvard O
School O
of O
Public O
Health O
, O
Boston O
, O
Massachusetts O
, O
USA O
; O
2Channing O
Laboratory O
, O
Department O
of O
Medicine O
, O
Harvard O
Medical O
School O
and O
Brigham O
and O
Women B
' O
s O
Hospital O
, O
Boston O
, O
Massachusetts O
, O
USA O

Recent O
studies O
have O
shown O
that O
particulate O
air O
pollution O
is O
a O
risk O
factor O
for O
hospitalization O
for O
heart O
and O
lung O
disease O
; O
however O
, O
little O
is O
known O
about O
what O
subpopulations O
are O
most O
sensitive O
to O
this O
pollutant O
. O

We O
analyzed O
Medicare O
hospital O
admissions O
for O
heart O
disease O
, O
chronic O
obstructive O
pulmonary O
disorders O
( O
COPD O
) O
and O
pneumonia O
in O
Chicago O
, O
Cook O
County O
, O
Illinois O
, O
between O
1985 O
and O
1994 O
. O

We O
examined O
whether O
previous O
admissions O
or O
secondary O
diagnoses O
for O
selected O
conditions O
predisposed O
persons B
to O
having O
a O
greater O
risk O
from O
air O
pollution O
. O

We O
also O
considered O
effect O
modification O
by O
age O
, O
sex O
, O
and O
race O
. O

We O
found O
that O
the O
air O
- O
pollution O
- O
associated O
increase O
in O
hospital O
admissions O
for O
cardiovascular O
diseases O
was O
almost O
doubled O
in O
subjects O
with O
concurrent O
respiratory O
infections O
. O

The O
risk O
was O
also O
increased O
by O
a O
previous O
admission O
for O
conduction O
disorders O
. O

For O
COPD O
and O
pneumonia O
admissions O
, O
diagnosis O
of O
conduction O
disorders O
or O
dysrhythmias O
increased O
the O
risk O
of O
particulate O
matter O
< O
10 O
< O
FFFD O
> O
m O
in O
aerodynamic O
diameter O
( O
PM10 O
) O
- O
associated O
admissions O
. O

Persons B
with O
asthma O
had O
twice O
the O
risk O
of O
a O
PM10 O
- O
associated O
pneumonia O
admission O
and O
persons B
with O
heart O
failure O
had O
twice O
the O
risk O
of O
PM10 O
- O
induced O
COPD O
admissions O
. O

The O
PM10 O
effect O
did O
not O
vary O
by O
sex O
, O
age O
, O
and O
race O
. O

These O
results O
suggest O
that O
patients B
with O
acute O
respiratory O
infections O
or O
defects O
in O
the O
electrical O
control O
of O
the O
heart O
are O
a O
risk O
group O
for O
particulate O
matter O
effects O
. O

Key O
words O
: O
effect O
modification O
, O
hospital O
admissions O
, O
particulate O
air O
pollution O
. O

Environ O
Health O
Perspect O
108 O
: O
841 O
< O
FFFD O
> O
845 O
( O
2000 O
) O
. O

[ O
Online O
28 O
July O
2000 O
] O
http O
: O
/ O
/ O
ehpnet1 O
. O
niehs O
. O
nih O
. O
gov O
/ O
docs O
/ O
2000 O
/ O
108p841 O
- O
845zanobetti O
/ O
abstract O
. O
html O

Particulate O
air O
pollution O
has O
been O
associated O
with O
increases O
in O
daily O
deaths O
and O
hospital O
admissions O
in O
studies O
all O
over O
the O
world O
( O
1 O
< O
FFFD O
> O
15 O
) O
. O

These O
associations O
are O
now O
well O
documented O
but O
little O
is O
known O
, O
as O
yet O
, O
of O
the O
characteristics O
of O
persons B
that O
put O
them O
at O
increased O
risk O
of O
adverse O
events O
related O
to O
particulate O
air O
pollution O
. O

This O
has O
been O
identified O
as O
a O
key O
data O
gap O
( O
16 O
) O
. O

Schwartz O
and O
Dockery O
( O
17 O
) O
reported O
that O
persons B
older O
than O
65 O
years O
of O
age O
had O
a O
somewhat O
increased O
risk O
of O
death O
, O
and O
this O
has O
been O
confirmed O
in O
other O
studies O
( O
18 O
) O
. O

A O
more O
detailed O
examination O
of O
particulate O
matter O
- O
related O
risk O
by O
deciles O
of O
age O
( O
19 O
) O
showed O
the O
risk O
beginning O
to O
increase O
at O
approximately O
40 O
years O
of O
age O
and O
reaching O
its O
maximum O
for O
those O
75 O
years O
of O
age O
and O
older O
. O

In O
addition O
to O
age O
, O
several O
studies O
suggest O
that O
persons B
with O
respiratory O
illness O
are O
at O
increased O
risk O
for O
cardiovascular O
effects O
associated O
with O
air O
pollution O
. O

An O
examination O
of O
death O
certificates O
on O
high O
- O
and O
low O
- O
air O
pollution O
days O
reported O
a O
substantial O
difference O
in O
the O
proportion O
of O
deaths O
from O
cardiovascular O
causes O
that O
had O
respiratory O
disease O
as O
a O
contributing O
cause O
of O
death O
( O
19 O
) O
. O

A O
recent O
follow O
- O
up O
study O
of O
a O
cohort O
of O
persons B
with O
chronic O
obstructive O
pulmonary O
disease O
( O
COPD O
) O
in O
Barcelona O
, O
Spain O
, O
found O
an O
association O
between O
particulate O
air O
pollution O
and O
all O
- O
cause O
mortality O
in O
the O
cohort O
( O
20 O
) O
. O

The O
magnitude O
of O
the O
risk O
per O
microgram O
per O
cubic O
meter O
of O
exposure O
was O
substantially O
greater O
than O
that O
for O
the O
general O
population O
. O

Environmental O
Health O
Perspectives O

Controlled O
exposure O
of O
animals O
with O
chronic O
bronchitis O
and O
control O
animals O
to O
concentrated O
air O
particles O
also O
demonstrated O
a O
potentiating O
effect O
of O
chronic O
lung O
disease O
in O
the O
response O
to O
airborne O
particles O
( O
21 O
) O
. O

This O
has O
led O
to O
the O
hypothesis O
that O
the O
cardiovascular O
effects O
of O
air O
pollution O
are O
predominantly O
in O
persons B
with O
chronic O
lung O
disease O
. O

There O
has O
been O
even O
less O
done O
to O
examine O
potential O
modifiers O
of O
the O
effects O
of O
airborne O
particles O
on O
hospital O
admissions O
. O

The O
existing O
literature O
on O
comorbidity O
shows O
that O
comorbidity O
per O
se O
seems O
to O
increase O
the O
risk O
of O
adverse O
outcomes O
( O
22 O
< O
FFFD O
> O
30 O
) O
. O

Little O
is O
known O
about O
the O
role O
of O
these O
comorbidities O
as O
effect O
modifiers O
for O
the O
effects O
of O
air O
pollution O
. O

This O
study O
uses O
data O
from O
the O
Medicare O
system O
to O
examine O
potential O
short O
- O
term O
and O
long O
- O
term O
medical O
conditions O
that O
may O
increase O
a O
person B
' O
s O
risk O
of O
hospital O
admissions O
associated O
with O
particulate O
air O
pollution O
. O

In O
addition O
, O
we O
examine O
potential O
effect O
modification O
by O
age O
, O
race O
, O
and O
sex O
. O

Materials O
and O
Methods O
Health O
data O
. O

The O
Health O
Care O
Financing O
Administration O
( O
Baltimore O
, O
MD O
) O
maintains O
records O
of O
every O
hospital O
admission O
for O
Medicare O
participants B
in O
the O
United O
States O
. O

Persons B
in O
this O
database O
have O
a O
unique O
identifier O
. O

Using O
this O
identifier O
, O
we O
traced O
every O
hospital O
admission O
for O
heart O
and O
lung O
disease O
for O
each O
person B
in O
Cook O
County O
, O
Illinois O
, O
between O
1985 O
and O
1994 O
. O

We O
chose O
Cook O
County O
because O
it O
is O
the O
most O
populous O

county O
in O
the O
United O
States O
with O
daily O
monitoring O
for O
particulate O
matter O
with O
aerodynamic O
diameter O
< O
10 O
< O
FFFD O
> O
m O
( O
PM10 O
) O
. O

The O
data O
were O
then O
analyzed O
to O
look O
at O
effect O
modification O
by O
concurrent O
and O
preexisting O
conditions O
as O
well O
as O
by O
age O
, O
race O
, O
and O
sex O
. O

To O
establish O
a O
baseline O
risk O
, O
we O
computed O
daily O
counts O
of O
hospital O
admissions O
for O
cardiovascular O
disease O
( O
CVD O
) O
[ O
International O
Classification O
of O
Disease O
, O
9th O
edition O
, O
World O
Health O
Organization O
, O
Geneva O
( O
ICD O
- O
9 O
) O
code O
390 O
< O
FFFD O
> O
429 O
] O
, O
pneumonia O
( O
ICD O
- O
9 O
code O
480 O
< O
FFFD O
> O
487 O
) O
, O
and O
COPD O
( O
ICD O
- O
9 O
code O
490 O
< O
FFFD O
> O
496 O
, O
excluding O
493 O
) O
. O

The O
association O
between O
these O
daily O
counts O
and O
PM10 O
was O
examined O
for O
the O
years O
1988 O
< O
FFFD O
> O
1994 O
, O
when O
daily O
PM10 O
monitoring O
data O
were O
available O
in O
Chicago O
. O

Once O
our O
baseline O
risks O
were O
established O
, O
we O
examined O
three O
classes O
of O
potential O
effect O
modifiers O
. O

First O
, O
we O
looked O
at O
whether O
previous O
admissions O
for O
selected O
conditions O
predisposed O
persons B
to O
having O
a O
greater O
risk O
from O
air O
pollution O
. O

For O
each O
of O
the O
three O
admission O
categories O
( O
CVD O
, O
pneumonia O
, O
and O
COPD O
) O
, O
we O
considered O
10 O
causes O
( O
defined O
by O
a O
previous O
admission O
) O
as O
effect O
modifiers O
: O
COPD O
( O
ICD O
- O
9 O
code O
490 O
< O
FFFD O
> O
496 O
except O
493 O
) O
, O
asthma O
( O
ICD O
- O
9 O
code O
493 O
) O
, O
acute O
bronchitis O
( O
ICD O
- O
9 O
code O
466 O
) O
, O
acute O
respiratory O
illness O
( O
ICD O
- O
9 O
code O
460 O
< O
FFFD O
> O
466 O
) O
, O
pneumonia O
( O
ICD O
- O
9 O
code O
480 O
< O
FFFD O
> O
487 O
) O
, O
CVD O
( O
ICD O

- O
9 O
code O
390 O
< O
FFFD O
> O
429 O
) O
, O
myocardial O
infarction O
( O
ICD O
- O
9 O
code O
410 O
) O
, O
congestive O
heart O
failure O
( O
ICD O
- O
9 O
code O
428 O
) O
, O
conduction O
disorders O
( O
ICD O
- O
9 O
code O
426 O
) O
, O
and O
dysrhythmias O
( O
ICD9 O
code O
427 O
) O
. O

To O
test O
the O
hypothesis O
that O
persons B
with O
these O
conditions O
had O
higher O
risks O
of O
subsequent O
PM10 O
- O
related O
admissions O
, O
we O
computed O
separate O
daily O
counts O
of O
admissions O
for O
our O
three O
target O
causes O
, O
stratified O
by O
whether O
or O
not O
the O
person B
admitted O
had O
been O
previously O
admitted O
for O
the O
hypothesized O
predisposing O
condition O
. O

Separate O
analyses O
were O
then O
performed O
within O
each O
strata O
to O
see O
if O
the O
effects O
of O
PM10 O
differed O
by O
strata O
. O

Address O
correspondence O
to O
A O
. O

Zanobetti O
, O
Department O
of O
Environmental O
Health O
, O
Environmental O
Epidemiology O
Program O
, O
Harvard O
School O
of O
Public O
Health O
, O
665 O
Huntington O
Avenue O
, O
Boston O
, O
MA O
02115 O
USA O
. O

Telephone O
: O
( O
617 O
) O
4324642 O
. O

Fax O
: O
( O
617 O
) O
277 O
- O
2382 O
. O

E O
- O
mail O
: O
azanob O
@ O
sparc6a O
. O
harvard O
. O
edu O
Supported O
by O
NIEHS O
grant O
ES07937 O
. O

Received O
18 O
January O
2000 O
; O
accepted O
18 O
April O
2000 O
. O

< O
FFFD O
> O
VOLUME O
108 O
| O
NUMBER O
9 O
| O
September O
2000 O

841 O

Articles O

< O
FFFD O
> O

Zanobetti O
et O
al O
. O

The O
second O
set O
of O
potential O
predisposing O
conditions O
included O
secondary O
diagnoses O
associated O
with O
the O
index O
admission O
. O

These O
could O
represent O
the O
presence O
of O
a O
chronic O
condition O
( O
e O
. O
g O
. O
, O
COPD O
) O
that O
has O
not O
resulted O
in O
a O
previous O
hospital O
admission O
. O

They O
could O
also O
represent O
acute O
conditions O
that O
may O
have O
increased O
the O
subjects O
' O
sensitivity O
to O
air O
pollution O
. O

For O
example O
, O
if O
respiratory O
infections O
modified O
the O
effect O
of O
particulate O
matter O
on O
the O
cardiovascular O
health O
of O
persons B
with O
underlying O
heart O
disease O
, O
then O
the O
risk O
of O
a O
hospital O
admission O
for O
heart O
disease O
might O
be O
different O
in O
persons B
with O
infections O
. O

If O
this O
were O
true O
, O
then O
the O
risk O
ratio O
of O
a O
10 O
- O
< O
FFFD O
> O
g O
/ O
m3 O
increase O
of O
PM10 O
on O
cardiovascular O
admissions O
of O
persons B
with O
a O
concurrent O
respiratory O
infection O
would O
be O
different O
from O
the O
ratio O
in O
persons B
without O
respiratory O
infection O
. O

To O
test O
these O
hypotheses O
, O
we O
computed O
separate O
daily O
counts O
of O
admissions O
for O
events O
with O
and O
without O
the O
concurrent O
conditions O
hypothesized O
to O
increase O
sensitivity O
to O
air O
pollution O
. O

These O
were O
taken O
as O
the O
same O
10 O
conditions O
in O
the O
first O
analysis O
with O
certain O
exclusions O
for O
pairing O
that O
would O
be O
illogical O
. O

That O
is O
, O
the O
concurrent O
diagnosis O
of O
a O
specific O
cardiac O
condition O
was O
not O
treated O
as O
an O
effect O
modifier O
for O
admissions O
for O
any O
cardiovascular O
condition O
. O

Likewise O
, O
pneumonia O
and O
COPD O
were O
not O
possible O
concurrent O
conditions O
for O
each O
other O
. O

The O
third O
set O
of O
predisposing O
conditions O
considered O
was O
being O
older O
than O
75 O
years O
of O
age O
, O
nonwhite O
, O
and O
female O
. O

These O
were O
examined O
for O
all O
three O
outcomes O
. O

We O
obtained O
weather O
data O
for O
O O
' O
Hare O
Airport O
from O
the O
EarthInfo O
CD O
- O
ROM O
( O
EarthInfo O
CD O
NCDC O
Surface O
Airways O
, O
EarthInfo O
Inc O
. O
, O
Boulder O
, O
CO O
) O
, O
and O
we O
obtained O
air O
pollution O
data O
from O
the O
U O
. O
S O
. O

Environmental O
Protection O
Agency O
Aerometric O
Information O
Retrieval O
System O
network O
( O
31 O
) O
. O

running O
- O
line O
smoother O
, O
loess O
( O
35 O
) O
, O
was O
chosen O
to O
estimate O
the O
smooth O
function O
. O

To O
control O
for O
weather O
variables O
and O
day O
of O
the O
week O
, O
we O
chose O
the O
smoothing O
parameter O
that O
minimized O
the O
Akaike O
' O
s O
information O
criterion O
( O
36 O
) O
. O

To O
model O
seasonality O
we O
chose O
the O
smoothing O
parameter O
that O
minimized O
the O
sum O
of O
the O
autocorrelation O
of O
the O
residuals O
while O
removing O
seasonal O
patterns O
. O

Two O
autoregressive O
terms O
( O
37 O
) O
were O
added O
in O
the O
model O
to O
eliminate O
the O
remaining O
serial O
correlation O
from O
the O
residuals O
. O

We O
used O
the O
mean O
of O
PM10 O
on O
the O
day O
of O
the O
admission O
and O
the O
day O
before O
the O
admission O
as O
our O
exposure O
variable O
. O

This O
gives O
results O
that O
are O
similar O
to O
those O
obtained O
fitting O
a O
full O
distributed O
lag O
model O
( O
38 O
) O
. O

PM10 O
was O
treated O
linearly O
. O

Our O
baseline O
models O
used O
the O
daily O
counts O
of O
CVD O
, O
pneumonia O
, O
and O
COPD O
admissions O
as O
outcomes O
. O

We O
then O
subdivided O
those O
counts O
by O
the O
presence O
or O
absence O
of O
the O
potential O
effect O
modifier O
and O
reestimated O
our O
regressions O
on O
those O
subgroups O
. O

We O
considered O
effect O
modification O
to O
be O
indicated O
when O
the O
estimates O
of O
PM10 O
in O
the O
group O
with O
the O
condition O
was O
outside O
of O
the O
95 O
% O
confidence O
interval O
( O
CI O
) O
of O
the O
effect O
estimate O
in O
persons B
without O
the O
condition O
. O

Results O
Table O
1 O
shows O
the O
mean O
daily O
admissions O
for O
COPD O
, O
cardiovascular O
, O
and O
pneumonia O
both O
overall O
and O
in O
the O
presence O
of O
the O
potential O
effect O
modifiers O
. O

For O
some O
effect O
modifiers O
such O
as O
conduction O
disorders O
or O
myocardial O
infarctions O
, O
the O
counts O
in O
conjunction O
with O
our O
respiratory O
outcomes O
are O

low O
, O
which O
limits O
power O
. O

In O
general O
, O
the O
numbers O
are O
lower O
for O
examining O
effect O
modification O
by O
previous O
admissions O
than O
for O
effect O
modification O
by O
concurrent O
diagnosis O
. O

This O
is O
as O
expected O
because O
many O
clinically O
relevant O
comorbidities O
may O
never O
have O
resulted O
in O
a O
hospital O
admission O
. O

Table O
2 O
shows O
the O
25th O
, O
50th O
, O
and O
75th O
percentile O
values O
for O
the O
environmental O
variables O
. O

The O
mean O
value O
for O
PM10 O
is O
33 O
< O
FFFD O
> O
g O
/ O
m3 O
. O

The O
daily O
values O
for O
PM10 O
were O
computed O
as O
the O
average O
of O
10 O
monitors O
, O
two O
of O
which O
measured O
PM10 O
almost O
every O
day O
and O
the O
others O
less O
frequently O
( O
38 O
) O
. O

Table O
3 O
shows O
the O
mean O
daily O
counts O
of O
CVD O
, O
COPD O
, O
and O
pneumonia O
by O
sex O
, O
age O
groups O
, O
and O
race O
. O

The O
distribution O
by O
sex O
is O
almost O
even O
, O
although O
the O
counts O
of O
admissions O
for O
males O
are O
generally O
lower O
( O
approximately O
10 O
% O
) O
than O
for O
females O
, O
particularly O
for O
cardiovascular O
diseases O
. O

The O
counts O
of O
CVD O
, O
COPD O
, O
and O
pneumonia O
admissions O
were O
similar O
for O
people B
65 O
< O
FFFD O
> O
75 O
or O
75 O
years O
of O
age O
and O
older O
. O

Tables O
4 O
< O
FFFD O
> O
6 O
show O
the O
results O
for O
the O
effect O
PM10 O
overall O
and O
stratifying O
by O
concurrent O
diagnosis O
and O
previous O
admissions O
. O

These O
are O
expressed O
as O
the O
percentage O
increase O
for O
10 O
< O
FFFD O
> O
g O
/ O
m3 O
PM10 O
. O

Table O
4 O
shows O
the O
results O
for O
CVD O
. O

A O
10 O
- O
< O
FFFD O
> O
g O
/ O
m3 O
increase O
in O
PM10 O
was O
associated O
with O
a O
1 O
. O
31 O
% O
( O
5 O
% O
CI O
, O
0 O
. O
97 O
% O
; O
95 O
% O
CI O
, O
1 O
. O
66 O
% O
) O
increase O
in O
hospital O
admissions O
for O
heart O
disease O
in O
all O
elderly O
persons B
. O

A O
concurrent O
( O
not O
previous O
) O
diagnosis O
of O
COPD O
modified O
the O
risk O
of O
PM10 O
- O
associated O
admissions O
for O
heart O
disease O
. O

However O
, O
significant O
associations O
were O
still O
seen O
between O
PM10 O

Table O
1 O
. O

Mean O
daily O
counts O
of O
admissions O
, O
Chicago O
1986 O
< O
FFFD O
> O
1994 O
, O
for O
COPD O
, O
CVD O
, O
and O
pneumonia O
overall O
and O
by O
concurrent O
diagnosis O
and O
by O
previous O
admissions O
. O

By O
concurrent O
diagnosis O
COPD O
CVD O
Pneumonia O
Overall O
Respiratory O
disease O
Acute O
bronchitis O
Acute O
respiratory O
infections O
Pneumonia O
Asthma O
COPD O
Cardiovascular O
disease O
CVD O
Conduction O
disorders O
Cardiac O
dysrhythmias O
Congestive O
heart O
failure O
Myocardial O
infarction O
NA O
, O
not O
applicable O
. O

By O
previous O
admissions O
COPD O
CVD O
Pneumonia O
7 O
. O
8 O
0 O
. O
8 O
0 O
. O
9 O
1 O
. O
6 O
0 O
. O
9 O
2 O
. O
7 O
2 O
. O
1 O
0 O
. O
0 O
0 O
. O
4 O
0 O
. O
9 O
0 O
. O
3 O
102 O
. O
1 O
1 O
. O
6 O
1 O
. O
8 O
7 O
. O
3 O
1 O
. O
5 O
2 O
. O
0 O
54 O
. O
7 O
1 O
. O
0 O
9 O
. O
9 O
24 O
. O
2 O
11 O
. O
4 O
26 O
. O
5 O
0 O
. O
9 O
1 O
. O
0 O
6 O
. O
4 O
0 O
. O
7 O
1 O
. O
4 O
7 O
. O
2 O
0 O
. O
2 O
1 O
. O
5 O
3 O
. O
1 O
1 O
. O
0 O

Methods O
We O
analyzed O
the O
data O
with O
a O
generalized O
additive O
robust O
Poisson O
regression O
model O
( O
32 O
) O
. O

This O
approach O
has O
become O
the O
norm O
in O
such O
studies O
( O
14 O
, O
33 O
, O
34 O
) O
. O

In O
the O
generalized O
additive O
model O
the O
outcome O
is O
assumed O
to O
depend O
on O
a O
sum O
of O
nonparametric O
smooth O
functions O
for O
each O
variable O
that O
models O
the O
potential O
nonlinear O
dependence O
of O
daily O
admission O
on O
weather O
and O
season O
. O

The O
model O
is O
of O
the O
form O
: O
log O
[ O
E O
( O
Yt O
) O
] O
= O
0 O
+ O
S1 O
( O
X1 O
) O
+ O
. O
. O
. O

+ O
Sp O
( O
Xp O
) O
where O
E O
( O
Yt O
) O
is O
the O
expected O
value O
of O
the O
daily O
count O
of O
admissions O
Yt O
and O
Si O
are O
the O
smooth O
functions O
of O
the O
covariates O
Xi O
. O

We O
examined O
temperature O
, O
previous O
day O
' O
s O
temperature O
, O
relative O
humidity O
, O
barometric O
pressure O
, O
and O
day O
of O
week O
covariates O
. O

The O
locally O
weighted O

7 O
. O
8 O
0 O
. O
1 O
0 O
. O
3 O
0 O
. O
4 O
0 O
. O
1 O
NA O
4 O
. O
7 O
0 O
. O
2 O
1 O
. O
4 O
1 O
. O
8 O
0 O
. O
1 O

102 O
. O
1 O
0 O
. O
9 O
1 O
. O
3 O
4 O
. O
0 O
1 O
. O
8 O
13 O
. O
4 O
NA O
NA O
NA O
NA O
NA O

26 O
. O
5 O
0 O
. O
3 O
0 O
. O
3 O
NA O
0 O
. O
9 O
6 O
. O
9 O
14 O
. O
7 O
0 O
. O
6 O
4 O
. O
6 O
7 O
. O
3 O
0 O
. O
4 O

Table O
2 O
. O

25th O
, O
50th O
, O
and O
75th O
percentile O
values O
for O
the O
environmental O
variables O
in O
Chicago O
, O
1988 O
< O
FFFD O
> O
1994 O
. O

Temperature O
( O
< O
FFFD O
> O
F O
) O
35 O
51 O
67 O
Relative O
humidity O
62 O
70 O
79 O
Barometric O
pressure O
29 O
. O
2 O
29 O
. O
3 O
29 O
. O
4 O
PM10 O
( O
< O
FFFD O
> O
g O
/ O
m3 O
) O
23 O
33 O
46 O

Table O
3 O
. O

Mean O
daily O
counts O
of O
admissions O
by O
sex O
, O
race O
, O
and O
age O
groups O
, O
Chicago O
, O
1986 O
< O
FFFD O
> O
1994 O
. O

Group O
Overall O
Female O
Nonwhite O
Age O
> O
75 O
years O
COPD O
7 O
. O
8 O
4 O
. O
2 O
1 O
. O
6 O
3 O
. O
7 O
CVD O
102 O
. O
1 O
59 O
. O
4 O
21 O
. O
0 O
55 O
. O
1 O
Pneumonia O
26 O
. O
5 O
14 O
. O
7 O
5 O
. O
2 O
17 O
. O
4 O

842 O

VOLUME O

108 O
| O
NUMBER O
9 O
| O
September O
2000 O
< O
FFFD O
> O
Environmental O
Health O
Perspectives O

Articles O

< O
FFFD O
> O

Effects O
of O
particles O
on O
sensitive O
subgroups O

and O
heart O
disease O
admissions O
in O
persons B
without O
COPD O
listed O
as O
either O
a O
comorbidity O
or O
a O
cause O
of O
previous O
admission O
( O
Table O
4 O
) O
. O

A O
significant O
association O
was O
also O
seen O
in O
persons B
without O
any O
respiratory O
disease O
as O
a O
concurrent O
diagnosis O
, O
although O
the O
risk O
is O
much O
lower O
than O
in O
persons B
with O
respiratory O
disease O
. O

However O
, O
the O
risk O
associated O
with O
PM10 O
was O
roughly O
doubled O
in O
subjects O
with O
concurrent O
respiratory O
infections O
and O
the O
risk O
estimates O
in O
those O
subjects O
were O
outside O
the O
95 O
% O
CI O
of O
the O
risk O
in O
patients B
without O
concurrent O
respiratory O
infections O
. O

A O
previous O
admission O
for O
conduction O
disorders O
( O
e O
. O
g O
. O
, O
heart O
block O
) O
increased O
the O
risk O
of O
a O
PM10 O
- O
related O
subsequent O
admission O
for O
any O
heart O
condition O
, O
and O
a O
weaker O
indication O
of O
effect O
modification O
was O
seen O
for O
persons B
with O
previous O
admission O
for O
dysrhythmias O
. O

In O
contrast O
heart O
failure O
and O
previous O
myocardial O
infarctions O
were O
highly O
insignificant O
as O
effect O
modifiers O
. O

Table O
5 O
shows O
the O
results O
for O
COPD O
. O

Overall O
, O
there O
is O
a O
1 O
. O
89 O
% O
( O
95 O
% O
CI O
, O
0 O
. O
8 O
< O
FFFD O
> O
3 O
. O
0 O
) O
increase O
in O
COPD O
admissions O
for O
a O
10 O
< O
FFFD O
> O
g O
/ O
m3 O
increase O
in O
PM10 O
. O

The O
results O
of O
the O
stratified O
analysis O
suggest O
that O
preexisting O
heart O
disease O
modifies O
Table O
4 O
. O

Percentage O
increase O
in O
hospital O
admissions O
for O
CVD O
in O
all O
persons B
and O
by O
concurrent O
diagnosis O
and O
previous O
admissions O
. O

PM10 O
2 O
. O
5 O
% O
CI O
97 O
. O
5 O
% O
CI O
All O
persons B
1 O
. O
31 O
By O
concurrent O
diagnosis O
Respiratory O
disease O
All O
respiratory O
disease O
With O
1 O
. O
65 O
Without O
0 O
. O
98 O
Acute O
bronchitis O
With O
2 O
. O
50 O
Without O
1 O
. O
07 O
Acute O
respiratory O
infections O
With O
2 O
. O
71 O
Without O
1 O
. O
06 O
Pneumonia O
With O
1 O
. O
95 O
Without O
1 O
. O
03 O
COPD O
With O
1 O
. O
59 O
Without O
1 O
. O
08 O
By O
previous O
admissions O
Respiratory O
disease O
All O
respiratory O
disease O
With O
1 O
. O
18 O
Without O
1 O
. O
08 O
COPD O
With O
1 O
. O
48 O
Without O
1 O
. O
09 O
Asthma O
With O
1 O
. O
71 O
Without O
1 O
. O
08 O

Cardiovascular O
disease O
Conduction O
disorders O
With O
2 O
. O
89 O
Without O
1 O
. O
07 O
Cardiac O
dyshrethmias O
With O
1 O
. O
61 O
Without O
1 O
. O
04 O
0 O
. O
97 O
1 O
. O
66 O

the O
risk O
of O
COPD O
admissions O
on O
high O
particle O
days O
. O

Previous O
admissions O
for O
any O
cardiovascular O
disease O
increased O
the O
risk O
of O
a O
PM10associated O
COPD O
admission O
approximately O
2 O
. O
5 O
- O
fold O
. O

A O
previous O
heart O
failure O
admission O
caused O
an O
even O
more O
striking O
increase O
in O
the O
PM10 O
effect O
. O

Previous O
admissions O
for O
dysrhythmias O
and O
conduction O
defects O
were O
rare O
( O
Table O
1 O
) O
with O
no O
power O
to O
examine O
effect O
modifications O
. O

Listings O
as O
concurrent O
diagnoses O
were O
more O
common O
and O
here O
they O
joined O
heart O
failure O
in O
increasing O
the O
risk O
of O
PM O
10 O
- O
associated O
COPD O
admissions O
. O

For O
COPD O
there O
was O
also O
some O
indication O
that O
concurrent O
pneumonia O
or O
an O
acute O
respiratory O
infection O
admission O
in O
the O
last O
year O
increased O
risk O
. O

The O
low O
numbers O
made O
these O
estimates O
less O
precise O
, O
however O
. O

The O
percentage O
increase O
in O
pneumonia O
admission O
( O
Table O
6 O
) O
for O
10 O
< O
FFFD O
> O
g O
/ O
m3 O
PM10 O
is O
higher O
than O
for O
COPD O
or O
CVD O
with O
an O
increase O
of O
2 O
. O
34 O
% O
( O
95 O
% O
CI O
, O
1 O
. O
66 O
< O
FFFD O
> O
3 O
. O
0 O
) O
. O

As O
with O
COPD O
, O
persons B
with O
heart O
disease O
appeared O
at O
higher O
risk O
of O
pneumonia O
hospital O
admissions O
associated O
with O
particulate O
air O
pollution O
. O

Here O
diagnoses O
suggestive O
of O
impaired O
autonomic O
control O
of O
the O
heart O
, O
such O
as O
conduction O
disorders O
or O
dysrhythmias O
, O
were O
associated O
with O
increased O
risk O
for O
PM O
10 O
effects O
on O
pneumonia O
admissions O
. O

Unlike O
COPD O
, O
no O
difference O
was O
seen O
for O
congestive O
heart O
failure O
. O

Persons B
with O
asthma O
Table O
5 O
. O

Percentage O
increase O
in O
hospital O
admissions O
for O
COPD O
in O
all O
persons B
and O
by O
concurrent O
diagnosis O
and O
previous O
admissions O
. O

PM10 O
2 O
. O
5 O
% O
CI O
97 O
. O
5 O
% O
CI O
All O
persons B
1 O
. O
89 O
By O
concurrent O
diagnosis O
Respiratory O
disease O
Pneumonia O
With O
4 O
. O
00 O
Without O
1 O
. O
51 O
Cardiovascular O
disease O
Conduction O
disorders O
With O
2 O
. O
34 O
Without O
1 O
. O
60 O
Cardiac O
dysrhythmias O
With O
3 O
. O
09 O
Without O
1 O
. O
43 O
Congestive O
heart O
failure O
With O
2 O
. O
90 O
Without O
1 O
. O
39 O
By O
previous O
admissions O
Respiratory O
disease O
Acute O
respiratory O
infections O
With O
3 O
. O
20 O
Without O
1 O
. O
70 O
Cardiovascular O
disease O
CVD O
With O
2 O
. O
90 O
Without O
1 O
. O
18 O
Congestive O
heart O
failure O
With O
4 O
. O
37 O
Without O

1 O
. O
14 O
Within O
1 O
year O
6 O
. O
04 O
0 O
. O
80 O
2 O
. O
99 O

had O
twice O
the O
risk O
of O
a O
PM10 O
- O
induced O
pneumonia O
admission O
as O
persons B
without O
asthma O
. O

Table O
7 O
shows O
the O
results O
by O
sex O
, O
age O
, O
and O
race O
. O

None O
of O
the O
effect O
size O
estimates O
for O
any O
of O
the O
stratification O
variables O
were O
outside O
of O
the O
95 O
% O
CI O
for O
the O
opposite O
strata O
. O

There O
was O
a O
tendency O
for O
the O
effect O
of O
PM10 O
on O
CVD O
admissions O
to O
be O
higher O
for O
females B
, O
whereas O
the O
effect O
on O
pneumonia O
admissions O
was O
higher O
for O
males O
. O

In O
general O
, O
we O
found O
somewhat O
larger O
effects O
on O
whites O
compared O
to O
nonwhites O
, O
and O
for O
persons B
older O
than O
75 O
years O
of O
age O
compared O
to O
younger O
persons B
. O

Discussion O
In O
this O
analysis O
we O
examined O
whether O
the O
effect O
of O
PM10 O
on O
the O
risk O
of O
hospital O
admission O
for O
heart O
and O
lung O
disease O
was O
different O
depending O
on O
the O
presence O
of O
comorbidities O
. O

We O
found O
that O
PM10 O
was O
associated O
with O
hospital O
admissions O
for O
all O
three O
causes O
( O
CVD O
, O
COPD O
, O
and O
pneumonia O
) O
and O
we O
found O
not O
a O
general O
increase O
in O
PM10 O
related O
risk O
with O
comorbidities O
, O
but O
a O
specific O
pattern O
that O
is O
suggestive O
of O
potential O
mechanisms O
and O
consistent O
with O
other O
recent O
epidemiologic O
and O
toxicologic O
findings O
. O

One O
major O
finding O
of O
this O
study O
is O
that O
preexisting O
cardiovascular O
disease O
, O
particularly O
impaired O
autonomic O
control O
( O
conduction O
defects O
and O
dysrhythmias O
) O
and O
heart O
failure O
, O
substantially O
increased O
the O
risk O
of O
respiratory O
admissions O
associated O
with O
airborne O
particles O
. O

In O
fact O
, O
recent O
human B
studies O
have O
shown O
that O
exposure O
to O
particulate O
air O
pollution O
is O
a O
risk O
factor O
for O
reduced O
heart O
rate O
variability O
( O
39 O
< O
FFFD O
> O
41 O
) O
. O

Reduced O
heart O
rate O
variability O
is O
an O
adverse O
response O
and O
a O
risk O
factor O
for O
arrhythmia O
. O

A O
new O
study O
of O
defibrillator O
discharges O
in O
patients B
with O
implanted O
cardioverter O
defibrillators O
found O
that O
discharges O
were O
associated O
with O
air O
pollution O
( O
42 O
) O
. O

Exposure O
to O
combustion O
Table O
6 O
. O

Percentage O
increase O
in O
hospital O
admissions O
for O
pneumonia O
in O
all O
persons B
and O
by O
concurrent O
diagnosis O
and O
previous O
admissions O
. O

PM10 O
All O
persons B
By O
concurrent O
diagnosis O
Respiratory O
disease O
Asthma O
With O
Without O
Cardiovascular O
disease O
Conduction O
disorders O
With O
Without O
Cardiac O
dysrhythmias O
With O
Without O
By O
previous O
admissions O
Cardiovascular O
disease O
Cardiac O
dysrhythmias O
With O
Without O
2 O
. O
34 O
2 O
. O
5 O
% O
CI O
97 O
. O
5 O
% O
CI O
1 O
. O
66 O
3 O
. O
02 O

1 O
. O
10 O
0 O
. O
64 O
< O
FFFD O
> O
0 O
. O
47 O
0 O
. O
76 O
0 O
. O
18 O
0 O
. O
76 O
0 O
. O
55 O
0 O
. O
72 O
0 O
. O
85 O
0 O
. O
75 O

2 O
. O
20 O
1 O
. O
33 O
5 O
. O
55 O
1 O
. O
37 O
5 O
. O
30 O
1 O
. O
37 O
3 O
. O
36 O
1 O
. O
35 O
2 O
. O
34 O
1 O
. O
41 O

< O
FFFD O
> O
0 O
. O
45 O
0 O
. O
47 O
< O
FFFD O
> O
4 O
. O
42 O
0 O
. O
58 O
0 O
. O
64 O
0 O
. O
33 O
0 O
. O
77 O
0 O
. O
24 O

8 O
. O
65 O
2 O
. O
57 O
9 O
. O
59 O
2 O
. O
64 O
5 O
. O
60 O
2 O
. O
55 O
5 O
. O
08 O
2 O
. O
55 O

0 O
. O
45 O
0 O
. O
76 O
< O
FFFD O
> O
0 O
. O
40 O
0 O
. O
78 O
< O
FFFD O
> O
0 O
. O
43 O
0 O
. O
77 O
0 O
. O
22 O
0 O
. O
76 O
0 O
. O
75 O
0 O
. O
72 O

1 O
. O
91 O
1 O
. O
41 O
3 O
. O
40 O
1 O
. O
40 O
3 O
. O
89 O
1 O
. O
39 O
5 O
. O
63 O
1 O
. O
38 O
2 O
. O
48 O
1 O
. O
36 O

4 O
. O
18 O
2 O
. O
07 O
7 O
. O
92 O
1 O
. O
99 O
< O
FFFD O
> O
< O
FFFD O
> O

1 O
. O
01 O
1 O
. O
46 O
4 O
. O
28 O
1 O
. O
37 O
< O
FFFD O
> O
< O
FFFD O
> O

7 O
. O
46 O
2 O
. O
69 O
11 O
. O
69 O
2 O
. O
61 O
< O
FFFD O
> O
< O
FFFD O
> O

< O
FFFD O
> O
1 O
. O
38 O
0 O
. O
66 O
0 O
. O
99 O
< O
FFFD O
> O
0 O
. O
01 O
1 O
. O
43 O
0 O
. O
05 O
2 O
. O
10 O

8 O
. O
01 O
2 O
. O
76 O
4 O
. O
85 O
2 O
. O
39 O
7 O
. O
40 O
2 O
. O
24 O
10 O
. O
14 O

3 O
. O
47 O
2 O
. O
08 O

1 O
. O
21 O
1 O
. O
45 O

5 O
. O
79 O
2 O
. O
71 O

Increases O
are O
for O
a O
10 O
- O
< O
FFFD O
> O
g O
/ O
m3 O
increase O
in O
PM10 O
. O

Increases O
are O
for O
a O
10 O
- O
< O
FFFD O
> O
g O
/ O
m3 O
increase O
in O
PM10 O
. O

Increases O
are O
for O
a O
10 O
- O
< O
FFFD O
> O
g O
/ O
m3 O
increase O
in O
PM10 O
. O

Environmental O
Health O
Perspectives O

< O
FFFD O
> O
VOLUME O
108 O
| O
NUMBER O
9 O
| O
September O
2000 O

843 O

Articles O

< O
FFFD O
> O

Zanobetti O
et O
al O
. O

Table O
7 O
. O

Effect O
modification O
by O
sex O
, O
race O
, O
and O
age O
groups O
for O
10 O
< O
FFFD O
> O
g O
/ O
m3 O
PM10 O
. O

% O
All O
persons B
Male O
Female O
White O
Non O
- O
white O
Age O
> O
75 O
Age O
75 O
1 O
. O
89 O
1 O
. O
34 O
2 O
. O
19 O
1 O
. O
65 O
1 O
. O
07 O
2 O
. O
20 O
1 O
. O
33 O
COPD O
( O
95 O
% O
CI O
) O
( O
0 O
. O
80 O
, O
2 O
. O
99 O
) O
( O
< O
FFFD O
> O
0 O
. O
14 O
, O
2 O
. O
84 O
) O
( O
0 O
. O
81 O
, O
3 O
. O
59 O
) O
( O
0 O
. O
51 O
, O
2 O
. O
81 O
) O
( O
< O
FFFD O
> O
1 O
. O
11 O
, O
3 O
. O
3 O
) O
( O
0 O
. O
72 O
, O
3 O
. O
69 O
) O
( O
0 O
. O
03 O
, O
2 O
. O
65 O
) O
% O
1 O
. O
31 O
1 O
. O
07 O
1 O
. O
21 O

1 O
. O
20 O
0 O
. O
70 O
1 O
. O
28 O
0 O
. O
93 O
CVD O
( O
95 O
% O
CI O
) O
( O
0 O
. O
97 O
, O
1 O
. O
66 O
) O
( O
0 O
. O
62 O
, O
1 O
. O
51 O
) O
( O
0 O
. O
83 O
, O
1 O
. O
6 O
) O
( O
0 O
. O
86 O
, O
1 O
. O
55 O
) O
( O
0 O
. O
1 O
, O
1 O
. O
3 O
) O
( O
0 O
. O
88 O
, O
1 O
. O
69 O
) O
( O
0 O
. O
51 O
, O
1 O
. O
35 O
) O
% O
2 O
. O
34 O
2 O
. O
65 O
1 O
. O
91 O
2 O
. O
45 O
1 O
. O
91 O
2 O
. O
12 O
2 O
. O
52 O
Pneumonia O
( O
95 O
% O
CI O
) O
( O
1 O
. O
66 O
, O
3 O
. O
02 O
) O
( O
1 O

. O
81 O
, O
3 O
. O
5 O
) O
( O
1 O
. O
11 O
, O
2 O
. O
72 O
) O
( O
1 O
. O
77 O
, O
3 O
. O
14 O
) O
( O
0 O
. O
69 O
, O
3 O
. O
14 O
) O
( O
1 O
. O
38 O
, O
2 O
. O
86 O
) O
( O
1 O
. O
57 O
, O
3 O
. O
48 O
) O

Figures O
shown O
are O
the O
percentage O
increase O
in O
admissions O
( O
95 O
% O
CI O
) O
. O

particles O
has O
also O
been O
associated O
with O
arrhythmia O
in O
an O
animal O
model O
( O
43 O
) O
and O
changes O
in O
ST O
segments O
have O
been O
noted O
as O
well O
( O
44 O
) O
. O

This O
is O
the O
first O
study O
to O
suggest O
persons B
with O
defects O
in O
the O
electrical O
control O
of O
the O
heart O
are O
also O
at O
higher O
risk O
of O
respiratory O
illness O
after O
exposure O
to O
airborne O
particles O
. O

These O
data O
also O
suggest O
that O
persons B
admitted O
to O
hospitals O
for O
pneumonia O
during O
an O
air O
pollution O
episode O
may O
be O
at O
high O
risk O
for O
clinically O
significant O
conduction O
disorders O
during O
that O
hospital O
admission O
. O

Patients B
with O
congestive O
heart O
failure O
were O
at O
greater O
risk O
of O
hospital O
admissions O
for O
COPD O
in O
association O
with O
airborne O
particles O
. O

Heart O
failure O
and O
COPD O
is O
not O
an O
uncommon O
combination O
. O

The O
finding O
that O
these O
patients B
are O
at O
higher O
risk O
for O
admissions O
associated O
with O
particulate O
air O
pollution O
is O
new O
but O
is O
also O
consistent O
with O
several O
other O
recent O
reports O
. O

The O
spontaneous O
hypertensive O
rat B
develops O
a O
model O
of O
heart O
failure O
, O
and O
recent O
studies O
have O
reported O
greater O
sensitivity O
to O
particulate O
air O
pollution O
in O
these O
rats B
. O

These O
include O
both O
electrocardiogram O
abnormalities O
( O
44 O
) O
and O
pulmonary O
toxicity O
( O
45 O
, O
46 O
) O
. O

Similarly O
, O
in O
an O
epidemiologic O
study O
, O
Hoek O
et O
al O
. O

( O
47 O
) O
, O
found O
a O
higher O
relative O
risk O
of O
death O
with O
an O
increase O
in O
PM10 O
for O
congestive O
heart O
failure O
deaths O
than O
other O
deaths O
. O

The O
potential O
role O
of O
COPD O
in O
those O
heart O
failure O
deaths O
was O
not O
examined O
. O

Another O
consistent O
pattern O
in O
our O
data O
is O
of O
acute O
respiratory O
infections O
increasing O
susceptibility O
to O
airborne O
particles O
. O

Acute O
bronchitis O
, O
or O
more O
generally O
acute O
upper O
respiratory O
illnesses O
, O
as O
well O
as O
pneumonia O
, O
increased O
susceptibility O
to O
particle O
- O
associated O
admissions O
for O
CVD O
and O
COPD O
. O

The O
notion O
that O
air O
pollution O
exacerbates O
acute O
respiratory O
infections O
is O
well O
supported O
by O
studies O
which O
report O
associations O
between O
airborne O
particles O
and O
hospital O
admissions O
for O
respiratory O
infections O
( O
48 O
, O
49 O
) O
. O

Zelikoff O
et O
al O
. O

( O
50 O
) O
exposed O
rats B
infected O
with O
streptococcus O
to O
concentrated O
air O
particles O
and O
reported O
a O
significant O
increase O
in O
bacterial O
burdens O
and O
in O
the O
extent O
of O
pneumonia O
compared O
to O
animals O
exposed O
to O
filtrated O
air O
. O

This O
suggests O
an O
impaired O
immune O
response O
. O

Similarly O
, O
exposure O
to O
combustion O

particles O
enhances O
influenza O
infections O
in O
mice B
( O
51 O
) O
. O

An O
impaired O
defense O
to O
respiratory O
infection O
is O
a O
major O
reason O
that O
persons B
with O
COPD O
require O
hospital O
admission O
. O

If O
airborne O
particles O
result O
in O
further O
impairment O
the O
effect O
modification O
we O
observe O
makes O
good O
sense O
. O

The O
effect O
modification O
for O
heart O
disease O
admissions O
is O
more O
relevant O
. O

This O
modification O
is O
consistent O
with O
the O
earlier O
report O
of O
Schwartz O
( O
19 O
) O
, O
who O
found O
greater O
reports O
of O
respiratory O
complications O
on O
death O
certificates O
with O
an O
underlying O
cause O
of O
heart O
disease O
if O
the O
death O
occurred O
on O
a O
day O
with O
high O
levels O
of O
airborne O
particles O
. O

Although O
airborne O
particle O
exposure O
has O
been O
associated O
with O
increased O
exacerbation O
of O
asthma O
( O
2 O
, O
12 O
, O
48 O
, O
52 O
< O
FFFD O
> O
59 O
) O
, O
this O
paper O
is O
the O
first O
to O
suggest O
that O
asthmatics O
are O
more O
susceptible O
to O
PM10 O
- O
induced O
pneumonia O
exacerbation O
or O
to O
cardiovascular O
effects O
. O

The O
effects O
on O
pneumonia O
admissions O
are O
plausible O
, O
given O
the O
impaired O
ability O
to O
fight O
off O
infections O
in O
asthmatics O
with O
mucus O
plugs O
and O
the O
evidence O
the O
airborne O
particles O
impair O
the O
lungs O
' O
ability O
to O
fight O
off O
bacterial O
and O
viral O
infections O
, O
as O
noted O
earlier O
. O

The O
increased O
cardiovascular O
sensitivity O
, O
albeit O
weaker O
, O
is O
interesting O
. O

If O
airborne O
particles O
affect O
the O
cardiovascular O
system O
via O
the O
role O
of O
the O
lung O
in O
autonomic O
control O
, O
it O
is O
possible O
that O
asthmatics O
would O
be O
more O
sensitive O
to O
those O
effects O
. O

Animal O
models O
of O
asthma O
showed O
that O
combustion O
particles O
enhance O
the O
asthmatic O
response O
to O
aeroallergen O
challenges O
( O
59 O
) O
. O

This O
suggests O
an O
enhancement O
of O
pulmonary O
response O
in O
asthmatics O
. O

On O
the O
other O
hand O
, O
the O
diagnosis O
of O
asthma O
is O
problematic O
in O
the O
elderly O
, O
and O
crossover O
with O
COPD O
is O
possible O
. O

The O
possibility O
that O
this O
explains O
our O
results O
is O
reduced O
by O
our O
failure O
to O
find O
previous O
hospital O
admission O
for O
COPD O
was O
an O
effect O
modifier O
for O
the O
effect O
of O
particles O
on O
cardiovascular O
admissions O
. O

We O
must O
acknowledge O
several O
potential O
limitations O
of O
this O
study O
. O

First O
, O
we O
considered O
only O
previous O
admissions O
that O
occurred O
within O
Cook O
County O
. O

Hence O
persons B
with O
previous O
admissions O
elsewhere O
would O
be O
misclassified O
to O
our O
reference O
group O
. O

The O
effect O
of O
this O
would O
be O
to O
reduce O
the O
difference O
in O
PM O
10 O
effect O
between O
the O
two O
groups O
. O

VOLUME O

Nevertheless O
, O
we O
identified O
some O
interesting O
interactions O
. O

We O
cannot O
exclude O
the O
possibility O
that O
there O
are O
areas O
we O
missed O
for O
this O
reason O
. O

We O
also O
examined O
interactions O
in O
a O
log O
relative O
risk O
model O
, O
which O
is O
inherently O
multiplicative O
. O

Although O
we O
believe O
this O
is O
justified O
because O
doubling O
the O
population O
exposed O
would O
be O
expected O
to O
double O
the O
pollution O
associated O
admissions O
, O
it O
results O
in O
a O
more O
conservative O
definition O
of O
interaction O
than O
would O
an O
additive O
risk O
model O
. O

Finally O
, O
our O
exposure O
is O
clearly O
measured O
with O
error O
. O

Most O
of O
this O
error O
is O
Berkson O
error O
( O
60 O
) O
and O
hence O
will O
introduce O
no O
bias O
, O
and O
Zeger O
et O
al O
. O

( O
60 O
) O
showed O
that O
the O
remaining O
error O
would O
have O
to O
have O
pathologic O
correlations O
with O
other O
variables O
to O
result O
in O
an O
upward O
bias O
. O

Another O
important O
result O
from O
this O
study O
, O
of O
course O
, O
is O
an O
estimate O
of O
the O
magnitude O
of O
the O
effect O
of O
airborne O
particles O
on O
public O
health O
. O

The O
PM10 O
concentrations O
in O
Chicago O
during O
this O
period O
were O
associated O
with O
approximately O
1 O
, O
600 O
additional O
admissions O
per O
year O
for O
heart O
disease O
, O
740 O
additional O
admissions O
per O
year O
for O
pneumonia O
, O
and O
170 O
additional O
admissions O
per O
year O
for O
COPD O
. O

These O
are O
not O
trivial O
increases O
in O
serious O
morbidity O
. O

The O
results O
of O
our O
study O
should O
be O
replicated O
in O
additional O
cities O
, O
although O
they O
do O
begin O
to O
fill O
in O
some O
missing O
information O
about O
the O
effects O
of O
airborne O
particles O
on O
health O
. O

More O
generally O
airborne O
particles O
have O
been O
associated O
with O
a O
broad O
range O
of O
systemic O
changes O
including O
heart O
rate O
variability O
( O
39 O
< O
FFFD O
> O
41 O
) O
, O
increased O
peripheral O
neutrophils O
( O
61 O
< O
FFFD O
> O
63 O
) O
, O
increased O
plasma O
viscosity O
( O
64 O
) O
, O
an O
increase O
in O
blood O
pressure O
( O
65 O
) O
, O
and O
the O
outcomes O
mentioned O
previously O
. O

The O
role O
of O
these O
systemic O
changes O
as O
potential O
sources O
of O
the O
specific O
effect O
modifications O
we O
have O
seen O
should O
be O
an O
area O
of O
fruitful O
research O
in O
the O
future O
. O

REFERENCES O
AND O
NOTES O
1 O
. O

Katsouyanni O
K O
, O
Touloumi O
G O
, O
Spix O
C O
, O
Schwartz O
J O
, O
Balducci O
F O
, O
Medina O
S O
, O
Rossi O
G O
, O
Wojtyniak O
D O
, O
Sunyer O
J O
, O
Bacharova O
L O
, O
et O
al O
. O

Short O
term O
effects O
of O
ambient O
sulphur O
dioxide O
and O
particulate O
matter O
on O
mortality O
in O
12 O
European O
cities O
: O
results O
from O
time O
series O
data O
from O
the O
APHEA O
project O
. O

Br O
Med O
J O
314 O
: O
1658 O
< O
FFFD O
> O
1663 O
( O
1997 O
) O
. O

Pope O
CA O
, O
Dockery O
DW O
, O
Schwartz O
J O
. O
Review O
of O
epidemiologic O
evidence O
of O
health O
effects O
of O
particulate O
air O
pollution O
. O

Inhal O
Toxicol O
7 O
: O
1 O
< O
FFFD O
> O
18 O
( O
1995 O
) O
. O

Schwartz O
J O
. O

Air O
pollution O
and O
daily O
mortality O
: O
a O
review O
and O
meta O
analysis O
. O

Environ O
Res O
64 O
: O
36 O
< O
FFFD O
> O
52 O
( O
1994 O
) O
. O

Dominici O
F O
, O
Samet O
J O
, O
Zeger O
SL O
. O

Combining O
evidence O
on O
air O
pollution O
and O
daily O
mortality O
from O
the O
largest O
20 O
US O
cities O
: O
a O
hierarchical O
modeling O
strategy O
. O

R O
Stat O
Soc O
Ser O
A O
, O
in O
press O
. O

Burnett O
RT O
, O
Dales O
RE O
, O
Raizenne O
ME O
, O
Krewski O
D O
, O
Summers O
PW O
, O
Roberts O
GR O
, O
Raad O
- O
Young O
M O
, O
Dann O
T O
, O
Brooke O
T O
. O
Effects O
of O
low O
ambient O
levels O
of O
ozone O
and O
sulfates O
on O
the O
frequency O
of O
respiratory O
admissions O
to O
Ontario O
hospitals O
. O

Environ O
Res O
65 O
: O
172 O
< O
FFFD O
> O
194 O
( O
1994 O
) O
. O

Anderson O
HR O
, O
Spix O
C O
, O
Medina O
S O
, O
Schouten O
JP O
, O
Castellsague O
J O
, O
Rossi O
G O
, O
Zmirou O
D O
, O
Touloumi O
G O
, O
Wojtyniak O
B O
, O
Ponka O
A O
, O
et O
al O
. O

Air O
pollution O
and O
daily O
admissions O
for O

2 O
. O

3 O
. O

4 O
. O

5 O
. O

6 O
. O

844 O

108 O
| O
NUMBER O
9 O
| O
September O
2000 O
< O
FFFD O
> O
Environmental O
Health O
Perspectives O

Articles O

< O
FFFD O
> O

Effects O
of O
particles O
on O
sensitive O
subgroups O

7 O
. O

8 O
. O

9 O
. O

10 O
. O

11 O
. O

12 O
. O

13 O
. O

14 O
. O

15 O
. O

16 O
. O

17 O
. O

18 O
. O

19 O
. O

20 O
. O

21 O
. O

22 O
. O

23 O
. O

24 O
. O

25 O
. O

26 O
. O

27 O
. O

28 O
. O

29 O
. O

30 O
. O

chronic O
obstructive O
pulmonary O
disease O
in O
6 O
European O
cities O
: O
results O
from O
the O
APHEA O
project O
. O

Eur O
Respir O
J O
10 O
: O
1064 O
< O
FFFD O
> O
1071 O
( O
1997 O
) O
. O

Schwartz O
J O
. O
Short O
term O
fluctuations O
in O
air O
pollution O
and O
hospital O
admissions O
of O
the O
elderly O
for O
respiratory O
disease O
. O

Thorax O
50 O
: O
531 O
< O
FFFD O
> O
538 O
( O
1995 O
) O
. O

Schwartz O
J O
. O

Air O
pollution O
and O
hospital O
admissions O
for O
heart O
disease O
in O
eight O
U O
. O
S O
. O
counties O
. O

Epidemiology O
10 O
: O
17 O
< O
FFFD O
> O
22 O
( O
1999 O
) O
. O

Schwartz O
J O
. O

Air O
pollution O
and O
hospital O
admissions O
for O
the O
elderly O
in O
Minneapolis O
. O

Arch O
Environ O
Health O
49 O
: O
366 O
< O
FFFD O
> O
374 O
( O
1994 O
) O
. O

Schwartz O
J O
. O

Air O
pollution O
and O
hospital O
admissions O
for O
the O
elderly O
in O
Birmingham O
, O
Alabama O
. O

Am O
J O
Epidemiol O
139 O
: O
589 O
< O
FFFD O
> O
598 O
( O
1994 O
) O
. O

Schwartz O
J O
. O

Air O
pollution O
and O
hospital O
admissions O
for O
the O
elderly O
in O
Detroit O
, O
MI O
. O

Am O
J O
Respir O
Crit O
Care O
Med O
150 O
: O
648 O
< O
FFFD O
> O
655 O
( O
1994 O
) O
. O

Pope O
CA O
III O
. O

Respiratory O
disease O
associated O
with O
community O
air O
pollution O
and O
a O
steel O
mill O
, O
Utah O
valley O
. O

Am O
J O
Public O
Health O
79 O
: O
623 O
< O
FFFD O
> O
628 O
( O
1989 O
) O
. O

Saldiva O
PH O
, O
Pope O
CA O
, O
Schwartz O
J O
, O
Dockery O
DW O
, O
Lichtenfels O
AJ O
, O
Salge O
JM O
, O
Barone O
I O
, O
Bohm O
GM O
. O

Air O
pollution O
and O
mortality O
in O
elderly O
people B
: O
a O
time O
series O
study O
in O
Sao O
Paulo O
, O
Brazil O
. O

Arch O
Environ O
Health O
50 O
: O
159 O
< O
FFFD O
> O
163 O
( O
1995 O
) O
. O

Schwartz O
, O
J O
. O

Air O
pollution O
and O
hospital O
admissions O
for O
cardiovascular O
disease O
in O
Tucson O
. O

Epidemiology O
8 O
: O
371 O
< O
FFFD O
> O
177 O
( O
1997 O
) O
. O

Delfino O
RJ O
, O
Murphy O
Moulton O
AM O
, O
Becklake O
MR O
. O

Emergency O
room O
visits O
for O
respiratory O
illnesses O
among O
the O
elderly O
in O
Montreal O
: O
association O
with O
low O
level O
ozone O
exposure O
. O

Environ O
Res O
76 O
: O
67 O
< O
FFFD O
> O
77 O
( O
1998 O
) O
. O

National O
Research O
Council O
. O

Research O
Priorities O
for O
Airborne O
Particulate O
Matter O
. O

Washington O
, O
DC O
: O
National O
Academy O
Press O
, O
1998 O
. O

Schwartz O
J O
, O
Dockery O
DW O
. O

Increased O
mortality O
in O
Philadelphia O
associated O
with O
daily O
air O
pollution O
concentrations O
. O

Am O
Rev O
Respir O
Dis O
145 O
: O
600 O
< O
FFFD O
> O
604 O
( O
1992 O
) O
. O

Samet O
JM O
, O
Zeger O
SL O
, O
Berhane O
K O
. O

The O
association O
of O
mortality O
and O
particulate O
air O
pollution O
. O

In O
: O
Particulate O
Air O
Pollution O
and O
Daily O
Mortality O
. O

The O
Phase O
I O
Report O
of O
the O
Particle O
Epidemiology O
Evaluation O
Project O
. O

Boston O
, O
MA O
: O
Health O
Effects O
Institute O
, O
1995 O
. O

Schwartz O
J O
. O
What O
are O
people B
dying O
of O
on O
high O
air O
pollution O
days O
? O

Environ O
Res O
64 O
: O
26 O
< O
FFFD O
> O
35 O
( O
1994 O
) O
. O

Sunyer O
J O
, O
Schwartz O
J O
, O
Tobias O
A O
, O
MacFarlane O
D O
, O
Garcia O
J O
, O
Anto O
JM O
. O

Patients B
with O
chronic O
obstructive O
pulmonary O
disease O
are O
a O
susceptible O
population O
of O
dying O
due O
to O
urban O
particles O
. O

Am O
J O
Epidemiol O
151 O
( O
1 O
) O
: O
50 O
< O
FFFD O
> O
56 O
( O
2000 O
) O
. O

Godleski O
JJ O
, O
Sioutas O
C O
, O
Katler O
M O
, O
Koutrakis O
P O
. O
Death O
from O
inhalation O
of O
concentrated O
air O
particles O
in O
animal O
models O
of O
pulmonary O
disease O
. O

Am O
J O
Respir O
Crit O
Care O
Med O
153 O
: O
A15 O
( O
1996 O
) O
. O

Matsui O
K O
, O
Goldman O
L O
. O
Comorbidity O
as O
a O
correlate O
of O
length O
of O
stay O
for O
hospitalized O
patients B
with O
acute O
chest O
pain O
. O

J O
Gen O
Intern O
Med O
11 O
: O
262 O
< O
FFFD O
> O
268 O
( O
1996 O
) O
. O

Charlson O
M O
, O
Szatrowshi O
TP O
, O
Peterson O
J O
, O
Gold O
J O
. O
Validation O
of O
a O
combined O
comorbidity O
index O
. O

J O
Clin O
Epidemiol O
47 O
: O
1245 O
< O
FFFD O
> O
1251 O
( O
1994 O
) O
. O

Monane O
M O
, O
Kanter O
DS O
, O
Glynn O
RJ O
, O
Avorn O
J O
. O
Variability O
in O
length O
of O
hospitalization O
for O
stroke O
. O

The O
role O
of O
managed O
care O
in O
an O
elderly O
population O
. O

Arch O
Neurol O
53 O
: O
848 O
( O
1996 O
) O
. O

Hallstrom O
AP O
, O
Cobb O
LA O
, O
Yu O
BH O
. O

Influence O
of O
comorbidity O
on O
the O
outcome O
of O
patients B
treated O
for O
out O
- O
of O
- O
hospital O
ventricular O
fibrillation O
. O

Circulation O
93 O
: O
2019 O
< O
FFFD O
> O
2022 O
( O
1996 O
) O
. O

Malenka O
DJ O
, O
Mclerran O
D O
, O
Roos O
N O
, O
Fisher O
ES O
, O
Wennberg O
JE O
. O

Using O
administrative O
data O
to O
describe O
case O
- O
mix O
: O
a O
comparison O
with O
the O
medical O
record O
. O

J O
Clin O
Epidemiol O
47 O
: O
1027 O
< O
FFFD O
> O
1032 O
( O
1994 O
) O
. O

Romano O
PS O
, O
Roos O
LL O
, O
Jollis O
JG O
. O

Adapting O
a O
clinical O
comorbidity O
index O
for O
use O
with O
ICD O
- O
9 O
- O
CM O
administrative O
data O
: O
differing O
perspectives O
. O

J O
Clin O
Epidemiol O
46 O
: O
1075 O
< O
FFFD O
> O
1079 O
( O
1993 O
) O
. O

Deyo O
RA O
, O
Cherkin O
DC O
, O
Ciol O
MA O
. O

Adapting O
a O
clinical O
comorbidity O
index O
for O
use O
with O
ICD O
- O
9CM O
administrative O
databases O
. O

J O
Clin O
Epidemiol O
45 O
: O
613 O
< O
FFFD O
> O
619 O
( O
1992 O
) O
. O

Charlson O
ME O
, O
Pompei O
P O
, O
Ales O
KL O
, O
MacKenzie O
CR O
. O

A O
new O
method O
of O
classifying O
prognostic O
comorbidity O
in O
longitudinal O
studies O
: O
development O
and O
validation O
. O

J O
Chronic O
Dis O
40 O
: O
373 O
< O
FFFD O
> O
383 O
( O
1987 O
) O
. O

Librero O
J O
, O
Peir O
< O
FFFD O
> O
S O
, O
Ordi O
< O
FFFD O
> O
ana O
R O
. O

Chronic O
comorbidity O
and O
outcomes O
of O
hospital O
care O
: O
length O
of O
stay O
, O
mortality O
, O
and O
readmission O
at O
30 O
and O
365 O
days O
. O

J O
Clin O
Epidemiol O
52 O
: O
171 O
< O
FFFD O
> O
179 O
( O
1999 O
) O
. O

31 O
. O

Nehls O
GJ O
, O
Akland O
GG O
. O

Procedures O
for O
handling O
aerometric O
data O
. O

J O
Air O
Pollut O
Control O
Assoc O
23 O
: O
180 O
< O
FFFD O
> O
184 O
( O
1973 O
) O
. O

32 O
. O

Hastie O
T O
, O
Tibshirani O
R O
. O
Generalized O
Additive O
Models O
. O

London O
: O
Chapman O
and O
Hall O
, O
1990 O
. O

33 O
. O

Schwartz O
J O
. O
Generalized O
additive O
models O
in O
epidemiology O
. O

In O
: O
International O
Biometric O
Society O
, O
Invited O
Papers O
. O

17th O
International O
Biometric O
Conference O
, O
8 O
< O
FFFD O
> O
12 O
August O
1994 O
, O
Hamilton O
, O
Ontario O
, O
Canada O
. O

Washington O
, O
DC O
: O
International O
Biometric O
Society O
, O
1994 O
; O
55 O
< O
FFFD O
> O
80 O
. O

34 O
. O

Rossi O
G O
, O
Vigotti O
MA O
, O
Zanobetti O
A O
, O
Repetto O
F O
, O
Giannelle O
V O
, O
Schwartz O
J O
. O

Air O
pollution O
and O
cause O
specific O
mortality O
in O
Milan O
, O
Italy O
, O
1980 O
< O
FFFD O
> O
1989 O
. O

Arch O
Environ O
Health O
54 O
: O
158 O
< O
FFFD O
> O
164 O
( O
1999 O
) O
. O

35 O
. O

Cleveland O
WS O
, O
Devlin O
SJ O
. O

Robust O
locally O
- O
weighted O
regression O
and O
smoothing O
scatterplots O
. O

J O
Am O
Stat O
Assoc O
74 O
: O
829 O
< O
FFFD O
> O
836 O
( O
1988 O
) O
. O

36 O
. O

Akaike O
H O
. O
Information O
theory O
and O
an O
extension O
of O
the O
maximum O
likelihood O
principal O
. O

In O
: O
2nd O
International O
Symposium O
on O
Information O
Theory O
( O
Petrov O
BN O
, O
Csaki O
F O
, O
eds O
) O
. O

Budapest O
: O
Akademiai O
Kaiado O
, O
1973 O
; O
267 O
< O
FFFD O
> O
281 O
. O

37 O
. O

Brumback O
BA O
, O
Ryan O
LM O
, O
Schwartz O
J O
, O
Neas O
LM O
, O
Stark O
PC O
, O
Burge O
HA O
. O

Transitional O
regression O
models O
with O
application O
to O
environmental O
time O
series O
. O

J O
Acoust O
Soc O
Am O
95 O
( O
449 O
) O
: O
16 O
< O
FFFD O
> O
28 O
( O
2000 O
) O
. O

38 O
. O

Schwartz O
J O
. O

The O
distributed O
lag O
between O
air O
pollution O
and O
daily O
deaths O
. O

Epidemiology O
11 O
: O
320 O
< O
FFFD O
> O
326 O
( O
2000 O
) O
. O

39 O
. O

Pope O
CA O
III O
, O
Verrier O
RL O
, O
Lovett O
EG O
, O
Larson O
AC O
, O
Raizenne O
ME O
, O
Kanner O
RE O
, O
Schwartz O
J O
, O
Villegas O
GM O
, O
Dockery O
DW O
. O

Heart O
rate O
variability O
associated O
with O
particulate O
air O
pollution O
. O

Am O
Heart O
J O
138 O
: O
890 O
< O
FFFD O
> O
899 O
( O
1999 O
) O
. O

40 O
. O

Gold O
DR O
, O
Litonjua O
A O
, O
Schwartz O
J O
, O
Lovett O
E O
, O
Larson O
A O
, O
Nearing O
B O
, O
Allen O
G O
, O
Verrier O
M O
, O
Cherry O
R O
, O
Verrier O
R O
. O
Ambient O
pollution O
and O
heart O
rate O
variability O
. O

Circulation O
101 O
( O
11 O
) O
: O
1267 O
< O
FFFD O
> O
1273 O
( O
2000 O
) O
. O

41 O
. O

Liao O
D O
, O
Creason O
J O
, O
Shy O
C O
, O
Williams O
R O
, O
Watts O
R O
, O
Zweidinger O
R O
. O
Daily O
variation O
of O
particulate O
air O
pollution O
and O
poor O
cardiac O
autonomic O
control O
in O
the O
elderly O
. O

Environ O
Health O
Perspect O
107 O
: O
521 O
< O
FFFD O
> O
525 O
( O
1999 O
) O
. O

42 O
. O

Peters O
A O
, O
Liu O
E O
, O
Verrier O
RL O
, O
Schwartz O
J O
, O
Gold O
DR O
, O
Mittelman O
M O
, O
Baliff O
J O
, O
Allen O
G O
, O
Monahan O
K O
, O
Dockery O
DW O
. O

Air O
pollution O
and O
incidences O
of O
cardiac O
arrhythmia O
. O

Epidemiology O
11 O
( O
1 O
) O
: O
11 O
< O
FFFD O
> O
17 O
( O
2000 O
) O
. O

43 O
. O

Godleski O
JJ O
, O
Verrier O
RL O
, O
Koutrakis O
P O
, O
Catalano O
P O
. O
Mechanisms O
of O
Morbidity O
and O
Mortality O
from O
Exposure O
to O
Ambient O
Air O
Particles O
. O

Health O
Effects O
Institute O
Research O
Report O
91 O
. O

Cambridge O
, O
MA O
: O
Health O
Effects O
Institute O
, O
2000 O
. O

44 O
. O

Watkinson O
WP O
, O
Campen O
MJ O
, O
Kodavanti O
UP O
, O
Ledbetter O
AD O
, O
Costa O
DL O
. O

Effects O
of O
inhaled O
residual O
oil O
fly O
ash O
particles O
on O
electrocardiographic O
and O
thermoregulatory O
parameters O
in O
normal O
and O
compromised O
rats B
[ O
Abstract O
] O
. O

Am O
J O
Respir O
Crit O
Care O
Med O
157 O
: O
A150 O
( O
1998 O
) O
. O

45 O
. O

Watkinson O
WP O
, O
Campen O
MJ O
, O
Costa O
DL O
. O

Cardiac O
arrhythmia O
induction O
after O
exposure O
to O
residual O
oil O
fly O
ash O
particles O
in O
a O
rodent B
model O
of O
pulmonary O
hypertension O
. O

Toxicol O
Sci O
41 O
: O
209 O
< O
FFFD O
> O
216 O
( O
1998 O
) O
. O

46 O
. O

Kodavanti O
UP O
, O
Jackson O
MC O
, O
Richards O
J O
, O
Ledbetter O
A O
, O
Costa O
DL O
. O

Differential O
pulmonary O
responses O
to O
inhaled O
emission O
particulate O
matter O
( O
PM O
) O
in O
systemically O
hypertensive O
vs O
. O
normotensive O
rats B
[ O
Abstract O
] O
. O

Am O
J O
Respir O
Crit O
Care O
Med O
157 O
: O
A260 O
( O
1998 O
) O
. O

47 O
. O

Hoek O
G O
, O
Brunekreef O
B O
, O
van O
Wijnen O
JH O
. O

Cardiovascular O
mortality O
response O
to O
air O
pollution O
is O
strongest O
for O
heart O
failure O
and O
thrombotic O
causes O
of O
death O
[ O
Abstract O
] O
. O

Epidemiology O
10 O
: O
S177 O
( O
1999 O
) O
. O

48 O
. O

Bates O
DV O
, O
Szito O
R O
. O
Hospital O
admissions O
and O
air O
pollutants O
in O
southern O
Ontario O
: O
the O
acid O
summer O
haze O
effect O
. O

Environ O
Res O
43 O
: O
317 O
< O
FFFD O
> O
331 O
( O
1987 O
) O
. O

49 O
. O

Pope O
CA O
III O
. O

Respiratory O
disease O
associated O
with O
community O
air O
pollution O
and O
a O
steel O
mill O
, O
Utah O
valley O
. O

Am O
J O
Public O
Health O
79 O
: O
623 O
< O
FFFD O
> O
628 O
( O
1989 O
) O
. O

50 O
. O

Zelikoff O
JT O
, O
Nadziejko O
C O
, O
Fang O
T O
, O
Gordon O
C O
, O
Premdass O
C O
, O
Cohen O
MD O
. O

Short O
term O
, O
low O
- O
dose O
inhalation O
of O
ambient O
particulate O
matter O
exacerbates O
ongoing O
pneumococcal O
infections O
in O
Streptococcus B
Pneumoniae I
- O
infected O
rates O
. O

In O
: O
Proceedings O
of O
the O
Third O
Colloquium O
on O
Particulate O
Air O
Pollution O
and O
Human B
Health O
( O
Phalen O
RF O
, O
Bell O
YM O
, O
eds O
) O
. O

Irvine O
, O
CA O
: O
Air O
Pollution O
Health O
Effects O
Laboratory O
, O
University O
of O
California O
, O
1999 O
; O
8 O
- O
94 O
< O
FFFD O
> O
8 O
- O
101 O
. O

51 O
. O

Clarke O
RW O
, O
Hemenway O
DR O
, O
Frank O
R O
, O
Kleeberger O
SR O
, O
Longphre O
MV O
, O
Jakab O
GJ O
. O

Particle O
associated O
sulfate O
exposure O
enhances O
murine B
influenza O
mortality O
[ O
Abstract O
] O
. O

Am O
J O
Respir O
Crit O
Care O
Med O
155 O
: O
A245 O
( O
1997 O
) O
. O

52 O
. O

Pope O
CA O
, O
Dockery O
DW O
, O
Spengler O
JD O
, O
Raizenne O
ME O
. O

Respiratory O
health O
and O
PM10 O
pollution O
: O
a O
daily O
time O
series O
analysis O
. O

Am O
Rev O
Respir O
Dis O
144 O
: O
668 O
< O
FFFD O
> O
674 O
( O
1991 O
) O
. O

53 O
. O

Schwartz O
J O
, O
Koenig O
J O
, O
Slater O
D O
, O
Larson O
T O
. O
Particulate O
air O
pollution O
and O
hospital O
emergency O
visits O
for O
asthma O
in O
Seattle O
. O

Am O
Rev O
Respir O
Dis O
147 O
: O
826 O
< O
FFFD O
> O
831 O
( O
1993 O
) O
. O

54 O
. O

Thurston O
GD O
, O
Ito O
K O
, O
Lippman O
M O
, O
Hayes O
CG O
, O
Bates O
DV O
. O

Respiratory O
hospital O
admissions O
and O
summertime O
haze O
air O
pollution O
in O
Toronto O
, O
Ontario O
: O
consideration O
of O
the O
role O
of O
acid O
aerosols O
. O

Environ O
Res O
65 O
: O
271 O
< O
FFFD O
> O
290 O
( O
1994 O
) O
. O

55 O
. O

Norris O
G O
, O
YoungPong O
SN O
, O
Koenig O
JQ O
, O
Larson O
TV O
, O
Sheppard O
L O
, O
Stout O
JW O
. O

An O
association O
between O
fine O
particles O
and O
asthma O
emergency O
department O
visits O
for O
children B
in O
Seattle O
. O

Environ O
Health O
Perspect O
107 O
: O
489 O
< O
FFFD O
> O
493 O
( O
1999 O
) O
. O

56 O
. O

Hamada O
K O
, O
Goldsmith O
CW O
, O
Kobzik O
L O
. O

Air O
pollutant O
aerosols O
allow O
airway O
sensitization O
to O
allergen O
in O
juvenile O
mice B
. O

Am O
J O
Resp O
Crit O
Care O
Med O
A28 O
( O
1999 O
) O
. O

57 O
. O

Lambert O
AL O
, O
Selgrade O
M O
, O
Dong O
W O
, O
Winsett O
D O
, O
Gilmour O
M O
. O
Enhanced O
allergic O
sensitization O
by O
residual O
oil O
fly O
ash O
particles O
is O
mediated O
by O
soluble O
metal O
constituents O
[ O
Abstract O
] O
. O

Am O
J O
Respir O
Crit O
Care O
Med O
159 O
: O
A26 O
( O
1999 O
) O
. O

58 O
. O

Dailey O
LA O
, O
Madden O
MC O
, O
Devlin O
RB O
. O

Do O
airway O
epithelial O
cells O
from O
normal O
and O
asthmatic O
donors O
respond O
differently O
to O
an O
in O
vitro O
challenge O
with O
a O
particulate O
pollutant O
? O

[ O
Abstract O
] O
. O

Am O
J O
Respir O
Crit O
Care O
Med O
157 O
: O
A598 O
( O
1998 O
) O
. O

59 O
. O

Gilmour O
MI O
, O
Winsett O
D O
, O
Selgrade O
MJ O
, O
Costa O
DL O
. O

Residual O
oil O
fly O
ash O
exposure O
enhances O
allergic O
sensitization O
to O
house O
dust O
mite O
in O
rats B
and O
augments O
immune O
- O
mediated O
inflammation O
[ O
Abstract O
] O
. O

Am O
J O
Respir O
Crit O
Care O
Med O
155 O
: O
A244 O
( O
1997 O
) O
. O

60 O
. O

Zeger O
SL O
, O
Thomas O
D O
, O
Dominici O
F O
, O
Samet O
JM O
, O
Schwartz O
JM O
, O
Dockery O
D O
, O
Cohen O
A O
. O
Exposure O
measurement O
error O
in O
time O
< O
FFFD O
> O
series O
studies O
of O
air O
pollution O
: O
concepts O
and O
consequences O
. O

Environ O
Health O
Perspect O
108 O
: O
419 O
< O
FFFD O
> O
426 O
( O
2000 O
) O
. O

61 O
. O

Salvi O
S O
, O
Blomberg O
A O
, O
Rudell O
B O
, O
Kelly O
F O
, O
Sandstrom O
T O
, O
Holgate O
ST O
, O
Frew O
A O
. O
Acute O
inflammatory O
responses O
in O
the O
airways O
and O
peripheral O
blood O
after O
short O
- O
term O
exposure O
to O
diesel O
exhaust O
in O
healthy O
human B
volunteers B
. O

Am O
J O
Respir O
Crit O
Care O
Med O
159 O
: O
702 O
< O
FFFD O
> O
709 O
( O
1999 O
) O
. O

62 O
. O

Tan O
WC O
, O
van O
Eeden O
S O
, O
Qiu O
DW O
, O
Liam O
BL O
, O
Dyachokova O
Y O
, O
Hogg O
JL O
. O

Particulate O
air O
pollution O
, O
bone O
marrow O
stimulation O
and O
the O
pathogenesis O
of O
excess O
cardiovascular O
and O
pulmonary O
deaths O
. O

Am O
J O
Respir O
Crit O
Care O
Med O
155 O
: O
1441 O
< O
FFFD O
> O
1447 O
( O
1997 O
) O
. O

63 O
. O

Gordon O
T O
, O
Nadziejko O
C O
, O
Schlesinger O
R O
, O
Chen O
LC O
. O

Pulmonary O
and O
cardiovascular O
effects O
of O
acute O
exposure O
to O
concentrated O
ambient O
particulate O
matter O
in O
rats B
. O

Toxicol O
Lett O
96 O
< O
FFFD O
> O
97 O
: O
285 O
< O
FFFD O
> O
288 O
( O
1998 O
) O
. O

64 O
. O

Peters O
A O
, O
Doering O
A O
, O
Wichmann O
HE O
, O
Koenig O
W O
. O
Increased O
plasma O
viscosity O
during O
an O
air O
pollution O
episode O
: O
a O
link O
to O
mortality O
? O

Lancet O
349 O
( O
9065 O
) O
: O
1582 O
< O
FFFD O
> O
1587 O
( O
1997 O
) O
. O

65 O
. O

Peters O
A O
, O
Stieberv O
J O
, O
Doering O
A O
, O
Wichmann O
HE O
. O

Is O
systolic O
blood O
pressure O
associated O
with O
air O
pollution O
? O

[ O
Abstract O
] O
. O

Epidemiology O
10 O
( O
4 O
) O
: O
S177 O
( O
1999 O
) O
. O

Environmental O
Health O
Perspectives O

< O
FFFD O
> O
VOLUME O
108 O
| O
NUMBER O
9 O
| O
September O
2000 O

845 O

Syndrome O
of O
arachnomelia O
in O
Simmental O
cattle B

Abstract O

Background O

The O
syndrome O
of O
arachnomelia O
is O
an O
inherited O
malformation O
mainly O
of O
limbs O
, O
back O
and O
head O
in O
cattle B
. O

At O
present O
the O
arachnomelia O
syndrome O
has O
been O
well O
known O
mainly O
in O
Brown O
Swiss O
cattle B
. O

Nevertheless O
, O
the O
arachnomelia O
syndrome O
had O
been O
observed O
in O
the O
Hessian O
Simmental O
population O
during O
the O
decade O
1964 O
- O
1974 O
. O

Recently O
, O
stillborn O
Simmental O
calves B
were O
observed O
having O
a O
morphology O
similar O
to O
the O
arachnomelia O
syndrome O
. O

The O
goal O
of O
this O
work O
was O
the O
characterization O
of O
the O
morphology O
and O
genealogy O
of O
the O
syndrome O
in O
Simmental O
to O
establish O
the O
basis O
for O
an O
effective O
management O
of O
the O
disease O
. O

Results O

The O
first O
pathologically O
confirmed O
arachnomelia O
syndrome O
- O
cases O
in O
the O
current O
Simmental O
population O
appeared O
in O
the O
year O
2005 O
. O

By O
2007 O
, O
an O
additional O
140 O
calves B
with O
the O
arachnomelia O
syndrome O
were O
identified O
. O

The O
major O
pathological O
findings O
were O
malformed O
bones O
affecting O
the O
head O
, O
long O
bones O
of O
the O
legs O
and O
the O
vertebral O
column O
. O

It O
could O
be O
shown O
that O
, O
with O
the O
exception O
of O
two O
cases O
that O
were O
considered O
as O
phenocopies O
, O
all O
of O
the O
paternal O
and O
about O
two O
- O
third O
of O
the O
maternal O
pedigrees O
of O
the O
affected O
calves B
could O
be O
traced O
back O
to O
one O
common O
founder O
. O

Together O
with O
the O
data O
from O
experimental O
matings O
, O
the O
pedigree O
data O
support O
an O
autosomal O
recessive O
mutation O
being O
the O
etiology O
of O
the O
arachnomelia O
syndrome O
. O

The O
frequency O
of O
the O
mutation O
in O
the O
current O
population O
was O
estimated O
to O
be O
3 O
. O
32 O
% O
. O

Conclusion O

We O
describe O
the O
repeated O
occurrence O
of O
the O
arachnomelia O
syndrome O
in O
Simmental O
calves B
. O

It O
resembles O
completely O
the O
same O
defect O
occurring O
in O
the O
Brown O
Swiss O
breed O
. O

The O
mutation O
became O
relatively O
widespread O
amongst O
the O
current O
population O
. O

Therefore O
, O
a O
control O
system O
has O
to O
be O
established O
and O
it O
is O
highly O
desirable O
to O
map O
the O
disease O
and O
develop O
a O
genetic O
test O
system O
. O

Background O

In O
the O
year O
2006 O
a O
syndrome O
was O
described O
in O
the O
German O
and O
Austrian O
Simmental O
( O
Fleckvieh O
, O
as O
it O
is O
locally O
called O
, O
is O
the O
main O
dual O
- O
purpose O
breed O
in O
Germany O
, O
in O
short O
called O
Simmental O
in O
the O
further O
text O
) O
population O
, O
that O
was O
pathologically O
similar O
to O
the O
arachnomelia O
syndrome O
in O
Brown B
Swiss I
cattle B
[ O
1 O
] O
. O

The O
congenital O
arachnomelia O
syndrome O
( O
AS O
, O
OMIA O
Phene O
ID O
139 O
, O
Group O
000059 O
) O
is O
mainly O
a O
malformation O
of O
the O
skeletal O
system O
in O
cattle B
that O
was O
initially O
described O
by O
Rieck O
and O
Schade O
[ O
2 O
] O
in O
Holstein B
Friesian I
, O
Red O
Holstein O
and O
Simmental O
. O

The O
main O
pathological O
changes O
are O
skeletal O
malformations O
of O
the O
legs O
, O
the O
spinal O
column O
and O
the O
skull O
. O

The O
legs O
are O
thinner O
and O
appear O
longer O
than O
normal O
( O
dolichostenomelia O
, O
arachnomelia O
) O
since O
the O
diameter O
of O
the O
diaphyses O
is O
reduced O
. O

These O
long O
bones O
are O
more O
fragile O
and O
, O
in O
combination O
with O
stiffened O
joints O
, O
they O
tend O
to O
fracture O
during O
calving O
. O

The O
fetlock O
joints O
are O
deformed O
, O
often O
stiffened O
and O
show O
hyperextension O
. O

The O
malformation O
of O
the O
spinal O
column O
leads O
to O
kyphosis O
and O
scoliosis O
. O

The O
skull O
malformations O
are O
characterized O
by O
a O
shortened O
lower O
jaw O
( O
brachygnathia O
inferior O
) O
, O
convex O
rounding O
of O
the O
frontal O
bone O
leading O
to O
a O
marked O
stop O
( O
" O
pointer O
head O
" O
) O
and O
rotation O
of O
the O
anterior O
cranium O
. O

In O
some O
cases O
, O
additional O
malformations O
like O
hydrocephalus O
externus O
develop O
[ O
2 O
- O
5 O
] O
. O

Since O
the O
report O
of O
Rieck O
and O
Schade O
[ O
2 O
] O
no O
further O
cases O
were O
reported O
in O
Simmental O
cattle B
, O
but O
in O
the O
1980s O
the O
syndrome O
was O
dispersed O
in O
another O
breed O
, O
the O
European O
Brown O
Swiss O
cattle B
, O
by O
the O
use O
of O
American O
Brown O
Swiss O
sires O
[ O
4 O
, O
6 O
] O
. O

In O
Brown O
Swiss O
an O
autosomal O
recessive O
mode O
of O
inheritance O
was O
supposed O
and O
a O
control O
program O
based O
on O
the O
identification O
of O
carriers O
by O
pedigree O
analyses O
was O
established O
[ O
5 O
] O
. O

Recently O
, O
four O
cases O
of O
arachnomelia O
syndrome O
were O
reported O
in O
Italy O
[ O
3 O
] O
. O

In O
this O
study O
, O
we O
present O
the O
data O
of O
152 O
pathologically O
confirmed O
cases O
of O
arachnomelia O
syndrome O
in O
Simmental O
that O
were O
collected O
from O
October O
2005 O
to O
March O
2007 O
. O

We O
describe O
the O
pathological O
findings O
, O
the O
familial O
occurrence O
and O
an O
estimate O
of O
the O
frequency O
of O
the O
diseases O
allele O
in O
Simmental O
cattle B
. O

Additional O
support O
for O
the O
mode O
of O
inheritance O
and O
the O
genetic O
basis O
of O
the O
arachnomelia O
syndrome O
is O
given O
by O
the O
result O
of O
experimental O
matings O
of O
obligate O
carriers O
. O

Results O
and O
discussion O

AS O
has O
not O
been O
reported O
again O
in O
Simmental O
since O
its O
first O
description O
in O
the O
1970s O
, O
more O
than O
thirteen O
years O
ago O
. O

In O
autumn O
2004 O
a O
number O
of O
stillborn O
calves B
with O
similar O
malformations O
of O
the O
legs O
and O
head O
were O
recorded O
within O
the O
monitoring O
system O
of O
anomalies O
in O
Simmental O
. O

Some O
of O
these O
calves B
were O
sent O
to O
the O
veterinary O
service O
laboratory O
for O
examination O
, O
and O
in O
December O
2005 O
the O
first O
15 O
cases O
of O
AS O
were O
pathologically O
confirmed O
. O

Subsequently O
, O
farmers O
and O
veterinarians O
had O
been O
encouraged O
to O
report O
cases O
by O
an O
information O
leaflet O
and O
various O
articles O
in O
local O
trade O
journals O
. O

An O
increasing O
number O
of O
suspected O
cases O
was O
reported O
and O
an O
additional O
136 O
affected O
calves B
were O
identified O
by O
pathological O
examination O
by O
June O
2007 O
. O

Familial O
occurrence O
and O
case O
presentation O
of O
the O
syndrome O
of O
arachnomelia O

The O
geographical O
origins O
of O
the O
cases O
were O
the O
southern O
part O
of O
Germany O
and O
Austria O
, O
reflecting O
the O
regional O
distribution O
of O
the O
Simmental O
breed O
. O

Both O
sexes O
were O
equally O
represented O
in O
the O
152 O
( O
80 O
male O
, O
72 O
female O
, O
chi O
2 O
= O
0 O
. O
21 O
, O
p O
= O
0 O
. O
64 O
) O
affected O
calves B
. O

The O
largest O
number O
of O
cases O
was O
registered O
in O
2006 O
( O
Figure O
1 O
) O
. O

In O
retrospect O
, O
it O
could O
be O
shown O
that O
the O
main O
reason O
for O
the O
rapid O
increase O
of O
cases O
in O
the O
years O
2005 O
and O
2006 O
was O
the O
high O
popularity O
of O
certain O
sires O
carrying O
the O
AS O
mutation O
( O
ROMEL O
, O
ISO O
- O
Nr O
. O
276000911043667 O
, O
born O
in O
1995 O
; O
EGEL O
, O
276000915512806 O
, O
1985 O
; O
REXON O
, O
276000913008210 O
, O
1989 O
) O
. O

The O
latter O
two O
sires O
represent O
the O
key O
- O
nodes O
of O
the O
pedigree O
pathways O
of O
the O
mutation O
from O
the O
founder O
into O
the O
current O
population O
( O
Figure O
2 O
) O
. O

ROMEL O
, O
for O
example O
, O
sired O
more O
than O
40 O
, O
000 O
cows B
4 O
to O
6 O
years O
ago O
. O

Furthermore O
, O
115 O
sons O
of O
ROMEL O
born O
from O
2001 O
to O
2005 O
are O
registered O
and O
listed O
in O
the O
breeding O
database O
[ O
7 O
] O
. O

These O
progeny O
were O
now O
mated O
to O
ROMEL O
and O
sons O
or O
grandsons O
of O
EGEL O
and O
REXON O
resulting O
in O
a O
high O
probability O
for O
the O
occurrence O
of O
affected O
calves B
. O

Increasing O
awareness O
of O
the O
disease O
and O
abandoning O
of O
selling O
the O
semen O
from O
carriers O
led O
to O
a O
sharp O
drop O
of O
cases O
in O
2007 O
. O

The O
disease O
was O
successfully O
managed O
by O
efficient O
collaboration O
of O
the O
Institute O
for O
Animal O
Breeding O
of O
the O
Bavarian O
State O
Research O
Centre O
for O
Agriculture O
( O
LfL O
) O
, O
the O
Landeskuratorium O
der O
Erzeugerringe O
f O
u O
r O
tierische O
Veredelung O
in O
Bayern O
e O
. O
V O
( O
LKV O
) O
, O
the O
Bavarian O
Animal O
Health O
Service O
( O
TGD O
) O
and O
breeding O
organizations O
. O

Pathological O
findings O

Calves B
under O
suspicion O
of O
the O
arachnomelia O
syndrome O
were O
sent O
to O
the O
pathology O
department O
of O
the O
TGD O
for O
macroscopic O
examination O
. O

The O
observed O
major O
pathological O
findings O
were O
( O
1 O
) O
facial O
deformation O
, O
including O
brachygnathia O
inferior O
and O
concave O
rounding O
of O
the O
maxilla O
forming O
a O
dent O
( O
' O
pointer O
- O
head O
' O
) O
; O
( O
2 O
) O
abnormally O
thin O
diaphyses O
of O
the O
long O
bones O
( O
the O
outer O
diameter O
of O
the O
diaphyses O
is O
diminished O
, O
whereas O
the O
width O
of O
the O
substantia O
compacta O
is O
normal O
) O
leading O
to O
frequent O
fractures O
of O
the O
metacarpus O
and O
metatarsus O
in O
the O
course O
of O
forced O
birth O
assistance O
( O
' O
spider O
- O
legs O
' O
, O
dolichostenomelia O
) O
. O

The O
deformations O
of O
other O
bones O
of O
the O
legs O
were O
less O
apparent O
and O
the O
scapula O
was O
usually O
unaffected O
; O
( O
3 O
) O
angular O
deformations O
of O
the O
distal O
parts O
of O
the O
legs O
characterized O
by O
bilateral O
stiff O
and O
hyperextended O
fetlocks O
with O
the O
extremity O
of O
the O
toe O
forward O
and O
parallel O
to O
the O
trunk O
of O
the O
body O
; O
and O
( O
4 O
) O
defects O
of O
the O
vertebral O
column O
( O
kyphosis O
and O
scoliosis O
) O
, O
but O
not O
of O
the O
ribs O
( O
Figure O
3A O
- O
C O
) O
. O

Additionally O
, O
inconsistent O
pathological O
findings O
included O
cerebral O
herniation O
combined O
with O
a O
malformed O
foramen O
magnum O
, O
microphthalmia O
, O
and O
external O
and O
internal O
hydrocephalus O
. O

The O
latter O
seem O
to O
develop O
secondarily O
, O
due O
to O
the O
enlarged O
foramen O
magnum I
. O

Histological O
examination O
of O
selected O
cases O
revealed O
the O
presence O
of O
hemorrhages O
at O
the O
osteochondral O
junction O
of O
the O
epiphysis O
and O
an O
abrupt O
transmission O
from O
chondral O
to O
osteogenic O
tissue O
. O

Cases O
never O
showed O
isolated O
malformations O
, O
e O
. O
g O
. O
of O
the O
head O
or O
legs O
, O
but O
usually O
a O
combination O
of O
all O
pathological O
findings O
that O
are O
characteristic O
for O
the O
syndrome O
. O

Nevertheless O
, O
the O
degree O
of O
the O
lesions O
ranged O
from O
obvious O
spider O
- O
leg O
cases O
to O
moderate O
or O
mild O
changes O
, O
making O
a O
definite O
diagnosis O
difficult O
. O

The O
latter O
cases O
( O
3 O
) O
were O
excluded O
from O
the O
initial O
pedigree O
analyses O
. O

Meanwhile O
, O
an O
indirect O
gene O
test O
is O
available O
that O
has O
been O
developed O
at O
the O
Institute O
for O
Animal O
Breeding O
of O
the O
Bavarian O
State O
Research O
Centre O
for O
Agriculture O
( O
ITZ O
) O
and O
it O
could O
be O
shown O
that O
these O
cases O
are O
most O
probably O
not O
genetically O
affected O
( O
Buitkamp O
et O
al O
. O
, O
in O
preparation O
) O
. O

Carrier O
identification O

Two O
criteria O
were O
used O
for O
carrier O
identification O
. O

The O
first O
was O
the O
presence O
of O
a O
calf B
that O
was O
diagnosed O
by O
pathological O
investigation O
. O

In O
many O
cases O
more O
than O
one O
affected O
calf B
per O
sire O
was O
identified O
[ O
8 O
] O
. O

Some O
sires O
had O
only O
one O
affected O
calf B
, O
but O
a O
large O
number O
of O
risk O
- O
matings O
. O

In O
these O
cases O
a O
second O
criterion O
, O
the O
statistical O
evaluation O
of O
risk O
- O
matings O
, O
was O
used O
to O
identify O
potential O
phenocopies O
. O

For O
this O
purpose O
, O
the O
probability O
of O
observing O
only O
a O
single O
affected O
calf B
among O
a O
certain O
number O
of O
risk O
- O
matings O
of O
the O
sire O
in O
question O
was O
calculated O
. O

Risk O
- O
matings O
were O
defined O
as O
matings O
with O
direct O
progenies O
of O
identified O
AS O
carriers O
. O

The O
probability O
of O
observing O
an O
affected O
calf B
depends O
also O
on O
the O
probability O
that O
such O
a O
calf B
is O
reported O
to O
the O
LKV O
. O

We O
assumed O
this O
probability O
to O
be O
50 O
% O
. O

Under O
these O
conditions O
, O
the O
probability O
of O
observing O
only O
one O
affected O
calf B
is O
lower O
than O
1 O
. O
0 O
percent O
, O
if O
at O
least O
104 O
risk O
- O
matings O
are O
given O
for O
a O
single O
sire O
. O

In O
this O
case O
it O
is O
very O
likely O
that O
the O
single O
affected O
calf B
is O
a O
phenocopy O
. O

In O
2006 O
and O
2007 O
this O
was O
the O
case O
for O
two O
sires O
used O
for O
artificial O
insemination O
that O
had O
no O
pedigree O
connection O
to O
SEMPER O
( O
see O
below O
) O
. O

Experimental O
matings O
of O
obligate O
carriers O

Four O
out O
of O
seven O
cows B
that O
were O
known O
AS O
carriers O
brought O
to O
the O
facilities O
of O
the O
ITZ O
were O
used O
for O
embryo O
transfer O
( O
Table O
1 O
) O
. O

33 O
of O
the O
60 O
recipients O
( O
55 O
% O
) O
were O
confirmed O
pregnant O
on O
day O
35 O
. O

Four O
of O
the O
33 O
pregnant O
heifers B
( O
12 O
% O
) O
aborted O
between O
days O
36 O
and O
49 O
of O
pregnancy O
. O

Of O
the O
remaining O
29 O
recipients O
, O
6 O
were O
slaughtered O
on O
day O
150 O
, O
6 O
on O
day O
200 O
, O
and O
17 O
animals O
on O
day O
225 O
of O
pregnancy O
( O
Table O
1 O
) O
. O

Four O
fetuses O
( O
three O
male O
and O
one O
female O
) O
out O
of O
29 O
( O
14 O
% O
) O
showed O
the O
typical O
pathological O
changes O
of O
the O
arachnomelia O
syndrome O
as O
described O
above O
( O
Figure O
4C O
, O
D O
) O
. O

All O
other O
fetuses O
showed O
no O
signs O
of O
AS O
( O
Figure O
4A O
, O
B O
) O
. O

Male O
fetuses O
represented O
76 O
% O
( O
22 O
of O
29 O
) O
of O
pregnancies O
( O
chi O
2 O
= O
3 O
. O
123 O
, O
p O
= O
0 O
. O
077 O
, O
Yates O
corrected O
for O
sample O
size O
< O
30 O
) O
. O

Female O
weight O
( O
Table O
2 O
) O
, O
crown O
- O
rump O
length O
at O
day O
225 O
and O
chest O
circumference O
at O
day O
225 O
of O
normal O
fetuses O
were O
lower O
than O
that O
of O
male O
fetuses O
. O

Fetuses O
that O
were O
affected O
by O
the O
arachnomelia O
syndrome O
showed O
lower O
weight O
than O
normal O
fetuses O
. O

The O
affected O
and O
the O
normal O
fetuses O
had O
similar O
crown O
- O
rump O
length O
, O
but O
the O
chest O
circumference O
of O
affected O
fetuses O
was O
higher O
than O
that O
of O
normal O
fetuses O
( O
Table O
2 O
) O
. O

Due O
to O
the O
small O
number O
of O
affected O
female O
fetuses O
, O
a O
comparison O
with O
unaffected O
animals O
for O
sex O
was O
not O
possible O
. O

To O
compare O
unaffected O
and O
affected O
animals O
in O
total O
, O
the O
data O
were O
analyzed O
for O
statistical O
differences O
by O
the O
non O
parametric O
Mann O
- O
Whitney O
- O
Test O
. O

The O
only O
trait O
that O
was O
significantly O
different O
between O
unaffected O
and O
affected O
fetuses O
was O
the O
chest O
circumference O
( O
p O
= O
0 O
. O
004 O
, O
Table O
2 O
) O
. O

The O
body O
weight O
of O
AS O
affected O
calves B
was O
tendentially O
lower O
than O
that O
of O
normal O
calves B
. O

Since O
affected O
calves B
did O
not O
have O
different O
crown O
- O
rump O
- O
length O
and O
their O
chest O
circumference O
was O
even O
higher O
, O
this O
can O
best O
be O
explained O
by O
a O
reduced O
bone O
mass O
. O

Pedigree O
Analysis O
and O
mode O
of O
inheritance O

Eight O
- O
generation O
pedigrees O
of O
all O
cases O
were O
extracted O
from O
the O
joint O
German O
and O
Austria O
pedigree O
data O
and O
screened O
for O
common O
ancestors O
. O

The O
pedigree O
of O
the O
majority O
of O
affected O
calves B
( O
paternal O
line O
150 O
, O
maternal O
line O
106 O
out O
of O
152 O
, O
Table O
3 O
) O
could O
be O
traced O
back O
to O
one O
founder O
, O
SEMPER O
( O
ISO O
- O
Nr O
. O
27000979299305 O
) O
, O
a O
sire O
born O
in O
1964 O
, O
6 O
- O
9 O
generations O
before O
the O
affected O
calves B
were O
born O
( O
Figure O
2 O
) O
. O

Most O
of O
the O
affected O
calves B
inherited O
the O
AS O
mutation O
via O
REXON O
or O
EGEL O
( O
Table O
3 O
, O
Figure O
2 O
) O
. O

In O
44 O
cases O
the O
maternal O
paths O
were O
not O
linked O
to O
the O
common O
pedigree O
( O
Table O
3 O
) O
. O

One O
explanation O
would O
be O
the O
existence O
of O
additional O
, O
hereto O
unknown O
origins O
of O
the O
mutation O
. O

This O
could O
happen O
if O
the O
AS O
mutation O
is O
much O
more O
ancient O
and O
additional O
pedigree O
paths O
exist O
or O
if O
an O
independent O
mutation O
event O
happened O
leading O
to O
the O
same O
phenotype O
. O

An O
alternative O
, O
more O
plausible O
explanation O
could O
be O
the O
occurrence O
of O
misparentages O
. O

It O
is O
well O
known O
that O
in O
the O
pre O
parentage O
- O
test O
era O
, O
the O
frequency O
of O
false O
paternity O
, O
especially O
of O
the O
cows B
, O
was O
reasonable O
high O
( O
up O
to O
23 O
% O
[ O
9 O
] O
) O
. O

Therefore O
, O
there O
is O
a O
good O
chance O
of O
a O
false O
registry O
within O
6 O
- O
9 O
generations O
. O

There O
is O
strong O
support O
for O
the O
assumption O
that O
the O
AS O
is O
regulated O
by O
a O
single O
autosomal O
locus O
acting O
in O
a O
recessive O
manner O
. O

First O
of O
all O
, O
the O
pedigree O
structure O
of O
the O
affected O
calves B
in O
Simmental O
can O
best O
be O
explained O
by O
a O
recessive O
mode O
of O
inheritance O
. O

The O
paternal O
branch O
of O
the O
pedigree O
could O
be O
traced O
back O
to O
one O
sire O
, O
SEMPER O
, O
for O
all O
affected O
calves B
, O
the O
maternal O
branch O
in O
the O
majority O
of O
the O
cases O
. O

Inbreeding O
loops O
over O
a O
few O
generations O
are O
present O
in O
several O
pedigrees O
of O
affected O
calves B
, O
e O
. O
g O
. O
cases O
P3364 O
and O
P1787 O
( O
Figure O
2 O
) O
. O

Sex O
- O
dependent O
inheritance O
can O
obviously O
be O
excluded O
and O
a O
dominant O
mode O
with O
reduced O
penetrance O
seems O
to O
be O
unlikely O
. O

Secondly O
, O
the O
experimental O
matings O
resulted O
in O
4 O
affected O
and O
25 O
unaffected O
fetuses O
, O
a O
result O
that O
most O
closely O
resembles O
the O
expectation O
of O
a O
recessive O
mode O
of O
inheritance O
. O

Thirdly O
, O
the O
occurrence O
of O
cases O
corresponded O
well O
with O
the O
numbers O
expected O
under O
the O
assumption O
of O
a O
recessively O
acting O
mutation O
. O

We O
tested O
this O
on O
the O
progeny O
of O
ROMEL O
, O
the O
largest O
dataset O
available O
from O
one O
carrier O
. O

We O
analyzed O
the O
period O
from O
the O
beginning O
of O
the O
recording O
system O
for O
malformations O
to O
May O
2007 O
. O

In O
that O
period O
44 O
, O
170 O
calves B
were O
born O
that O
were O
sired O
by O
ROMEL O
. O

From O
these O
, O
662 O
were O
considered O
as O
risk O
pairings O
, O
i O
. O
e O
. O
the O
mother O
had O
a O
risk O
of O
0 O
. O
5 O
to O
be O
a O
carrier O
( O
i O
. O
e O
. O
one O
of O
the O
grandparents O
was O
an O
obligate O
carrier O
) O
and O
35 O
calves B
out O
of O
these O
were O
diagnosed O
as O
affected O
. O

Since O
it O
is O
expected O
that O
about O
1 O
/ O
2 O
to O
1 O
/ O
3 O
of O
the O
affected O
calves B
were O
recorded O
, O
this O
result O
is O
very O
close O
to O
the O
expected O
1 O
: O
7 O
ratio O
of O
affected O
to O
unaffected O
calves B
. O

Moreover O
, O
these O
findings O
are O
concordant O
with O
the O
historical O
description O
of O
the O
arachnomelia O
syndrome O
in O
Simmental O
[ O
2 O
] O
and O
the O
analyses O
of O
cases O
in O
Brown O
Swiss O
[ O
5 O
] O
. O

Finally O
, O
when O
applying O
linkage O
analyses O
using O
microsatellite O
markers O
, O
evaluations O
with O
a O
model O
assuming O
recessive O
autosomal O
inheritance O
gave O
the O
highest O
lod O
scores O
( O
Buitkamp O
et O
al O
. O
, O
in O
preparation O
) O
. O

Allelic O
frequency O
of O
carriers O
in O
the O
present O
Simmental O
cow B
population O

Since O
the O
arachnomelia O
syndrome O
- O
allele O
was O
passed O
to O
the O
current O
population O
through O
two O
parental O
lines O
( O
REXON O
and O
EGEL O
) O
and O
the O
main O
carriers O
are O
known O
, O
it O
is O
possible O
to O
estimate O
the O
frequency O
of O
the O
disease O
allele O
by O
an O
allele O
- O
counting O
method O
[ O
10 O
] O
. O

The O
allelic O
frequency O
was O
calculated O
for O
all O
cows B
from O
the O
breeding O
population O
who O
were O
alive O
in O
June O
2007 O
. O

In O
10 O
. O
4 O
percent O
of O
the O
pedigrees O
of O
540 O
, O
725 O
cows B
an O
identified O
carrier O
was O
found O
and O
the O
probability O
that O
individual O
cows B
were O
carrier O
of O
the O
arachnomelia O
syndrome O
- O
allele O
was O
calculated O
. O

E O
. O
g O
. O
in O
14 O
, O
740 O
and O
41 O
, O
032 O
cases O
a O
known O
carrier O
appeared O
as O
sire O
and O
grandsire O
, O
respectively O
. O

In O
these O
cases O
the O
probability O
of O
transmitting O
the O
allele O
is O
50 O
and O
25 O
percent O
, O
respectively O
, O
if O
no O
further O
carrier O
is O
present O
in O
the O
two O
generation O
pedigree O
. O

The O
averaged O
rate O
of O
the O
arachnomelia O
syndrome O
carriers O
based O
on O
known O
carriers O
over O
all O
cows B
alive O
in O
Bavarian O
Simmental O
was O
3 O
. O
32 O
percent O
. O

Using O
this O
approach O
, O
the O
frequency O
of O
carriers O
was O
calculated O
for O
each O
year O
( O
always O
based O
on O
the O
actual O
datasets O
from O
August O
2008 O
) O
from O
2003 O
to O
2008 O
( O
Figure O
1 O
) O
. O

The O
calculations O
were O
done O
twice O
, O
considering O
all O
known O
carriers O
together O
, O
and O
also O
by O
using O
only O
ROMEL O
as O
a O
carrier O
to O
show O
the O
numeric O
contribution O
of O
his O
progeny O
( O
Figure O
1 O
) O
. O

For O
these O
analyses O
, O
the O
sires O
that O
are O
designated O
to O
be O
non O
- O
carriers O
by O
the O
number O
of O
risk O
pairings O
without O
having O
a O
case O
or O
the O
indirect O
gene O
test O
are O
set O
as O
non O
- O
carrier O
. O

Therefore O
, O
these O
frequencies O
are O
slightly O
lower O
than O
the O
initial O
frequency O
estimate O
of O
3 O
. O
32 O
percent O
. O

Conclusion O

The O
cases O
of O
malformed O
Simmental O
calves B
presented O
here O
showed O
the O
same O
morphology O
described O
in O
the O
arachnomelia O
syndrome O
in O
Brown O
Swiss O
[ O
e O
. O
g O
. O
[ O
3 O
] O
] O
, O
even O
though O
there O
is O
a O
certain O
morphological O
variation O
from O
mild O
to O
severe O
malformations O
. O

The O
main O
findings O
, O
brachygnathia O
inferior O
and O
convex O
frontal O
bone O
of O
the O
face O
, O
deformation O
of O
vertebrae O
, O
and O
dysplasia O
of O
the O
limbs O
, O
namely O
the O
diaphyses O
of O
metatarsus O
and O
- O
carpus O
and O
the O
fetlocks O
, O
can O
best O
be O
explained O
by O
irregularly O
developed O
bone O
structure O
at O
the O
corresponding O
locations O
. O

Without O
pathological O
examination O
it O
is O
difficult O
to O
distinguish O
the O
arachnomelia O
syndrome O
from O
other O
malformations O
of O
the O
limbs O
. O

Therefore O
, O
low O
numbers O
of O
cases O
in O
Simmental O
probably O
passed O
unrecognized O
before O
2005 O
. O

In O
that O
year O
the O
allelic O
frequency O
of O
the O
disease O
in O
the O
cow B
population O
increased O
sharply O
because O
some O
sires O
that O
had O
been O
carriers O
of O
the O
mutation O
had O
become O
very O
popular O
2 O
- O
4 O
years O
before O
. O

The O
identification O
of O
a O
common O
ancestor O
, O
the O
results O
from O
the O
experimental O
matings O
and O
the O
analyses O
of O
numbers O
of O
cases O
from O
risk O
matings O
strongly O
support O
the O
hypothesis O
of O
an O
autosomal O
recessively O
inherited O
disease O
. O

Furthermore O
, O
this O
assumption O
is O
concordant O
with O
the O
historical O
description O
of O
the O
syndrome O
in O
Simmental O
and O
Brown O
Swiss O
. O

The O
allelic O
frequency O
of O
the O
arachnomelia O
syndrome O
in O
the O
current O
population O
is O
well O
above O
3 O
percent O
and O
a O
substantial O
number O
of O
progeny O
from O
known O
carriers O
with O
superior O
genetic O
merit O
shall O
be O
used O
as O
sires O
during O
the O
next O
years O
. O

Therefore O
, O
a O
control O
system O
has O
to O
be O
established O
and O
the O
arachnomelia O
syndrome O
- O
gene O
should O
be O
mapped O
as O
a O
prerequisite O
for O
the O
development O
of O
an O
indirect O
gene O
test O
for O
carrier O
identification O
. O

The O
availability O
of O
pathologically O
well O
characterized O
cases O
from O
the O
field O
and O
from O
the O
ET O
- O
generated O
full O
- O
sib O
families O
will O
be O
an O
excellent O
material O
for O
a O
genetic O
mapping O
procedure O
. O

Methods O

Recording O
system O
for O
congenital O
malformations O

A O
system O
for O
monitoring O
inherited O
congenital O
malformations O
in O
Bavarian O
cattle B
populations O
was O
established O
by O
the O
Institute O
for O
Animal O
Breeding O
of O
the O
Bavarian O
State O
Research O
Centre O
for O
Agriculture O
( O
ITZ O
) O
in O
cooperation O
with O
the O
Bavarian O
milk O
recording O
organization O
( O
LKV O
) O
[ O
11 O
] O
. O

In O
short O
, O
a O
questionnaire O
was O
developed O
for O
detailed O
recording O
of O
malformed O
calves B
. O

The O
malformation O
was O
described O
according O
to O
its O
location O
( O
e O
. O
g O
. O
head O
, O
legs O
) O
and O
its O
characteristics O
( O
e O
. O
g O
. O
hernia O
) O
. O

The O
standardized O
data O
were O
stored O
in O
a O
database O
at O
the O
LKV O
, O
that O
is O
evaluated O
monthly O
for O
a O
potential O
genetic O
background O
of O
malformations O
. O

Sires O
that O
fit O
into O
the O
pedigree O
( O
progeny O
of O
REXON O
or O
EGEL O
) O
with O
at O
least O
one O
affected O
calf B
with O
confirmed O
paternity O
were O
defined O
as O
obligate O
carriers O
and O
marked O
in O
the O
breeding O
information O
system O
[ O
7 O
] O
. O

In O
cases O
without O
connection O
to O
the O
pedigree O
and O
only O
one O
recorded O
calf B
the O
number O
of O
" O
risk O
pairings O
" O
( O
matings O
to O
cows B
where O
at O
least O
one O
parent O
is O
a O
known O
carrier O
, O
enabling O
the O
calculation O
of O
the O
probability O
for O
the O
occurrence O
of O
cases O
) O
was O
calculated O
. O

When O
the O
probability O
that O
a O
case O
occurs O
was O
above O
99 O
% O
for O
the O
sire O
in O
question O
the O
case O
was O
considered O
to O
be O
a O
phenocopy O
. O

The O
number O
of O
calves B
affected O
by O
the O
arachnomelia O
syndrome O
and O
their O
parentage O
is O
routinely O
published O
[ O
8 O
] O
. O

Pathological O
examinations O

Pathological O
examinations O
followed O
standard O
procedures O
. O

Calves B
were O
photographed O
and O
size O
and O
weight O
measurements O
were O
recorded O
. O

Tissue O
specimens O
from O
the O
condyle O
( O
epiphysis O
) O
and O
from O
the O
diaphysis O
of O
the O
femur O
were O
collected O
for O
histological O
examination O
. O

Specimens O
were O
fixed O
in O
10 O
% O
formalin O
and O
kept O
in O
a O
decalcifying O
solution O
( O
Ossafixonafor O
) O
for O
24 O
hours O
. O

Thereafter O
, O
specimens O
were O
processed O
in O
an O
automated O
embedding O
system O
, O
sectioned O
at O
4 O
- O
6 O
microns O
and O
finally O
stained O
with O
haematoxyline O
and O
eosin O
. O

Experimental O
matings O
and O
embryo O
transfer O

Known O
carriers O
of O
the O
arachnomelia O
syndrome O
( O
seven O
cows B
that O
had O
produced O
at O
least O
one O
affected O
calf B
) O
were O
brought O
to O
the O
facilities O
of O
the O
ITZ O
for O
embryo O
transfer O
( O
Table O
1 O
) O
. O

Late O
morulae O
and O
blastocysts O
collected O
on O
day O
7 O
( O
day O
0 O
= O
estrus O
) O
from O
superovulated O
donor O
cows B
were O
nonsurgically O
transferred O
to O
heifers B
[ O
12 O
] O
. O

Mode O
of O
inheritance O
and O
allele O
frequency O

The O
pedigree O
of O
all O
cases O
was O
constructed O
from O
the O
pedigree O
that O
is O
used O
for O
the O
joint O
breeding O
evaluation O
of O
Germany O
and O
Austria O
. O

The O
graphical O
presentation O
of O
the O
pedigree O
was O
performed O
with O
the O
Pedigraph O
TM O
software O
[ O
13 O
] O
. O

The O
allelic O
frequency O
of O
the O
AS O
mutation O
in O
the O
current O
cow B
population O
was O
estimated O
from O
ancestors O
with O
known O
genotypes O
following O
the O
allele O
- O
counting O
method O
[ O
10 O
] O
. O

For O
this O
reason O
two O
generation O
pedigrees O
of O
herd O
book O
cows B
in O
Bavarian O
Simmental O
were O
analyzed O
for O
obligate O
carriers O
. O

We O
considered O
all O
cows B
that O
were O
alive O
in O
June O
2007 O
and O
included O
in O
the O
herd O
book O
. O

All O
animals O
were O
bred O
by O
the O
use O
of O
artificial O
insemination O
. O

Statistical O
analyses O

The O
non O
parametric O
Mann O
- O
Whitney O
- O
Test O
was O
performed O
using O
SPSS O
Version O
14 O
. O
0 O
, O
the O
Chi O
- O
square O
test O
was O
performed O
using O
R O
2 O
. O
4 O
. O
0 O
[ O
14 O
] O
. O

Authors O
' O
contributions O

JB O
drafted O
the O
manuscript O
and O
analyzed O
the O
pedigrees O
. O

BL O
conceived O
the O
monitoring O
system O
for O
inherited O
diseases O
. O

RE O
extracted O
the O
data O
from O
the O
database O
and O
estimated O
the O
allelic O
frequencies O
of O
the O
arachnomelia O
syndrome O
. O

HR O
and O
MW O
performed O
the O
embryo O
collection O
, O
transfer O
and O
recorded O
the O
morpho O
- O
metrical O
data O
of O
the O
experimental O
matings O
. O

BS O
examined O
the O
calves B
pathologically O
. O

NM O
and O
KG O
participated O
in O
study O
design O
and O
coordination O
and O
critically O
revised O
the O
manuscript O
. O

All O
authors O
read O
and O
approved O
the O
final O
manuscript O
. O

Suggestions O
Concerning O
the O
Use O
of O
the O
Subclavian O
which O
Arises O
from O
the O
Aorta O
in O
the O
Treatment O
of O
the O
Tetralogy O
of O
Fallot O
* O

Abstract O

Images O

SUGGESTIONS O
CONCERNING O
THE O
USE O
OF O
THE O
SUBCLAVIAN O

WHICH O
ARISES O
FROM O
THE O
AORTA O
IN O
THE O
TREATMENT O

OF O
THE O
TETRALOGY O
OF O
FALLOT O
* O

HARRIS O
B O
. O

SHUMACKER O
, O
JR O
. O
* O
* O

In O
Blalock O
' O
s O
early O
experience O
with O
the O
operative O
treatment O
of O
the O
tetralogy O
of O
Fallot O
, O
the O
two O
subclavian O
, O
the O
innominate O
, O
and O
carotid O
arteries O
were O
all O
used O
for O
anastomosis O
to O
the O
pulmonary O
artery O
. O

Soon O
, O
however O
, O
it O
became O
evident O
that O
use O
of O
the O
innominate O
or O
carotid O
was O
followed O
by O
a O
relatively O
high O
incidence O
of O
complications O
resulting O
from O
cerebral O
ischemia O
. O

Blalock O
suggested O
, O
therefore O
, O
that O
the O
subclavian O
be O
used O
by O
preference O
. O

The O
subclavian O
branch O
of O
the O
innominate O
does O
not O
become O
kinked O
or O
badly O
angulated O
when O
it O
is O
turned O
down O
for O
the O
anastomosis O
, O
and O
a O
good O
functional O
result O
almost O
invariably O
follows O
the O
completion O
of O
a O
satisfactory O
anastomosis O
. O

The O
subclavian O
which O
arises O
directly O
from O
the O
aorta O
, O
on O
the O
other O
hand O
, O
tends O
to O
form O
a O
bad O
angle O
when O
it O
is O
brought O
down O
for O
the O
anastomosis O
, O
and O
, O
indeed O
, O
near O
its O
point O
of O
origin O
it O
may O
be O
so O
flattened O
out O
against O
the O
relatively O
rigid O
aortic O
wall O
as O
to O
obstruct O
all O
blood O
flow O
through O
it O
. O

These O
considerations O
led O
Blalock O
to O
recommend O
the O
use O
of O
the O
former O
except O
in O
infants B
under O
two O
years O
of O
age O
in O
whom O
this O
artery O
may O
be O
too O
small O
and O
in O
adults O
or O
children B
over O
twelve O
years O
old O
or O
five O
feet O
tall O
with O
a O
left O
aortic O
arch O
in O
whom O
it O
is O
often O
too O
short O
to O
permit O
a O
satisfactory O
anastomosis O
. O
7 O
' O

In O
contrast O
, O
Paine O
and O
Varco O
, O
' O
Lam O
, O
6 O
Holman O
, O
' O
Olim O
, O
7 O
and O
others O
have O
preferred O
to O
use O
the O
subclavian O
which O
arises O
from O
the O
aorta O
and O
have O
obtained O
generally O
excellent O
results O
. O

They O
point O
out O
that O
the O
tendency O
to O
kinking O
of O
the O
artery O
is O
more O
a O
theoretical O
than O
a O
practical O
disadvantage O
, O
that O
the O
exposure O
and O
dissection O
of O
both O
the O
pulmonary O
artery O
and O
the O
subclavian O
are O
easier O
on O
the O
side O
of O
the O
aortic O
arch O
, O
that O
both O
vessels O
are O
generally O
longer O
than O
on O
the O
opposite O
side O
and O
that O
, O
as O
a O
general O
rule O
, O
the O
anastomosis O
can O
be O
accomplished O
more O
readily O
. O

The O
practical O
usefulness O
of O
a O
third O
systemic O
vessel O
, O
namely O
the O
aorta O
, O
was O
demonstrated O
by O
the O
development O
of O
a O
technique O
for O
side O
- O
to O
- O
side O
aortic O
pulmonary O
anastomosis O
by O
Potts O
and O
his O
associates O
. O
" O
0 O
In O
spite O
of O
the O
preferences O
held O
by O
individual O
operators O
for O
use O
of O
one O
vessel O
or O
another O
, O
the O
fact O
remains O
, O
as O
Blalock O
has O
emphasized O
, O
that O
there O
is O
always O
present O
a O
usable O
systemic O
vessel O
provided O
a O
suitable O
pulmonary O
artery O
is O
available O
. O

Nevertheless O
, O
the O
choice O
of O
the O
systemic O
artery O
in O
each O
individual O
patient B
is O
important O
since O
in O
certain O
cases O
one O
or O
another O
is O
unsuitable O
. O

* O
From O
the O
Department O
of O
Surgery O
, O
the O
Indiana O
University O
Medical O
Center O
, O
Indianapolis O
, O
Indiana O
. O

Aided O
by O
a O
contract O
between O
the O
Office O
of O
Naval O
Research O
, O
Department O
of O
the O
Navy O
, O
and O
Indiana O
University O
. O

* O
* O
Resident O
Surgeon O
, O
New O
Haven O
Hospital O
, O
1937 O
- O
1938 O
. O

Received O
for O
publication O
April O
26 O
, O
1951 O
. O

TREATMENT O
OF O
TETRALOGY O
OF O
FALLOT O

It O
has O
been O
my O
custom O
to O
follow O
a O
policy O
rather O
similar O
to O
that O
outlined O
by O
Blalock O
. O

The O
subclavian O
branch O
of O
the O
innominate O
has O
generally O
been O
preferred O
in O
patients B
ranging O
between O
two O
and O
twelve O
years O
of O
age O
and O
in O
older O
individuals O
in O
whom O
there O
is O
a O
right O
aortic O
arch O
. O

In O
others O
the O
thoracotomy O
is O
usually O
made O
on O
the O
side O
of O
the O
aortic O
arch O
, O
and O
either O
the O
aorta O
itself O
or O
the O
subclavian O
arising O
from O
the O
aorta O
is O
used O
for O
the O
anastomosis O
. O

My O
experience O
with O
the O
use O
of O
the O
subclavian O
originating O
from O
the O
aorta O
is O
small O
and O
the O
results O
have O
not O
been O
as O
good O
as O
those O
which O
others O
have O
reported O
. O

One O
eighteen O
- O
month O
- O
old O
child B
died O
the O
day O
after O
operation O
and O
was O
found O
at O
post O
- O
mortem O
examination O
to O
have O
an O
occluded O
anastomosis O
. O

In O
one O
three O
- O
year O
- O
old O
child B
and O
one O
twenty O
- O
year O
- O
old O
woman B
a O
poor O
result O
was O
obtained O
and O
a O
second O
operation O
upon O
the O
other O
side O
was O
necessary O
. O

In O
two O
additional O
cases O
the O
result O
was O
only O
fair O
. O

Excluding O
the O
patients B
mentioned O
in O
the O
present O
report O
, O
only O
in O
two O
was O
an O
unquestionably O
good O
result O
obtained O
. O

Recently O
, O
in O
several O
cases O
in O
which O
failure O
seemed O
evident O
a O
modification O
has O
been O
required O
in O
order O
to O
obtain O
a O
functioning O
shunt O
. O

Since O
these O
modifications O
proved O
successful O
and O
since O
these O
threats O
of O
failure O
tax O
one O
' O
s O
ingenuiity O
at O
the O
time O
of O
operation O
, O
I O
have O
thought O
it O
might O
be O
helpful O
to O
describe O
the O
procedures O
employed O
and O
to O
illustrate O
them O
with O
case O
reports O
. O

One O
is O
a O
method O
which O
obviously O
has O
very O
limited O
applicability O
, O
while O
the O
otlher O
would O
seem O
to O
be O
rather O
generally O
applicable O
. O

The O
first O
consists O
of O
the O
transplantation O
of O
the O
origin O
of O
the O
subclavian O
to O
a O
more O
suitable O
portion O
of O
the O
aorta O
. O

Case O
report O

The O
patient B
was O
a O
19 O
- O
year O
- O
old O
girl B
who O
had O
been O
cyanotic O
since O
the O
age O
of O
six O
months O
. O

She O
had O
developed O
normally O
but O
was O
always O
limited O
in O
exercise O
capacity O
. O

In O
her O
early O
years O
of O
school O
she O
was O
taken O
to O
school O
by O
her O
parents O
and O
carried O
to O
and O
from O
her O
seat O
in O
the O
classroom O
. O

She O
did O
reasonably O
well O
in O
high O
school O
, O
being O
driven O
to O
and O
from O
the O
school O
, O
but O
she O
got O
into O
severe O
difficulty O
when O
she O
attempted O
to O
attend O
college O
. O

The O
longer O
distances O
between O
classes O
and O
the O
necessity O
for O
climbing O
stairs O
precipitated O
a O
downhill O
course O
, O
with O
increasing O
dyspnea O
, O
more O
marked O
cyanosis O
and O
fatigue O
, O
and O
the O
onset O
of O
bouts O
of O
loss O
of O
consciousness O
. O

The O
latter O
occurred O
several O
times O
daily O
. O

One O
which O
was O
witnessed O
by O
her O
physician O
lasted O
45 O
minutes O
; O
he O
feared O
it O
would O
prove O
fatal O
. O

Ordinarily O
, O
she O
could O
not O
walk O
more O
than O
half O
a O
block O
. O

There O
was O
marked O
cyanosis O
of O
the O
nails O
and O
mucous O
membranes O
and O
to O
a O
lesser O
extent O
of O
the O
skin O
. O

Clubbing O
was O
very O
prominent O
. O

All O
the O
results O
of O
physical O
and O
laboratory O
examination O
fitted O
in O
with O
a O
diagnosis O
of O
tetralogy O
of O
Fallot O
. O

The O
hematocrit O
ranged O
between O
83 O
and O
90 O
. O

Oxygen O
saturation O
of O
arterial O
blood O
was O
65 O
per O
cent O
at O
complete O
rest O
. O

The O
aortic O
arch O
was O
on O
the O
left O
side O
. O

On O
October O
26 O
, O
1949 O
a O
left O
lateral O
thoracotomy O
was O
performed O
, O
the O
pleural O
cavity O
being O
entered O
through O
the O
bed O
of O
the O
fifth O
rib O
. O

There O
was O
evident O
greatly O
increased O
collateral O
circulation O
in O
the O
mediastinum O
and O
hilum O
. O

The O
pulmonary O
artery O
appeared O
to O
have O
markedly O
reduced O
blood O
flow O
, O
was O
very O
short O
, O
small O
, O
and O
thin O
- O
walled O
. O

Its O
diameter O
was O
less O
than O
5 O
mm O
. O

The O
aorta O
seemed O
to O
be O
bowed O
out O
laterally O
in O
an O
unusual O
fashion O
and O
the O
short O
pulmonary O
artery O
could O
not O
possibly O
be O
brought O
out O
over O
it O
. O

The O
subclavian O
artery O
was O
relatively O
small O
, O
having O

487 O

YALE O
JOURNAL O
OF O
BIOLOGY O
AND O
MEDICINE O

a O
diameter O
of O
only O
about O
4 O
mm O
. O
but O
it O
seemed O
rather O
long O
. O

It O
was O
apparent O
that O
a O
Pott O
' O
s O
procedure O
was O
impossible O
and O
that O
an O
end O
- O
to O
- O
side O
subclavian O
pulmonary O
artery O
anastomosis O
would O
be O
doomed O
to O
failure O
. O

Hence O
, O
the O
subclavian O
artery O
was O
divided O
at O
the O
level O
of O
its O
first O
branch O
, O
the O
pulmonary O
artery O
was O
divided O
proximally O
, O
and O
an O
end O
- O
to O
- O
end O
subclavian O
- O
pulmonary O
anastomosis O
was O
carried O
out O
. O

This O
was O
done O
with O
ease O
. O

When O
the O
clamps O
were O
removed O
, O
however O
, O
no O
blood O
flowed O
through O
it O
. O

The O
softwalled O
subclavian O
was O
completely O
flattened O
out O
against O
the O
relatively O
rigid O
aorta O
( O
Fig O
. O
1 O
) O
. O

When O
the O
hilar O
structures O
were O
forcibly O
elevated O
in O
a O
cephalad O
direction O
the O
subclavian O
immediately O
filled O
and O
pulsated O
normally O
and O
a O
thrill O
could O
be O
felt O
. O

Simple O
expansion O
of O
the O
lungs O
, O
however O
, O
failed O
to O
achieve O
this O
result O
and O
I O
could O
conceive O
of O
no O
way O
in O
which O
the O
hilar O
region O
could O
be O
held O
in O
a O
more O
cephalad O
direction O
. O

The O
subclavian O
was O
then O
ligated O
at O
the O
point O
where O
it O
came O
off O
the O
aorta O
and O
its O
proximal O
end O
was O
anastomosed O
end O
- O
to O
- O
side O
to O
the O
descending O
aorta O
. O

It O
now O
pulsated O
vigorously O
as O
did O
the O
pulmonary O
artery O
. O

In O
spite O
of O
the O
good O
pulsation O
no O
thrill O
was O
palpable O
. O

The O
patient B
had O
an O
uneventful O
but O
disappointing O
convalescence O
. O

She O
remained O
markedly O
cyanotic O
and O
no O
continuous O
bruit O
was O
audible O
. O

When O
she O
left O
the O
hospital O
14 O
days O
after O
operation O
there O
was O
no O
improvement O
in O
her O
appearance O
, O
hematocrit O
, O
or O
arterial O
oxygen O
saturation O
. O

Surprisingly O
enough O
she O
reported O
progress O
on O
each O
follow O
- O
up O
examination O
. O

By O
the O
end O
of O
seven O
weeks O
she O
was O
obviously O
less O
cyanotic O
, O
her O
hematocrit O
was O
77 O
, O
and O
she O
was O
able O
to O
walk O
a O
number O
of O
blocks O
and O
to O
climb O
a O
flight O
of O
stairs O
without O
difficulty O
. O

She O
reported O
that O
she O
had O
danced O
and O
roller O
- O
skated O
without O
much O
trouble O
. O

Shortly O
thereafter O
a O
continuous O
murmur O
was O
audible O
in O
the O
left O
chest O
. O

She O
continued O
to O
improve O
and O
in O
January O
entered O
a O
southern O
college O
. O

She O
did O
well O
. O

At O
least O
once O
a O
day O
, O
often O
twice O
, O
she O
walked O
without O
difficulty O
from O
the O
campus O
into O
town O
and O
back O
, O
a O
distance O
of O
ten O
blocks O
each O
way O
. O

She O
played O
a O
little O
tennis O
, O
learned O
to O
swim O
, O
and O
began O
to O
dance O
, O
including O
jitterbugging O
. O

When O
she O
xvas O
seen O
in O
June O
, O
her O
color O
was O
good O
, O
although O
there O
was O
still O
slight O
cyanosis O
of O
lips O
and O
nail O
beds O
. O

There O
was O
a O
very O
loud O
, O
continuous O
murmur O
in O
the O
left O
chest O
. O

The O
following O
fall O
she O
transferred O
to O
a O
midwestern O
university O
. O

She O
got O
along O
reasonably O
well O
but O
not O
as O
well O
as O
she O
had O
in O
a O
warmer O
climate O
and O
on O
more O
level O
terrain O
. O

She O
walked O
four O
or O
five O
blocks O
up O
and O
down O
hills O
between O
classes O
without O
difficulty O
in O
good O
weather O
but O
complained O
of O
some O
dyspnea O
and O
fatigue O
on O
cold O
, O
windy O
days O
. O

She O
attended O
dances O
and O
often O
danced O
each O
number O
throughout O
the O
evening O
without O
trouble O
. O

When O
seen O
in O
December O
she O
looked O
well O
. O

Her O
color O
was O
good O
and O
clubbing O
was O
definitely O
less O
marked O
than O
it O
had O
been O
previously O
. O

She O
had O
a O
severe O
cold O
at O
the O
time O
. O

Her O
oxygen O
saturation O
of O
arterial O
blood O
was O
75 O
per O
cent O
at O
rest O
and O
it O
did O
not O
fall O
when O
she O
stood O
or O
still O
- O
walked O
. O

The O
hematocrit O
was O
69 O
. O

There O
was O
audible O
the O
same O
loud O
continuous O
murmur O
in O
the O
left O
chest O
. O

Though O
the O
patient B
has O
been O
markedly O
improved O
, O
it O
is O
recognized O
that O
the O
result O
is O
not O
as O
good O
as O
is O
commonly O
obtained O
when O
patients B
with O
more O
adequate O
pulmonary O
arteries O
are O
treated O
by O
the O
conventional O
anastomosis O
of O
a O
systemic O
artery O
to O
the O
side O
of O
the O
pulmonary O
artery O
. O

Nevertheless O
, O
the O
patient B
has O
thus O
far O
been O
given O
such O
good O
health O
and O
relatively O
normal O
capacity O
for O
ordinary O
activity O
that O
further O
operation O
has O
seemed O
unwarranted O
, O
though O
the O
possibility O
of O
some O
future O
attempt O
at O
creation O
of O
an O
additional O
shunt O
is O
being O
kept O
in O
mind O
. O

The O
second O
procedure O
embodies O
the O
cephalad O
transplantation O
of O
the O
pulmonary O
artery O
by O
a O
plastic O
repair O
of O
the O
incision O
in O
the O
hilar O
and O
mediastinal O
pleural O
structures O
. O

488 O

FIG O
. O

1 O
. O

Drawing O
illustrating O
the O
condition O
which O
existed O
in O
the O
first O
case O
after O
completion O
of O
the O
anastomosis O
( O
A O
) O
and O
its O
correction O
by O
transplantation O
of O
the O
origin O
of O
the O
subclavian O
( O
B O
) O
. O

FIG O
. O

2 O
. O

Drawing O
illustrating O
correction O
of O
obstruction O
to O
blood O
flow O
through O
the O
subclavian O
artery O
by O
inverted O
T O
or O
L O
plastic O
closure O
of O
the O
defect O
in O
the O
hilar O
and O
mediastinal O
structures O
. O

TREATMENT O
OF O
TETRALOGY O
OF O
FALLOT O

Case O
reports O

The O
first O
patient B
was O
a O
fully O
grown O
young O
man B
of O
17 O
with O
tetralogy O
of O
Fallot O
which O
caused O
considerable O
incapacity O
. O

He O
was O
admitted O
to O
the O
hospital O
on O
July O
18 O
, O
1950 O
and O
was O
operated O
upon O
two O
days O
later O
. O

He O
was O
known O
to O
have O
a O
left O
aortic O
arch O
, O
and O
a O
left O
lateral O
thoracotomy O
was O
performed O
. O

The O
aorta O
was O
freed O
for O
a O
side O
- O
to O
- O
side O
anastomosis O
with O
the O
pulmonary O
artery O
. O

So O
much O
difficulty O
was O
encountered O
, O
however O
, O
in O
placing O
the O
aortic O
clamp O
in O
proper O
position O
for O
making O
a O
satisfactory O
incision O
in O
the O
aorta O
that O
the O
procedure O
was O
abandoned O
and O
an O
end O
- O
to O
- O
side O
subclavian O
- O
pulmonary O
artery O
anastomosis O
accomplished O
instead O
. O

The O
pulmonary O
artery O
was O
of O
fair O
size O
, O
having O
an O
estimated O
diameter O
of O
1 O
cm O
. O

The O
subclavian O
artery O
appeared O
to O
be O
quite O
long O
and O
it O
was O
of O
very O
satisfactory O
size O
, O
having O
a O
diameter O
of O
about O
6 O
mm O
. O

To O
my O
dismay O
, O
the O
first O
portion O
of O
the O
soft O
- O
walled O
subclavian O
artery O
was O
completely O
flattened O
out O
against O
the O
rather O
rigid O
wall O
of O
the O
aorta O
and O
no O
blood O
flow O
through O
it O
could O
be O
demonstrated O
, O
there O
being O
no O
subclavian O
pulsation O
nor O
thrill O
in O
the O
pulmonary O
artery O
. O

If O
one O
forcefully O
elevated O
the O
hilum O
of O
the O
lung O
in O
a O
cephalad O
direction O
, O
the O
obstruction O
disappeared O
, O
the O
subclavian O
artery O
began O
to O
pulsate O
, O
and O
a O
thrill O
could O
be O
palpated O
. O

When O
the O
lung O
was O
allowed O
to O
assume O
its O
usual O
position O
, O
the O
subclavian O
obstruction O
was O
again O
evident O
and O
could O
not O
be O
prevented O
by O
full O
expansion O
of O
the O
lung O
. O

It O
was O
found O
that O
the O
hilar O
region O
and O
the O
pulmonary O
artery O
could O
be O
maintained O
in O
a O
satisfactory O
cephalad O
position O
by O
traction O
upward O
upon O
the O
cuff O
of O
hilar O
pleura O
and O
the O
adjacent O
vascular O
sheath O
and O
that O
these O
structures O
were O
sufficiently O
strong O
so O
that O
traction O
could O
be O
maintained O
upon O
them O
with O
a O
small O
hemostat O
. O

The O
defect O
in O
the O
mediastinal O
and O
hilar O
tissues O
was O
then O
repaired O
using O
a O
sort O
of O
inverted O
T O
- O
plastic O
closure O
. O

By O
this O
maneuver O
success O
was O
achieved O
in O
elevating O
the O
pulmonary O
artery O
in O
a O
cephalad O
direction O
so O
that O
the O
subclavian O
obstruction O
was O
relieved O
and O
excellent O
pulsation O
was O
evident O
( O
Fig O
. O
2B O
) O
. O

A O
fairly O
good O
continuous O
thrill O
was O
palpable O
. O

The O
patient B
had O
an O
uneventful O
convalescence O
. O

When O
last O
seen O
on O
January O
16 O
, O
1951 O
he O
had O
excellent O
color O
without O
any O
visible O
cyanosis O
. O

The O
clubbing O
seemed O
to O
have O
decreased O
somewhat O
. O

A O
continuous O
murmur O
was O
audible O
. O

He O
stated O
that O
he O
noted O
no O
limitation O
of O
exercise O
capacity O
. O

In O
outlining O
his O
activities O
he O
said O
that O
, O
among O
other O
things O
, O
he O
was O
doing O
a O
great O
deal O
of O
ice O
- O
skating O
and O
was O
playing O
ice O
hockey O
regularly O
. O

He O
was O
planning O
to O
start O
college O
work O
the O
following O
month O
. O

The O
second O
patient B
was O
a O
16 O
- O
year O
- O
old O
boy B
who O
had O
been O
cyanotic O
since O
birth O
. O

His O
physical O
development O
was O
somewhat O
retarded O
and O
he O
was O
slow O
in O
learning O
to O
sit O
and O
walk O
. O

Until O
he O
had O
grown O
old O
enough O
to O
be O
self O
- O
conscious O
about O
it O
he O
had O
always O
squatted O
when O
he O
was O
tired O
. O

By O
perseverance O
he O
had O
managed O
to O
do O
more O
than O
one O
would O
have O
suspected O
he O
could O
from O
the O
degree O
of O
his O
cyanosis O
. O

He O
could O
walk O
as O
much O
as O
five O
or O
six O
blocks O
at O
a O
slow O
pace O
. O

He O
was O
fond O
of O
drums O
and O
managed O
to O
play O
occasionally O
with O
an O
orchestra O
in O
a O
somewhat O
restricted O
fashion O
. O

For O
the O
past O
few O
months O
he O
had O
had O
more O
dyspnea O
, O
fatigability O
, O
and O
seemed O
to O
be O
going O
downhill O
generally O
. O

The O
results O
of O
physical O
and O
laboratory O
studies O
were O
rather O
typical O
of O
the O
tetralogy O
of O
Fallot O
. O

Clubbing O
was O
marked O
, O
the O
nailbeds O
and O
mucous O
membranes O
were O
a O
rather O
deep O
purplish O
- O
blue O
color O
, O
and O
the O
skin O
had O
a O
dusky O
cyanotic O
tint O
. O

The O
aortic O
arch O
was O
determined O
to O
be O
on O
the O
left O
. O

On O
July O
21 O
, O
1950 O
a O
left O
lateral O
thoracotomy O
was O
performed O
, O
the O
pleural O
cavity O
being O
entered O
through O
the O
bed O
of O
the O
fourth O
rib O
. O

There O
was O
a O
rather O
marked O
increase O
in O
the O
collateral O
circulation O
in O
the O
mediastinum O
and O
the O
hilar O
region O
. O

The O
pulmonary O
artery O
was O
easily O
dissected O
free O
. O

It O
was O
fairly O
long O
and O
was O
about O
1 O
cm O
. O
in O
diameter O
. O

The O
subclavian O
artery O
seemed O
quite O
long O
and O
was O
of O
adequate O
size O
, O
having O
a O
diameter O
of O
about O
5 O
mm O
. O

An O
end O
- O
to O
- O
side O
sub9lavian O
pulmonary O
anastomosis O
was O
performed O
. O

Again O
in O
this O
case O
, O
however O
, O
the O
first O
part O
of O
the O
subclavian O
was O
acutely O
angulated O
and O
completely O
flattened O
out O
against O
the O
aortic O
wall O
. O

There O
was O
no O

489 O

YALE O
JOURNAL O
OF O
BIOLOGY O
AND O
MEDICINE O

pulsation O
in O
the O
subclavian O
and O
no O
thrill O
in O
the O
pulmonary O
. O

In O
this O
instance O
also O
, O
good O
blood O
flow O
through O
the O
subclavian O
artery O
was O
evident O
whenever O
the O
hilar O
structures O
were O
elevated O
in O
a O
cephalad O
direction O
but O
no O
pulsation O
was O
demonstrable O
simply O
by O
expanding O
fully O
the O
lung O
. O

Again O
, O
a O
sort O
of O
inverted O
T O
- O
plastic O
closure O
of O
the O
defect O
in O
the O
hilum O
and O
mediastinum O
brought O
about O
a O
cephalad O
elevation O
of O
the O
pulmonary O
artery O
so O
that O
tension O
was O
released O
and O
there O
was O
excellent O
pulsation O
in O
the O
subclavian O
artery O
. O

A O
thrill O
was O
now O
palpable O
. O

Convalescence O
was O
uneventful O
. O

Improvement O
in O
color O
was O
evident O
within O
a O
few O
days O
and O
his O
color O
was O
excellent O
by O
the O
time O
he O
was O
discharged O
from O
the O
hospital O
on O
the O
thirteenth O
postoperative O
day O
. O

He O
rapidly O
found O
that O
he O
was O
now O
able O
to O
lead O
a O
quite O
normal O
sort O
of O
life O
. O

When O
he O
was O
seen O
on O
December O
9 O
he O
stated O
that O
he O
had O
no O
limitation O
in O
exercise O
capacity O
. O

He O
could O
walk O
rapidly O
without O
fatigue O
. O

He O
was O
going O
to O
school O
and O
was O
playing O
the O
drums O
in O
a O
professional O
orchestra O
three O
or O
four O
nights O
each O
week O
. O

There O
was O
some O
diminution O
in O
the O
clubbing O
and O
his O
color O
was O
excellent O
. O

There O
was O
a O
loud O
continuous O
bruit O
audible O
in O
the O
chest O
. O

On O
March O
27 O
the O
arterial O
oxygen O
saturation O
was O
88 O
. O
2 O
per O
cent O
. O

When O
I O
first O
used O
this O
procedure O
I O
was O
rather O
surprised O
that O
such O
tissues O
would O
hold O
sutures O
and O
serve O
satisfactorily O
to O
elevate O
the O
hilum O
. O

On O
occasions O
I O
had O
previously O
toyed O
with O
the O
idea O
of O
suturing O
hilar O
pleura O
to O
the O
mediastinal O
pleura O
but O
had O
abandoned O
it O
as O
impractical O
because O
the O
sutures O
seemed O
to O
pull O
out O
whenever O
there O
was O
any O
tension O
whatsoever O
. O

If O
the O
procedure O
is O
to O
be O
successful O
, O
it O
is O
essential O
that O
the O
sutures O
encompass O
any O
adjacent O
areolar O
and O
fibrous O
tissue O
and O
especially O
the O
so O
- O
called O
vascular O
sheath O
which O
surrounds O
both O
pulmonary O
artery O
and O
aorta O
and O
is O
dissected O
free O
during O
the O
course O
of O
the O
operation O
. O

Fortunately O
, O
in O
its O
proximal O
portion O
the O
sheath O
about O
the O
pulmonary O
artery O
gains O
added O
strength O
from O
the O
extension O
into O
it O
of O
a O
reflection O
of O
the O
fibrous O
pericardium O
. O

Sutures O
through O
the O
mediastinal O
pleura O
in O
the O
region O
of O
the O
aortic O
arch O
purposely O
include O
the O
perivascular O
tissues O
which O
have O
been O
stripped O
off O
the O
aorta O
and O
subclavian O
artery O
and O
also O
any O
other O
available O
tissue O
which O
may O
lend O
strength O
, O
such O
as O
the O
divided O
ends O
of O
the O
supreme O
intercostal O
vein O
or O
other O
vessels O
which O
lhave O
been O
transected O
and O
ligated O
. O

The O
exact O
method O
of O
repair O
will O
vary O
from O
case O
to O
case O
. O

By O
placing O
the O
sutures O
properly O
it O
would O
seem O
possible O
sometimes O
to O
displace O
the O
pulmonary O
artery O
laterally O
as O
well O
as O
in O
a O
cephalad O
direction O
if O
such O
a O
maneuver O
was O
thought O
to O
be O
desirable O
( O
Fig O
. O
2C O
) O
. O

On O
occasions O
one O
would O
close O
the O
pleural O
defect O
fairly O
snugly O
, O
on O
others O
leave O
it O
wide O
open O
in O
places O
. O

Discussion O

Though O
there O
is O
always O
available O
some O
suitable O
systemic O
vessel O
and O
though O
the O
major O
concern O
in O
the O
operative O
treatment O
of O
the O
tetralogy O
of O
Fallot O
is O
the O
adequacy O
of O
the O
pulmonary O
artery O
, O
from O
time O
to O
time O
one O
may O
find O
the O
achievement O
of O
a O
satisfactory O
result O
thwarted O
by O
the O
local O
anatomical O
characteristics O
regardless O
of O
one O
' O
s O
choice O
of O
procedure O
. O

Consequently O
, O
those O
modifications O
which O
may O
add O
to O
the O
likelihood O
of O
a O
successful O
outcome O
are O
important O
. O

Blalock O
" O
' O
pointed O
out O
the O
practicability O
of O
performing O
an O
end O
- O
toend O
subclavian O
- O
pulmonary O
artery O
anastomosis O
whenever O
the O
pulmonary O
artery O
is O
judged O
too O
small O
or O
the O
subclavian O
too O
short O
for O
a O
satisfactory O
end O

490 O

TREATMENT O
OF O
TETRALOGY O
OF O
FALLOT O
491 O

to O
- O
side O
anastomosis O
. O

Holman O
' O
feels O
that O
a O
poorly O
functioning O
shunt O
after O
end O
- O
to O
- O
side O
anastomosis O
can O
usually O
be O
corrected O
by O
proximal O
division O
of O
the O
subclavian O
artery O
, O
thus O
in O
effect O
converting O
the O
procedure O
into O
an O
end O
- O
to O
- O
end O
anastomosis O
. O

If O
a O
satisfactory O
end O
- O
to O
- O
side O
suture O
of O
subclavian O
and O
pulmonary O
arteries O
or O
side O
- O
to O
- O
side O
aortic O
pulmonary O
anastomosis O
seems O
difficult O
to O
achieve O
, O
one O
may O
occasionally O
find O
it O
useful O
to O
divide O
the O
upper O
lobe O
branch O
of O
the O
pulmonary O
artery O
and O
carry O
out O
an O
end O
- O
to O
- O
end O
suture O
of O
the O
subclavian O
and O
the O
proximal O
end O
of O
the O
upper O
lobe O
branch O
. O

Potts O
and O
Smith O
' O
performed O
an O
anastomosis O
between O
the O
proximal O
end O
of O
the O
upper O
lobe O
branch O
and O
the O
side O
of O
the O
aorta O
in O
a O
case O
in O
which O
complete O
temporary O
occlusion O
of O
the O
main O
pulmonary O
artery O
was O
withstood O
poorly O
. O

I O
have O
found O
this O
principle O
of O
division O
of O
the O
upper O
lobe O
branch O
and O
use O
of O
its O
proximal O
end O
of O
value O
in O
obtaining O
a O
suitable O
subclavian O
pulmonary O
anastomosis O
when O
the O
subclavian O
seemed O
to O
have O
inadequate O
length O
. O

On O
occasions O
when O
the O
systemic O
vessel O
seems O
too O
short O
, O
one O
may O
elect O
to O
interpose O
a O
free O
vascular O
transplant O
, O
3 O
" O
' O
a O
modification O
I O
first O
employed O
in O
1946 O
though O
unfortunately O
not O
with O
success O
in O
this O
instance O
. O

The O
operation O
performed O
in O
my O
first O
patient B
constitutes O
in O
reality O
the O
use O
of O
the O
subclavian O
artery O
as O
an O
autogenous O
graft O
between O
the O
aorta O
and O
pulmonary O
artery O
. O

It O
will O
obviously O
not O
often O
be O
the O
procedure O
of O
choice O
but O
sometimes O
may O
be O
found O
a O
useful O
measure O
in O
converting O
an O
apparently O
inadequate O
functional O
shunt O
into O
a O
good O
one O
. O

If O
my O
initial O
experiences O
with O
the O
plastic O
repair O
of O
the O
defect O
in O
the O
mediastinal O
and O
hilar O
structures O
are O
characteristic O
of O
what O
may O
be O
expected O
of O
this O
procedure O
, O
it O
would O
seem O
to O
have O
wide O
applicability O
whenever O
a O
poorly O
functioning O
subclavian O
- O
pulmonary O
shunt O
seems O
correctable O
by O
cephalad O
transplantation O
of O
the O
hilar O
structures O
and O
the O
consequent O
release O
of O
tension O
. O

I O
was O
unaware O
of O
any O
reference O
in O
the O
literature O
to O
its O
use O
until O
belatedly O
I O
discovered O
that O
I O
had O
overlooked O
a O
statement O
in O
the O
legend O
of O
one O
of O
the O
excellent O
drawings O
in O
Blalock O
' O
s O
paper O
on O
surgical O
procedures O
in O
pulmonic O
stenosis O
. O
' O
Here O
he O
states O
that O
suture O
of O
the O
pleura O
of O
the O
superior O
aspect O
of O
the O
hilum O
to O
the O
mediastinal O
pleura O
may O
effectively O
elevate O
a O
little O
the O
pulmonary O
artery O
. O

Perhaps O
our O
more O
detailed O
consideration O
of O
this O
maneuver O
may O
add O
to O
its O
general O
usefulness O
. O

REFERENCES O

1 O
Blalock O
, O
A O
. O
: O
The O
technique O
of O
creation O
of O
an O
artificial O
ductus O
arteriosus O
in O
the O

treatment O
of O
pulmonic O
stenosis O
. O

J O
. O

Thorac O
. O

Surg O
. O
, O
1947 O
, O
16 O
, O
244 O
. O

2 O
Blalock O
, O
A O
. O
: O
Surgical O
procedures O
employed O
and O
anatomical O
variations O
encountered O

in O
the O
treatment O
of O
congenital O
pulmonic O
stenosis O
. O

Surg O
. O
, O
Gyn O
. O

Obst O
. O
, O
1948 O
, O
87 O
, O
385 O
. O

3 O
Gross O
, O
R O
. O

E O
. O
, O
Bill O
, O
A O
. O

H O
. O
, O
Jr O
. O
, O
and O
Pierce O
, O
E O
. O

C O
. O
: O
Methods O
for O
preservation O
and O

transplantation O
of O
aortic O
grafts O
. O

Observation O
on O
arterial O
grafts O
in O
dogs B
. O

Report O
of O
transplantation O
of O
preserved O
arterial O
grafts O
in O
nine O
human B
cases O
. O

Surg O
. O
, O
Gyn O
. O

Obst O
. O
, O
1949 O
, O
88 O
, O
689 O
. O

4 O
Holman O
, O
E O
. O
: O
The O
surgery O
of O
pulmonary O
stenosis O
. O

Experiences O
with O
left O
subclavian O

to O
left O
pulmonary O
artery O
anastomosis O
. O

J O
. O

Thorac O
. O

Surg O
. O
, O
1949 O
, O
18 O
, O
827 O
. O

5 O
Johnson O
, O
J O
. O
, O
Kirby O
, O
C O
. O

K O
. O
, O
Greifenstein O
, O
F O
. O

E O
. O
, O
and O
Costillo O
, O
A O
. O
: O
The O
experimental O

and O
clinical O
use O
of O
vein O
grafts O
to O
replace O
defects O
of O
large O
arteries O
. O

Surgery O
, O
1949 O
, O
26 O
, O
945 O
. O

492 O
YALE O
JOURNAL O
OF O
BIOLOGY O
AND O
MEDICINE O

6 O
Lam O
, O
C O
. O

R O
. O
: O
The O
choice O
of O
the O
side O
for O
approach O
in O
operations O
for O
pulmonary O

stenosis O
. O

J O
. O

Thorac O
. O

Surg O
. O
, O
1949 O
, O
18 O
, O
661 O
. O

7 O
Olim O
, O
C O
. O

B O
. O
: O
Experiences O
in O
the O
surgical O
treatment O
of O
congenital O
pulmonary O

stenosis O
. O

American O
Surgeon O
, O
1951 O
, O
17 O
, O
245 O
. O

8 O
Paine O
, O
J O
. O

R O
. O
and O
Varco O
, O
R O
. O

C O
. O
: O
Experiences O
with O
the O
surgical O
treatment O
of O
pul O

monic O
stenosis O
. O

Surgery O
, O
1948 O
, O
24 O
, O
355 O
. O

9 O
Potts O
, O
W O
. O

J O
. O
and O
Smith O
, O
S O
. O
: O
New O
surgical O
procedures O
in O
certain O
cases O
of O
congenital O

pulmonary O
stenosis O
. O

Arch O
. O

Surg O
. O
, O
1949 O
, O
59 O
, O
491 O
. O

10 O
Potts O
, O
W O
. O

J O
. O
, O
Smith O
S O
. O
, O
and O
Gibson O
, O
S O
. O
: O
Anastomosis O
of O
the O
aorta O
to O
a O
pulmonary O

artery O
; O
certain O
types O
in O
congenital O
heart O
disease O
. O

J O
. O

Am O
. O

M O
. O

Ass O
. O
, O
1946 O
, O
132 O
, O
627 O
. O

Tissue O
remodeling O
: O
a O
mating O
- O
induced O
differentiation O
program O
for O
the O
Drosophila B
oviduct O

Abstract O

Background O

In O
both O
vertebrates O
and O
invertebrates O
, O
the O
oviduct O
is O
an O
epithelial O
tube O
surrounded O
by O
visceral O
muscles O
that O
serves O
as O
a O
conduit O
for O
gamete O
transport O
between O
the O
ovary O
and O
uterus O
. O

While O
Drosophila B
is O
a O
model O
system O
for O
tubular O
organ O
development O
, O
few O
studies O
have O
addressed O
the O
development O
of O
the O
fly B
' O
s O
oviduct O
. O

Recent O
studies O
in O
Drosophila B
have O
identified O
mating O
- O
responsive O
genes O
and O
proteins O
whose O
levels O
in O
the O
oviduct O
are O
altered O
by O
mating O
. O

Since O
many O
of O
these O
molecules O
( O
e O
. O
g O
. O
Muscle O
LIM O
protein O
84B O
, O
Coracle O
, O
Neuroglian O
) O
have O
known O
roles O
in O
the O
differentiation O
of O
muscle O
and O
epithelia O
of O
other O
organs O
, O
mating O
may O
trigger O
similar O
differentiation O
events O
in O
the O
oviduct O
. O

This O
led O
us O
to O
hypothesize O
that O
mating O
mediates O
the O
last O
stages O
of O
oviduct O
differentiation O
in O
which O
organ O
- O
specific O
specializations O
arise O
. O

Results O

Using O
electron O
- O
and O
confocal O
- O
microscopy O
we O
identified O
tissue O
- O
wide O
post O
- O
mating O
changes O
in O
the O
oviduct O
including O
differentiation O
of O
cellular O
junctions O
, O
remodeling O
of O
extracellular O
matrix O
, O
increased O
myofibril O
formation O
, O
and O
increased O
innervation O
. O

Analysis O
of O
once O
- O
and O
twice O
- O
mated O
females O
reveals O
that O
some O
mating O
- O
responsive O
proteins O
respond O
only O
to O
the O
first O
mating O
, O
while O
others O
respond O
to O
both O
matings O
. O

Conclusion O

We O
uncovered O
ultrastructural O
changes O
in O
the O
mated O
oviduct O
that O
are O
consistent O
with O
the O
roles O
that O
mating O
- O
responsive O
proteins O
play O
in O
muscle O
and O
epithelial O
differentiation O
elsewhere O
. O

This O
suggests O
that O
mating O
triggers O
the O
late O
differentiation O
of O
the O
oviduct O
. O

Furthermore O
, O
we O
suggest O
that O
mating O
- O
responsive O
proteins O
that O
respond O
only O
to O
the O
first O
mating O
are O
involved O
in O
the O
final O
maturation O
of O
the O
oviduct O
while O
proteins O
that O
remain O
responsive O
to O
later O
matings O
are O
also O
involved O
in O
maintenance O
and O
ongoing O
function O
of O
the O
oviduct O
. O

Taken O
together O
, O
our O
results O
establish O
the O
oviduct O
as O
an O
attractive O
system O
to O
address O
mechanisms O
that O
regulate O
the O
late O
stages O
of O
differentiation O
and O
maintenance O
of O
a O
tubular O
organ O
. O

Background O

Most O
internal O
organs O
, O
including O
the O
vascular O
and O
respiratory O
systems O
and O
the O
gastro O
- O
intestinal O
and O
urinary O
- O
genital O
tracts O
are O
comprised O
of O
a O
single O
epithelial O
tube O
or O
a O
network O
of O
tubes O
. O

Tubular O
organs O
serve O
as O
conduits O
for O
the O
transport O
of O
gases O
, O
liquids O
, O
or O
solutes O
, O
and O
serve O
as O
barriers O
between O
biological O
compartments O
. O

To O
create O
tubes O
with O
specific O
flow O
and O
barrier O
properties O
, O
the O
morphology O
of O
the O
tube O
must O
be O
precisely O
specified O
during O
development O
and O
modulated O
by O
physiology O
. O

To O
accommodate O
specific O
physiological O
roles O
, O
tissue O
- O
specific O
programs O
for O
differentiation O
are O
employed O
at O
the O
last O
stages O
of O
development O
. O

While O
much O
is O
known O
about O
the O
molecular O
and O
cellular O
basis O
of O
tube O
formation O
[ O
1 O
- O
6 O
] O
, O
little O
is O
known O
about O
the O
mechanisms O
that O
regulate O
the O
late O
stages O
of O
differentiation O
in O
which O
organ O
- O
specific O
specializations O
arise O
. O

The O
conservation O
of O
genes O
and O
similarity O
in O
tubular O
organ O
design O
across O
taxa O
make O
Drosophila B
an O
excellent O
model O
for O
understanding O
organogenesis O
in O
higher O
animals O
. O

In O
Drosophila B
, O
the O
best O
understood O
tubular O
organs O
from O
a O
developmental O
point O
of O
view O
are O
the O
trachea O
and O
salivary O
gland O
. O

Studies O
of O
these O
organs O
reveal O
a O
general O
program O
for O
tubular O
organ O
development O
, O
in O
which O
combinatorial O
expression O
of O
global O
patterning O
genes O
specifies O
positions O
within O
the O
embryo O
for O
the O
subsequent O
activation O
of O
tissue O
- O
specific O
early O
genes O
and O
transcription O
factors O
. O

This O
program O
results O
in O
the O
activation O
of O
downstream O
genes O
involved O
in O
terminal O
differentiation O
of O
organ O
- O
specific O
specializations O
such O
as O
the O
cuticle O
that O
lines O
the O
tracheal O
lumen O
[ O
1 O
, O
2 O
, O
4 O
, O
7 O
- O
9 O
] O
. O

The O
Drosophila B
female O
reproductive O
tract O
is O
another O
tubular O
system O
, O
consisting O
of O
the O
uterus O
and O
a O
common O
oviduct O
( O
the O
main O
tube O
) O
that O
branches O
into O
two O
lateral O
oviducts O
. O

Regional O
differences O
in O
function O
are O
observed O
along O
the O
length O
of O
the O
tract O
, O
with O
egg O
activation O
occurring O
largely O
at O
the O
proximal O
end O
, O
in O
the O
lateral O
oviducts O
, O
and O
fertilization O
occurring O
at O
the O
distal O
end O
, O
in O
the O
uterus O
[ O
10 O
, O
11 O
] O
. O

Unlike O
other O
tubular O
organs O
, O
little O
is O
known O
about O
the O
development O
of O
the O
female O
reproductive O
tract O
. O

However O
, O
regional O
differences O
in O
function O
suggest O
the O
presence O
of O
region O
- O
specific O
differentiation O
programs O
within O
the O
female O
reproductive O
tract O
. O

In O
Drosophila B
, O
mating O
induces O
changes O
in O
female O
behavior O
and O
physiology O
via O
molecules O
transmitted O
in O
the O
seminal O
fluid O
. O

These O
changes O
are O
rapid O
and O
lead O
to O
a O
mated O
female O
state O
which O
is O
profoundly O
different O
from O
the O
unmated O
female O
state O
. O

While O
an O
unmated O
female O
lays O
few O
eggs O
and O
readily O
accepts O
the O
courtship O
efforts O
of O
a O
male O
, O
a O
mated O
female O
exhibits O
increased O
egg O
- O
laying O
and O
actively O
rejects O
males O
[ O
12 O
- O
19 O
] O
. O

Microarray O
studies O
of O
whole O
flies O
reveal O
that O
the O
changes O
in O
egg O
- O
laying O
rate O
are O
accompanied O
by O
a O
change O
in O
gene O
expression O
. O

Within O
three O
hours O
of O
mating O
there O
is O
an O
increase O
in O
expression O
of O
a O
small O
number O
of O
genes O
[ O
20 O
, O
21 O
] O
. O

Rapid O
changes O
in O
gene O
expression O
, O
as O
well O
as O
protein O
abundance O
, O
have O
also O
been O
observed O
in O
the O
female O
reproductive O
tract O
[ O
22 O
, O
23 O
] O
. O

In O
the O
upper O
reproductive O
tract O
( O
lateral O
and O
common O
oviducts O
, O
hereafter O
, O
oviduct O
) O
, O
mating O
induces O
an O
increase O
in O
immune O
related O
transcripts O
and O
down O
regulates O
transcription O
factors O
involved O
in O
cell O
growth O
and O
differentiation O
. O

At O
the O
protein O
level O
mating O
induces O
increased O
abundance O
of O
proteins O
associated O
with O
muscle O
assembly O
and O
function O
and O
cytoskeletal O
proteins O
associated O
with O
epithelial O
morphogenesis O
[ O
23 O
] O
. O

Since O
many O
of O
these O
mating O
- O
responsive O
proteins O
act O
in O
late O
differentiation O
pathways O
of O
muscle O
and O
epithelia O
elsewhere O
( O
e O
. O
g O
. O
Bent O
, O
Muscle O
LIM O
protein O
84B O
( O
Mlp84B O
) O
, O
Neuroglian O
( O
Nrg O
) O
, O
Coracle O
( O
Cora O
) O
) O
, O
we O
hypothesize O
that O
mating O
triggers O
similar O
differentiation O
in O
the O
oviduct O
. O

To O
test O
our O
hypothesis O
we O
characterized O
the O
ultrastructure O
of O
oviduct O
epithelia O
and O
muscle O
, O
as O
well O
as O
the O
pattern O
of O
innervation O
before O
and O
after O
mating O
. O

We O
then O
examined O
the O
effect O
of O
different O
mating O
regimes O
on O
oviduct O
mating O
- O
responsive O
cytoskeletal O
proteins O
and O
on O
female O
reproductive O
output O
. O

Our O
results O
suggest O
that O
active O
tissue O
remodeling O
takes O
place O
in O
the O
oviduct O
epithelia O
and O
musculature O
in O
response O
to O
mating O
. O

Furthermore O
, O
we O
found O
a O
striking O
increase O
in O
innervation O
of O
the O
oviduct O
after O
mating O
. O

Our O
results O
show O
that O
the O
reproductive O
tract O
is O
an O
attractive O
system O
to O
address O
mechanisms O
that O
regulate O
the O
late O
stages O
of O
tissue O
differentiation O
in O
a O
tubular O
organ O
. O

Unlike O
other O
tubular O
organs O
, O
the O
last O
differentiation O
stage O
of O
the O
oviduct O
is O
triggered O
by O
an O
extrinsic O
cue O
( O
mating O
) O
. O

This O
makes O
it O
possible O
to O
experimentally O
control O
the O
onset O
of O
differentiation O
, O
with O
an O
opportunity O
to O
independently O
examine O
the O
effects O
of O
mating O
and O
age O
. O

In O
addition O
, O
it O
allows O
us O
to O
examine O
processes O
essential O
for O
reproduction O
. O

Results O

Mating O
induces O
changes O
in O
oviduct O
lumen O

Our O
previous O
molecular O
profiling O
showed O
that O
mating O
promotes O
changes O
in O
actin O
- O
based O
cytoskeletal O
molecules O
and O
suggests O
that O
mating O
triggers O
molecular O
changes O
and O
tissue O
remodeling O
in O
the O
female O
reproductive O
tract O
that O
mediate O
its O
progression O
to O
a O
mature O
functional O
stage O
[ O
23 O
] O
. O

To O
gain O
insight O
into O
the O
mechanisms O
that O
underlie O
this O
progression O
, O
we O
used O
light O
and O
electron O
microscopy O
to O
determine O
the O
morphological O
status O
of O
the O
oviduct O
in O
unmated O
and O
mated O
3 O
- O
day O
old O
females O
. O

In O
nearly O
all O
mated O
reproductive O
tracts O
processed O
for O
microscopy O
( O
8 O
/ O
9 O
) O
, O
an O
egg O
was O
located O
in O
one O
of O
the O
lateral O
oviducts O
, O
whereas O
an O
egg O
was O
never O
observed O
in O
the O
oviduct O
of O
unmated O
reproductive O
tracts O
( O
5 O
/ O
5 O
) O
( O
Figure O
1 O
) O
. O

This O
observation O
is O
consistent O
with O
previous O
studies O
that O
report O
increased O
ovulation O
and O
egg O
- O
laying O
at O
6 O
h O
post O
- O
mating O
[ O
24 O
] O
. O

In O
all O
unmated O
reproductive O
tracts O
examined O
, O
the O
region O
between O
the O
lateral O
oviducts O
and O
the O
middle O
of O
the O
common O
oviduct O
was O
either O
tapered O
or O
constricted O
, O
whereas O
this O
region O
appeared O
relaxed O
in O
the O
mated O
reproductive O
tract O
( O
Figure O
1A O
and O
1D O
) O
. O

These O
observations O
raise O
the O
possibility O
that O
the O
lumen O
is O
narrow O
in O
the O
unmated O
oviduct O
and O
larger O
in O
the O
mated O
oviduct O
. O

To O
address O
this O
possibility O
, O
we O
collected O
serial O
1 O
mu O
m O
longitudinal O
sections O
through O
the O
reproductive O
tracts O
of O
unmated O
and O
mated O
females O
and O
stained O
these O
sections O
with O
toluidine O
blue O
to O
survey O
the O
appearance O
of O
the O
lumen O
along O
the O
entire O
length O
of O
the O
oviduct O
( O
Figure O
1B O
and O
1E O
) O
. O

Our O
examination O
reveals O
that O
, O
in O
unmated O
reproductive O
tracts O
, O
the O
lateral O
oviduct O
lumen O
has O
an O
irregular O
shape O
, O
while O
the O
common O
oviduct O
lumen O
appears O
straight O
. O

Moreover O
, O
in O
all O
the O
unmated O
reproductive O
tracts O
sectioned O
, O
we O
detected O
patches O
of O
darkly O
stained O
material O
in O
the O
lumen O
of O
the O
lower O
common O
oviduct O
. O

In O
the O
mated O
reproductive O
tract O
, O
the O
lumen O
of O
the O
upper O
oviduct O
( O
defined O
as O
the O
lateral O
oviduct O
and O
upper O
part O
of O
the O
common O
oviduct O
) O
has O
an O
irregular O
shape O
, O
and O
the O
lumen O
of O
the O
lower O
oviduct O
( O
defined O
as O
the O
lower O
part O
of O
common O
oviduct O
) O
appears O
straight O
. O

In O
addition O
, O
the O
lumen O
of O
the O
lower O
oviduct O
appears O
wider O
in O
mated O
than O
unmated O
reproductive O
tracts O
( O
Figure O
1C O
and O
1F O
) O
. O

Interestingly O
, O
darkly O
stained O
material O
was O
not O
detected O
in O
the O
oviduct O
lumen O
of O
mated O
females O
. O

This O
observation O
appears O
to O
be O
consistent O
with O
the O
description O
made O
by O
Mahowald O
et O
al O
. O

[ O
25 O
] O
, O
who O
reported O
that O
the O
oviduct O
lumen O
of O
unmated O
females O
is O
nearly O
filled O
with O
an O
intima O
- O
like O
matrix O
and O
that O
this O
matrix O
is O
reduced O
after O
mating O
. O

Because O
3 O
- O
day O
- O
old O
mated O
females O
lay O
eggs O
, O
it O
is O
unclear O
whether O
the O
lack O
of O
lumenal O
material O
and O
increase O
in O
lumen O
size O
in O
mated O
females O
occurred O
before O
or O
after O
the O
passage O
of O
eggs O
. O

We O
suggest O
that O
mating O
directly O
or O
indirectly O
induces O
morphological O
changes O
in O
the O
oviduct O
that O
facilitate O
egg O
passage O
through O
the O
duct O
. O

Taken O
together O
, O
our O
observations O
lead O
us O
to O
propose O
that O
the O
oviduct O
lumen O
is O
closed O
and O
/ O
or O
obstructed O
in O
the O
unmated O
reproductive O
tract O
, O
and O
that O
mating O
induces O
changes O
in O
the O
epithelia O
and O
/ O
or O
muscle O
that O
" O
open O
" O
the O
oviduct O
lumen O
. O

Initial O
formation O
of O
cell O
- O
cell O
junctions O
in O
oviduct O
epithelia O
is O
mating O
- O
independent O

To O
determine O
whether O
mating O
induces O
specific O
morphological O
changes O
in O
the O
oviduct O
epithelia O
post O
- O
mating O
, O
we O
next O
examined O
the O
ultrastructure O
of O
the O
oviduct O
epithelia O
in O
unmated O
and O
mated O
reproductive O
tracts O
. O

Since O
molecular O
profiling O
demonstrates O
that O
proteins O
associated O
with O
cellular O
junctions O
such O
as O
alpha O
- O
and O
beta O
- O
Spectrin O
( O
Spec O
) O
, O
Cora O
, O
and O
Nrg O
[ O
23 O
] O
increase O
post O
- O
mating O
, O
we O
first O
determined O
the O
status O
of O
the O
cellular O
junctions O
in O
the O
oviduct O
epithelia O
of O
unmated O
females O
, O
and O
whether O
these O
junctions O
change O
post O
- O
mating O
. O

In O
Drosophila B
, O
most O
ectodermally O
derived O
epithelia O
( O
such O
as O
the O
epidermis O
and O
trachea O
) O
, O
with O
a O
few O
exceptions O
, O
are O
joined O
apically O
by O
a O
belt O
- O
like O
adherens O
junction O
called O
the O
zonal O
adherens O
junction O
( O
ZA O
) O
followed O
basally O
by O
a O
septate O
junction O
( O
SJ O
) O
[ O
26 O
] O
. O

Our O
analysis O
reveals O
that O
the O
oviduct O
is O
lined O
, O
along O
its O
entire O
length O
, O
by O
a O
monolayered O
epithelium O
comprised O
of O
squamous O
- O
type O
cells O
. O

Although O
region O
- O
specific O
differences O
in O
morphology O
were O
observed O
, O
all O
oviduct O
epithelia O
examined O
, O
in O
both O
unmated O
and O
mated O
females O
, O
are O
joined O
along O
their O
lateral O
membranes O
by O
an O
extensive O
SJ O
and O
lack O
an O
apical O
ZA O
( O
Figure O
2 O
) O
. O

SJs O
and O
ZAs O
form O
complete O
belts O
that O
surround O
the O
epithelial O
cell O
, O
thus O
making O
these O
junctions O
easily O
visible O
in O
transverse O
sections O
through O
the O
epithelium O
. O

Because O
ZAs O
were O
not O
detected O
in O
our O
transverse O
sections O
through O
the O
oviduct O
, O
this O
implies O
that O
ZAs O
never O
formed O
, O
or O
developed O
earlier O
and O
were O
lost O
( O
Figure O
2D O
) O
. O

Interestingly O
, O
we O
did O
not O
detect O
any O
ultrastructural O
differences O
in O
the O
SJs O
at O
6 O
h O
post O
- O
mating O
, O
but O
we O
did O
uncover O
differences O
in O
SJ O
ultrastructure O
in O
different O
regions O
of O
the O
oviduct O
. O

Based O
on O
their O
ultrastructure O
, O
two O
types O
of O
SJs O
, O
smooth O
and O
pleated O
, O
can O
be O
distinguished O
in O
Drosophila B
[ O
26 O
] O
. O

Smooth O
SJs O
are O
distinguished O
by O
the O
lack O
of O
visible O
septae O
and O
the O
appearance O
of O
electron O
dense O
material O
in O
the O
intercellular O
space O
, O
while O
pleated O
SJs O
are O
distinguished O
by O
the O
ladder O
- O
like O
appearance O
of O
septae O
. O

In O
the O
lateral O
oviducts O
and O
upper O
common O
oviduct O
, O
septa O
were O
not O
detected O
in O
the O
SJ O
, O
thus O
these O
SJs O
represent O
smooth O
SJs O
or O
an O
immature O
stage O
of O
pleated O
SJ O
( O
Figure O
2A O
, O
2A O
' O
, O
2A O
" O
) O
. O

In O
contrast O
, O
a O
ladder O
- O
like O
arrangement O
of O
septae O
was O
often O
visible O
in O
the O
SJs O
of O
the O
lower O
common O
oviduct O
( O
Figure O
2C O
' O
) O
, O
thus O
these O
SJs O
can O
be O
classified O
as O
pleated O
. O

Unlike O
the O
smooth O
- O
like O
SJs O
of O
the O
upper O
oviduct O
, O
the O
pleated O
SJs O
of O
the O
lower O
oviduct O
are O
followed O
basally O
by O
spot O
type O
adherens O
junction O
( O
spot O
AJs O
) O
( O
Figure O
2B O
, O
2B O
' O
; O
additional O
file O
1 O
) O
. O

Further O
analysis O
is O
necessary O
to O
determine O
if O
the O
SJs O
of O
the O
upper O
and O
lower O
oviduct O
represent O
different O
types O
or O
different O
developmental O
stages O
. O

Our O
findings O
demonstrate O
that O
the O
initial O
formation O
of O
SJs O
, O
as O
well O
as O
spot O
AJs O
in O
the O
lower O
oviduct O
, O
are O
mating O
- O
independent O
. O

This O
raises O
an O
interesting O
question O
. O

Why O
are O
SJ O
proteins O
such O
as O
Cora O
and O
Nrg O
up O
- O
regulated O
post O
- O
mating O
if O
SJs O
are O
formed O
prior O
to O
mating O
? O

It O
is O
possible O
that O
the O
increased O
expression O
of O
SJ O
proteins O
is O
associated O
with O
functional O
changes O
in O
polarized O
secretion O
post O
- O
mating O
. O

Recent O
studies O
have O
shown O
that O
SJs O
play O
an O
unexpected O
role O
in O
regulating O
the O
apical O
secretion O
of O
specialized O
extracellular O
matrix O
molecules O
in O
the O
trachea O
[ O
27 O
, O
28 O
] O
, O
and O
that O
these O
molecules O
are O
important O
regulators O
of O
lumen O
size O
. O

Mating O
modulates O
apical O
secretory O
activity O
in O
the O
oviduct O

Given O
the O
presence O
of O
extensive O
SJs O
in O
the O
oviduct O
and O
the O
role O
of O
SJs O
in O
regulating O
apical O
secretion O
of O
extracellular O
matrix O
molecules O
in O
other O
epithelia O
( O
e O
. O
g O
. O
trachea O
) O
, O
we O
asked O
if O
mating O
modulates O
apical O
secretion O
in O
the O
oviduct O
epithelia O
. O

Our O
ultrastructural O
analysis O
reveals O
that O
different O
regions O
of O
the O
oviduct O
display O
different O
apical O
membrane O
morphology O
( O
i O
. O
e O
microvilli O
or O
pleats O
) O
( O
see O
Figures O
2 O
and O
additional O
file O
2A O
and O
2A O
) O
, O
but O
all O
epithelia O
are O
covered O
by O
an O
electron O
dense O
apical O
extracellular O
matrix O
( O
AECM O
) O
and O
a O
thin O
layer O
of O
cuticle O
. O

We O
found O
that O
mating O
induces O
ultrastructural O
changes O
in O
the O
AECM O
and O
cuticle O
in O
both O
the O
upper O
and O
lower O
oviduct O
. O

In O
the O
upper O
oviduct O
of O
the O
unmated O
female O
, O
the O
AECM O
varies O
in O
thickness O
along O
the O
apical O
surface O
( O
Figure O
3A O
) O
. O

Some O
areas O
have O
little O
AECM O
, O
while O
other O
areas O
are O
covered O
by O
a O
distinct O
layer O
of O
AECM O
( O
~ O
1 O
- O
2 O
mu O
m O
in O
thickness O
; O
Figure O
3B O
) O
. O

However O
, O
the O
AECM O
of O
mated O
females O
is O
more O
evenly O
distributed O
along O
the O
apical O
surface O
, O
( O
~ O
2 O
mu O
m O
in O
thickness O
; O
Figure O
3E O
) O
. O

Strikingly O
, O
the O
AECM O
and O
cuticle O
of O
mated O
females O
have O
a O
ruffled O
appearance O
, O
suggesting O
that O
the O
AECM O
and O
cuticle O
have O
increased O
in O
surface O
area O
( O
Figure O
3F O
) O
. O

Electron O
dense O
granules O
up O
to O
~ O
1 O
. O
5 O
mu O
m O
in O
diameter O
were O
occasionally O
observed O
in O
both O
the O
AECM O
and O
cell O
cytoplasm O
( O
Figure O
3F O
) O
. O

Although O
further O
analysis O
is O
needed O
to O
determine O
the O
role O
of O
these O
granules O
in O
the O
oviduct O
epithelia O
, O
it O
is O
possible O
that O
these O
granules O
participate O
in O
the O
secretion O
and O
deposition O
of O
the O
AECM O
. O

Taken O
together O
, O
our O
findings O
suggest O
that O
polarized O
secretion O
via O
the O
AECM O
, O
while O
ongoing O
in O
the O
upper O
oviduct O
of O
the O
unmated O
female O
is O
enhanced O
and O
/ O
or O
modulated O
post O
- O
mating O
. O

Post O
- O
mating O
changes O
in O
AECM O
ultrastructure O
are O
also O
observed O
in O
the O
lower O
common O
oviduct O
. O

However O
, O
unlike O
the O
AECM O
of O
the O
upper O
oviduct O
, O
the O
AECM O
of O
the O
lower O
oviduct O
is O
well O
developed O
prior O
to O
mating O
. O

In O
the O
lower O
oviduct O
of O
the O
unmated O
female O
, O
the O
AECM O
consists O
of O
an O
amorphous O
electron O
dense O
material O
and O
is O
unevenly O
distributed O
, O
forming O
a O
thick O
, O
bulbous O
layer O
above O
the O
plasma O
membrane O
in O
some O
regions O
( O
additional O
file O
2 O
) O
. O

Matrix O
- O
like O
material O
is O
also O
observed O
in O
the O
lumen O
, O
but O
this O
material O
is O
more O
electron O
dense O
than O
the O
AECM O
( O
additional O
file O
2A O
and O
2A O
) O
. O

In O
the O
mated O
female O
, O
the O
AECM O
is O
flattened O
against O
the O
plasma O
membrane O
and O
is O
uniformly O
distributed O
along O
the O
apical O
surface O
( O
additional O
file O
2B O
and O
2B O
) O
. O

Matrix O
- O
like O
material O
was O
not O
observed O
in O
the O
center O
of O
the O
lumen O
, O
but O
small O
pools O
of O
very O
electron O
dense O
material O
were O
detected O
in O
the O
spaces O
between O
the O
epithelial O
folds O
( O
additional O
file O
2C O
and O
2C O
) O
. O

This O
may O
explain O
why O
lumenal O
matrix O
was O
not O
detected O
at O
the O
light O
microscopic O
level O
in O
the O
mated O
female O
oviduct O
( O
see O
Figure O
1E O
and O
1F O
) O
. O

Taken O
together O
, O
our O
observations O
suggest O
that O
the O
lower O
common O
oviduct O
is O
a O
site O
of O
active O
apical O
secretion O
in O
both O
mated O
and O
unmated O
females O
, O
and O
that O
matrix O
secretion O
, O
particularly O
in O
the O
lumen O
, O
is O
reduced O
post O
- O
mating O
. O

These O
findings O
raise O
the O
intriguing O
possibility O
that O
the O
AECM O
and O
lumenal O
matrix O
function O
as O
a O
plug O
in O
the O
lower O
oviduct O
, O
and O
that O
mating O
induces O
the O
breakdown O
of O
this O
plug O
. O

Mating O
induces O
changes O
in O
hemi O
- O
adherens O
junctions O
in O
upper O
oviduct O

In O
addition O
to O
modulating O
secretion O
at O
the O
apical O
membrane O
, O
mating O
induces O
changes O
at O
the O
basolateral O
membrane O
. O

In O
many O
epithelia O
, O
one O
of O
the O
last O
steps O
of O
differentiation O
is O
the O
development O
of O
a O
layer O
of O
extracellular O
matrix O
( O
ECM O
) O
called O
the O
basal O
lamina O
that O
covers O
the O
apical O
and O
/ O
or O
basal O
membranes O
and O
the O
concomitant O
development O
of O
hemi O
- O
adherens O
junctions O
( O
HAJs O
) O
. O

HAJs O
connect O
the O
cell O
cytoskeleton O
with O
the O
ECM O
and O
are O
formed O
at O
virtually O
all O
cell O
surfaces O
that O
contact O
an O
ECM O
. O

HAJs O
can O
be O
distinguished O
at O
the O
ultrastructural O
level O
as O
a O
patch O
- O
like O
, O
electron O
dense O
undercoat O
of O
the O
plasma O
membrane O
that O
opposes O
the O
basal O
lamina O
( O
[ O
26 O
] O
; O
Figure O
3G O
, O
3H O
) O
. O

In O
the O
Drosophila B
embryo O
, O
the O
HAJs O
and O
basal O
lamina O
are O
formed O
at O
the O
same O
time O
[ O
26 O
] O
. O

Because O
the O
basal O
lamina O
is O
established O
at O
a O
time O
when O
the O
majority O
of O
extracellular O
matrix O
molecules O
are O
actively O
secreted O
[ O
26 O
, O
29 O
] O
, O
this O
suggests O
that O
the O
formation O
of O
HAJs O
is O
tightly O
coordinated O
with O
the O
secretion O
of O
the O
ECM O
. O

One O
of O
the O
most O
striking O
post O
- O
mating O
changes O
observed O
in O
the O
oviduct O
epithelia O
was O
the O
appearance O
of O
numerous O
HAJs O
along O
the O
basolateral O
membrane O
in O
the O
upper O
oviduct O
( O
Figure O
3 O
) O
. O

The O
importance O
of O
HAJs O
, O
particularly O
in O
the O
upper O
oviduct O
, O
is O
underscored O
by O
the O
extensive O
infolding O
of O
the O
basolateral O
membrane O
that O
is O
observed O
in O
both O
unmated O
and O
mated O
females O
( O
Figure O
3A O
and O
3E O
) O
. O

The O
infolded O
membrane O
gives O
rise O
to O
a O
highly O
branched O
intercellular O
space O
that O
is O
filled O
with O
an O
ECM O
( O
Figure O
3C O
and O
3G O
) O
. O

This O
ECM O
is O
contiguous O
with O
the O
basal O
lamina O
that O
surrounds O
the O
epithelia O
. O

Few O
HAJs O
were O
observed O
in O
the O
upper O
oviduct O
of O
the O
unmated O
female O
, O
and O
these O
were O
largely O
restricted O
to O
the O
basal O
membrane O
, O
and O
not O
observed O
along O
the O
basolateral O
infolding O
( O
Figure O
3C O
) O
. O

In O
contrast O
, O
numerous O
HAJs O
appear O
along O
the O
basolateral O
infolding O
post O
- O
mating O
in O
this O
region O
of O
the O
oviduct O
( O
Figure O
3F O
- O
3H O
) O
. O

In O
addition O
, O
the O
intercellular O
space O
appears O
wider O
post O
- O
mating O
( O
Figure O
3C O
- O
3D O
and O
3G O
- O
3H O
) O
, O
suggesting O
that O
mating O
induces O
increased O
secretion O
and O
/ O
or O
deposition O
of O
the O
ECM O
in O
this O
cellular O
compartment O
and O
brings O
the O
ECM O
to O
a O
threshold O
concentration O
that O
can O
support O
the O
development O
of O
HAJs O
. O

HAJs O
were O
also O
detected O
along O
the O
basal O
membrane O
, O
but O
they O
were O
not O
detected O
along O
the O
apical O
membrane O
even O
though O
this O
membrane O
was O
covered O
by O
an O
ECM O
. O

Interestingly O
, O
while O
the O
basolateral O
membrane O
forms O
very O
shallow O
folds O
in O
the O
lower O
oviduct O
( O
see O
additional O
file O
2 O
) O
, O
HAJs O
were O
observed O
along O
this O
membrane O
in O
unmated O
reproductive O
tracts O
( O
data O
not O
shown O
) O
, O
thus O
suggesting O
that O
the O
epithelia O
is O
more O
differentiated O
in O
this O
region O
of O
the O
oviduct O
, O
and O
that O
the O
differentiation O
of O
the O
upper O
and O
lower O
oviduct O
may O
be O
under O
different O
control O
. O

Muscle O
differentiation O
is O
enhanced O
post O
- O
mating O

While O
we O
uncovered O
post O
- O
mating O
changes O
in O
the O
oviduct O
epithelia O
that O
might O
facilitate O
its O
transition O
to O
a O
high O
egg O
- O
laying O
state O
, O
this O
transition O
may O
also O
be O
mediated O
by O
changes O
in O
oviduct O
muscle O
properties O
and O
/ O
or O
activity O
. O

The O
oviduct O
is O
lined O
by O
circular O
muscle O
fibers O
with O
supercontractile O
characteristics O
[ O
30 O
] O
. O

Our O
previous O
studies O
showed O
that O
mef2 O
and O
mlp84B O
genes O
that O
regulate O
muscle O
differentiation O
, O
are O
expressed O
and O
increased O
post O
- O
mating O
in O
the O
oviduct O
, O
as O
well O
as O
in O
the O
sperm O
storage O
regions O
of O
the O
reproductive O
tract O
[ O
22 O
, O
23 O
] O
. O

This O
suggests O
that O
mating O
induces O
muscle O
differentiation O
in O
the O
reproductive O
tract O
. O

Muscle O
differentiation O
is O
characterized O
by O
the O
assembly O
of O
myofilaments O
into O
bundles O
called O
myofibrils O
. O

As O
muscles O
differentiate O
, O
myofibrils O
and O
z O
- O
bodies O
appear O
simultaneously O
, O
and O
increase O
in O
number O
until O
the O
cytoplasm O
is O
filled O
with O
myofibrils O
[ O
31 O
] O
. O

Like O
epithelia O
, O
one O
of O
the O
last O
steps O
of O
muscle O
differentiation O
is O
the O
secretion O
of O
a O
basal O
lamina O
that O
surrounds O
the O
muscle O
fiber O
. O

To O
determine O
if O
mating O
induces O
structural O
changes O
in O
muscles O
( O
such O
as O
increased O
myofibrils O
) O
we O
examined O
the O
ultrastructure O
of O
muscle O
fibers O
in O
the O
upper O
and O
lower O
parts O
of O
the O
oviduct O
. O

Our O
analysis O
revealed O
that O
, O
in O
both O
unmated O
and O
mated O
reproductive O
tracts O
, O
the O
muscles O
of O
the O
lower O
oviduct O
are O
highly O
differentiated O
as O
evidenced O
by O
the O
high O
density O
of O
myofibrils O
, O
well O
developed O
and O
aligned O
z O
- O
bodies O
, O
and O
secretion O
of O
a O
thick O
, O
electron O
dense O
basal O
lamina O
( O
Figure O
4E O
and O
4F O
) O
. O

In O
contrast O
, O
muscle O
fibers O
in O
the O
lateral O
oviducts O
and O
upper O
common O
oviduct O
appear O
less O
differentiated O
than O
muscles O
in O
the O
lower O
common O
oviduct O
, O
as O
evidenced O
by O
the O
lesser O
density O
of O
myofibrils O
and O
z O
- O
bodies O
, O
and O
little O
or O
no O
basal O
lamina O
( O
Figure O
4A O
and O
4B O
) O
. O

Moreover O
, O
the O
muscles O
of O
the O
upper O
oviduct O
appear O
more O
differentiated O
in O
the O
mated O
than O
in O
unmated O
reproductive O
tracts O
( O
Figure O
4A O
- O
4D O
) O
. O

Interestingly O
, O
we O
observed O
neighboring O
muscle O
fibers O
in O
different O
states O
of O
differentiation O
in O
the O
lateral O
oviducts O
in O
both O
unmated O
and O
mated O
reproductive O
tracts O
, O
but O
not O
elsewhere O
in O
the O
oviduct O
( O
Figure O
4B O
) O
. O

These O
results O
suggest O
that O
mating O
enhances O
the O
rate O
of O
muscle O
differentiation O
in O
the O
upper O
oviduct O
, O
and O
that O
muscle O
differentiation O
is O
delayed O
in O
the O
upper O
as O
compared O
to O
the O
lower O
oviduct O
. O

The O
increased O
muscle O
differentiation O
in O
the O
upper O
oviduct O
is O
not O
dramatic O
and O
likely O
reflects O
the O
short O
post O
- O
mating O
period O
examined O
in O
this O
study O
. O

The O
delayed O
differentiation O
of O
the O
upper O
oviduct O
muscles O
resembles O
the O
delay O
in O
the O
onset O
of O
development O
between O
the O
adult O
thoracic O
muscles O
and O
abdominal O
muscles O
during O
metamorphosis O
[ O
32 O
] O
. O

Since O
the O
ovaries O
and O
the O
other O
parts O
of O
the O
reproductive O
tract O
are O
known O
to O
have O
different O
segmental O
origins O
[ O
33 O
] O
, O
we O
hypothesize O
that O
different O
parts O
of O
the O
oviduct O
develop O
at O
different O
rates O
or O
begin O
development O
at O
different O
times O
. O

Increased O
innervation O
in O
the O
oviduct O
post O
- O
mating O

Nerve O
- O
muscle O
interactions O
play O
an O
important O
role O
in O
regulating O
adult O
muscle O
development O
and O
refining O
the O
final O
pattern O
of O
innervation O
[ O
34 O
, O
35 O
] O
. O

Given O
that O
oviduct O
muscle O
differentiation O
is O
enhanced O
post O
- O
mating O
, O
we O
predicted O
that O
mating O
either O
directly O
or O
indirectly O
induces O
changes O
in O
innervation O
. O

To O
address O
this O
prediction O
, O
we O
quantified O
the O
number O
of O
nerve O
terminals O
or O
boutons O
innervating O
the O
lateral O
oviducts O
and O
common O
oviduct O
in O
unmated O
and O
mated O
reproductive O
tracts O
. O

Studies O
of O
oviduct O
innervation O
in O
Drosophila B
reveal O
that O
the O
fly B
' O
s O
oviduct O
receives O
aminergic O
, O
peptidergic O
and O
glutamatergic O
input O
[ O
30 O
, O
36 O
- O
39 O
] O
. O

In O
both O
larval O
and O
adult O
Drosophila B
, O
different O
types O
of O
boutons O
are O
formed O
by O
neurons O
that O
express O
different O
neurotransmitters O
and O
modulators O
[ O
40 O
- O
42 O
] O
. O

By O
similarity O
to O
the O
boutons O
described O
at O
the O
larval O
and O
adult O
neuromuscular O
junction O
, O
Middleton O
et O
al O
. O

[ O
30 O
] O
report O
that O
the O
fly B
' O
s O
oviduct O
is O
innervated O
by O
glutamatergic O
type O
I O
boutons O
and O
tyraminergic O
/ O
octopaminergic O
type O
II O
boutons O
. O

Rodgriguez O
- O
Valentin O
et O
al O
. O

[ O
43 O
] O
further O
report O
that O
the O
oviduct O
type O
II O
boutons O
co O
- O
express O
octopamine O
and O
glutamate O
. O

The O
neurons O
that O
give O
rise O
to O
the O
type O
I O
innervation O
have O
not O
been O
identified O
. O

However O
, O
it O
is O
well O
established O
that O
type O
II O
innervation O
arises O
from O
octopaminergic O
neurons O
located O
in O
the O
abdominal O
ganglion O
[ O
30 O
, O
43 O
] O
. O

In O
addition O
, O
it O
has O
been O
shown O
that O
some O
or O
all O
of O
these O
neurons O
express O
a O
GAL4 O
insertion O
line O
for O
the O
bullwinkle O
( O
bwk O
) O
gene O
[ O
43 O
] O
. O

Bwk O
encodes O
a O
HMG O
- O
box O
containing O
putative O
transcription O
factor O
[ O
44 O
] O
. O

To O
determine O
if O
mating O
induces O
any O
changes O
in O
the O
number O
of O
type O
I O
and O
II O
boutons O
innervating O
the O
oviduct O
muscles O
, O
we O
used O
the O
pan O
- O
neural O
marker O
, O
anti O
- O
HRP O
to O
label O
all O
oviduct O
boutons O
in O
unmated O
and O
mated O
females O
. O

To O
distinguish O
between O
type O
I O
and O
II O
boutons O
we O
used O
an O
antibody O
against O
the O
Disc O
Large O
( O
DLG O
) O
protein O
[ O
45 O
] O
. O

Type O
I O
boutons O
were O
identified O
by O
their O
DLG O
postsynaptic O
staining O
and O
large O
size O
( O
> O
8 O
mu O
m O
in O
diameter O
) O
( O
Figure O
4G O
) O
, O
while O
type O
II O
boutons O
were O
distinguished O
by O
their O
absence O
of O
DLG O
staining O
and O
smaller O
size O
( O
< O
2 O
mu O
m O
) O
( O
Figure O
4H O
) O
. O

We O
find O
that O
type O
I O
and O
II O
boutons O
innervate O
the O
lateral O
oviducts O
and O
common O
oviduct O
, O
and O
that O
the O
type O
I O
innervation O
is O
restricted O
to O
a O
few O
axons O
that O
run O
parallel O
to O
the O
length O
of O
the O
oviduct O
, O
while O
the O
type O
II O
innervation O
is O
more O
widespread O
. O

We O
quantified O
the O
number O
of O
boutons O
in O
the O
lateral O
oviducts O
and O
common O
oviduct O
and O
observed O
a O
74 O
% O
increase O
in O
bouton O
number O
in O
the O
lateral O
oviduct O
and O
a O
66 O
% O
increase O
in O
the O
common O
oviduct O
post O
- O
mating O
( O
Figure O
4I O
) O
. O

More O
over O
, O
we O
observed O
no O
significant O
change O
in O
the O
number O
of O
type O
I O
boutons O
in O
the O
lateral O
oviduct O
and O
common O
oviduct O
. O

However O
, O
we O
detected O
a O
1 O
. O
5 O
- O
fold O
increase O
in O
the O
number O
of O
type O
II O
boutons O
in O
the O
lateral O
oviduct O
and O
a O
1 O
. O
8 O
- O
fold O
increase O
in O
type O
II O
innervation O
in O
the O
common O
oviduct O
. O

Dramatic O
increases O
in O
bouton O
growth O
are O
also O
observed O
during O
development O
. O

For O
example O
, O
a O
ten O
- O
fold O
increase O
in O
bouton O
number O
is O
observed O
at O
the O
neuromuscular O
junction O
during O
the O
larval O
period O
[ O
46 O
] O
. O

To O
determine O
if O
the O
increase O
in O
type O
II O
innervation O
was O
specific O
to O
mating O
or O
reflected O
normal O
growth O
in O
3 O
day O
- O
old O
females O
, O
we O
quantified O
type O
I O
and O
II O
innervation O
in O
the O
oviducts O
of O
5 O
day O
- O
old O
unmated O
females O
. O

We O
found O
no O
significant O
difference O
in O
type O
I O
and O
II O
innervation O
in O
unmated O
3 O
day O
- O
old O
and O
5 O
- O
day O
- O
old O
females O
, O
indicating O
that O
mating O
, O
either O
directly O
or O
indirectly O
, O
induces O
a O
dramatic O
increase O
in O
type O
II O
innervation O
( O
Figure O
4I O
) O
. O

To O
determine O
if O
the O
post O
- O
mating O
increase O
in O
innervation O
is O
unique O
to O
the O
oviduct O
, O
we O
asked O
if O
mating O
induces O
a O
global O
change O
in O
innervation O
. O

We O
quantified O
bouton O
number O
in O
the O
adult O
ventral O
midline O
muscles O
of O
the O
5th O
abdominal O
segment O
. O

These O
muscles O
are O
innervated O
by O
boutons O
that O
increase O
in O
number O
during O
metamorphosis O
[ O
47 O
] O
. O

No O
significant O
difference O
in O
bouton O
number O
was O
detected O
at O
these O
muscles O
in O
unmated O
and O
mated O
females O
( O
additional O
file O
3 O
) O
. O

Though O
further O
analysis O
is O
needed O
, O
this O
suggests O
that O
the O
post O
- O
mating O
increase O
in O
innervation O
is O
oviduct O
- O
specific O
. O

Because O
the O
type O
II O
boutons O
are O
octopaminergic O
, O
the O
increased O
type O
II O
innervation O
may O
result O
in O
increased O
octopamine O
( O
OA O
) O
release O
in O
the O
oviduct O
. O

In O
support O
of O
this O
possibility O
, O
we O
have O
preliminary O
evidence O
that O
OA O
is O
released O
in O
the O
oviduct O
post O
- O
mating O
( O
Heifetz O
and O
Wolfner O
, O
in O
preparation O
) O
. O

Studies O
in O
locust O
and O
Drosophila B
demonstrate O
that O
OA O
inhibits O
oviduct O
contraction O
, O
while O
glutamate O
activates O
oviduct O
contraction O
[ O
30 O
, O
43 O
, O
48 O
] O
. O

In O
Drosophila B
, O
electrical O
stimulation O
of O
the O
posterior O
abdominal O
nerve O
gives O
rise O
to O
a O
series O
of O
muscle O
contractions O
in O
the O
oviduct O
followed O
by O
a O
period O
of O
muscle O
fatigue O
or O
relaxation O
[ O
43 O
] O
. O

This O
pattern O
of O
muscle O
contraction O
and O
relaxation O
may O
facilitate O
the O
proper O
movement O
of O
the O
egg O
through O
the O
oviduct O
. O

In O
their O
study O
of O
bwk O
expressing O
neurons O
that O
innervate O
the O
oviduct O
, O
Rodriguez O
- O
Valentin O
et O
al O
. O

[ O
43 O
] O
show O
that O
OA O
and O
glutamate O
interact O
to O
produce O
the O
pattern O
of O
oviduct O
contraction O
and O
relaxation O
described O
above O
. O

It O
is O
therefore O
possible O
that O
the O
post O
- O
mating O
increase O
in O
type O
II O
innervation O
in O
the O
oviduct O
plays O
an O
important O
role O
in O
the O
increased O
rate O
of O
ovulation O
and O
egg O
- O
laying O
observed O
post O
- O
mating O
. O

Female O
mating O
history O
affects O
the O
enrichment O
of O
cytosekeletal O
proteins O
in O
the O
oviduct O

To O
gain O
insights O
into O
the O
role O
of O
cytoskeletal O
protein O
enrichment O
( O
[ O
23 O
] O
; O
additional O
file O
4 O
) O
in O
mediating O
the O
morphological O
changes O
detected O
in O
this O
study O
, O
we O
examined O
the O
effect O
of O
different O
mating O
regimes O
on O
cytoskeletal O
protein O
abundance O
. O

We O
focused O
on O
a O
subset O
of O
mating O
- O
responsive O
cytoskeletal O
proteins O
with O
well O
established O
roles O
in O
the O
differentiation O
of O
muscle O
and O
epithelia O
. O

These O
include O
: O
( O
i O
) O
Mlp84B O
which O
regulates O
the O
late O
differentiation O
pathway O
of O
muscle O
[ O
49 O
] O
; O
( O
ii O
) O
Cora O
and O
Nrg O
which O
are O
required O
for O
the O
formation O
of O
septate O
junctions O
in O
epithelia O
[ O
50 O
] O
, O
and O
( O
iii O
) O
Hu O
- O
li O
tai O
shao O
( O
Hts O
) O
, O
also O
known O
as O
adducin O
- O
like O
protein O
, O
which O
functions O
in O
assembly O
of O
the O
cytoskeletal O
network O
. O

Na O
+ O
pump O
alpha O
subunit O
( O
ATP O
alpha O
) O
, O
another O
protein O
associated O
with O
septate O
junctions O
in O
epithelia O
, O
is O
not O
a O
mating O
- O
responsive O
protein O
and O
was O
used O
as O
a O
control O
. O

Using O
western O
blots O
, O
we O
first O
determined O
the O
abundance O
of O
the O
cytoskeletal O
proteins O
in O
oviducts O
of O
3 O
- O
day O
- O
old O
unmated O
and O
mated O
females O
at O
6 O
hrs O
post O
- O
mating O
. O

We O
confirmed O
the O
proteomic O
results O
of O
Kalpenikov O
et O
al O
. O

[ O
23 O
] O
and O
found O
that O
mating O
increases O
the O
abundance O
of O
all O
proteins O
, O
except O
ATP O
alpha O
in O
mated O
oviducts O
relative O
to O
their O
abundance O
in O
unmated O
oviducts O
( O
Figure O
5A O
) O
. O

To O
determine O
whether O
the O
increased O
abundance O
of O
mating O
- O
responsive O
proteins O
persists O
for O
longer O
times O
post O
- O
mating O
, O
we O
examined O
oviducts O
of O
10 O
- O
day O
- O
old O
females O
that O
mated O
once O
at O
3 O
days O
of O
age O
, O
and O
calculated O
the O
abundance O
of O
the O
mating O
- O
responsive O
proteins O
relative O
to O
their O
level O
in O
oviducts O
of O
3 O
- O
day O
- O
old O
unmated O
females O
. O

We O
found O
no O
change O
or O
a O
slight O
increase O
in O
the O
relative O
abundance O
of O
all O
cytoskeletal O
proteins O
except O
Mlp84B O
at O
7 O
days O
post O
- O
mating O
( O
Figure O
5A O
) O
. O

Strikingly O
, O
the O
level O
of O
Mlp84B O
declines O
by O
7 O
days O
post O
- O
mating O
to O
the O
level O
observed O
prior O
to O
mating O
. O

Thus O
Mlp84B O
levels O
rise O
and O
fall O
after O
mating O
, O
while O
the O
epithelial O
- O
related O
proteins O
rapidly O
rise O
and O
are O
maintained O
at O
a O
high O
level O
after O
mating O
. O

This O
raises O
the O
possibility O
that O
a O
second O
mating O
might O
trigger O
an O
increase O
in O
Mlp84B O
protein O
expression O
as O
observed O
in O
3 O
- O
day O
- O
old O
females O
at O
6 O
h O
post O
- O
mating O
. O

To O
test O
this O
possibility O
, O
females O
were O
mated O
twice O
( O
once O
at O
day O
3 O
, O
and O
once O
on O
day O
10 O
of O
age O
) O
, O
and O
their O
oviducts O
were O
examined O
at O
6 O
hrs O
after O
the O
second O
mating O
. O

We O
calculated O
the O
abundance O
of O
the O
cytoskeletal O
proteins O
in O
the O
twice O
mated O
oviducts O
relative O
to O
their O
abundance O
in O
oviducts O
of O
3 O
- O
day O
- O
old O
unmated O
females O
. O

Our O
results O
show O
that O
a O
second O
mating O
has O
little O
or O
no O
effect O
on O
Mlp84B O
abundance O
. O

Thus O
, O
Mlp84B O
may O
represent O
a O
class O
of O
mating O
- O
responsive O
proteins O
that O
is O
only O
needed O
after O
the O
first O
mating O
. O

Interestingly O
, O
the O
effect O
of O
the O
second O
mating O
on O
the O
epithelial O
- O
related O
mating O
- O
responsive O
proteins O
appears O
to O
be O
different O
for O
each O
protein O
. O

While O
Cora O
levels O
drop O
to O
the O
level O
observed O
in O
3 O
- O
day O
- O
old O
unmated O
females O
, O
Nrg O
and O
Hts O
are O
maintained O
at O
a O
high O
level O
. O

To O
determine O
if O
the O
changes O
in O
cytoskeletal O
protein O
abundance O
are O
mating O
- O
dependent O
we O
measured O
their O
abundance O
in O
the O
oviducts O
of O
unmated O
5 O
- O
and O
10 O
- O
day O
- O
old O
females O
. O

We O
calculated O
their O
abundance O
relative O
to O
their O
level O
in O
oviducts O
of O
unmated O
3 O
- O
day O
- O
old O
females O
( O
Figure O
5B O
) O
. O

We O
rationalized O
that O
if O
the O
change O
in O
cytoskeletal O
protein O
abundance O
is O
mating O
- O
dependent O
we O
will O
not O
see O
similar O
changes O
in O
unmated O
females O
. O

We O
observed O
a O
slow O
increase O
in O
the O
relative O
abundance O
of O
all O
mating O
- O
responsive O
proteins O
with O
time O
post O
- O
eclosion O
( O
Figure O
5B O
) O
. O

Because O
unmated O
females O
lay O
more O
eggs O
as O
they O
age O
( O
see O
additional O
file O
5C O
) O
one O
possible O
interpretation O
of O
the O
increased O
level O
of O
cytoskeletal O
proteins O
in O
unmated O
females O
is O
that O
these O
proteins O
are O
associated O
with O
an O
intrinsic O
program O
for O
oviduct O
maturation O
and O
that O
mating O
accelerates O
this O
process O
to O
maximize O
egg O
- O
laying O
efficacy O
. O

Alternatively O
, O
it O
is O
possible O
that O
the O
slow O
increase O
in O
protein O
abundance O
observed O
in O
unmated O
females O
is O
due O
to O
the O
passage O
of O
eggs O
through O
the O
oviduct O
. O

Taken O
together O
, O
our O
results O
suggest O
that O
mating O
is O
essential O
to O
fine O
- O
tune O
the O
levels O
of O
the O
mating O
- O
responsive O
proteins O
examined O
in O
this O
study O
. O

Because O
the O
changes O
in O
cytoskeletal O
protein O
abundance O
are O
different O
in O
unmated O
and O
mated O
females O
, O
this O
suggests O
that O
the O
post O
- O
mating O
changes O
are O
mating O
- O
dependent O
. O

Furthermore O
, O
we O
suggest O
that O
these O
post O
- O
mating O
changes O
are O
linked O
to O
changes O
in O
oviduct O
function O
. O

Early O
or O
prior O
mating O
increases O
fecundity O

In O
Drosophila B
, O
female O
fecundity O
decreases O
with O
age O
[ O
51 O
- O
54 O
] O
. O

It O
has O
been O
proposed O
that O
this O
decrease O
is O
due O
, O
in O
part O
, O
to O
the O
loss O
of O
germline O
and O
somatic O
stem O
cells O
[ O
55 O
] O
. O

Since O
the O
expression O
of O
the O
oviduct O
cytoskeletal O
proteins O
examined O
in O
this O
study O
change O
with O
age O
and O
mating O
experience O
, O
the O
state O
of O
the O
oviduct O
may O
also O
play O
a O
role O
in O
fecundity O
. O

To O
separate O
the O
effects O
of O
age O
and O
mating O
, O
we O
measured O
the O
fecundity O
of O
females O
that O
mated O
twice O
, O
first O
at O
3 O
days O
post O
- O
eclosion O
and O
again O
at O
10 O
days O
, O
and O
compared O
that O
to O
the O
fecundity O
of O
females O
that O
mated O
once O
at O
3 O
days O
and O
females O
that O
mated O
once O
at O
10 O
days O
. O

Fecundity O
was O
measured O
as O
the O
number O
of O
eggs O
laid O
per O
day O
per O
female O
during O
the O
first O
three O
days O
after O
mating O
. O

Once O
- O
mated O
3 O
- O
day O
- O
old O
females O
laid O
nearly O
twice O
as O
many O
eggs O
as O
once O
- O
mated O
10 O
- O
day O
- O
old O
females O
during O
the O
three O
days O
examined O
( O
24 O
. O
5 O
+ O
/ O
- O
0 O
. O
7 O
versus O
13 O
. O
3 O
+ O
/ O
- O
0 O
. O
9 O
, O
p O
< O
0 O
. O
0001 O
) O
. O

Twice O
- O
mated O
10 O
- O
day O
- O
old O
females O
also O
laid O
about O
50 O
% O
more O
eggs O
than O
once O
- O
mated O
females O
of O
the O
same O
age O
( O
19 O
. O
3 O
+ O
/ O
- O
0 O
. O
9 O
versus O
13 O
. O
3 O
+ O
/ O
- O
0 O
. O
9 O
, O
p O
< O
0 O
. O
0001 O
) O
, O
but O
about O
20 O
% O
fewer O
than O
laid O
by O
once O
- O
mated O
3 O
- O
day O
- O
old O
females B
during O
the O
three O
days O
examined O
( O
19 O
. O
3 O
+ O
/ O
- O
0 O
. O
9 O
versus O
24 O
. O
5 O
+ O
/ O
- O
0 O
. O
7 O
, O
p O
< O
0 O
. O
0001 O
) O
( O
Figure O
6A O
, O
see O
also O
additional O
file O
5A O
and O
5B O
) O
. O

Thus O
, O
the O
difference O
in O
fecundity O
between O
once O
- O
mated O
10 O
- O
day O
- O
old O
and O
once O
- O
mated O
3 O
- O
day O
- O
old O
females O
is O
not O
a O
result O
of O
age O
alone O
. O

Rather O
the O
main O
determinant O
of O
fecundity O
at O
10 O
days O
is O
whether O
there O
had O
been O
a O
prior O
mating O
at O
3 O
days O
. O

We O
also O
calculated O
the O
fertility O
( O
number O
of O
adults O
eclosed O
) O
of O
once O
- O
and O
twice O
- O
mated O
females O
. O

Once O
- O
mated O
3 O
- O
day O
- O
old O
females O
are O
more O
fertile O
than O
once O
- O
mated O
10 O
- O
day O
- O
old O
females O
( O
69 O
. O
5 O
+ O
/ O
- O
1 O
. O
6 O
% O
versus O
56 O
. O
1 O
+ O
/ O
- O
3 O
. O
0 O
% O
, O
p O
< O
0 O
. O
0001 O
) O
and O
slightly O
more O
fertile O
than O
twice O
- O
mated O
10 O
- O
day O
- O
old O
females B
( O
69 O
. O
5 O
+ O
/ O
- O
1 O
. O
6 O
% O
versus O
62 O
. O
7 O
+ O
/ O
- O
2 O
. O
4 O
% O
, O
p O
< O
0 O
. O
015 O
) O
( O
Figure O
6B O
, O
see O
also O
additional O
file O
5D O
) O
. O

Because O
there O
is O
no O
significant O
difference O
in O
fertility O
between O
once O
- O
mated O
and O
twice O
- O
mated O
10 O
- O
day O
- O
old O
females O
, O
this O
suggests O
that O
( O
1 O
) O
a O
prior O
mating O
has O
no O
significant O
effect O
on O
the O
fertility O
of O
10 O
- O
day O
- O
old O
mated O
females O
and O
( O
2 O
) O
fertility O
decreases O
with O
age O
. O

One O
intriguing O
interpretation O
of O
these O
results O
is O
that O
an O
early O
mating O
increases O
fecundity O
which O
partially O
compensates O
for O
the O
age O
- O
related O
decrease O
in O
fertility O
. O

Thus O
, O
cytoskeletal O
mating O
- O
responsive O
protein O
changes O
may O
be O
associated O
with O
structural O
changes O
in O
the O
oviduct O
that O
result O
in O
increased O
fecundity O
. O

This O
may O
counteract O
the O
effects O
of O
decreased O
fertility O
on O
the O
reproductive O
output O
. O

Discussion O

Despite O
the O
use O
of O
Drosophila B
as O
a O
model O
system O
for O
organ O
- O
level O
biology O
and O
the O
emerging O
parallels O
between O
mammalian O
and O
Drosophila B
reproductive O
biology O
[ O
56 O
] O
, O
this O
is O
the O
first O
integrative O
tissue O
- O
wide O
study O
of O
post O
- O
mating O
changes O
in O
the O
Drosophila B
oviduct O
. O

Our O
results O
provide O
several O
lines O
of O
evidence O
at O
the O
molecular O
, O
morphological O
and O
physiological O
levels O
suggesting O
that O
mating O
induces O
tissue O
- O
wide O
differentiation O
in O
the O
oviduct O
. O

Moreover O
, O
we O
identify O
ultrastructural O
changes O
in O
the O
mated O
oviduct O
that O
are O
consistent O
with O
the O
roles O
that O
some O
of O
the O
mating O
- O
responsive O
proteins O
examined O
in O
this O
study O
( O
e O
. O
g O
. O
Mlp84B O
, O
Cora O
, O
Nrg O
) O
are O
reported O
to O
play O
in O
muscle O
and O
epithelial O
differentiation O
elsewhere O
. O

For O
example O
, O
the O
increased O
abundance O
of O
Mlp84B O
, O
a O
major O
regulator O
of O
the O
late O
differentiation O
pathway O
of O
muscle O
[ O
49 O
] O
is O
consistent O
with O
the O
increased O
muscle O
differentiation O
in O
the O
upper O
oviduct O
post O
- O
mating O
( O
Figure O
4A O
- O
4F O
) O
. O

Similarly O
, O
the O
increased O
abundance O
of O
Cora O
and O
Nrg O
, O
molecules O
that O
are O
essential O
for O
SJ O
development O
and O
function O
[ O
50 O
] O
is O
consistent O
with O
the O
observation O
that O
SJs O
in O
the O
upper O
oviduct O
are O
immature O
and O
/ O
or O
that O
mating O
induces O
changes O
in O
the O
apical O
extracellular O
matrix O
whose O
secretion O
may O
be O
regulated O
, O
in O
part O
, O
by O
SJs O
as O
occurs O
in O
the O
trachea O
[ O
27 O
, O
28 O
] O
. O

Other O
post O
- O
mating O
changes O
that O
indicate O
that O
mating O
induces O
tissue O
- O
wide O
differentiation O
include O
increased O
HAJs O
along O
the O
basolateral O
membrane O
and O
increased O
innervation O
. O

Analysis O
of O
protein O
abundance O
following O
different O
mating O
regimes O
( O
unmated O
, O
once O
- O
mated O
, O
twice O
- O
mated O
) O
gave O
us O
further O
insights O
into O
the O
possible O
roles O
that O
mating O
- O
responsive O
proteins O
play O
in O
the O
oviduct O
. O

For O
example O
, O
Mlp84B O
is O
only O
responsive O
to O
the O
first O
mating O
, O
while O
the O
epithelial O
proteins O
examined O
( O
Cora O
, O
Nrg O
and O
Hts O
) O
are O
responsive O
to O
the O
first O
and O
second O
mating O
. O

Furthermore O
, O
the O
response O
to O
the O
second O
mating O
is O
different O
from O
the O
response O
to O
the O
first O
mating O
. O

Taken O
together O
, O
these O
results O
suggest O
that O
Mlp84B O
is O
required O
for O
the O
final O
maturation O
of O
the O
oviduct O
, O
while O
the O
epithelial O
proteins O
examined O
are O
required O
for O
both O
the O
maturation O
and O
maintenance O
of O
the O
oviduct O
at O
a O
high O
functional O
state O
. O

Moreover O
, O
the O
post O
- O
mating O
pattern O
of O
Mlp84B O
supports O
the O
idea O
that O
the O
first O
mating O
induces O
the O
final O
maturation O
of O
the O
oviduct O
. O

The O
rise O
and O
fall O
of O
Mlp84B O
abundance O
after O
the O
first O
mating O
( O
Figure O
5 O
) O
parallels O
the O
expression O
pattern O
of O
Mlp84B O
during O
development O
where O
peaks O
in O
Mlp84B O
transcription O
occur O
during O
periods O
of O
embryogenesis O
and O
metamorphosis O
when O
muscle O
is O
differentiating O
[ O
49 O
] O
. O

Using O
different O
mating O
regimes O
we O
tested O
whether O
the O
first O
/ O
early O
mating O
is O
essential O
for O
maintenance O
of O
high O
reproductive O
output O
in O
the O
second O
mating O
. O

Our O
results O
suggest O
that O
mating O
at O
an O
early O
age O
is O
essential O
to O
achieve O
maximum O
reproductive O
output O
( O
i O
. O
e O
. O
high O
fecundity O
and O
fertility O
) O
. O

Since O
the O
first O
mating O
increases O
reproductive O
output O
( O
evidence O
from O
our O
mating O
regime O
experiments O
) O
, O
it O
is O
likely O
that O
the O
ultrastructural O
changes O
detected O
in O
mated O
3 O
- O
day O
- O
old O
females O
lead O
to O
a O
highly O
functional O
oviduct O
. O

We O
suggest O
that O
the O
final O
maturation O
of O
the O
oviduct O
includes O
a O
mating O
- O
dependent O
stage O
. O

We O
propose O
that O
during O
the O
first O
few O
days O
post O
- O
eclosion O
, O
the O
oviduct O
undergoes O
the O
first O
phase O
of O
differentiation O
, O
after O
which O
the O
oviduct O
is O
developmentally O
poised O
for O
a O
rapid O
response O
to O
an O
extrinsic O
cue O
( O
mating O
) O
. O

Mating O
then O
triggers O
the O
second O
phase O
of O
maturation O
( O
tissue O
remodeling O
and O
modulation O
) O
which O
is O
essential O
for O
proper O
oviduct O
function O
( O
Figure O
7 O
) O
. O

We O
further O
propose O
that O
the O
second O
phase O
of O
maturation O
consists O
of O
processes O
that O
are O
mating O
- O
independent O
and O
- O
dependent O
, O
and O
that O
both O
of O
these O
pathways O
are O
essential O
to O
produce O
a O
functional O
oviduct O
. O

For O
example O
, O
initial O
formation O
of O
SJs O
occurs O
prior O
to O
mating O
( O
mating O
- O
independent O
) O
while O
the O
increased O
apical O
secretion O
and O
development O
of O
HAJs O
are O
mating O
- O
dependent O
. O

The O
oviduct O
musculature O
is O
an O
example O
where O
both O
mating O
- O
independent O
and O
- O
dependent O
processes O
play O
a O
role O
. O

Muscles O
are O
highly O
differentiated O
in O
the O
lower O
oviduct O
prior O
to O
mating O
, O
while O
muscle O
differentiation O
is O
ongoing O
in O
the O
upper O
oviduct O
and O
increases O
after O
mating O
. O

Although O
the O
onset O
of O
muscle O
differentiation O
in O
both O
regions O
is O
mating O
- O
independent O
, O
the O
further O
differentiation O
of O
muscle O
in O
the O
upper O
oviduct O
is O
mating O
- O
dependent O
. O

Another O
possible O
interpretation O
of O
the O
role O
of O
mating O
on O
oviduct O
maturation O
is O
that O
mating O
accelerates O
and O
synchronizes O
processes O
that O
are O
essential O
for O
the O
functional O
maturation O
of O
the O
oviduct O
. O

It O
will O
therefore O
be O
interesting O
to O
examine O
the O
oviducts O
of O
older O
females O
to O
determine O
the O
status O
of O
oviduct O
maturation O
. O

What O
is O
the O
benefit O
of O
mating O
- O
induced O
differentiation O
of O
the O
oviduct O
tissues O
? O

Unmated O
females O
are O
capable O
of O
laying O
eggs O
, O
albeit O
at O
a O
reduced O
rate O
as O
compared O
to O
mated O
females O
of O
the O
same O
age O
. O

One O
possible O
interpretation O
is O
that O
reproduction O
is O
energetically O
costly O
, O
thus O
delaying O
oviduct O
maturation O
until O
sperm O
is O
available O
is O
advantageous O
to O
the O
female O
. O

This O
may O
reflect O
the O
evolution O
of O
a O
mechanism O
to O
optimize O
reproductive O
capacity O
in O
early O
adulthood O
in O
short O
- O
lived O
animals O
. O

In O
summary O
, O
we O
have O
identified O
events O
at O
the O
cellular O
, O
molecular O
and O
physiological O
levels O
that O
are O
part O
of O
an O
efficient O
and O
specific O
program O
for O
reproduction O
. O

Drosophila B
affords O
us O
the O
opportunity O
to O
uncover O
the O
signaling O
pathways O
that O
coordinate O
these O
events O
to O
produce O
a O
physiologically O
functional O
organ O
. O

Methods O

Flies O

Wild O
- O
type O
Canton O
- O
S O
flies O
were O
used O
for O
the O
fecundity O
/ O
fertility O
experiments O
and O
confocal O
analysis O
. O

Wild O
- O
type O
Canton O
- O
S5 O
[ O
57 O
] O
flies O
were O
used O
for O
electron O
microscopy O
. O

All O
flies O
were O
kept O
in O
a O
12 O
hrs O
light O
/ O
dark O
cycle O
at O
23 O
+ O
/ O
- O
2 O
degrees O
C O
. O

Upon O
eclosion O
, O
females O
and O
males O
were O
collected O
on O
ice O
and O
held O
separately O
until O
3 O
( O
females O
and O
males O
) O
or O
10 O
( O
females O
) O
days O
of O
age O
. O

Sample O
preparation O

For O
all O
assays O
( O
unless O
described O
differently O
) O
unmated O
females O
were O
placed O
with O
3 O
- O
day O
- O
old O
unmated O
males O
and O
observed O
until O
mating O
initiated O
. O

At O
the O
end O
of O
mating O
, O
females O
were O
aspirated O
into O
fresh O
vials O
and O
held O
for O
6 O
hrs O
. O

At O
6 O
hrs O
after O
the O
start O
of O
mating O
, O
females O
were O
placed O
on O
ice O
for O
dissection O
. O

Previous O
molecular O
studies O
indicate O
changes O
in O
protein O
abundance O
at O
3 O
hrs O
post O
- O
mating O
. O

We O
hypothesize O
that O
these O
molecular O
changes O
will O
translate O
into O
morphological O
changes O
in O
the O
next O
few O
hours O
. O

We O
chose O
to O
analyze O
the O
morphology O
of O
mated O
females O
at O
6 O
h O
post O
- O
mating O
as O
opposed O
to O
later O
times O
post O
- O
mating O
because O
the O
changes O
observed O
at O
later O
times O
may O
be O
due O
, O
in O
part O
, O
to O
the O
high O
rate O
of O
eggs O
passing O
through O
the O
oviduct O
. O

Electron O
microscopy O

Reproductive O
tracts O
were O
dissected O
in O
Schneider O
' O
s O
Drosophila O
medium O
( O
Sigma O
) O
on O
ice O
and O
processed O
for O
electron O
microscopy O
as O
described O
in O
[ O
42 O
] O
. O

Tracts O
were O
flat O
- O
embedded O
between O
two O
sheets O
of O
Aclar O
( O
Electron O
Microscopy O
Sciences O
) O
, O
which O
allowed O
us O
to O
image O
the O
entire O
tract O
at O
the O
light O
microscopic O
level O
prior O
to O
sectioning O
. O

Sections O
were O
cut O
on O
a O
Reichart O
Ultracut O
microtome O
. O

One O
- O
mu O
m O
thick O
sections O
were O
stained O
with O
1 O
% O
toluidine O
blue O
, O
and O
viewed O
with O
a O
Zeiss O
Axoplan O
microscope O
. O

Ultrathin O
sections O
( O
~ O
100 O
nm O
) O
were O
mounted O
on O
formvar O
grids O
, O
stained O
with O
lead O
citrate O
, O
and O
viewed O
with O
a O
Philips O
/ O
FEI O
Morgagni O
268 O
TEM O
at O
80 O
kV O
. O

Our O
analysis O
is O
based O
on O
4 O
unmated O
samples O
and O
3 O
mated O
samples O
. O

Two O
unmated O
samples O
were O
cut O
in O
the O
longitudinal O
plane O
and O
two O
additional O
unmated O
samples O
were O
cut O
in O
the O
transverse O
plane O
, O
while O
one O
mated O
sample O
was O
cut O
in O
the O
longitudinal O
plane O
and O
two O
additional O
mated O
samples O
were O
cut O
in O
the O
transverse O
plane O
. O

For O
longitudinal O
sections O
, O
the O
entire O
tract O
was O
re O
- O
embedded O
and O
cut O
. O

For O
samples O
cut O
in O
the O
transverse O
plane O
, O
the O
flat O
- O
embedded O
reproductive O
tract O
was O
divided O
into O
three O
regions O
: O
( O
1 O
) O
lateral O
oviducts O
and O
upper O
common O
oviducts O
, O
( O
2 O
) O
middle O
common O
oviduct O
, O
and O
( O
3 O
) O
lower O
common O
oviduct O
. O

Each O
region O
was O
re O
- O
embedded O
and O
sectioned O
. O

It O
is O
beyond O
the O
scope O
of O
this O
paper O
to O
describe O
all O
three O
regions O
, O
and O
our O
analysis O
focuses O
on O
the O
uppermost O
and O
lowermost O
regions O
. O

Immunocytochemistry O

Reproductive O
tracts O
were O
dissected O
in O
Yamamoto O
' O
s O
Ringer O
( O
10 O
mM O
MOPS O
; O
80 O
mM O
NaCl O
; O
10 O
mM O
KCL O
; O
0 O
. O
2 O
mM O
MgCl2 O
; O
0 O
. O
1 O
mM O
CaCl2 O
) O
with O
5 O
% O
( O
w O
/ O
v O
) O
sucrose O
on O
ice O
, O
fixed O
in O
4 O
% O
paraphormaldehyde O
in O
PBS O
( O
phosphate O
- O
buffered O
saline O
; O
0 O
. O
85 O
% O
NaCl O
, O
1 O
. O
4 O
mM O
KH2 O
PO4 O
, O
8 O
mM O
Na2 O
HPO4 O
, O
pH O
7 O
. O
4 O

) O
for O
45 O
min O
and O
then O
washed O
in O
PBS O
. O

The O
reproductive O
tracts O
were O
then O
incubated O
in O
blocking O
solution O
( O
0 O
. O
5 O
% O
Triton O
x O
- O
100 O
, O
3 O
% O
NGS O
, O
0 O
. O
1 O
% O
BSA O
) O
for O
2 O
hrs O
at O
room O
temperature O
. O

The O
following O
primary O
antibodies O
, O
reagents O
, O
and O
dilutions O
were O
used O
: O
Cy3 O
- O
conjugated O
goat B
anti O
- O
HRP O
, O
1 O
: O
200 O
( O
Jackson O
Immunochemicals O
, O
West O
Grove O
, O
PA O
) O
; O
mouse B
anti O
- O
Disc O
Large O
( O
DLG O
) O
, O
1 O
: O
1000 O
( O
Developmental O
Hybridoma O
Bank O
) O
, O
Alexa O
Fluor O
488 O
- O
phalloidin O
, O
1 O
: O
200 O
( O
Invitrogen O
, O
Molecular O
Probes O
, O
Scotland O
) O
. O

Secondary O
antibodies O
were O
Alexa O
Flour O
488 O
- O
conjugated O
Goat B
anti O
- O
mouse B
, O
1 O
: O
200 O
and O
Alexa O
Flour O
546 O
- O
conjugated O
Goat B
anti O
- O
rabbit B
, O
1 O
: O
200 O
( O
Invitrogen O
, O
Molecular O
Probes O
, O
Scotland O
) O
. O

Reproductive O
tracts O
were O
incubated O
with O
the O
different O
primary O
antibodies O
( O
diluted O
in O
PBS O
+ O
0 O
. O
2 O
% O
Triton O
x O
- O
100 O
) O
for O
2 O
hr O
at O
room O
temperature O
, O
washed O
with O
PBST O
, O
incubated O
with O
secondary O
antibodies O
for O
2 O
hrs O
at O
room O
temperature O
and O
washed O
with O
PBS O
. O

Reproductive O
tracts O
of O
the O
different O
treatments O
were O
mounted O
with O
Antifade O
media O
[ O
58 O
] O
on O
a O
multi O
- O
well O
glass O
slide O
( O
Hendley O
- O
Essex O
, O
UK O
) O
. O

For O
each O
treatment O
( O
unmated O
, O
mated O
) O
and O
antibody O
/ O
reagent O
( O
HRP O
, O
DLG O
, O
phalloidin O
) O
a O
minimum O
of O
ten O
reproductive O
tracts O
from O
at O
least O
two O
independent O
biological O
replicates O
were O
prepared O
. O

Confocal O
microscopy O

Reproductive O
tracts O
were O
viewed O
with O
a O
Zeiss O
510 O
laser O
scanning O
confocal O
microscope O
using O
20 O
x O
and O
60 O
x O
objective O
with O
additional O
zooming O
. O

Optical O
sections O
from O
different O
focal O
plans O
of O
each O
reproductive O
tract O
region O
( O
lateral O
oviducts O
, O
common O
oviduct O
, O
uterus O
) O
were O
collected O
and O
projected O
as O
a O
reconstructed O
three O
- O
dimensional O
image O
using O
LSM O
image O
browser O
( O
version O
3 O
, O
5 O
, O
0 O
, O
376 O
) O
software O
. O

Image O
collections O
were O
identical O
for O
each O
of O
the O
different O
reproductive O
tract O
regions O
analyzed O
. O

Quantitation O
of O
bouton O
number O

To O
quantify O
the O
number O
of O
boutons O
in O
the O
lateral O
and O
common O
oviducts O
we O
used O
ImageJ O
software O
( O
1 O
. O
37b O
, O
National O
Institutes O
of O
Health O
) O
to O
analyze O
confocal O
images O
of O
anti O
- O
HRP O
and O
anti O
- O
DLG O
labeled O
boutons O
in O
the O
oviduct O
. O

The O
number O
of O
boutons O
per O
unit O
area O
was O
quantified O
with O
the O
Particle O
Analysis O
Tool O
. O

Briefly O
, O
to O
differentiate O
between O
the O
boutons O
, O
the O
particle O
analysis O
tool O
requires O
the O
image O
to O
be O
a O
" O
binary O
" O
image O
( O
i O
. O
e O
. O
, O
black O
or O
white O
) O
, O
thus O
we O
first O
converted O
the O
images O
to O
gray O
scale O
. O

We O
then O
set O
a O
" O
threshold O
" O
range O
so O
that O
pixels O
in O
the O
image O
whose O
value O
lies O
in O
this O
range O
are O
converted O
to O
black O
; O
pixels O
with O
values O
outside O
this O
range O
are O
converted O
to O
white O
. O

We O
next O
defined O
a O
region O
of O
interest O
( O
ROI O
) O
within O
the O
oviduct O
to O
count O
particles O
( O
i O
. O
e O
. O
count O
boutons O
) O
. O

This O
ROI O
was O
saved O
and O
served O
to O
measure O
the O
number O
of O
boutons O
per O
unit O
area O
in O
each O
treatment O
. O

For O
each O
oviduct O
we O
counted O
the O
number O
of O
anti O
- O
HRP O
and O
anti O
- O
DLG O
labeled O
boutons O
in O
two O
ROIs O
within O
the O
lateral O
oviducts O
and O
two O
ROIs O
in O
the O
common O
oviduct O
. O

One O
- O
way O
ANOVA O
( O
SPSS O
15 O
. O
0 O
) O
was O
used O
to O
measure O
the O
difference O
in O
bouton O
number O
per O
unit O
area O
in O
different O
regions O
of O
the O
oviducts O
, O
in O
both O
unmated O
and O
mated O
females O
. O

Quantitation O
of O
cytoskeleton O
proteins O

Sample O
preparation O

To O
evaluate O
the O
effect O
of O
mating O
on O
the O
abundance O
of O
the O
cytoskeleton O
proteins O
tested O
, O
females O
were O
: O
( O
i O
) O
aged O
for O
3 O
days O
, O
mated O
with O
3 O
- O
day O
- O
old O
unmated O
males O
and O
their O
oviducts O
were O
dissected O
after O
6 O
hrs O
post O
- O
mating O
( O
Once3 O
) O
; O
( O
ii O
) O
aged O
for O
3 O
days O
, O
mated O
with O
3 O
- O
day O
- O
old O
unmated O
males O
and O
their O
oviducts O
were O
dissected O
after O
7 O
days O
( O
Once3 O
day10 O
) O
; O
( O
iii O
) O
aged O
for O
3 O
days O
, O
mated O
first O
with O
3 O
- O
day O
- O
old O
unmated O
males O
and O
held O

singly O
for O
7 O
days O
. O

At O
10 O
days O
of O
age O
, O
female O
were O
mated O
again O
with O
3 O
- O
day O
- O
old O
unmated O
males O
. O

Oviducts O
were O
dissected O
at O
6 O
hrs O
post O
- O
second O
mating O
( O
Twice3 O
& O
10 O
) O
. O

We O
also O
examined O
5 O
- O
day O
- O
old O
and O
10 O
- O
day O
- O
old O
unmated O
females O
( O
UM5 O
, O
UM10 O
respectively O
) O
. O

SDS O
polyacrylamide O
gel O
electrophoresis O
( O
SDS O
- O
PAGE O
) O
and O
Western O
blotting O

For O
each O
mating O
regime O
, O
sixty O
oviducts O
were O
pooled O
and O
30 O
mu O
l O
of O
SDS O
- O
PAGE O
sample O
buffer O
was O
added O
as O
described O
in O
[ O
59 O
] O
. O

Samples O
were O
boiled O
, O
and O
then O
frozen O
at O
- O
20 O
degrees O
C O
until O
loading O
. O

SDS O
- O
PAGE O
was O
performed O
on O
12 O
% O
polyacrylamide O
gels O
and O
western O
blotted O
as O
in O
[ O
60 O
] O
. O

Proteins O
were O
cross O
- O
linked O
to O
the O
filter O
. O

The O
following O
primary O
antibodies O
and O
dilutions O
were O
used O
: O
mouse B
anti O
- O
Neuroglian O
( O
kindly O
provided O
by O
M O
. O
Hortsch O
) O
1 O
: O
250 O
; O
Guinea B
pig I
anti O
- O
Coracle O
( O
kindly O
provided O
by O
R O
. O
G O
. O
Fehon O
) O
1 O
: O
2500 O
; O
rabbit B
anti O
- O
Mlp84B O
( O
kindly O
provided O
by O
M O
. O
Beckerle O
) O
1 O
: O
1000 O
; O
mouse B
anti O
- O
hts O
( O
1B1 O
, O
Developmental O
Studies O
Hybridoma O
Bank O
, O
DSHB O
) O
1 O
: O
75 O
; O
mouse B
anti O
- O
Na O
, O
K O
- O
ATPase O
( O
alpha O
5 O
, O
DSHB O
) O
1 O
: O
100 O
. O

Secondary O
antibodies O
included O
: O
anti O
- O
Guinea B
pig I
IgG O
( O
peroxidase O
conjugated O
) O
, O
anti O
- O
Rabbit B
IgG O
and O
anti O
- O
Mouse B
IgG O
( O
developed O
in O
goat B
, O
Sigma O
, O
Israel O
) O
1 O
: O
10 O
, O
000 O
. O

Proteins O
were O
visualized O
using O
an O
enhanced O
chemiluminescence O
( O
ECL O
) O
detection O
system O
( O
Amersham O
Piscataway O
, O
NJ O
) O
. O

Analysis O

The O
developed O
film O
was O
scanned O
and O
the O
signal O
intensity O
( O
protein O
abundance O
) O
of O
each O
band O
was O
determined O
using O
ImageJ O
software O
( O
1 O
. O
37d O
, O
National O
Institutes O
of O
Health O
) O
. O

We O
evaluated O
protein O
abundance O
by O
measuring O
the O
mean O
gray O
value O
of O
a O
specific O
band O
and O
the O
background O
. O

The O
mean O
gray O
value O
of O
the O
background O
was O
then O
subtracted O
from O
that O
of O
the O
measured O
band O
. O

Relative O
protein O
abundance O
in O
mated O
oviduct O
vs O
. O
3 O
- O
day O
- O
old O
unmated O
oviduct O
was O
then O
calculated O
. O

Four O
independent O
biological O
replicates O
were O
prepared O
for O
each O
mating O
status O
. O

The O
reported O
abundance O
( O
see O
Figure O
5 O
) O
is O
the O
relative O
ratio O
( O
mated O
/ O
unmated O
or O
unmated O
/ O
unmated O
) O
of O
at O
least O
three O
replicates O
that O
showed O
the O
same O
trend O
. O

Examination O
of O
female O
reproductive O
output O

Mating O
regimes O

To O
evaluate O
the O
effect O
of O
mating O
on O
reproductive O
output O
females O
were O
treated O
as O
follows O
: O
( O
i O
) O
aged O
for O
3 O
days O
and O
mated O
with O
3 O
- O
day O
- O
old O
unmated O
males O
( O
Once3 O
) O
; O
( O
ii O
) O
aged O
for O
10 O
days O
and O
mated O
with O
3 O
- O
day O
- O
old O
unmated O
males O
( O
Once10 O
) O
; O
( O
iii O
) O
aged O
for O
3 O
days O
, O
mated O
first O
with O
3 O
- O
day O
- O
old O
unmated O
males O
, O
held O
for O
7 O
days O
and O
mated O
again O
with O
3 O
- O
day O
- O
old O
unmated O
males O
( O
Twice3 O
& O
10 O
) O
. O

In O
all O
cases O
male O
and O
female O
pairs O
were O
observed O
to O
record O
mating O
initiation O
and O
termination O
. O

Analysis O

Following O
mating O
, O
females O
were O
aspirated O
into O
fresh O
vials O
, O
held O
singly O
and O
allowed O
to O
lay O
eggs O
for O
6 O
hrs O
, O
then O
transferred O
daily O
( O
each O
24 O
hrs O
) O
to O
fresh O
vials O
. O

The O
number O
of O
eggs O
laid O
and O
the O
number O
of O
eclosed O
adults O
were O
counted O
from O
vials O
created O
at O
6 O
hrs O
, O
1 O
, O
2 O
and O
3 O
days O
post O
- O
mating O
. O

To O
ascertain O
the O
baseline O
of O
female O
egg O
- O
laying O
, O
we O
also O
included O
in O
our O
experiment O
unmated O
females O
that O
were O
kept O
in O
the O
same O
conditions O
as O
mated O
females O
. O

The O
number O
of O
eggs O
laid O
by O
unmated O
females O
was O
counted O
from O
vials O
created O
at O
6 O
hrs O
, O
1 O
, O
2 O
and O
3 O
days O
after O
placing O
the O
females O
in O
the O
holding O
vials O
. O

In O
addition O
, O
we O
also O
recorded O
the O
pattern O
of O
unmated O
female O
egg O
- O
laying O
for O
10 O
days O
. O

To O
determine O
the O
effect O
of O
different O
mating O
regimes O
on O
female O
reproductive O
output O
( O
i O
. O
e O
. O
fecundity O
and O
fertility O
) O
, O
we O
used O
One O
- O
way O
ANOVA O
( O
SPSS O
15 O
. O
0 O
) O
. O

Abbreviations O

Nrg O
: O
Neuroglian O
; O
Cora O
: O
Coracle O
; O
Spec O
: O
alpha O
- O
and O
beta O
- O
Spectrin O
; O
SJ O
: O
septate O
junction O
; O
SSJ O
: O
smooth O
septate O
junction O
; O
PSJ O
: O
pleated O
septate O
junction O
; O
ZA O
: O
zonal O
adherens O
junction O
; O
AJ O
: O
adherens O
junction O
; O
AECM O
: O
apical O
extracellular O
matrix O
; O
ECM O
: O
extracellular O
matrix O
; O
HAJ O
: O
hemi O
- O
adherens O
junction O
; O
SAJ O
: O
spot O
adherens O
junction O
; O
OA O
: O
Octopamine O
; O
Hts O
: O
Hu O
- O
li O
tai O
shao O
; O

ATP O
alpha O
: O
Na O
+ O
pump O
alpha O
subunit O
; O
Mlp84B O
: O
Muscle O
LIM O
protein O
at O
84B O
; O
DLG O
: O
Disc O
Large O
; O
HRP O
: O
horseradish O
peroxidas O
; O
UM O
: O
unmated O
; O
M O
: O
mated O
. O

Authors O
' O
contributions O

AK O
and O
PKR O
contributed O
equally O
to O
this O
manuscript O
. O

AK O
, O
PKR O
and O
YH O
conceived O
and O
designed O
the O
project O
and O
analyzed O
the O
data O
. O

AK O
performed O
the O
confocal O
, O
Western O
blots O
and O
fertility O
assays O
. O

PKR O
and O
AK O
performed O
the O
light O
microscopy O
. O

PKR O
conducted O
the O
electron O
microscopy O
. O

AK O
, O
PKR O
and O
YH O
wrote O
the O
manuscript O
. O

RRH O
contributed O
to O
design O
of O
the O
study O
and O
revision O
of O
the O
manuscript O
. O

All O
authors O
participated O
in O
the O
discussion O
and O
approval O
of O
the O
final O
manuscript O
. O

Supplementary O
Material O

Efficacy O
of O
intra O
- O
articular O
hyaluronan O
( O
Synvisc O
( O
R O
) O
) O
for O
the O
treatment O
of O
osteoarthritis O
affecting O
the O
first O
metatarsophalangeal O
joint O
of O
the O
foot O
( O
hallux O
limitus O
) O
: O
study O
protocol O
for O
a O
randomised O
placebo O
controlled O
trial O

Abstract O

Background O

Osteoarthritis O
of O
the O
first O
metatarsophalangeal O
joint O
( O
MPJ O
) O
of O
the O
foot O
, O
termed O
hallux O
limitus O
, O
is O
common O
and O
painful O
. O

Numerous O
non O
- O
surgical O
interventions O
have O
been O
proposed O
for O
this O
disorder O
, O
however O
there O
is O
limited O
evidence O
for O
their O
efficacy O
. O

Intra O
- O
articular O
injections O
of O
hyaluronan O
have O
shown O
beneficial O
effects O
in O
case O
- O
series O
and O
clinical O
trials O
for O
the O
treatment O
of O
osteoarthritis O
of O
the O
first O
metatarsophalangeal O
joint O
. O

However O
, O
no O
study O
has O
evaluated O
the O
efficacy O
of O
this O
form O
of O
treatment O
using O
a O
randomised O
placebo O
controlled O
trial O
. O

This O
article O
describes O
the O
design O
of O
a O
randomised O
placebo O
controlled O
trial O
to O
evaluate O
the O
efficacy O
of O
intra O
- O
articular O
hyaluronan O
( O
Synvisc O
( O
R O
) O
) O
to O
reduce O
pain O
and O
improve O
function O
in O
people B
with O
hallux O
limitus O
. O

Methods O

One O
hundred O
and O
fifty O
community O
- O
dwelling O
men B
and O
women B
aged O
18 O
years O
and O
over O
with O
hallux O
limitus O
( O
who O
satisfy O
inclusion O
and O
exclusion O
criteria O
) O
will O
be O
recruited O
. O

Participants B
will O
be O
randomised O
, O
using O
a O
computer O
- O
generated O
random O
number O
sequence O
, O
to O
receive O
a O
single O
intra O
- O
articular O
injection O
of O
up O
to O
1 O
ml O
hyaluronan O
( O
Synvisc O
( O
R O
) O
) O
or O
sterile O
saline O
( O
placebo O
) O
into O
the O
first O
MPJ O
. O

The O
injections O
will O
be O
performed O
by O
an O
interventional O
radiologist O
using O
fluoroscopy O
to O
ensure O
accurate O
deposition O
of O
the O
hyaluronan O
in O
the O
joint O
. O

Participants B
will O
be O
given O
the O
option O
of O
a O
second O
and O
final O
intra O
- O
articular O
injection O
( O
of O
Synvisc O
( O
R O
) O
or O
sterile O
saline O
according O
to O
the O
treatment O
group O
they O
are O
in O
) O
either O
1 O
or O
3 O
months O
post O
- O
treatment O
if O
there O
is O
no O
improvement O
in O
pain O
and O
the O
participant B
has O
not O
experienced O
severe O
adverse O
effects O
after O
the O
first O
injection O
. O

The O
primary O
outcome O
measures O
will O
be O
the O
pain O
and O
function O
subscales O
of O
the O
Foot O
Health O
Status O
Questionnaire O
. O

The O
secondary O
outcome O
measures O
will O
be O
pain O
at O
the O
first O
MPJ O
( O
during O
walking O
and O
at O
rest O
) O
, O
stiffness O
at O
the O
first O
MPJ O
, O
passive O
non O
- O
weightbearing O
dorsiflexion O
of O
the O
first O
MPJ O
, O
plantar O
flexion O
strength O
of O
the O
toe O
- O
flexors O
of O
the O
hallux O
, O
global O
satisfaction O
with O
the O
treatment O
, O
health O
- O
related O
quality O
of O
life O
( O
assessed O
using O
the O
Short O
- O
Form O
- O
36 O
version O
two O
questionnaire O
) O
, O
magnitude O
of O
symptom O
change O
, O
use O
of O
pain O
- O
relieving O
medication O
and O
changes O
in O
dynamic O
plantar O
pressure O
distribution O
( O
maximum O
force O
and O
peak O
pressure O
) O
during O
walking O
. O

Data O
will O
be O
collected O
at O
baseline O
, O
then O
1 O
, O
3 O
and O
6 O
months O
post O
- O
treatment O
. O

Data O
will O
be O
analysed O
using O
the O
intention O
to O
treat O
principle O
. O

Discussion O

This O
study O
is O
the O
first O
randomised O
placebo O
controlled O
trial O
to O
evaluate O
the O
efficacy O
of O
intra O
- O
articular O
hyaluronan O
( O
Synvisc O
( O
R O
) O
) O
for O
the O
treatment O
of O
osteoarthritis O
of O
the O
first O
MPJ O
( O
hallux O
limitus O
) O
. O

The O
study O
has O
been O
pragmatically O
designed O
to O
ensure O
that O
the O
study O
findings O
can O
be O
implemented O
into O
clinical O
practice O
if O
this O
form O
of O
treatment O
is O
found O
to O
be O
an O
effective O
treatment O
strategy O
. O

Trial O
registration O

Australian O
New O
Zealand O
Clinical O
Trials O
Registry O
: O
ACTRN12607000654459 O

Background O

Osteoarthritis O
( O
OA O
) O
is O
a O
degenerative O
joint O
disease O
that O
commonly O
presents O
within O
the O
first O
metatarsophalangeal O
joint O
( O
MPJ O
) O
of O
the O
foot O
. O

The O
terms O
hallux O
limitus O
and O
hallux O
rigidus I
have O
frequently O
been O
used O
interchangeably O
to O
describe O
differing O
severities O
of O
pain O
and O
limitation O
of O
motion O
associated O
with O
OA O
at O
the O
first O
MPJ O
[ O
1 O
] O
. O

Hallux O
limitus O
is O
a O
progressive O
osteoarthritic O
condition O
of O
the O
first O
MPJ O
that O
may O
advance O
to O
an O
end O
- O
stage O
presentation O
of O
hallux O
rigidus I
where O
the O
joint O
fuses O
and O
there O
is O
a O
complete O
restriction O
of O
motion O
[ O
1 O
] O
. O

First O
MPJ O
OA O
is O
the O
second O
most O
common O
disorder O
affecting O
the O
foot O
after O
hallux O
valgus O
[ O
2 O
] O
. O

The O
prevalence O
of O
the O
condition O
increases O
with O
age O
, O
and O
it O
has O
been O
reported O
that O
radiographic O
changes O
in O
the O
first O
MPJ O
affect O
are O
evident O
in O
approximately O
46 O
% O
of O
women B
and O
32 O
% O
of O
men B
at O
60 O
years O
of O
age O
[ O
3 O
] O
. O

Osteoarthritis O
at O
the O
first O
MPJ O
is O
characterised O
by O
the O
symptoms O
of O
pain O
and O
stiffness O
at O
the O
joint O
[ O
1 O
] O
. O

Secondary O
painful O
symptoms O
relate O
to O
compensations O
during O
gait O
that O
may O
occur O
due O
to O
the O
reduced O
motion O
of O
the O
first O
MPJ O
[ O
1 O
] O
. O

The O
presence O
of O
pain O
associated O
with O
first O
MPJ O
OA O
impacts O
on O
normal O
walking O
and O
quality O
of O
life O
[ O
4 O
] O
. O

Treatment O
of O
hallux O
limitus O
involves O
conservative O
measures O
( O
such O
as O
physical O
therapy O
, O
foot O
orthoses O
, O
footwear O
modification O
, O
joint O
manipulation O
and O
injection O
with O
corticosteroid O
) O
[ O
5 O
] O
, O
or O
surgical O
intervention O
( O
either O
joint O
- O
salvage O
or O
joint O
- O
destructive O
procedures O
) O
[ O
6 O
] O
. O

Pharmacological O
treatment O
is O
also O
often O
undertaken O
as O
an O
adjunct O
for O
pain O
relief O
in O
the O
management O
of O
hallux O
limitus O
[ O
6 O
] O
. O

However O
, O
although O
non O
- O
steroidal O
anti O
- O
inflammatory O
drugs O
( O
NSAIDs O
) O
and O
cyclooxygenase O
- O
2 O
inhibitors O
have O
been O
found O
to O
be O
effective O
in O
the O
management O
of O
various O
forms O
of O
OA O
, O
gastrointestinal O
complications O
remain O
a O
concern O
[ O
7 O
] O
. O

In O
light O
of O
these O
limitations O
with O
existing O
treatments O
, O
an O
alternative O
treatment O
termed O
' O
viscosupplementation O
' O
- O
the O
intra O
- O
articular O
injection O
of O
hyaluronan O
into O
arthritic O
joints O
with O
the O
aim O
of O
restoring O
the O
viscoelasticity O
of O
the O
synovial O
fluid O
[ O
8 O
] O
- O
has O
been O
proposed O
and O
has O
attracted O
considerable O
attention O
in O
the O
medical O
literature O
as O
a O
treatment O
for O
OA O
[ O
9 O
] O
. O

In O
particular O
, O
both O
the O
American O
College O
of O
Rheumatology O
( O
ACR O
) O
and O
European O
League O
Against O
Rheumatism O
( O
EULAR O
) O
recommend O
hyaluronan O
in O
the O
management O
of O
OA O
of O
the O
knee O
[ O
10 O
, O
11 O
] O
. O

Although O
the O
results O
of O
systematic O
reviews O
investigating O
the O
effectiveness O
of O
this O
type O
of O
treatment O
for O
knee O
OA O
are O
controversial O
, O
the O
most O
recent O
update O
of O
the O
Cochrane O
systematic O
review O
evaluating O
viscosupplementation O
for O
the O
treatment O
of O
knee O
OA O
concluded O
that O
viscosupplementation O
was O
both O
safe O
and O
effective O
for O
the O
treatment O
of O
OA O
and O
was O
superior O
or O
equivalent O
to O
any O
form O
of O
systemic O
intervention O
or O
intra O
- O
articular O
corticosteroids O
[ O
9 O
, O
12 O
] O
. O

Despite O
there O
being O
a O
large O
number O
of O
studies O
investigating O
the O
effectiveness O
of O
hyaluronan O
for O
knee O
OA O
, O
few O
studies O
have O
investigated O
the O
effects O
of O
this O
form O
of O
treatment O
for O
OA O
at O
the O
first O
MPJ O
[ O
13 O
] O
. O

In O
a O
case O
- O
series O
retrospective O
study O
, O
14 O
patients B
with O
radiographically O
confirmed O
OA O
at O
the O
first O
MPJ O
that O
received O
up O
to O
3 O
intra O
- O
articular O
injections O
of O
1 O
ml O
hyaluronan O
( O
Ostenil O
( O
R O
) O
Mini O
) O
( O
sodium O
hyaluronate O
) O
reported O
a O
statistically O
significant O
reduction O
in O
pain O
( O
reported O
using O
a O
visual O
analogue O
scale O
) O
after O
6 O
months O
[ O
14 O
] O
. O

The O
treatment O
was O
well O
tolerated O
, O
with O
3 O
/ O
14 O
( O
21 O
% O
) O
participants B
reporting O
mild O
adverse O
reactions O
at O
the O
injection O
site O
. O

In O
another O
study O
, O
Pons O
et O
al O
[ O
13 O
] O
compared O
a O
single O
intra O
- O
articular O
injection O
of O
1 O
ml O
Ostenil O
( O
R O
) O
Mini O
( O
sodium O
hyaluronate O
) O
with O
1 O
ml O
Trigon O
depot O
( O
R O
) O
( O
triamcinolone O
acetonide O
, O
a O
corticosteroid O
) O
for O
the O
treatment O
of O
painful O
, O
grade O
1 O
hallux O
limitus O
( O
Karasick O
and O
Wapner O
[ O
15 O
] O
scale O
) O
in O
37 O
participants B
( O
40 O
feet O
) O
[ O
13 O
] O
. O

Both O
treatment O
groups O
showed O
statistically O
significant O
reductions O
in O
pain O
at O
rest O
or O
on O
palpation O
for O
up O
to O
12 O
weeks O
post O
- O
injection O
. O

However O
, O
hyaluronan O
treatment O
resulted O
in O
a O
statistically O
significant O
greater O
reduction O
in O
pain O
during O
walking O
and O
greater O
improvement O
in O
the O
American O
Orthopaedic O
Foot O
and O
Ankle O
Society O
( O
AOFAS O
) O
hallux O
MPJ O
score O
compared O
to O
treatment O
with O
triamcinolone O
acetonide O
. O

The O
treatment O
with O
hyaluronan O
was O
well O
tolerated O
, O
with O
2 O
/ O
20 O
( O
10 O
% O
) O
participants B
reporting O
mild O
adverse O
reactions O
at O
the O
injection O
site O
. O

Although O
both O
of O
these O
studies O
suggest O
that O
intra O
- O
articular O
hyaluronan O
is O
safe O
and O
effective O
for O
the O
treatment O
of O
hallux O
limitus O
, O
neither O
used O
a O
placebo O
control O
group O
[ O
13 O
, O
14 O
] O
. O

This O
limitation O
is O
significant O
as O
a O
placebo O
effect O
can O
account O
for O
79 O
% O
of O
the O
efficacy O
of O
intra O
- O
articular O
hyaluronan O
treatment O
[ O
16 O
] O
. O

Further O
, O
both O
studies O
are O
limited O
in O
that O
neither O
of O
the O
studies O
used O
blinding O
of O
both O
the O
participants B
and O
assessors O
in O
their O
protocols O
. O

It O
is O
therefore O
possible O
that O
the O
positive O
effects O
of O
hyaluronan O
may O
have O
been O
overestimated O
. O

Accordingly O
, O
the O
aims O
of O
this O
project O
are O
to O
conduct O
a O
double O
blind O
randomised O
controlled O
trial O
to O
determine O
the O
effectiveness O
of O
intra O
- O
articular O
hyaluronan O
( O
Synvisc O
( O
R O
) O
) O
on O
( O
i O
) O
foot O
pain O
and O
function O
; O
( O
ii O
) O
the O
range O
of O
motion O
of O
the O
first O
MPJ O
; O
( O
iii O
) O
the O
strength O
of O
the O
plantarflexor O
muscles O
of O
the O
first O
MPJ O
; O
( O
iv O
) O
the O
health O
related O
quality O
of O
life O
; O
and O
( O
v O
) O
the O
use O
of O
pain O
- O
relieving O
medications O
in O
people B
with O
hallux O
limitus O
. O

The O
study O
protocol O
is O
presented O
in O
this O
paper O
, O
consistent O
with O
the O
recommendations O
of O
Editorial O
Board O
of O
BioMed O
Central O
[ O
17 O
] O
. O

Methods O

Design O

This O
study O
is O
a O
parallel O
group O
, O
participant B
and O
assessor O
blinded O
, O
randomised O
controlled O
trial O
with O
a O
6 O
month O
follow O
- O
up O
( O
Figure O
1 O
) O
. O

It O
has O
been O
developed O
using O
the O
principles O
described O
by O
Osteoarthritis O
Research O
Society O
International O
( O
OARSI O
) O
Clinical O
Trials O
Task O
Force O
guidelines O
[ O
18 O
] O
. O

Participants B
will O
be O
randomised O
to O
receive O
a O
single O
intra O
- O
articular O
injection O
of O
up O
to O
1 O
ml O
hyaluronan O
( O
Synvisc O
( O
R O
) O
) O
or O
sterile O
saline O
( O
placebo O
) O
into O
the O
first O
MPJ O
. O

Allocation O
to O
either O
the O
Synvisc O
( O
R O
) O
or O
placebo O
groups O
will O
be O
achieved O
using O
a O
computer O
- O
generated O
random O
number O
sequence O
. O

The O
allocation O
sequence O
will O
be O
generated O
and O
held O
by O
an O
external O
person B
not O
directly O
involved O
in O
the O
trial O
. O

Concealment O
of O
the O
allocation O
sequence O
will O
be O
ensured O
as O
each O
participant B
' O
s O
allocation O
will O
be O
contained O
in O
a O
sealed O
opaque O
envelope O
. O

Envelopes O
will O
be O
made O
opaque O
by O
using O
a O
sheet O
of O
aluminium O
foil O
inside O
the O
envelope O
. O

In O
addition O
, O
a O
system O
using O
carbon O
paper O
will O
be O
employed O
so O
the O
details O
( O
name O
and O
date O
of O
recruitment O
) O
are O
transferred O
from O
the O
outside O
of O
the O
envelope O
to O
the O
paper O
inside O
the O
envelope O
containing O
the O
allocation O
prior O
to O
opening O
the O
seal O
. O

Assessors O
and O
participants B
will O
be O
blinded O
to O
group O
allocation O
. O

Participants B
will O
be O
given O
the O
option O
of O
a O
second O
and O
final O
intra O
- O
articular O
injection O
( O
of O
Synvisc O
( O
R O
) O
or O
sterile O
saline O
according O
to O
the O
treatment O
group O
they O
are O
in O
) O
on O
days O
30 O
or O
90 O
if O
there O
is O
no O
improvement O
in O
pain O
and O
the O
participant B
has O
not O
experienced O
severe O
adverse O
effects O
after O
the O
first O
injection O
) O
. O

Participants B

The O
Human B
Studies O
Ethics O
Committee O
at O
La O
Trobe O
University O
( O
Human B
Ethics O
Committee O
Application O
No O
. O
07 O
- O
45 O
) O
and O
the O
Radiation O
Advisory O
Committee O
of O
the O
Victorian O
Department O
of O
Human B
Services O
have O
given O
approval O
for O
the O
study O
. O

Written O
informed O
consent O
will O
be O
obtained O
from O
all O
participants B
prior O
to O
their O
participation O
. O

People B
with O
hallux O
limitus O
will O
be O
recruited O
from O
a O
number O
of O
sources O
: O

( O
i O
) O
advertisements O
in O
relevant O
Melbourne O
( O
Australia O
) O
newspapers O
; O

( O
ii O
) O
mail O
- O
out O
advertisements O
to O
health O
- O
care O
practitioners O
in O
Melbourne O
; O

( O
iii O
) O
advertisements O
using O
relevant O
internet O
web O
- O
sites O
( O
including O
) O
; O

( O
iv O
) O
posters O
displayed O
in O
local O
retirement O
villages O
, O
community O
centres O
and O
universities O
located O
in O
Melbourne O
. O

Respondents O
will O
initially O
be O
screened O
by O
telephone O
interview O
to O
ensure O
they O
are O
suitable O
for O
the O
study O
. O

Suitable O
individuals O
will O
then O
be O
invited O
to O
participate O
in O
the O
study O
and O
attend O
an O
initial O
assessment O
. O

To O
be O
included O
in O
the O
study O
, O
participants B
must O
meet O
the O
following O
inclusion O
criteria O
: O

( O
i O
) O
be O
aged O
at O
least O
18 O
years O
; O

( O
ii O
) O
report O
having O
symptoms O
of O
pain O
, O
during O
walking O
or O
rest O
, O
in O
the O
first O
MPJ O
for O
at O
least O
3 O
months O
; O

( O
iii O
) O
report O
having O
pain O
rated O
at O
least O
20 O
mm O
on O
a O
100 O
mm O
visual O
analogue O
pain O
scale O
( O
VAPS O
) O
; O

( O
iv O
) O
have O
pain O
upon O
palpation O
of O
the O
dorsal O
aspect O
of O
the O
first O
MPJ O
; O

( O
v O
) O
radiographic O
evidence O
of O
OA O
( O
score O
1 O
or O
2 O
for O
either O
osteophytes O
or O
joint O
space O
narrowing O
using O
a O
previously O
published O
radiographic O
classification O
) O
[ O
19 O
] O
at O
the O
first O
MPJ O
. O

( O
vi O
) O
able O
to O
walk O
household O
distances O
( O
> O
50 O
meters O
) O
without O
the O
aid O
of O
a O
walker O
, O
crutches O
or O
cane O
; O

( O
vii O
) O
be O
willing O
to O
attend O
the O
La O
Trobe O
University O
Medical O
Centre O
( O
Melbourne O
, O
Australia O
) O
for O
treatment O
with O
either O
Synvisc O
( O
R O
) O
or O
placebo O
( O
single O
intra O
- O
articular O
injection O
) O
and O
attend O
the O
Health O
Sciences O
Clinic O
at O
La O
Trobe O
University O
( O
Melbourne O
, O
Australia O
) O
for O
the O
initial O
assessment O
and O
the O
outcome O
measurements O
( O
at O
baseline O
and O
1 O
, O
3 O
and O
6 O
months O
post O
- O
treatment O
) O
; O

( O
viii O
) O
not O
receive O
other O
intra O
- O
articular O
injections O
into O
the O
first O
MPJ O
during O
the O
course O
of O
the O
study O
, O
apart O
from O
those O
dictated O
by O
the O
study O
; O

( O
ix O
) O
be O
willing O
to O
discontinue O
taking O
all O
pain O
- O
relieving O
medications O
( O
analgesics O
and O
non O
- O
steroidal O
anti O
- O
inflammatory O
medications O
( O
NSAIDs O
) O
, O
except O
paracetamol O
up O
to O
4 O
g O
/ O
day O
, O
taken O
by O
mouth O
or O
applied O
topically O
) O
: O

- O
for O
at O
least O
14 O
days O
prior O
to O
the O
baseline O
assessment O
; O

- O
during O
the O
study O
period O
( O
6 O
months O
after O
the O
final O
treatment O
with O
Synvisc O
( O
R O
) O
) O
. O

Participants B
who O
do O
take O
paracetamol O
need O
to O
discontinue O
its O
use O
at O
least O
24 O
hours O
prior O
to O
the O
baseline O
assessment O
and O
follow O
- O
up O
assessments O
at O
1 O
, O
3 O
and O
6 O
months O
after O
the O
treatment O
; O

( O
x O
) O
be O
willing O
to O
not O
receive O
any O
physical O
therapy O
on O
the O
involved O
MPJ O
or O
trial O
of O
shoe O
modifications O
or O
foot O
orthoses O
during O
the O
study O
period O
. O

Exclusion O
criteria O
for O
participants B
in O
this O
study O
will O
be O
: O

( O
i O
) O
Severe O
radiographic O
evidence O
of O
OA O
( O
score O
3 O
for O
either O
osteophytes O
or O
joint O
space O
narrowing O
) O
at O
the O
first O
MPJ O
using O
a O
previously O
published O
radiographic O
classification O
[ O
19 O
] O
; O

( O
ii O
) O
previous O
surgery O
on O
the O
first O
MPJ O
; O

( O
iii O
) O
intra O
- O
articular O
steroid O
, O
or O
any O
other O
intra O
- O
articular O
injection O
at O
the O
first O
MPJ O
in O
the O
previous O
6 O
months O
; O

( O
iv O
) O
treatment O
with O
systemic O
steroid O
( O
excluding O
inhalation O
or O
topical O
steroids O
) O
, O
immunosuppressives O
or O
anticoagulants O
( O
except O
for O
acetylsalicylic O
acid O
at O
dosages O
of O
up O
to O
325 O
mg O
/ O
day O
) O
; O

( O
v O
) O
presence O
of O
joint O
infection O
( O
s O
) O
of O
the O
foot O
; O

( O
vi O
) O
significant O
deformity O
of O
the O
first O
MPJ O
including O
hallux O
abducto O
valgus O
( O
grade O
of O
3 O
or O
4 O
scored O
using O
the O
Manchester O
Scale O
[ O
20 O
] O
; O

( O
vii O
) O
presence O
of O
peripheral O
vascular O
disease O
. O

Peripheral O
vascular O
disease O
will O
be O
considered O
to O
be O
present O
if O
any O
of O
the O
following O
are O
present O
[ O
21 O
] O
; O

* O
past O
history O
of O
, O
vascular O
surgery O
, O
Raynaud O
' O
s O
phenomenon O
, O
vasculitis O
associated O
with O
connective O
tissue O
diseases O
, O
Buerger O
' O
s O
disease O
, O
arterial O
emboli O
, O
deep O
vein O
thrombosis O
or O
lower O
limb O
ulcers O
; O

* O
history O
of O
intermittent O
claudication O
or O
rest O
pain O
; O

* O
presence O
of O
atrophy O
, O
ulcers O
or O
significant O
oedema O
; O

* O
inability O
to O
palpate O
at O
least O
one O
pedal O
pulse O
; O

* O
Ankle O
Brachial O
Pressure O
Index O
< O
0 O
. O
9 O
; O

( O
viii O
) O
presence O
of O
one O
or O
more O
conditions O
that O
can O
confound O
pain O
and O
functional O
assessments O
of O
the O
first O
MPJ O
, O
such O
as O
metatarsalgia O
, O
plantar O
fasciitis O
, O
pre O
- O
dislocation O
syndrome O
, O
sprains O
of O
the O
foot O
, O
Achilles O
tendinopathy O
, O
degenerative O
joint O
disease O
of O
the O
foot O
( O
other O
than O
the O
first O
MPJ O
) O
or O
painful O
corns O
and O
callus O
; O

( O
ix O
) O
planning O
to O
undergo O
any O
surgical O
procedure O
or O
receive O
any O
injections O
, O
apart O
from O
those O
dictated O
by O
the O
study O
, O
at O
the O
involved O
first O
MPJ O
during O
the O
study O
period O
; O

( O
x O
) O
presence O
of O
systemic O
inflammatory O
condition O
or O
infection O
, O
such O
as O
inflammatory O
arthritis O
, O
diagnosed O
with O
rheumatoid O
arthritis O
, O
ankylosing O
spondylitis O
, O
psoriatic O
arthritis O
, O
reactive O
arthritis O
, O
septic O
arthritis O
, O
acute O
pseudogout O
, O
or O
any O
other O
connective O
tissue O
disease O
; O

( O
xi O
) O
evidence O
of O
gout O
or O
other O
musculoskeletal O
disease O
other O
than O
OA O
within O
the O
feet O
. O

Gout O
will O
be O
screened O
for O
using O
clinical O
history O
and O
physical O
assessment O
( O
painful O
joint O
, O
abrupt O
onset O
, O
swelling O
) O
, O
radiographic O
assessment O
( O
asymmetrical O
joint O
swelling O
, O
subcortical O
cysts O
without O
erosion O
and O
tophi O
) O
as O
well O
as O
serum O
uric O
acid O
levels O
( O
hyperuricaemia O
= O
serum O
uric O
acid O
> O
mean O
+ O
2 O
SD O
from O
normal O
population O
) O
[ O
22 O
] O
; O

( O
xii O
) O
active O
skin O
disease O
or O
infection O
in O
the O
area O
of O
the O
injection O
site O
; O

( O
xiii O
) O
any O
medical O
condition O
that O
, O
in O
the O
opinion O
of O
the O
investigators O
, O
makes O
the O
participant B
unsuitable O
for O
inclusion O
( O
e O
. O
g O
. O
, O
severe O
progressive O
chronic O
disease O
, O
malignancy O
, O
bleeding O
disorder O
, O
clinically O
important O
pain O
in O
a O
part O
of O
the O
musculoskeletal O
system O
other O
than O
the O
first O
MPJ O
, O
or O
fibromyalgia O
) O
; O

( O
xiv O
) O
pregnant O
or O
lactating O
women B
, O
or O
women B
who O
are O
of O
child O
bearing O
age O
or O
have O
not O
undergone O
menopause O
( O
Synvisc O
( O
R O
) O
has O
not O
been O
tested O
in O
pregnant O
women B
or O
women B
who O
are O
nursing O
) O
; O

( O
xv O
) O
cognitive O
impairment O
( O
defined O
as O
a O
score O
of O
< O
7 O
on O
the O
Short O
Portable O
Mental O
Status O
Questionnaire O
) O
[ O
23 O
] O
; O

( O
xvi O
) O
known O
hypersensitivity O
( O
allergy O
) O
to O
hyaluronan O
preparations O
, O
or O
to O
avian O
proteins O
, O
feathers O
or O
egg O
products O
; O

( O
xvii O
) O
involvement O
in O
any O
clinical O
research O
study O
in O
the O
previous O
3 O
months O
that O
could O
be O
considered O
to O
affect O
the O
results O
of O
this O
study O
. O

Intra O
- O
articular O
injections O
for O
the O
treatment O
groups O

Participants B
will O
be O
randomised O
to O
receive O
a O
single O
intra O
- O
articular O
injection O
of O
up O
to O
1 O
ml O
of O
hyaluronan O
( O
Synvisc O
( O
R O
) O
; O
Genzyme O
Biosurgery O
, O
Genzyme O
Corporation O
, O
NJ O
, O
USA O
) O
or O
sterile O
saline O
( O
placebo O
) O
into O
the O
first O
MPJ O
. O

Each O
2 O
ml O
ampoule O
of O
Synvisc O
( O
R O
) O
contains O
16 O
mg O
of O
hylan O
G O
- O
F O
20 O
( O
cross O
- O
linked O
hylan O
polymers O
; O
hylan O
A O
and O
B O
) O
, O
17 O
mg O
sodium O
chloride O
, O
0 O
. O
32 O
mg O
disodium O
hydrogen O
phosphate O
, O
0 O
. O
08 O
mg O
sodium O
dihydrogen O
phosphate O
monohydrate O
. O

The O
hyaluronan O
is O
extracted O
from O
chicken B
combs O
and O
the O
purified O
material O
has O
an O
average O
molecular O
weight O
of O
6 O
, O
000 O
kDa O
. O

The O
injections O
will O
be O
performed O
by O
the O
same O
experienced O
interventional O
radiologist O
( O
AEZ O
) O
using O
fluoroscopic O
imaging O
to O
ensure O
accurate O
deposition O
of O
the O
hyaluronan O
within O
the O
joint O
. O

As O
the O
Synvisc O
( O
R O
) O
is O
provided O
in O
ampoules O
that O
are O
labelled O
with O
the O
product O
name O
, O
it O
will O
not O
be O
possible O
to O
blind O
the O
injector O
, O
however O
this O
person B
is O
not O
involved O
in O
generation O
of O
the O
allocation O
order O
, O
recruitment O
, O
assessment O
or O
data O
analysis O
. O

The O
intra O
- O
articular O
injection O
will O
be O
performed O
using O
a O
21 O
gauge O
( O
0 O
. O
80 O
x O
19 O
mm O
) O
Surflo O
( O
R O
) O
( O
Terumo O
( O
R O
) O
Corp O
. O
, O
Tokyo O
, O
Japan O
) O
winged O
infusion O
set O
under O
aseptic O
procedures O
. O

Either O
a O
dorso O
- O
lateral O
or O
dorso O
- O
medial O
approach O
for O
injection O
will O
be O
used O
at O
the O
discretion O
of O
the O
injector O
( O
depending O
on O
which O
approach O
provides O
minimum O
interference O
from O
the O
osteophytes O
at O
the O
first O
MPJ O
joint O
margins O
) O
. O

No O
anaesthetic O
will O
be O
used O
. O

If O
the O
participant B
has O
bilateral O
painful O
first O
MPJs O
, O
only O
one O
side O
( O
the O
most O
painful O
side O
) O
will O
be O
treated O
and O
used O
for O
data O
collection O
. O

The O
injector O
will O
record O
the O
volume O
of O
the O
agent O
that O
is O
injected O
. O

Participants B
will O
be O
given O
the O
option O
of O
a O
second O
and O
final O
intra O
- O
articular O
injection O
( O
of O
Synvisc O
( O
R O
) O
or O
sterile O
saline O
according O
to O
the O
treatment O
group O
they O
are O
in O
) O
on O
days O
30 O
or O
90 O
if O
there O
is O
no O
improvement O
in O
pain O
( O
assessed O
using O
the O
VAPS O
for O
pain O
during O
walking O
or O
at O
rest O
) O
and O
the O
participant B
has O
not O
experienced O
severe O
adverse O
effects O
after O
the O
first O
injection O
) O
. O

Assessments O

Initial O
assessments O

An O
initial O
assessment O
will O
be O
performed O
to O
determine O
the O
eligibility O
of O
participants B
for O
this O
study O
. O

Demographic O
data O
will O
be O
collected O
including O
the O
age O
, O
gender O
, O
height O
and O
weight O
of O
participants B
. O

Data O
will O
also O
be O
obtained O
concerning O
the O
presentation O
of O
symptoms O
( O
foot O
affected O
, O
duration O
of O
symptoms O
) O
. O

If O
the O
participant B
has O
bilateral O
painful O
first O
MPJs O
, O
the O
most O
painful O
side O
will O
be O
used O
for O
data O
collection O
and O
subsequent O
treatment O
. O

To O
establish O
eligibility O
for O
the O
study O
, O
participants B
will O
undergo O
a O
clinical O
assessment O
, O
have O
one O
set O
of O
dorso O
- O
plantar O
and O
lateral O
weight O
- O
bearing O
x O
- O
rays O
taken O
of O
their O
feet O
to O
grade O
the O
severity O
of O
first O
MPJ O
OA O
as O
well O
as O
undergo O
a O
blood O
test O
to O
assess O
serum O
uric O
acid O
levels O
( O
to O
exclude O
gout O
) O
. O

Weightbearing O
dorso O
- O
plantar O
and O
lateral O
radiographic O
views O
will O
be O
obtained O
from O
both O
feet O
with O
the O
participant B
standing O
in O
a O
relaxed O
bipedal O
stance O
position O
. O

All O
x O
- O
rays O
will O
be O
taken O
by O
the O
same O
medical O
imaging O
department O
using O
a O
Shimadzu O
UD150LRII O
50 O
kw O
/ O
30 O
kHz O
Generator O
and O
0 O
. O
6 O
/ O
1 O
. O
2 O
P18DE O
- O
80S O
high O
speed O
x O
- O
ray O
tube O
from O
a O
ceiling O
suspended O
tube O
mount O
. O

AGFA O
MD40 O
CR O
digital O
phosphor O
plates O
in O
a O
24 O
cm O
x O
30 O
cm O
cassette O
will O
be O
used O
. O

For O
dorso O
- O
plantar O
projections O
, O
the O
x O
- O
ray O
tube O
will O
be O
angled O
15 O
degrees O
cephalad O
and O
centered O
at O
the O
base O
of O
the O
third O
metatarsal O
. O

For O
lateral O
projections O
, O
the O
tube O
will O
be O
angled O
90 O
degrees O
and O
centered O
at O
the O
base O
of O
the O
third O
metatarsal O
. O

The O
film O
focus O
distance O
will O
be O
set O
at O
100 O
cm O
[ O
19 O
] O
. O

Baseline O
assessments O
and O
outcome O
measures O

Participants B
who O
are O
eligible O
for O
the O
study O
will O
be O
invited O
to O
attend O
a O
baseline O
assessment O
. O

During O
the O
baseline O
assessment O
, O
participants B
will O
undergo O
primary O
and O
secondary O
outcome O
measurements O
prior O
to O
receiving O
their O
injection O
. O

The O
outcome O
measurements O
have O
been O
developed O
in O
accordance O
of O
the O
recommendations O
of O
the O
OARSI O
Clinical O
Trials O
Task O
Force O
guidelines O
[ O
18 O
] O
. O

Primary O
outcome O
measures O

Outcome O
measurements O
( O
primary O
and O
secondary O
) O
will O
occur O
at O
four O
time O
- O
points O
at O
baseline O
, O
1 O
, O
3 O
and O
6 O
months O
post O
- O
treatment O
( O
after O
the O
intra O
- O
articular O
injection O
of O
Synvisc O
( O
R O
) O
or O
placebo O
) O
. O

The O
assessor O
performing O
the O
measurements O
will O
be O
blinded O
as O
to O
which O
treatment O
group O
participants B
have O
been O
allocated O
to O
. O

Participants B
who O
receive O
a O
second O
treatment O
at O
day O
30 O
or O
90 O
will O
be O
followed O
for O
a O
further O
30 O
days O
or O
90 O
days O
respectively O
and O
undergo O
outcome O
measurements O
at O
7 O
or O
9 O
months O
respectively O
. O

The O
primary O
outcome O
measures O
will O
be O
the O
Pain O
and O
Function O
subscales O
of O
the O
Foot O
Health O
Status O
Questionnaire O
( O
FHSQ O
) O
[ O
24 O
] O
. O

The O
FHSQ O
includes O
13 O
questions O
that O
assess O
four O
domains O
of O
foot O
health O
, O
Foot O
pain O
, O
Foot O
function O
, O
Footwear O
and O
General O
foot O
health O
. O

The O
FHSQ O
has O
been O
subjected O
to O
an O
extensive O
validation O
( O
content O
, O
criterion O
and O
construct O
validity O
) O
process O
. O

It O
has O
a O
high O
test O
- O
retest O
reliability O
( O
intraclass O
correlation O
coefficients O
ranging O
from O
0 O
. O
74 O
to O
0 O
. O
92 O
) O
and O
a O
high O
degree O
of O
internal O
consistency O
( O
Cronbach O
' O
s O
alpha O
ranging O
from O
0 O
. O
85 O
to O
0 O
. O
88 O
) O
[ O
24 O
] O
. O

Rigorous O
reviews O
have O
rated O
it O
as O
one O
of O
the O
highest O
quality O
foot O
health O
status O
measures O
currently O
available O
[ O
25 O
- O
27 O
] O
. O

Secondary O
outcome O
measures O

The O
secondary O
outcome O
measures O
will O
be O
: O

( O
i O
) O
Severity O
of O
pain O

Severity O
of O
pain O
at O
the O
first O
MPJ O
during O
walking O
, O
and O
during O
rest O
, O
over O
the O
past O
week O
will O
be O
assessed O
using O
a O
100 O
mm O
visual O
analogue O
pain O
scale O
. O

The O
left O
side O
of O
the O
scale O
( O
0 O
mm O
) O
will O
be O
labelled O
" O
no O
pain O
" O
and O
the O
right O
side O
of O
the O
scale O
( O
100 O
mm O
) O
will O
be O
labelled O
" O
worst O
pain O
possible O
" O
for O
each O
question O
[ O
25 O
, O
28 O
] O
. O

( O
ii O
) O
Severity O
and O
duration O
of O
stiffness O
at O
the O
first O
metatarsophalangeal O
joint O

The O
severity O
of O
stiffness O
at O
the O
first O
MPJ O
during O
walking O
over O
the O
past O
week O
will O
be O
assessed O
using O
a O
100 O
mm O
visual O
analogue O
scale O
. O

The O
left O
side O
of O
the O
scale O
( O
0 O
mm O
) O
will O
be O
labelled O
" O
not O
stiff O
at O
all O
" O
and O
the O
right O
side O
of O
the O
scale O
( O
100 O
mm O
) O
will O
be O
labelled O
" O
most O
stiff O
possible O
" O
. O

The O
average O
duration O
of O
stiffness O
at O
the O
first O
MPJ O
over O
the O
past O
week O
will O
be O
assessed O
using O
a O
four O
category O
scale O
response O
. O

The O
responses O
are O
: O
" O
none O
" O
, O
" O
1 O
- O
15 O
minutes O
" O
, O
" O
16 O
- O
30 O
minutes O
" O
and O
" O
greater O
than O
30 O
minutes O
" O
[ O
29 O
] O
. O

( O
iii O
) O
Passive O
, O
non O
- O
weightbearing O
dorsiflexion O
range O
of O
motion O
of O
the O
first O
metatarsophalangeal O
joint O

First O
MPJ O
dorsiflexion O
range O
of O
motion O
will O
be O
measured O
using O
a O
goniometer O
as O
the O
maximum O
angle O
at O
which O
the O
hallux O
cannot O
be O
passively O
moved O
into O
further O
extension O
in O
a O
non O
- O
weightbearing O
position O
( O
Figure O
2 O
) O
[ O
30 O
] O
. O

The O
test O
will O
be O
performed O
two O
times O
and O
the O
average O
will O
be O
used O
for O
analysis O
. O

This O
measurement O
technique O
shows O
high O
intra O
- O
reliability O
( O
ICC O
= O
0 O
. O
95 O
, O
standard O
error O
of O
mean O
= O
1 O
. O
3 O
degrees O
) O
[ O
30 O
] O
. O

( O
iv O
) O
Plantar O
flexion O
strength O
of O
the O
toe O
- O
flexors O
of O
the O
hallux O

Plantar O
flexion O
strength O
of O
the O
toe O
- O
flexors O
of O
the O
hallux O
will O
be O
measured O
using O
the O
Mat O
Scan O
( O
R O
) O
plantar O
pressure O
measurement O
device O
[ O
31 O
] O
. O

Participants B
will O
be O
seated O
with O
the O
hip O
, O
knee O
, O
and O
ankle O
at O
90 O
degrees O
and O
their O
foot O
placed O
over O
the O
Mat O
Scan O
( O
R O
) O
plantar O
pressure O
measurement O
device O
( O
Tekscan O
, O
Boston O
, O
MA O
, O
USA O
) O
( O
Figure O
3a O
) O
. O

This O
system O
consists O
of O
a O
5 O
- O
mm O
thick O
floor O
mat O
( O
432 O
x O
368 O
mm O
) O
incorporating O
2288 O
resistive O
sensors O
( O
1 O
. O
4 O
sensors O
/ O
cm2 O
) O
sampling O
at O
a O
rate O
of O
40 O
Hz O
. O

The O
mat O
will O
be O
calibrated O
for O
each O
participant B
using O
his O
or O
her O
own O
bodyweight O
before O
each O
testing O
session O
. O

Participants B
will O
be O
instructed O
to O
use O
their O
toe O
- O
flexor O
muscles O
to O
maximally O
push O
their O
hallux O
down O
on O
the O
MatScan O
( O
R O
) O
device O
and O
forces O
under O
the O
hallux O
will O
be O
recorded O
( O
Figure O
3b O
) O
. O

The O
test O
will O
be O
performed O
three O
times O
for O
the O
hallux O
and O
the O
maximal O
force O
will O
be O
used O
for O
analysis O
. O

The O
test O
- O
retest O
reliability O
of O
this O
measurement O
technique O
has O
previously O
been O
shown O
to O
be O
high O
, O
with O
intraclass O
correlation O
coefficients O
( O
ICCs O
) O
= O
0 O
. O
88 O
( O
95 O
% O
CI O
0 O
. O
81 O
- O
0 O
. O
93 O
) O
[ O
31 O
] O
. O

( O
vi O
) O
Plantar O
pressure O
measurement O

Plantar O
pressures O
will O
be O
recorded O
during O
level O
barefoot O
walking O
using O
the O
MatScan O
( O
R O
) O
system O
( O
Tekscan O
( O
R O
) O
, O
Boston O
, O
MA O
, O
USA O
) O
. O

The O
two O
- O
step O
gait O
initiation O
protocol O
will O
be O
used O
to O
obtain O
foot O
pressure O
data O
, O
as O
it O
requires O
fewer O
trials O
than O
the O
mid O
- O
gait O
protocol O
and O
has O
similar O
re O
- O
test O
reliability O
[ O
32 O
] O
. O

Three O
trials O
will O
be O
recorded O
, O
which O
has O
been O
found O
to O
be O
sufficient O
to O
ensure O
adequate O
reliability O
of O
pressure O
data O
[ O
32 O
, O
33 O
] O
. O

Following O
data O
collection O
, O
the O
Research O
Foot O
( O
R O
) O
software O
( O
version O
5 O
. O
24 O
) O
will O
be O
used O
to O
construct O
individual O
" O
masks O
" O
to O
determine O
maximum O
force O
( O
kg O
) O
and O
peak O
pressure O
( O
kg O
/ O
cm2 O
) O
under O
seven O
regions O
of O
the O
foot O
: O
hallux O
, O
lesser O
toes O
, O
1st O
MPJ O
, O
2nd O
MPJ O
, O
3rd O
to O
5th O
MPJs O
, O
midfoot O
and O
heel O
( O
Figure O
4a O
) O
. O

For O
each O
region O
, O
the O
median O
of O
the O
three O
trials O
will O
be O
used O
for O
analysis O
. O

Typical O
plantar O
pressure O
recordings O
from O
a O
participant B
are O
shown O
in O
Figure O
4b O
. O

( O
vi O
) O
Global O
satisfaction O
with O
the O
treatment O

Global O
satisfaction O
with O
the O
treatment O
will O
be O
assessed O
using O
a O
5 O
- O
point O
Likert O
scale O
, O
as O
well O
as O
a O
dichotomous O
( O
yes O
/ O
no O
) O
scale O
. O

The O
five O
point O
- O
Likert O
scale O
will O
ask O
" O
How O
satisfied O
are O
you O
with O
the O
treatment O
you O
received O
for O
your O
big O
- O
toe O
joint O
pain O
? O
" O
, O
and O
will O
have O
the O
following O
five O
responses O
: O
" O
Dissatisfied O
" O
, O
" O
Only O
moderately O
satisfied O
" O
, O
" O
Fairly O
satisfied O
" O
, O
" O
Clearly O
satisfied O
" O
and O
" O
Very O
satisfied O
" O
. O

The O
dichotomous O
scale O
of O
satisfaction O
will O
be O
answered O
as O
" O
Yes O
" O
' O
or O
" O
No O
" O
in O
response O
to O
the O
question O
: O
" O
Would O
you O
recommend O
the O
treatment O
that O
you O
received O
to O
someone O
else O
with O
big O
- O
toe O
joint O
pain O
" O
. O

( O
vii O
) O
Health O
related O
quality O
of O
life O

The O
Short O
- O
Form O
- O
36 O
( O
version O
two O
) O
( O
SF O
- O
36 O
) O
questionnaire O
will O
be O
used O
to O
assess O
health O
related O
quality O
of O
life O
. O

The O
SF O
- O
36 O
is O
a O
36 O
question O
survey O
that O
measures O
eight O
health O
concepts O
most O
affected O
by O
disease O
and O
treatment O
. O

The O
eight O
health O
concepts O
can O
then O
be O
used O
to O
form O
two O
summary O
measures O
: O
Physical O
health O
and O
Mental O
health O
. O

The O
Short O
Form O
- O
36 O
( O
SF O
- O
36 O
) O
has O
been O
extensively O
validated O
and O
is O
one O
of O
the O
most O
widely O
used O
instruments O
to O
measure O
health O
status O
. O

The O
SF O
- O
36 O
shows O
content O
, O
concurrent O
, O
criterion O
, O
construct O
, O
and O
predictive O
evidence O
of O
validity O
. O

The O
reliability O
of O
the O
eight O
concepts O
and O
two O
summary O
measures O
has O
been O
assessed O
using O
both O
internal O
consistency O
and O
test O
- O
retest O
methods O
. O

Reliability O
statistics O
have O
exceeded O
0 O
. O
80 O
[ O
34 O
- O
37 O
] O
. O

( O
viii O
) O
Self O
- O
reported O
magnitude O
of O
symptom O
change O

Self O
- O
reported O
magnitude O
of O
symptom O
change O
will O
be O
measured O
using O
a O
15 O
- O
point O
Likert O
scale O
. O

The O
scale O
will O
ask O
participants B
" O
how O
much O
have O
your O
symptoms O
in O
your O
big O
- O
toe O
joint O
have O
changed O
from O
the O
beginning O
of O
the O
study O
to O
now O
? O
" O
. O

The O
fifteen O
responses O
will O
range O
from O
" O
A O
very O
great O
deal O
better O
" O
to O
" O
A O
very O
great O
deal O
worse O
" O
. O

( O
ix O
) O
Use O
of O
rescue O
medications O
to O
relieve O
pain O
at O
the O
first O
metatarsophalangeal O
joint O

The O
number O
of O
participants B
who O
consumed O
rescue O
medication O
( O
e O
. O
g O
. O
, O
paracetamol O
) O
and O
mean O
consumption O
of O
rescue O
medication O
to O
relieve O
pain O
at O
the O
first O
MPJ O
( O
mean O
grams O
of O
paracetamol O
/ O
participant B
/ O
month O
] O
will O
be O
assessed O
using O
a O
medications O
diary O
that O
participants B
will O
self O
- O
complete O
[ O
38 O
, O
39 O
] O
. O

The O
diary O
will O
be O
returned O
to O
the O
assessor O
at O
monthly O
intervals O
for O
analysis O
. O

( O
x O
) O
Frequency O
and O
severity O
of O
adverse O
events O
as O
safety O
variables O

The O
frequency O
( O
number O
of O
participants B
affected O
and O
number O
of O
cases O
) O
and O
types O
of O
adverse O
events O
( O
including O
adverse O
drug O
reactions O
) O
in O
each O
treatment O
group O
during O
the O
trial O
will O
be O
recorded O
using O
a O
questionnaire O
that O
participants B
will O
complete O
during O
the O
follow O
- O
up O
appointments O
at O
1 O
, O
3 O
and O
6 O
months O
post O
- O
treatment O
[ O
40 O
] O
. O

To O
classify O
the O
' O
type O
' O
of O
adverse O
event O
, O
a O
blinded O
assessor O
will O
classify O
the O
adverse O
event O
as O
being O
serious O
or O
non O
- O
serious O
[ O
40 O
] O
. O

Any O
serious O
adverse O
events O
, O
defined O
as O
adverse O
events O
leading O
to O
serious O
disability O
, O
hospital O
admission O
, O
or O
prolongation O
of O
hospitalisation O
, O
life O
- O
threatening O
events O
; O
or O
death O
) O
will O
be O
further O
classified O
using O
the O
International O
Classification O
of O
Diseases O
( O
ICD O
) O
codes O
[ O
41 O
] O
. O

Non O
- O
serious O
adverse O
events O
will O
include O
both O
local O
( O
pain O
, O
effusion O
and O
heat O
, O
with O
each O
classified O
as O
mild O
, O
moderate O
, O
severe O
) O
and O
systemic O
adverse O
events O
. O

An O
open O
- O
response O
type O
format O
will O
also O
be O
available O
for O
participant B
responses O
. O

Sample O
size O

The O
sample O
size O
for O
the O
study O
has O
been O
pre O
- O
specified O
using O
an O
a O
priori O
power O
analysis O
using O
the O
primary O
outcome O
measure O
of O
the O
pain O
domain O
of O
the O
FHSQ O
[ O
42 O
] O
. O

One O
hundred O
and O
forty O
two O
participants B
( O
i O
. O
e O
. O
71 O
per O
group O
) O
will O
provide O
power O
of O
90 O
% O
to O
detect O
a O
minimally O
important O
difference O
in O
the O
pain O
domain O
of O
the O
FHSQ O
( O
i O
. O
e O
. O
14 O
points O
on O
the O
FHSQ O
questionnaire O
) O
with O
the O
significance O
level O
set O
at O
p O
< O
0 O
. O
05 O
. O

A O
difference O
of O
14 O
points O
was O
determined O
to O
be O
a O
clinically O
significant O
difference O
worth O
detecting O
[ O
43 O
] O
and O
a O
standard O
deviation O
of O
25 O
was O
derived O
from O
a O
previous O
report O
[ O
44 O
] O
. O

This O
calculation O
included O
a O
5 O
% O
drop O
- O
out O
rate O
[ O
13 O
] O
. O

However O
, O
we O
will O
aim O
to O
recruit O
150 O
participants B
( O
~ O
75 O
participants B
per O
intervention O
group O
) O
. O

Further O
, O
we O
have O
conservatively O
ignored O
the O
extra O
precision O
provided O
by O
covariate O
analysis O
when O
estimating O
the O
sample O
size O
. O

Statistical O
analysis O

Statistical O
analysis O
will O
be O
undertaken O
using O
SPSS O
version O
14 O
. O
0 O
( O
SPSS O
Corp O
, O
Chicago O
, O
Ill O
, O
USA O
) O
and O
STATA O
8 O
( O
Stata O
Corp O
, O
College O
Station O
, O
Tex O
. O
, O
USA O
) O
statistical O
software O
. O

All O
analyses O
will O
be O
conducted O
on O
an O
intention O
- O
to O
- O
treat O
principle O
using O
all O
randomised O
participants B
[ O
45 O
- O
47 O
] O
. O

Missing O
data O
will O
be O
replaced O
with O
the O
last O
score O
carried O
forward O
[ O
48 O
] O
. O

Standard O
tests O
for O
normal O
distribution O
will O
be O
used O
and O
transformation O
carried O
out O
if O
required O
. O

Demographic O
characteristics O
( O
gender O
, O
age O
, O
weight O
, O
height O
, O
body O
mass O
index O
) O
will O
be O
determined O
for O
the O
baseline O
visit O
for O
each O
treatment O
group O
. O

Summary O
statistics O
will O
be O
calculated O
for O
duration O
of O
symptoms O
, O
side O
affected O
( O
left O
, O
right O
, O
bilateral O
) O
, O
grade O
of O
OA O
at O
the O
first O
MPJ O
[ O
19 O
] O
as O
well O
as O
all O
primary O
and O
secondary O
outcome O
measurements O
for O
each O
treatment O
group O
. O

Analyses O
will O
be O
conducted O
on O
1 O
, O
3 O
and O
6 O
month O
outcome O
measures O
. O

The O
continuously O
- O
scored O
outcome O
measures O
at O
1 O
, O
3 O
and O
6 O
months O
will O
be O
compared O
using O
analysis O
of O
covariance O
with O
baseline O
scores O
and O
intervention O
group O
entered O
as O
independent O
variables O
[ O
49 O
, O
50 O
] O
. O

The O
exception O
to O
this O
will O
be O
the O
plantar O
pressure O
measurements O
which O
will O
be O
analysed O
at O
baseline O
, O
1 O
, O
3 O
and O
6 O
months O
using O
two O
- O
way O
repeated O
measures O
analysis O
of O
variance O
statistics O
. O

Post O
- O
hoc O
comparisons O
will O
be O
performed O
using O
Bonferroni O
- O
adjusted O
t O
- O
tests O
. O

Nominal O
and O
ordinal O
scaled O
data O
will O
be O
compared O
at O
1 O
, O
3 O
and O
6 O
months O
using O
Mann O
- O
Whitney O
U O
- O
tests O
and O
chi O
- O
square O
analyses O
( O
or O
Fisher O
' O
s O
exact O
test O
where O
appropriate O
) O
respectively O
. O

Effect O
sizes O
will O
be O
determined O
using O
Cohen O
' O
s O
d O
( O
continuous O
scaled O
data O
) O
or O
odds O
ratios O
( O
nominal O
scaled O
data O
and O
ordinal O
scaled O
data O
) O
as O
appropriate O
. O

The O
outcome O
measurements O
obtained O
at O
7 O
or O
9 O
months O
for O
participants B
that O
receive O
a O
second O
and O
final O
intra O
- O
articular O
injection O
( O
of O
Synvisc O
( O
R O
) O
or O
sterile O
saline O
according O
to O
the O
treatment O
group O
they O
are O
in O
) O
on O
days O
30 O
or O
90 O
respectively O
, O
will O
also O
be O
analysed O
as O
described O
above O
. O

These O
analyses O
will O
be O
classified O
as O
secondary O
outcomes O
. O

Discussion O

This O
study O
is O
a O
randomised O
placebo O
controlled O
trial O
designed O
to O
investigate O
the O
efficacy O
of O
intra O
- O
articular O
hyaluronan O
( O
Synvisc O
( O
R O
) O
) O
to O
reduce O
pain O
and O
improve O
function O
in O
people B
with O
OA O
of O
the O
first O
MPJ O
( O
hallux O
limitus O
) O
. O

Two O
studies O
have O
previously O
investigated O
the O
efficacy O
of O
intra O
- O
articular O
hyaluronan O
for O
the O
treatment O
of O
first O
MPJ O
OA O
[ O
13 O
, O
14 O
] O
. O

However O
, O
neither O
of O
these O
studies O
used O
a O
placebo O
control O
group O
. O

To O
our O
knowledge O
, O
this O
is O
the O
first O
randomised O
controlled O
trial O
using O
intra O
- O
articular O
hyaluronan O
for O
OA O
of O
the O
first O
MPJ O
. O

The O
use O
of O
a O
placebo O
control O
group O
is O
essential O
for O
studies O
evaluating O
the O
effects O
of O
intra O
- O
articular O
therapies O
as O
there O
is O
likely O
to O
be O
a O
large O
placebo O
response O
related O
to O
the O
injection O
procedure O
and O
this O
may O
inflate O
the O
results O
in O
uncontrolled O
evaluations O
[ O
51 O
] O
. O

Indeed O
, O
a O
recent O
meta O
- O
analysis O
of O
hyaluronan O
for O
knee O
OA O
concluded O
that O
a O
placebo O
effect O
accounted O
for O
79 O
% O
of O
the O
efficacy O
of O
intra O
- O
articular O
hyaluronan O
[ O
16 O
] O
. O

The O
study O
protocol O
and O
outcome O
measures O
have O
been O
developed O
in O
accordance O
of O
the O
recommendations O
of O
the O
OARSI O
Clinical O
Trials O
Task O
Force O
guidelines O
[ O
18 O
] O
. O

The O
outcome O
measures O
are O
pain O
and O
function O
subscales O
of O
the O
FHSQ O
, O
pain O
and O
stiffness O
at O
the O
first O
MPJ O
, O
range O
of O
motion O
( O
dorsiflexion O
) O
of O
the O
first O
MPJ O
, O
plantar O
flexion O
strength O
of O
muscles O
of O
the O
first O
MPJ O
, O
generic O
health O
related O
quality O
of O
life O
( O
SF O
- O
36 O
) O
, O
patient B
satisfaction O
with O
treatment O
, O
consumption O
of O
rescue O
medication O
as O
well O
as O
frequency O
and O
nature O
of O
adverse O
effects O
. O

These O
outcomes O
will O
be O
measured O
at O
baseline O
then O
at O
1 O
, O
3 O
and O
6 O
months O
after O
treatment O
. O

Previous O
research O
suggests O
that O
the O
effects O
of O
intra O
- O
articular O
hyaluronan O
persist O
for O
up O
to O
12 O
months O
following O
treatment O
[ O
9 O
, O
38 O
] O
. O

Thus O
, O
the O
use O
of O
follow O
- O
up O
assessments O
at O
6 O
month O
post O
- O
treatment O
will O
allow O
us O
to O
determine O
if O
the O
effects O
, O
if O
any O
, O
of O
intra O
- O
articular O
hyaluronan O
persist O
in O
the O
longer O
term O
. O

Participants B
will O
be O
given O
the O
option O
of O
a O
second O
and O
final O
intra O
- O
articular O
injection O
( O
of O
Synvisc O
( O
R O
) O
or O
sterile O
saline O
according O
to O
the O
treatment O
group O
they O
are O
in O
) O
on O
days O
30 O
or O
90 O
if O
there O
is O
no O
improvement O
in O
their O
symptoms O
. O

Although O
this O
has O
the O
potential O
to O
complicate O
the O
interpretation O
of O
the O
results O
of O
the O
study O
, O
this O
protocol O
was O
included O
as O
it O
is O
likely O
to O
be O
more O
reflective O
of O
clinical O
practice O
[ O
14 O
] O
, O
and O
this O
is O
in O
keeping O
with O
the O
pragmatic O
nature O
of O
this O
trial O
. O

In O
summary O
, O
this O
project O
is O
the O
first O
randomised O
controlled O
trial O
to O
be O
conducted O
to O
evaluate O
the O
efficacy O
of O
intra O
- O
articular O
hyaluronan O
for O
reducing O
pain O
and O
improving O
function O
in O
people B
with O
hallux O
limitus O
. O

The O
study O
protocol O
, O
including O
interventions O
, O
have O
been O
pragmatically O
designed O
to O
ensure O
that O
the O
study O
findings O
are O
generaliseable O
to O
clinical O
practice O
. O

Recruitment O
for O
the O
study O
will O
commence O
in O
June O
2008 O
, O
and O
we O
expect O
final O
results O
to O
be O
available O
in O
mid O
- O
2010 O
. O

Competing O
interests O

HBM O
and O
KBL O
are O
Editor O
- O
in O
- O
Chief O
and O
Deputy O
Editor O
- O
in O
- O
Chief O
, O
respectively O
, O
of O
Journal O
of O
Foot O
and O
Ankle O
Research O
. O

It O
is O
journal O
policy O
that O
editors O
are O
removed O
from O
the O
peer O
review O
and O
editorial O
decision O
making O
processes O
for O
papers O
they O
have O
co O
- O
authored O
. O

Authors O
' O
contributions O

SEM O
, O
HBM O
, O
KBL O
and O
CJH O
conceived O
the O
idea O
and O
obtained O
funding O
for O
the O
study O
. O

SEM O
, O
HBM O
, O
KBL O
, O
AEZ O
and O
JDL O
designed O
the O
trial O
protocol O
. O

SEM O
, O
HBM O
, O
KBL O
and O
GVZ O
drafted O
the O
manuscript O
. O

All O
authors O
have O
read O
and O
approved O
the O
final O
manuscript O
. O

p8 O
inhibits O
the O
growth O
of O
human B
pancreatic O
cancer O
cells O
and O
its O
expression O
is O
induced O
through O
pathways O
involved O
in O
growth O
inhibition O
and O
repressed O
by O
factors O
promoting O
cell O
growth O

Abstract O

Background O

p8 O
is O
a O
stress O
- O
induced O
protein O
with O
multiple O
functions O
and O
biochemically O
related O
to O
the O
architectural O
factor O
HMG O
- O
I O
/ O
Y O
. O

We O
analyzed O
the O
expression O
and O
function O
of O
p8 O
in O
pancreatic O
cancer O
- O
derived O
cells O
. O

Methods O

Expression O
of O
p8 O
was O
silenced O
in O
the O
human B
pancreatic O
cancer O
cell O
lines O
Panc O
- O
1 O
and O
BxPc O
- O
3 O
by O
infection O
with O
a O
retrovirus O
expressing O
p8 O
RNA O
in O
the O
antisense O
orientation O
. O

Cell O
growth O
was O
measured O
in O
control O
and O
p8 O
- O
silenced O
cells O
. O

Influence O
on O
p8 O
expression O
of O
the O
induction O
of O
intracellular O
pathways O
promoting O
cellular O
growth O
or O
growth O
arrest O
was O
monitored O
. O

Results O

p8 O
- O
silenced O
cells O
grew O
more O
rapidly O
than O
control O
cells O
transfected O
with O
the O
empty O
retrovirus O
. O

Activation O
of O
the O
Ras O
- O
- O
> O
Raf O
- O
- O
> O
MEK O
- O
- O
> O
ERK O
and O
JNK O
intracellular O
pathways O
down O
- O
regulated O
p8 O
expression O
. O

In O
addition O
, O
the O
MEK1 O
/ O
2 O
inhibitor O
U0126 O
and O
the O
JNK O
inhibitor O
SP600125 O
up O
- O
regulates O
expression O
of O
p8 O
. O

Conversely O
, O
p38 O
or O
TGF O
beta O
- O
1 O
induced O
p8 O
expression O
whereas O
the O
specific O
p38 O
inhibitor O
SB203580 O
down O
- O
regulated O
p8 O
expression O
. O

Finally O
, O
TGF O
beta O
- O
1 O
induction O
was O
in O
part O
mediated O
through O
p38 O
. O

Conclusions O

p8 O
inhibits O
the O
growth O
of O
human B
pancreatic O
cancer O
cells O
. O

p8 O
expression O
is O
induced O
through O
pathways O
involved O
in O
growth O
inhibition O
and O
repressed O
by O
factors O
that O
promote O
cell O
growth O
. O

These O
results O
suggest O
that O
p8 O
belongs O
to O
a O
pathway O
regulating O
the O
growth O
of O
pancreatic O
cancer O
cells O
. O

Background O

While O
studying O
the O
molecular O
response O
of O
the O
injured O
pancreas O
, O
we O
identified O
a O
new O
gene O
, O
called O
p8 O
, O
whose O
expression O
is O
strongly O
induced O
during O
the O
acute O
phase O
of O
pancreatitis O
[ O
1 O
] O
. O

Further O
experiments O
have O
shown O
that O
p8 O
mRNA O
is O
activated O
in O
almost O
all O
cells O
in O
response O
to O
several O
stresses O
[ O
2 O
] O
, O
including O
minimal O
stresses O
such O
as O
after O
routine O
change O
of O
the O
culture O
medium O
in O
the O
absence O
of O
any O
added O
substance O
[ O
3 O
] O
, O
indicating O
that O
p8 O
is O
a O
ubiquitous O
protein O
induced O
by O
cellular O
stress O
. O

The O
p8 O
gene O
was O
cloned O
in O
human B
, O
rat B
, O
mouse B
, O
and O
Xenopus B
laevis I
[ O
1 O
, O
4 O
- O
6 O
] O
, O
conceptually O
translated O
from O
the O
Drosophila B
melanogaster I
genome O
or O
deduced O
from O
EST O
libraries O
( O
Bos B
taurus I
, O
Xenopus B
tropicalis I
, O
Zebrafish B
, O
Orzzias O
latipes I
, O
Bombyx B
mori I
and O
Paralichthys O
olivaceous I
) O
. O

The O
overall O
degree O
of O
homology O
with O
human B
p8 O
ranged O
from O
81 O
to O
40 O
% O
. O

Secondary O
structure O
prediction O
methods O
indicated O
that O
within O
the O
homologous O
region O
of O
the O
eleven O
proteins O
, O
there O
is O
a O
basic O
Helix O
- O
Loop O
- O
Helix O
secondary O
structure O
motif O
, O
characteristic O
of O
some O
classes O
of O
transcription O
factors O
[ O
1 O
] O
. O

Even O
though O
a O
small O
protein O
such O
as O
p8 O
would O
not O
need O
a O
nuclear O
localization O
signal O
( O
NLS O
) O
to O
be O
transported O
to O
the O
nucleus O
, O
a O
clear O
NLS O
can O
be O
predicted O
for O
the O
eleven O
proteins O
comprising O
a O
bipartite O
domain O
of O
positively O
charged O
aminoacids O
. O

In O
addition O
, O
a O
nuclear O
/ O
cytoplasmic O
location O
has O
been O
demonstrated O
for O
human B
p8 O
upon O
overexpression O
of O
the O
recombinant O
protein O
and O
immunohistochemistry O
[ O
4 O
] O
, O
and O
for O
recombinant O
Xenopus B
laevis I
p8 O
fused O
to O
green O
fluorescent O
protein O
[ O
6 O
] O
. O

Homology O
searching O
in O
databases O
did O
not O
reveal O
significant O
similarity O
of O
p8 O
with O
other O
proteins O
of O
known O
function O
. O

However O
, O
biochemical O
properties O
of O
the O
mammalian O
p8 O
proteins O
are O
shared O
by O
some O
high O
mobility O
group O
proteins O
( O
HMG O
) O
[ O
7 O
] O
, O
particularly O
by O
the O
HMG O
- O
I O
/ O
Y O
family O
. O

The O
overall O
identity O
of O
human B
p8 O
with O
human B
HMG O
- O
I O
/ O
Y O
is O
only O
about O
35 O
% O
, O
but O
the O
molecular O
mass O
, O
isoelectric O
point O
, O
hydrophilicity O
plot O
, O
the O
resistance O
to O
denaturation O
after O
heating O
at O
100 O
degrees O
C O
and O
the O
charge O
separation O
are O
very O
similar O
[ O
8 O
] O
. O

The O
p8 O
protein O
seems O
to O
bind O
DNA O
weakly O
, O
as O
shown O
by O
electrophoretic O
mobility O
shift O
assay O
, O
without O
preference O
for O
DNA O
sequences O
. O

Finally O
, O
human B
p8 O
has O
also O
been O
shown O
to O
be O
a O
substrate O
for O
protein O
kinase O
A O
in O
vitro O
and O
phosphorylated O
p8 O
has O
a O
higher O
content O
of O
secondary O
structure O
and O
binding O
to O
DNA O
is O
highly O
increased O
[ O
8 O
] O
. O

An O
architectural O
role O
in O
transcription O
has O
been O
proposed O
for O
this O
protein O
, O
in O
analogy O
with O
the O
HMG O
- O
I O
/ O
Y O
proteins O
, O
and O
a O
recent O
work O
seems O
to O
confirm O
this O
hypothesis O
[ O
9 O
] O
. O

Functions O
of O
p8 O
appear O
to O
be O
multiple O
and O
complex O
. O

For O
example O
, O
p8 O
mRNA O
expression O
was O
strongly O
induced O
in O
3T3 O
cells O
upon O
TGF O
beta O
- O
1 O
treatment O
which O
in O
turn O
enhances O
the O
Smad O
- O
transactivating O
function O
responsible O
for O
TGF O
beta O
- O
1 O
activity O
[ O
10 O
] O
. O

We O
also O
found O
that O
p8 O
is O
involved O
in O
cell O
cycle O
regulation O
since O
p8 O
- O
deficient O
embryonic O
fibroblasts O
grew O
more O
rapidly O
and O
incorporated O
more O
[ O
3H O
] O
thymidine O
and O
BrdU O
than O
p8 O
- O
expressing O
cells O
[ O
11 O
] O
. O

Moreover O
, O
expression O
of O
p8 O
in O
breast O
cancer O
- O
derived O
cells O
seems O
to O
mediate O
the O
inhibition O
of O
cell O
growth O
induced O
by O
1 O
, O
25 O
- O
Dihydroxyvitamin O
D3 O
[ O
12 O
] O
. O

On O
the O
contrary O
, O
we O
also O
reported O
that O
p8 O
may O
promote O
cell O
growth O
when O
overexpressed O
in O
Cos O
- O
7 O
, O
AR42J O
and O
HeLa O
cells O
[ O
1 O
, O
4 O
] O
. O

In O
addition O
, O
p8 O
seems O
to O
be O
involved O
in O
other O
intracellular O
functions O
such O
as O
apoptosis O
since O
p8 O
- O
expressing O
fibroblasts O
are O
more O
sensitive O
than O
p8 O
- O
deficient O
fibroblasts O
to O
the O
apoptosis O
induced O
by O
DNA O
damage O
. O

Also O
, O
p8 O
is O
required O
for O
endothelin O
- O
induced O
mesangial O
cell O
hypertrophy O
in O
diabetic O
kidney O
, O
in O
a O
mechanism O
involving O
ERK O
, O
JNK O
and O
PI3 O
kinase O
[ O
13 O
] O
. O

p8 O
seems O
to O
play O
a O
functional O
role O
in O
the O
initiation O
of O
LH O
beta O
gene O
expression O
during O
embryonic O
cell O
differentiation O
[ O
14 O
] O
. O

Moreover O
, O
the O
Drosophila B
melanogaster I
p8 O
homologue O
is O
involved O
in O
response O
to O
starvation O
and O
might O
be O
activated O
to O
stop O
cell O
growth O
in O
case O
of O
nutrient O
deprivation O
[ O
15 O
] O
. O

Finally O
, O
a O
particularly O
attractive O
role O
in O
tumour O
progression O
was O
recently O
proposed O
for O
p8 O
[ O
16 O
] O
. O

Fibroblasts O
obtained O
from O
p8 O
- O
expressing O
or O
p8 O
- O
deficient O
animals O
were O
transformed O
with O
a O
retroviral O
vector O
expressing O
both O
the O
rasV12 O
mutated O
protein O
and O
the O
E1A O
adenoviral O
oncogene O
. O

In O
soft O
- O
agar O
assays O
, O
transformed O
p8 O
- O
expressing O
cells O
formed O
colonies O
at O
high O
frequency O
, O
as O
expected O
, O
but O
p8 O
- O
deficient O
transformed O
fibroblasts O
were O
unable O
to O
form O
colonies O
. O

Similarly O
, O
transformed O
p8 O
- O
expressing O
cells O
produced O
tumours O
in O
all O
athymic B
nude I
mice I
when O
injected O
subcutaneously O
or O
intraperitoneally O
, O
whereas O
transformed O
p8 O
- O
deficient O
fibroblasts O
did O
not O
. O

On O
the O
other O
hand O
, O
studies O
by O
another O
laboratory O
revealed O
that O
expression O
of O
the O
Com1 O
protein O
[ O
17 O
] O
, O
which O
is O
identical O
to O
human B
p8 O
, O
mediates O
the O
growth O
of O
tumour O
cells O
after O
metastatic O
establishment O
in O
a O
secondary O
organ O
, O
indicating O
that O
activated O
expression O
of O
Com1 O
/ O
p8 O
in O
metastatic O
cells O
is O
required O
for O
tumour O
progression O
. O

These O
results O
strongly O
suggest O
that O
p8 O
is O
involved O
in O
the O
cellular O
pathway O
( O
s O
) O
required O
for O
tumour O
progression O
and O
metastasis O
. O

Our O
aim O
is O
to O
check O
the O
relevance O
of O
p8 O
to O
cancer O
progression O
in O
human B
. O

As O
a O
first O
step O
, O
we O
investigated O
in O
the O
present O
study O
the O
function O
of O
p8 O
in O
two O
cell O
lines O
derived O
from O
human B
pancreatic O
cancer O
. O

We O
observed O
that O
inhibition O
of O
p8 O
expression O
increased O
the O
cells O
growth O
rate O
. O

In O
addition O
, O
activations O
of O
the O
Ras O
- O
- O
> O
Raf O
- O
- O
> O
MEK O
- O
- O
> O
ERK O
and O
JNK O
intracellular O
pathways O
, O
which O
promote O
the O
growth O
of O
pancreatic O
cells O
, O
down O
- O
regulated O
p8 O
expression O
, O
whereas O
activation O
of O
p38 O
or O
TGF O
beta O
- O
1 O
, O
which O
inhibit O
cell O
growth O
, O
induced O
its O
expression O
. O

It O
was O
concluded O
that O
i O
/ O
p8 O
inhibits O
the O
growth O
of O
human B
pancreatic O
cancer O
cell O
lines O
, O
ii O
/ O
p8 O
expression O
is O
induced O
through O
pathways O
involved O
in O
growth O
inhibition O
and O
, O
conversely O
, O
repressed O
by O
factors O
that O
promote O
cell O
growth O
. O

Results O

p8 O
is O
silenced O
in O
pancreatic O
cancer O
cells O
by O
infection O
with O
a O
retrovirus O
expressing O
p8 O
RNA O
in O
the O
antisense O
orientation O

Panc O
- O
1 O
and O
BxPc O
- O
3 O
pancreatic O
cells O
were O
chosen O
for O
this O
study O
because O
, O
on O
the O
one O
hand O
, O
both O
cells O
express O
higher O
level O
of O
p8 O
( O
Figure O
1 O
) O
and O
, O
on O
the O
other O
hand O
, O
because O
Panc O
- O
1 O
is O
wild O
- O
type O
for O
Smad4 O
/ O
DPC4 O
and O
mutated O
for O
K O
- O
ras O
, O
while O
BxPc O
- O
3 O
is O
Smad4 O
/ O
DPC4 O
mutated O
and O
K O
- O
ras O
wild O
- O
type O
[ O
18 O
, O
19 O
] O
, O
therefore O
representing O
different O
mechanisms O
of O
transformation O
and O
different O
genetic O
backgrounds O
. O

K O
- O
ras O
and O
Smad4 O
/ O
DPC4 O
mutations O
are O
the O
major O
mechanisms O
involved O
in O
pancreatic O
cancer O
development O
. O

We O
inhibited O
p8 O
expression O
in O
both O
Panc O
- O
1 O
and O
BxPc O
- O
3 O
pancreatic O
cells O
by O
infecting O
cells O
with O
a O
retrovirus O
expressing O
the O
p8 O
asRNA O
( O
antisense O
RNA O
) O
and O
carrying O
the O
puromycin O
resistance O
. O

The O
antibiotic O
- O
selected O
cells O
were O
analyzed O
by O
Western O
blotting O
to O
evaluate O
the O
intracellular O
amounts O
of O
p8 O
protein O
. O

As O
shown O
in O
Figure O
2 O
, O
the O
p8 O
protein O
was O
clearly O
visible O
in O
both O
Panc O
- O
1 O
and O
BxPc O
- O
3 O
pancreatic O
cells O
infected O
with O
the O
empty O
retrovirus O
but O
almost O
undetectable O
in O
cells O
infected O
with O
the O
retrovirus O
encoding O
the O
p8 O
asRNA O
showed O
, O
indicating O
that O
our O
anti O
- O
sense O
strategy O
is O
efficient O
to O
silence O
p8 O
gene O
expression O
in O
pancreatic O
cancer O
cells O
. O

Preliminary O
studies O
had O
been O
conducted O
to O
select O
the O
best O
strategy O
to O
inhibit O
p8 O
expression O
. O

We O
compared O
the O
efficacy O
of O
the O
stable O
transfection O
of O
a O
siRNA O
, O
using O
a O
retroviral O
expression O
vector O
to O
the O
asRNA O
strategy O
described O
above O
. O

In O
our O
hands O
, O
the O
antisense O
strategy O
worked O
best O
, O
as O
judged O
from O
Western O
blot O
assessment O
of O
p8 O
protein O
expression O
( O
data O
not O
shown O
) O
. O

p8 O
- O
silenced O
pancreatic O
cells O
grow O
more O
rapidly O

We O
compared O
in O
the O
two O
cell O
lines O
the O
influence O
on O
growth O
parameters O
of O
blocking O
p8 O
expression O
with O
the O
p8 O
asRNA O
. O

Figure O
3 O
shows O
that O
both O
Panc O
- O
1 O
and O
BxPc O
- O
3 O
cells O
in O
which O
p8 O
has O
been O
silenced O
grew O
more O
rapidly O
than O
cells O
infected O
with O
the O
empty O
vector O
suggesting O
that O
inhibition O
by O
p8 O
of O
pancreatic O
cancer O
cell O
growth O
is O
independent O
from O
the O
mechanism O
of O
transformation O
and O
genetic O
background O
. O

Serum O
- O
stimulated O
cellular O
growth O
down O
- O
regulates O
p8 O
expression O

Fetal O
calf B
serum O
, O
which O
contains O
a O
complex O
mix O
of O
growth O
factors O
, O
can O
be O
used O
as O
inductor O
of O
cell O
growth O
. O

As O
shown O
in O
Figure O
4 O
expression O
of O
p8 O
mRNA O
was O
down O
- O
regulated O
in O
both O
Panc O
- O
1 O
and O
BxPc O
- O
3 O
when O
the O
cells O
were O
shifted O
from O
culture O
media O
containing O
0 O
. O
1 O
% O
fetal O
calf B
serum O
to O
media O
containing O
10 O
% O
FCS O
. O

p8 O
protein O
showed O
a O
similar O
behavior O
. O

These O
results O
show O
that O
p8 O
expression O
is O
down O
- O
regulated O
in O
growing O
pancreatic O
cells O
. O

The O
Ras O
- O
- O
> O
Raf O
- O
- O
> O
MEK O
- O
- O
> O
ERK O
pathway O
down O
- O
regulates O
p8 O
expression O
in O
pancreatic O
cancer O
cells O

Most O
human B
pancreatic O
cancers O
harbor O
mutations O
in O
the O
K O
- O
ras O
oncogene O
, O
which O
happens O
relatively O
early O
in O
pancreatic O
tumorigenesis O
[ O
20 O
] O
. O

The O
oncogenic O
mutation O
of O
the O
K O
- O
ras O
gene O
stabilizes O
the O
Ras O
protein O
in O
a O
GTP O
- O
bound O
form O
, O
which O
is O
constitutively O
active O
and O
make O
the O
cells O
grow O
more O
rapidly O
. O

Contrary O
to O
the O
activated O
Ras O
protein O
, O
p8 O
inhibits O
cell O
growth O
( O
Figure O
3 O
) O
. O

We O
looked O
whether O
the O
Ras O
- O
- O
> O
Raf O
- O
- O
> O
MEK O
- O
- O
> O
ERK O
pathway O
was O
also O
involved O
in O
the O
regulation O
of O
p8 O
expression O
, O
and O
which O
step O
( O
s O
) O
were O
critical O
. O

Figure O
5 O
shown O
that O
expression O
of O
a O
mutated O
form O
of O
the O
Ras O
protein O
( O
rasV12 O
) O
in O
BxPc O
- O
3 O
cells O
, O
which O
are O
wild O
- O
type O
for O
ras O
, O
resulted O
in O
decreased O
p8 O
mRNA O
concentration O
and O
protein O
level O
suggesting O
that O
the O
activated O
ras O
inhibits O
p8 O
expression O
. O

Figure O
6 O
shows O
that O
overexpression O
of O
Raf O
, O
but O
not O
of O
Raf301 O
( O
a O
negative O
mutant O
of O
Raf O
) O
, O
and O
of O
ERK O
also O
inhibited O
the O
expression O
of O
the O
p8 O
- O
CAT O
construct O
in O
Panc O
- O
1 O
as O
well O
as O
in O
BxPc O
- O
3 O
. O

Finally O
, O
the O
MEK1 O
/ O
2 O
specific O
inhibitor O
U0126 O
[ O
21 O
] O
activated O
p8 O
mRNA O
expression O
in O
pancreatic O
cells O
whether O
they O
carry O
mutated O
ras O
( O
Panc O
- O
1 O
) O
or O
wild O
- O
type O
( O
BxPc O
- O
3 O
) O
. O

Similar O
results O
were O
observed O
when O
expression O
of O
the O
p8 O
protein O
was O
monitored O
by O
Western O
blotting O
( O
Figure O
7 O
) O
. O

Activation O
of O
the O
JNK O
pathway O
down O
- O
regulates O
p8 O
expression O
in O
pancreatic O
cancer O
cells O

c O
- O
Jun O
NH2 O
- O
terminal O
kinase O
( O
JNK O
) O
is O
another O
major O
MAPK O
pathway O
which O
converts O
extracellular O
signals O
into O
expression O
of O
specific O
target O
genes O
through O
phosphorylation O
and O
activation O
of O
transcription O
factors O
. O

JNK O
activation O
has O
been O
implicated O
in O
various O
, O
often O
opposite O
cellular O
responses O
, O
such O
as O
cell O
proliferation O
, O
transformation O
and O
apoptosis O
. O

As O
shown O
in O
Figure O
8 O
, O
overexpression O
of O
JNK O
down O
- O
regulates O
the O
gene O
reporter O
activity O
of O
the O
p8 O
- O
CAT O
construct O
in O
Panc O
- O
1 O
cells O
. O

Similar O
results O
were O
found O
in O
BxPc O
- O
3 O
cells O
. O

Treatment O
of O
these O
cells O
with O
the O
JNK O
specific O
inhibitor O
SP600125 O
[ O
22 O
] O
up O
- O
regulates O
expression O
of O
the O
p8 O
mRNA O
and O
p8 O
protein O
( O
Figure O
9 O
) O
. O

These O
results O
show O
that O
the O
JNK O
pathway O
is O
involved O
in O
the O
regulation O
of O
p8 O
expression O
. O

The O
p38 O
pathway O
up O
- O
regulates O
p8 O
expression O
in O
pancreatic O
cancer O
cells O

The O
p38 O
signal O
transduction O
pathway O
also O
plays O
an O
essential O
role O
in O
regulating O
several O
cell O
functions O
including O
growth O
, O
response O
to O
inflammation O
, O
differentiation O
and O
apoptosis O
. O

In O
fact O
, O
in O
pancreatic O
cancer O
cells O
, O
p38 O
is O
a O
strong O
inhibitor O
of O
proliferation O
[ O
23 O
] O
contrary O
to O
the O
Ras O
- O
- O
> O
Raf O
- O
- O
> O
MEK O
- O
- O
> O
ERK O
and O
JNK O
pathways O
. O

We O
therefore O
analyzed O
the O
putative O
role O
of O
the O
p38 O
pathway O
in O
regulating O
p8 O
expression O
in O
pancreatic O
cancer O
cells O
. O

Figure O
10 O
shows O
that O
over O
- O
expression O
of O
the O
plasmid O
encoding O
p38 O
significantly O
increases O
p8 O
- O
CAT O
activity O
in O
Panc O
- O
1 O
as O
well O
as O
in O
BxPc O
- O
3 O
cells O
. O

Then O
, O
cells O
were O
treated O
with O
SB203580 O
, O
a O
specific O
inhibitor O
of O
p38 O
[ O
24 O
] O
, O
and O
p8 O
expression O
was O
measured O
. O

p8 O
mRNA O
as O
well O
as O
the O
encoded O
protein O
were O
down O
- O
regulated O
after O
inhibition O
of O
the O
p38 O
activity O
( O
Figure O
11 O
) O
. O

These O
results O
indicate O
that O
the O
p38 O
pathway O
is O
a O
positive O
regulator O
of O
p8 O
expression O
in O
pancreatic O
cancer O
cells O
. O

TGF O
beta O
- O
1 O
up O
- O
regulates O
p8 O
expression O
in O
pancreatic O
cancer O
cells O

The O
most O
prominent O
biological O
activity O
of O
TGF O
beta O
- O
1 O
is O
its O
potent O
inhibition O
of O
cell O
growth O
in O
a O
wide O
variety O
of O
cell O
types O
including O
pancreatic O
cells O
. O

TGF O
beta O
- O
1 O
signals O
are O
sent O
through O
two O
types O
of O
transmembrane O
serine O
/ O
threonine O
kinase O
receptors O
. O

In O
fact O
, O
TGF O
beta O
- O
1 O
binds O
and O
brings O
together O
the O
type O
I O
and O
type O
II O
receptors O
. O

In O
the O
resulting O
complex O
, O
the O
constitutively O
active O
TGF O
beta O
- O
1 O
type O
II O
receptor O
phosphorylates O
the O
type O
I O
receptor O
, O
which O
then O
plays O
a O
major O
role O
in O
transducing O
the O
signal O
to O
downstream O
components O
to O
affect O
gene O
expression O
through O
phosphorylation O
of O
SMAD O
proteins O
. O

Phosphorylated O
receptor O
- O
regulated O
SMADs O
then O
form O
heteromeric O
complexes O
with O
the O
common O
partner O
SMAD4 O
. O

These O
heteromeric O
complexes O
then O
move O
to O
the O
nucleus O
, O
where O
SMAD4 O
will O
bind O
DNA O
and O
contribute O
to O
transcriptional O
activation O
. O

In O
general O
, O
pancreatic O
cancer O
cells O
present O
with O
defects O
in O
TGF O
beta O
- O
1 O
signaling O
and O
are O
resistant O
to O
TGF O
beta O
- O
1 O
- O
mediated O
growth O
suppression O
. O

Since O
TGF O
beta O
- O
1 O
and O
p8 O
are O
inhibitors O
of O
pancreatic O
cell O
growth O
we O
analyzed O
whether O
p8 O
could O
mediate O
, O
at O
least O
in O
part O
, O
the O
effect O
of O
TGF O
beta O
- O
1 O
. O

First O
, O
we O
found O
that O
treatment O
of O
Panc O
- O
1 O
cells O
with O
TGF O
beta O
- O
1 O
increased O
p8 O
mRNA O
levels O
and O
p8 O
protein O
as O
judged O
by O
Western O
blot O
( O
Figure O
12 O
) O
. O

Then O
, O
to O
confirm O
that O
overexpression O
is O
regulated O
at O
the O
transcriptional O
level O
, O
we O
analyzed O
the O
effect O
of O
some O
constructs O
expressing O
constitutively O
activated O
type O
I O
TGF O
beta O
receptor O
, O
dominant O
negative O
type O
II O
TGF O
beta O
receptor O
, O
a O
dominant O
negative O
of O
Smad4 O
and O
the O
wild O
- O
type O
Smad4 O
on O
the O
p8 O
- O
CAT O
activity O
. O

As O
expected O
, O
the O
constitutively O
activated O
type O
I O
TGF O
beta O
receptor O
but O
not O
the O
dominant O
negative O
type O
II O
TGF O
beta O
receptor O
increased O
CAT O
activity O
. O

Also O
, O
expression O
of O
the O
Smad4 O
, O
contrary O
to O
that O
of O
the O
negative O
mutant O
, O
induced O
p8 O
transcription O
( O
Figure O
13 O
) O
. O

Together O
, O
these O
results O
indicate O
that O
p8 O
is O
positively O
regulated O
by O
TGF O
beta O
- O
1 O
. O

Beside O
the O
Smad O
proteins O
, O
TGF O
beta O
- O
1 O
also O
activates O
the O
p38 O
MAPK O
pathway O
in O
pancreas O
- O
derived O
cells O
, O
which O
may O
play O
an O
important O
role O
in O
TGF O
beta O
- O
1 O
induced O
genes O
[ O
25 O
] O
. O

Therefore O
, O
we O
analyzed O
the O
p38 O
- O
dependent O
effect O
of O
TGF O
beta O
- O
1 O
on O
p8 O
transcription O
. O

As O
shown O
in O
Figure O
13 O
, O
inhibition O
of O
p38 O
activity O
with O
the O
SB203580 O
specific O
inhibitor O
decreased O
about O
40 O
% O
the O
activity O
of O
TGF O
beta O
- O
1 O
on O
the O
p8 O
promoter O
indicating O
that O
the O
effect O
of O
TGF O
beta O
- O
1 O
on O
p8 O
promoter O
is O
mediated O
by O
both O
p38 O
- O
dependent O
and O
p38 O
- O
independent O
pathways O
. O

Discussion O

Pancreatic O
adenocarcinoma O
is O
the O
fourth O
leading O
cause O
of O
death O
from O
malignant O
diseases O
[ O
26 O
] O
. O

The O
aggressive O
nature O
of O
the O
neoplasia O
, O
the O
lack O
of O
early O
detection O
, O
and O
the O
limited O
response O
to O
treatments O
such O
as O
chemotherapy O
and O
radiotherapy O
contribute O
to O
the O
high O
mortality O
rate O
of O
the O
disease O
. O

Therefore O
, O
a O
better O
understanding O
of O
the O
molecular O
mechanism O
leading O
to O
pancreatic O
cancer O
remains O
a O
major O
goal O
because O
it O
may O
help O
proposing O
strategies O
for O
earlier O
diagnosis O
and O
better O
treatment O
. O

The O
most O
commonly O
altered O
genes O
in O
pancreatic O
adenocarcinoma O
are O
K O
- O
ras O
( O
75 O
to O
100 O
% O
) O
, O
p16INK4a O
( O
95 O
% O
) O
, O
p53 O
( O
50 O
to O
75 O
% O
) O
and O
DPC4 O
( O
50 O
% O
) O
[ O
27 O
- O
31 O
] O
. O

Whereas O
K O
- O
ras O
is O
a O
proto O
- O
oncogene O
all O
the O
others O
are O
tumour O
suppressor O
genes O
. O

Additional O
genes O
have O
been O
found O
altered O
at O
lower O
frequency O
. O

Panc O
- O
1 O
and O
BxPc O
- O
3 O
pancreatic O
cells O
were O
chosen O
for O
this O
study O
because O
they O
both O
express O
high O
levels O
of O
p8 O
( O
Figure O
1 O
) O
and O
because O
they O
present O
with O
different O
mechanisms O
of O
transformation O
and O
genetic O
backgrounds O
, O
Panc O
- O
1 O
being O
wild O
- O
type O
for O
Smad4 O
/ O
DPC4 O
but O
mutated O
for O
K O
- O
ras O
and O
BxPc O
- O
3 O
mutated O
for O
Smad4 O
/ O
DPC4 O
and O
wild O
- O
type O
for O
K O
- O
ras O
[ O
18 O
, O
19 O
] O
. O

This O
work O
presents O
evidences O
that O
p8 O
inhibits O
the O
growth O
rate O
of O
pancreatic O
cancer O
- O
derived O
cells O
and O
that O
the O
intracellular O
pathways O
promoting O
cell O
growth O
down O
- O
regulate O
p8 O
expression O
whereas O
those O
promoting O
growth O
arrest O
up O
- O
regulate O
its O
expression O
. O

Together O
, O
these O
results O
suggest O
that O
p8 O
is O
downstream O
of O
some O
cell O
growth O
regulators O
and O
therefore O
regulation O
of O
p8 O
expression O
or O
its O
activity O
could O
be O
used O
as O
a O
target O
for O
treating O
pancreatic O
cancer O
. O

Silencing O
p8 O
expression O
was O
able O
to O
strongly O
promote O
cell O
growth O
in O
both O
cell O
types O
, O
Panc O
- O
1 O
and O
BxPc O
- O
3 O
, O
suggesting O
that O
p8 O
may O
act O
downstream O
of O
the O
ras O
- O
or O
Smad4 O
/ O
DPC4 O
- O
dependent O
ways O
. O

Also O
, O
we O
found O
that O
stimulating O
cell O
growth O
by O
the O
complex O
combination O
of O
growth O
factors O
contained O
in O
fetal O
calf B
serum O
down O
- O
regulated O
expression O
of O
p8 O
whereas O
, O
on O
the O
contrary O
, O
treating O
the O
cells O
with O
TGF O
beta O
- O
1 O
, O
which O
promotes O
cell O
cycle O
arrest O
, O
stimulates O
p8 O
expression O
. O

Therefore O
, O
p8 O
gene O
expression O
seems O
to O
be O
regulated O
in O
opposite O
directions O
by O
mechanisms O
promoting O
cell O
growth O
or O
cell O
cycle O
arrest O
. O

It O
is O
interesting O
to O
note O
that O
while O
p8 O
expression O
is O
under O
the O
control O
of O
cell O
growth O
regulatory O
pathways O
such O
as O
Ras O
- O
- O
> O
Raf O
- O
- O
> O
MEK O
- O
- O
> O
ERK O
, O
JNK O
, O
p38 O
and O
TGF O
beta O
- O
1 O
, O
p8 O
can O
affect O
cell O
cycle O
progression O
, O
suggesting O
that O
p8 O
is O
a O
target O
for O
factors O
regulating O
pancreatic O
cell O
growth O
. O

A O
mechanism O
by O
which O
p8 O
could O
regulate O
cell O
cycle O
progression O
in O
embryonic O
fibroblasts O
was O
previously O
proposed O
[ O
11 O
] O
. O

In O
fact O
, O
p8 O
seems O
to O
take O
action O
upstream O
from O
cyclin O
- O
dependent O
kinases O
because O
the O
intracellular O
levels O
and O
activities O
of O
Cdk2 O
and O
Cdk4 O
are O
decreased O
when O
p8 O
is O
expressed O
. O

Concomitantly O
, O
the O
cyclin O
- O
dependent O
kinase O
inhibitor O
p27 O
is O
expressed O
at O
a O
low O
level O
in O
p8 O
- O
deficient O
cells O
which O
may O
explain O
the O
increased O
activity O
of O
Cdk2 O
and O
Cdk4 O
. O

The O
mechanism O
by O
which O
p8 O
regulates O
the O
intracellular O
level O
of O
those O
proteins O
remains O
to O
be O
determined O
. O

However O
, O
because O
p8 O
is O
a O
transcriptional O
cofactor O
, O
it O
is O
possible O
that O
regulation O
of O
expression O
of O
these O
molecules O
takes O
place O
, O
at O
least O
in O
part O
, O
at O
the O
transcription O
level O
. O

Interestingly O
, O
expression O
of O
p8 O
mRNA O
seems O
to O
be O
regulated O
in O
a O
cell O
type O
- O
and O
stimulus O
- O
specific O
manner O
since O
, O
for O
example O
, O
p38 O
can O
induce O
p8 O
expression O
in O
response O
to O
stress O
in O
fibroblasts O
[ O
3 O
] O
but O
not O
in O
renal O
mesangial O
cells O
treated O
with O
endothelin O
[ O
13 O
] O
. O

In O
pancreatic O
cancer O
- O
derived O
cells O
p38 O
seems O
to O
play O
a O
major O
role O
since O
it O
is O
involved O
in O
p8 O
activation O
as O
judged O
by O
transient O
transfection O
assays O
and O
using O
a O
specific O
p38 O
inhibitor O
( O
Figures O
10 O
and O
11 O
) O
. O

In O
addition O
, O
p38 O
is O
also O
involved O
in O
TGF O
beta O
- O
1 O
- O
induced O
p8 O
expression O
because O
about O
40 O
% O
of O
the O
TGF O
beta O
- O
1 O
effect O
was O
abolished O
when O
p38 O
activity O
was O
specifically O
blocked O
( O
Figure O
13 O
) O
. O

On O
the O
other O
hand O
, O
ERK O
and O
JNK O
are O
inducers O
of O
p8 O
expression O
in O
mesangial O
cells O
treated O
with O
endothelin O
, O
but O
not O
involved O
in O
the O
activation O
of O
p8 O
in O
response O
to O
stress O
in O
fibroblasts O
[ O
3 O
] O
, O
and O
even O
repressors O
in O
pancreatic O
cells O
( O
Figures O
5 O
, O
6 O
, O
7 O
, O
8 O
and O
9 O
) O
. O

Finally O
, O
PI3 O
kinase O
is O
an O
inducer O
of O
p8 O
expression O
in O
both O
endothelin O
- O
mediated O
p8 O
activation O
in O
mesangial O
cells O
[ O
13 O
] O
and O
pancreatic O
cells O
( O
data O
not O
shown O
) O
. O

Based O
on O
these O
observations O
, O
overexpression O
of O
p8 O
could O
be O
considered O
a O
possible O
goal O
for O
treating O
pancreatic O
tumours O
, O
in O
order O
to O
limit O
their O
growth O
. O

However O
, O
we O
previously O
reported O
that O
p8 O
repression O
would O
prevent O
rasV12 O
/ O
E1A O
transformed O
fibroblasts O
from O
evolving O
as O
tumours O
in O
nude B
mice I
[ O
16 O
] O
. O

This O
apparent O
contradiction O
needs O
to O
be O
resolved O
before O
considering O
p8 O
as O
a O
target O
for O
treating O
cancer O
progression O
. O

Conclusions O

In O
conclusion O
( O
see O
Figure O
14 O
) O
, O
we O
report O
in O
this O
paper O
that O
inhibition O
of O
p8 O
expression O
by O
an O
anti O
- O
sense O
strategy O
increases O
the O
growth O
rate O
of O
both O
Panc O
- O
1 O
and O
BxPc O
- O
3 O
pancreatic O
cancer O
- O
derived O
cells O
. O

Moreover O
, O
ERK O
- O
and O
JNK O
- O
mediated O
pathways O
down O
- O
regulate O
p8 O
expression O
, O
whereas O
p38 O
and O
TGF O
beta O
- O
1 O
pathways O
induce O
p8 O
expression O
. O

Also O
, O
cell O
growth O
triggered O
by O
expression O
of O
a O
RAS O
mutated O
protein O
or O
by O
10 O
% O
fetal O
calf B
serum O
induces O
down O
- O
regulation O
of O
p8 O
expression O
. O

Together O
, O
these O
results O
indicate O
that O
p8 O
is O
an O
intracellular O
cell O
growth O
inhibitor O
and O
that O
it O
is O
oppositely O
regulated O
by O
growth O
- O
promoting O
or O
growth O
- O
inhibiting O
factors O
in O
pancreatic O
cancer O
- O
derived O
cells O
. O

Material O
and O
Methods O

Cell O
lines O
and O
cell O
culture O
conditions O

The O
human B
pancreatic O
cancer O
cell O
lines O
Panc O
- O
1 O
and O
BxPc O
- O
3 O
were O
a O
kind O
gift O
of O
Dr O
C O
. O

Susini O
( O
INSERM O
U O
. O
531 O
, O
Toulouse O
) O
and O
A O
. O

Hajri O
( O
IRCAD O
, O
Strasbourg O
) O
respectively O
. O

Panc O
- O
1 O
cells O
were O
grown O
in O
Dulbecco O
' O
s O
modified O
Eagle O
' O
s O
medium O
( O
DMEM O
) O
supplemented O
with O
10 O
% O
fetal O
calf O
serum O
, O
2 O
mM O
L O
- O
glutamine O
, O
100 O
IU O
/ O
ml O
penicilin O
G O
and O
100 O
mu O
g O
/ O
ml O
streptomycin O
. O

BxPc O
- O
3 O
were O
cultivated O
in O
RPMI O
1640 O
medium O
in O
the O
presence O
of O
2 O
mM O
L O
- O
glutamine O
, O
4 O
. O
5 O
g O
/ O
L O
glucose O
, O
10 O
mM O
Hepes O
, O
1 O
. O
0 O
mM O
sodium O
pyruvate O
, O
10 O
% O
fetal O
calf B
serum O
and O
100 O
IU O
/ O
ml O
penicilin O
G O
and O
100 O
mu O
g O
/ O
ml O
streptomycin O
. O

Human B
recombinant O
TGF O
beta O
- O
1 O
was O
obtained O
from O
Sigma O
, O
and O
specific O
SB203580 O
, O
U0126 O
and O
SP600125 O
inhibitors O
were O
from O
Calbiochem O
and O
utilized O
at O
10 O
mu O
M O
. O

Expression O
plasmids O

Expression O
plamids O
encoding O
p38 O
( O
pCEFL O
HA O
p38 O
) O
, O
Erk2 O
( O
pcDNAIII O
HA O
ERK2 O
) O
, O
JNK O
( O
pcDNAIIIB O
HA O
JNK O
) O
, O
the O
wild O
- O
type O
Raf O
( O
pcDNA O
RAF O
BXB O
) O
and O
the O
Raf O
dominant O
negative O
( O
pcDNA O
RAF O
301 O
K375W O
) O
were O
obtained O
from O
O O
Coso O
( O
University O
of O
Buenos O
Aires O
) O
. O

Plamids O
encoding O
the O
constitutively O
activated O
type O
I O
TGF O
beta O
receptor O
( O
RI O
ACT O
) O
, O
the O
dominant O
negative O
type O
II O
TGF O
beta O
receptor O
( O
RII O
DN O
) O
and O
the O
Smad4 O
dominant O
negative O
( O
DPC4 O
1 O
- O
514 O
a O
. O
a O
. O
) O
were O
obtained O
from O
R O
Urrutia O
( O
Mayo O
Clinic O
, O
Rochester O
) O
and O
the O
wild O
type O
Smad4 O
was O
from O
C O
Heldin O
( O
Ludwig O
Institute O
, O
Uppsala O
) O
. O

Pancreatic O
p8 O
- O
deficient O
cells O

To O
silence O
p8 O
expression O
in O
pancreatic O
cells O
, O
we O
infected O
these O
cells O
with O
a O
retrovirus O
expressing O
human B
p8 O
in O
the O
antisense O
orientation O
. O

The O
retroviral O
vector O
was O
constructed O
as O
follows O
: O
human B
p8 O
cDNA O
was O
subcloned O
in O
HindIII O
and O
XhoI O
restriction O
sites O
of O
the O
pLPC O
plasmid O
( O
obtained O
from O
S B
. O
Lowe O
) O
in O
the O
antisense O
orientation O
. O

Amphotrope O
human B
p8 O
expressing O
retrovirus O
was O
then O
generated O
by O
transient O
transfection O
using O
Phoenix O
amphotrope O
packaging O
cells O
. O

Viral O
supernatant O
was O
used O
to O
infect O
Panc O
- O
1 O
and O
BxPc O
- O
3 O
pancreatic O
cells O
and O
the O
population O
of O
p8 O
- O
silenced O
cells O
was O
isolated O
by O
selection O
in O
presence O
of O
puromycin O
( O
1 O
mu O
g O
/ O
ml O
) O
. O

As O
control O
, O
cells O
were O
infected O
with O
the O
pLPC O
empty O
vector O
. O

p8 O
expression O
in O
arrested O
and O
growing O
cells O

One O
million O
of O
Panc O
- O
1 O
or O
BxPc O
- O
3 O
cells O
were O
cultivated O
on O
10 O
- O
cm O
Petri O
plates O
in O
standard O
conditions O
( O
with O
10 O
% O
FCS O
) O
. O

After O
48 O
h O
, O
culture O
media O
were O
changed O
for O
fresh O
media O
with O
FCS O
restricted O
to O
0 O
. O
1 O
% O
, O
in O
order O
to O
stop O
growth O
. O

After O
24 O
hours O
of O
growth O
arrest O
, O
culture O
medium O
was O
replaced O
either O
by O
medium O
with O
10 O
% O
fetal O
calf B
serum O
to O
resume O
cell O
growth O
or O
, O
as O
control O
, O
by O
medium O
with O
0 O
. O
1 O
% O
fetal O
calf B
serum O
. O

Twenty O
four O
hours O
later O
cells O
were O
recovered O
and O
RNA O
and O
protein O
extracted O
. O

p8 O
expression O
in O
TGF O
beta O
- O
1 O
- O
treated O
Panc O
- O
1 O
cells O

One O
million O
of O
Panc O
- O
1 O
cells O
were O
cultivated O
in O
10 O
- O
cm O
culture O
dishes O
for O
48 O
hours O
under O
standard O
conditions O
before O
TGF O
beta O
- O
1 O
treatment O
. O

Human B
recombinant O
TGF O
beta O
- O
1 O
( O
5 O
ng O
/ O
ml O
) O
was O
added O
to O
cells O
, O
without O
changing O
the O
culture O
medium O
, O
and O
cells O
were O
collected O
12 O
hours O
later O
for O
RNA O
and O
protein O
preparation O
. O

BxPc O
- O
3 O
rasV12 O
- O
expressing O
cells O

pLPC O
- O
rasV12 O
and O
pLPC O
plasmids O
were O
obtained O
from O
S B
. O

Lowe O
. O

Phoenix O
amphotrope O
packaging O
cells O
( O
106 O
) O
were O
plated O
in O
a O
6 O
- O
well O
plate O
, O
incubated O
for O
24 O
hours O
, O
then O
transfected O
with O
PEI O
with O
5 O
mu O
g O
of O
retroviral O
plasmid O
. O

After O
48 O
hours O
, O
the O
medium O
containing O
virus O
was O
filtered O
( O
0 O
. O
45 O
mu O
m O
filter O
, O
Millipore O
) O
to O
obtain O
the O
viral O
supernatant O
. O

Target O
BxPc O
- O
3 O
were O
plated O
at O
2 O
x O
105 O
cells O
per O
35 O
- O
mm O
dish O
and O
incubated O
overnight O
. O

For O
infections O
, O
the O
culture O
medium O
was O
replaced O
by O
an O
appropriate O
mix O
of O
the O
viral O
supernatant O
and O
culture O
medium O
( O
V O
/ O
V O
) O
, O
supplemented O
with O
4 O
mu O
g O
/ O
ml O
polybrene O
( O
Sigma O
) O
, O
and O
cells O
were O
incubated O
at O
37 O
degrees O
C O
. O

BxPc O
- O
3 O
rasV12 O
- O
expressing O
cells O
were O
selected O
with O
puromycin O
( O
1 O
mu O
g O
/ O
ml O
) O
. O

Cells O
infected O
with O
the O
pLPC O
empty O
vector O
were O
used O
as O
control O
. O

Western O
- O
blot O
analyses O

One O
hundred O
mu O
g O
of O
total O
protein O
extracted O
from O
cells O
was O
separated O
with O
standard O
procedures O
on O
15 O
. O
0 O
% O
SDS O
- O
PAGE O
using O
the O
Mini O
Protean O
System O
( O
Bio O
- O
Rad O
) O
and O
transferred O
to O
a O
nitrocellulose O
membrane O
( O
Sigma O
) O
. O

The O
intracellular O
level O
of O
p8 O
was O
estimated O
by O
Western O
blot O
using O
a O
polyclonal O
antibody O
( O
1 O
: O
1000 O
) O
raised O
against O
human B
p8 O
[ O
4 O
] O
. O

Growth O
curves O

One O
hundred O
thousand O
cells O
per O
well O
were O
plated O
in O
a O
series O
of O
35 O
- O
mm O
culture O
dishes O
. O

The O
cell O
number O
was O
estimated O
daily O
in O
triplicate O
, O
during O
1 O
to O
5 O
days O
, O
in O
a O
haemocytometer O
. O

Within O
experiments O
, O
each O
point O
was O
determined O
at O
least O
two O
times O
. O

Cell O
transfection O
and O
gene O
reporter O
assays O

Panc O
- O
1 O
and O
BxPc O
- O
3 O
( O
105 O
) O
were O
cultivated O
in O
30 O
mm O
diameter O
culture O
dishes O
for O
24 O
hours O
then O
transiently O
transfected O
with O
0 O
. O
5 O
mu O
g O
of O
p8 O
- O
CAT O
reporter O
plasmid O
and O
0 O
. O
5 O
mu O
g O
of O
pCMV O
/ O
beta O
gal O
plasmid O
( O
to O
control O
transfection O
efficiency O
) O
using O
the O
Fugene O
reagent O
in O
accordance O
with O
the O
manufacturer O
' O
s O
protocol O
( O
Roche O
Molecular O
Biochemicals O
) O
. O

The O
p8 O
- O
CAT O
plasmid O
is O
the O
previously O
reported O
p O
- O
1471 O
/ O
+ O
37p8 O
- O
CAT O
promoter O
construct O
[ O
5 O
] O
. O

Reporter O
activities O
were O
measured O
as O
previously O
described O
[ O
5 O
] O
. O

Briefly O
, O
cell O
extracts O
were O
prepared O
with O
the O
reporter O
lysis O
buffer O
( O
Promega O
) O
24 O
hours O
after O
transfection O
and O
CAT O
activity O
was O
determined O
by O
the O
phase O
extraction O
procedure O
[ O
32 O
] O
and O
beta O
- O
galactosidase O
assay O
was O
performed O
essentially O
as O
described O
in O
Sambrook O
et O
al O
. O

[ O
33 O
] O
. O

CAT O
activity O
was O
normalized O
to O
beta O
- O
galactosidase O
activity O
. O

Experiments O
were O
carried O
out O
in O
triplicate O
and O
repeated O
at O
least O
two O
times O
. O

Expression O
plasmids O
( O
0 O
. O
5 O
mu O
g O
) O
were O
co O
- O
transfected O
with O
p8 O
- O
CAT O
and O
pCMV O
/ O
beta O
gal O
plasmids O
as O
indicated O
. O

RT O
- O
PCR O
analysis O

RNA O
was O
extracted O
using O
the O
Trizol O
( O
Life O
Technologies O
) O
procedure O
. O

Total O
RNA O
( O
1 O
mu O
g O
) O
was O
analyzed O
by O
RT O
- O
PCR O
with O
the O
SuperScript O
( O
TM O
) O
One O
- O
step O
RT O
- O
PCR O
System O
and O
the O
Platinum O
Taq O
kit O
( O
Life O
Technologies O
) O
. O

RT O
- O
PCR O
was O
performed O
using O
different O
numbers O
of O
cycles O
to O
verify O
that O
the O
conditions O
chosen O
were O
within O
the O
linear O
range O
. O

The O
mRNA O
coding O
for O
p8 O
was O
specifically O
amplified O
with O
sense O
( O
5 O
' O
GAAGAGAGGCAGGGAAGACA O
3 O
' O
) O
and O
antisense O
( O
5 O
' O
CTGCCGTGCGTGTCTATTTA O
3 O
' O
) O
primers O
, O
in O
positions O
72 O
and O
643 O
of O
the O
cDNA O
( O
accession O
# O
NM O
_ O
012385 O
) O
, O
respectively O
. O

As O
control O
, O
the O
transcript O
coding O
for O
the O
ribosomal O
protein O
RL3 O
was O
specifically O
amplified O
for O
22 O
cycles O
with O
sense O
( O
5 O
' O
GAAAGAAGTCGTGGAGGCTG O
' O
) O
and O
antisense O
( O
5 O
' O
ATCTCATCCTGCCCAAACAC O
' O
) O
primers O
, O
in O
positions O
216 O
and O
637 O
of O
the O
cDNA O
, O
respectively O
. O

Author O
' O
s O
contributions O

CM O
prepared O
cells O
and O
retrovirus O
, O
carried O
out O
RNA O
purification O
, O
RT O
- O
PCR O
, O
Western O
blots O
, O
and O
cell O
growth O
experiments O
, O
NL O
carried O
out O
CAT O
assays O
, O
SV O
participated O
in O
the O
design O
of O
the O
study O
and O
analysis O
of O
data O
, O
JLI O
participated O
in O
the O
analysis O
of O
data O
and O
wrote O
the O
manuscript O
. O

All O
authors O
read O
and O
approved O
the O
final O
manuscript O
. O

Enzymatic O
multiplex O
DNA O
sequencing O
. O

Abstract O

The O
problem O
of O
reading O
DNA O
sequence O
films O
has O
been O
reformulated O
using O
an O
easily O
implemented O
, O
multiplex O
version O
of O
enzymatic O
DNA O
sequencing O
. O

By O
utilizing O
a O
uniquely O
tagged O
primer O
for O
each O
base O
- O
specific O
sequencing O
reaction O
, O
the O
four O
reactions O
can O
be O
pooled O
and O
electrophoresed O
in O
a O
single O
lane O
. O

This O
approach O
has O
been O
previously O
proposed O
for O
use O
with O
fluorescently O
labelled O
probes O
( O
1 O
) O
, O
and O
is O
analogous O
to O
the O
principle O
used O
in O
four O
- O
dye O
fluorescence O
sequencing O
except O
that O
the O
signals O
are O
resolved O
following O
electrophoresis O
( O
2 O
) O
. O

After O
transfer O
to O
a O
nylon O
membrane O
, O
images O
are O
obtained O
separately O
for O
each O
of O
the O
four O
reactions O
by O
hybridization O
using O
oligonucleotide O
probes O
. O

The O
images O
can O
then O
be O
superimposed O
to O
reconstitute O
a O
complete O
sequence O
pattern O
. O

In O
this O
way O
the O
correction O
of O
gel O
distortion O
effects O
and O
accurate O
band O
registration O
are O
considerably O
simplified O
, O
as O
each O
of O
the O
four O
base O
- O
specific O
ladders O
require O
very O
similar O
corrections O
. O

The O
methods O
therefore O
provide O
the O
basis O
for O
a O
second O
generation O
of O
more O
accurate O
and O
reliable O
film O
reading O
programs O
, O
as O
well O
as O
being O
useful O
for O
conventional O
multiplex O
sequencing O
. O

Unlike O
the O
original O
multiplex O
protocol O
( O
3 O
) O
, O
the O
approach O
described O
is O
suitable O
for O
small O
projects O
, O
as O
multiple O
cloning O
vectors O
are O
not O
used O
. O

Although O
more O
than O
one O
vector O
can O
be O
utilized O
, O
only O
a O
library O
of O
fragments O
cloned O
into O
any O
single O
phage O
, O
phagemid O
or O
plasmid O
vector O
is O
actually O
required O
, O
together O
with O
a O
set O
of O
tagged O
oligonucleotide O
primers O
. O
Images O

Nucleic O
Acids O
Research O
, O
Vol O
. O

19 O
, O
No O
. O
12 O
3301 O

Enzymatic O
multiplex O
DNA O
sequencing O

Mark O
Chee O

Medical O
Research O
Council O
Laboratory O
of O
Molecular O
Biology O
, O
Hills O
Road O
, O
Cambridge O
CB2 O
20H O
, O
UK O

Received O
March O
15 O
, O
1991 O
; O
Revised O
and O
Accepted O
May O
2 O
, O
1991 O

ABSTRACT O

The O
problem O
of O
reading O
DNA O
sequence O
films O
has O
been O
reformulated O
using O
an O
easily O
implemented O
, O
multiplex O
version O
of O
enzymatic O
DNA O
sequencing O
. O

By O
utilizing O
a O
uniquely O
tagged O
primer O
for O
each O
base O
- O
specific O
sequencing O
reaction O
, O
the O
four O
reactions O
can O
be O
pooled O
and O
electrophoresed O
in O
a O
single O
lane O
. O

This O
approach O
has O
been O
previously O
proposed O
for O
use O
with O
fluorescently O
labelled O
probes O
( O
1 O
) O
, O
and O
is O
analogous O
to O
the O
principle O
used O
in O
four O
- O
dye O
fluorescence O
sequencing O
except O
that O
the O
signals O
are O
resolved O
following O
electrophoresis O
( O
2 O
) O
. O

After O
transfer O
to O
a O
nylon O
membrane O
, O
images O
are O
obtained O
separately O
for O
each O
of O
the O
four O
reactions O
by O
hybridization O
using O
oligonucleotide O
probes O
. O

The O
images O
can O
then O
be O
superimposed O
to O
reconstitute O
a O
complete O
sequence O
pattern O
. O

In O
this O
way O
the O
correction O
of O
gel O
distortion O
effects O
and O
accurate O
band O
registration O
are O
considerably O
simplified O
, O
as O
each O
of O
the O
four O
basespecific O
ladders O
require O
very O
similar O
corrections O
. O

The O
methods O
therefore O
provide O
the O
basis O
for O
a O
second O
generation O
of O
more O
accurate O
and O
reliable O
film O
reading O
programs O
, O
as O
well O
as O
being O
useful O
for O
conventional O
multiplex O
sequencing O
. O

Unlike O
the O
original O
multiplex O
protocol O
( O
3 O
) O
, O
the O
approach O
described O
is O
suitable O
for O
small O
projects O
, O
as O
multiple O
cloning O
vectors O
are O
not O
used O
. O

Although O
more O
than O
one O
vector O
can O
be O
utilized O
, O
only O
a O
library O
of O
fragments O
cloned O
into O
any O
single O
phage O
. O
phagemid O
or O
plasmid O
vector O
is O
actually O
required O
, O
together O
with O
a O
set O
of O
tagged O
oligonucleotide O
primers O
. O

INTRODUCTION O

The O
community O
of O
biologists O
is O
undertaking O
the O
sequencing O
of O
representative O
genomes O
of O
various O
free O
- O
living O
organisms O
, O
ranging O
in O
size O
from O
Mycoplasma O
( O
800kb O
) O
to O
mammals O
( O
3 O
Gb O
) O
( O
4 O
) O
. O

However O
, O
the O
largest O
contiguous O
DNA O
sequences O
which O
have O
been O
determined O
so O
far O
are O
the O
genomes O
of O
several O
dsDNA O
eukaryotic O
viruses O
( O
5 O
, O
6 O
, O
7 O
, O
8 O
, O
9 O
) O
and O
plant O
chloroplasts O
( O
10 O
, O
11 O
, O
12 O
) O
. O

The O
largest O
of O
these O
is O
the O
229kb O
genome O
of O
human B
cytomegalovirus O
( O
8 O
) O
. O

The O
difficulty O
in O
sequencing O
millions O
of O
base O
pairs O
of O
DNA O
is O
that O
several O
steps O
in O
the O
methods O
are O
relatively O
labour O
intensive O
, O
although O
the O
sequencing O
reactions O
themselves O
are O
rapid O
and O
easily O
performed O
. O

Two O
limiting O
steps O
in O
conventional O
procedures O
are O
the O
size O
fractionation O
of O
sequencing O
reaction O
products O
by O
gel O
electrophoresis O
and O
the O
subsequent O
reading O
of O
sequence O
ladders O
. O

The O
former O
problem O
can O
be O
overcome O
by O
multiplexing O
, O
which O
theoretically O
allows O
an O
enormous O
amount O
of O

data O
to O
be O
obtained O
from O
a O
single O
gel O
by O
processing O
clones O
as O
mixtures O
rather O
than O
individually O
( O
3 O
) O
. O

Each O
sequence O
in O
the O
mixture O
is O
labelled O
by O
a O
unique O
short O
oligonucleotide O
' O
tag O
' O
sequence O
. O

This O
allows O
the O
mixture O
to O
be O
resolved O
following O
electrophoresis O
: O
the O
superimposed O
sequence O
ladders O
are O
blotted O
from O
the O
gel O
to O
a O
nylon O
membrane O
, O
and O
detected O
one O
at O
a O
time O
by O
hybridization O
using O
tag O
- O
specific O
oligonucleotide O
probes O
. O

In O
practice O
, O
at O
least O
50 O
sets O
of O
sequences O
can O
be O
obtained O
from O
a O
single O
gel O
( O
3 O
) O
. O

Unfortunately O
, O
a O
bottleneck O
in O
the O
multiplex O
procedure O
is O
the O
reading O
of O
sequence O
films O
. O

In O
previous O
large O
- O
scale O
sequencing O
projects O
this O
task O
has O
been O
performed O
with O
the O
aid O
of O
a O
sonic O
digitizer O
( O
13 O
, O
14 O
) O
. O

Although O
film O
reading O
programs O
have O
been O
under O
development O
for O
some O
time O
( O
15 O
) O
, O
and O
some O
programs O
are O
commercially O
available O
, O
their O
error O
rates O
are O
presently O
more O
variable O
and O
unpredictable O
than O
that O
of O
a O
skilled O
person B
and O
the O
accurate O
interpretation O
of O
film O
- O
imaged O
sequence O
ladders O
by O
computer O
programs O
is O
difficult O
to O
achieve O
in O
routine O
practice O
. O

Programs O
specifically O
designed O
to O
read O
multiplex O
films O
have O
an O
advantage O
. O

This O
is O
because O
a O
sequence O
image O
can O
be O
used O
as O
an O
' O
internal O
standard O
' O
to O
help O
interpret O
other O
images O
derived O
from O
the O
same O
membrane O
( O
3 O
) O
. O

However O
, O
the O
original O
implementation O
of O
the O
multiplex O
strategy O
used O
chemical O
sequencing O
( O
16 O
) O
, O
which O
yields O
a O
more O
complex O
sequence O
ladder O
than O
the O
enzymatic O
dideoxynucleotide O
chain O
- O
termination O
method O
( O
17 O
) O
. O

Most O
successful O
large O
scale O
sequencing O
projects O
have O
used O
the O
chaintermination O
method O
and O
bacteriophage O
M13 O
vectors O
, O
which O
allows O
the O
routine O
production O
of O
clean O
and O
easily O
interpretable O
sequences O
( O
18 O
) O
. O

It O
was O
therefore O
decided O
to O
adapt O
the O
original O
multiplex O
protocol O
for O
use O
with O
enzymatic O
sequencing O
, O
using O
tagged O
primers O
. O

MATERIALS O
AND O
METHODS O

Eight O
oligonucleotide O
sequencing O
primers O
were O
synthesized O
, O
each O
37 O
nucleotides O
in O
length O
. O

The O
3 O
' O
end O
of O
each O
primer O
consists O
of O
the O
17 O
nucleotide O
M13 O
universal O
priming O
sequence O
[ O
GTAAAACGACGGCCAGT3 O
' O
] O
. O

The O
5 O
' O
ends O
of O
the O
primers O
bear O
different O
20mer O
tag O
sequences O
( O
Figure O
1 O
) O
. O

In O
four O
of O
the O
primers O
, O
UEO1C O
, O
UPOIC O
, O
UE02C O
and O
UP02C O
, O
these O
tags O
are O
complementary O
to O
the O
EO1 O
, O
PO1 O
, O
E02 O
and O
P02 O
probe O
sequences O
respectively O
( O
copied O
from O
the O
original O
' O
plex O
' O
vectors O
( O
3 O
) O
) O
. O

A O
second O
set O
of O
four O
primers O
, O
UJOL14C O
, O
UJOL15C O
, O
UJOL16C O
and O
UJOL17C O
, O
have O
the O
following O
tag O
sequences O
: O
5 O
' O
CAAGTTTGAAGGTACTCATT O
, O
TATCAATTAAATTGTllTGA O
, O
GTGTTGCTACCCAAGAAGCA O
, O
and O
TGTCACTAGAGCTGTCACTT O
, O
respectively O
. O

The O

? O
= O
) O
1991 O
Oxford O
University O
Press O

3302 O
Nucleic O
Acids O
Research O
, O
Vol O
. O

19 O
, O
No O
. O
12 O

oligonucleotides O
were O
gel O
- O
purified O
( O
19 O
) O
and O
used O
to O
sequence O
ssDNA O
templates O
prepared O
by O
phenol O
extraction O
( O
20 O
) O
or O
SDS O
denaturation O
( O
21 O
) O
. O

Conventional O
sequencing O
reactions O
were O
performed O
as O
previously O
described O
( O
20 O
) O
. O

For O
hybridization O
experiments O
, O
radioactively O
labelled O
nucleotides O
were O
omitted O
from O
the O
sequencing O
reactions O
. O

Instead O
, O
the O
21d O
of O
each O
nucleotide O
mix O
added O
to O
the O
reaction O
mixture O
consisted O
of O
the O
following O
: O
' O
A O
' O
mix O
: O
6 O
. O
25MtM O
dATP O
, O
62 O
. O
5lM O
ddATP O
; O
' O
C O
' O
mix O
: O
6 O
. O
25MM O
dCTP O
, O
40MtM O
ddCTP O
; O
' O
G O
' O
mix O
: O
6 O
. O
25MtM O
dGTP O
, O
80MtM O
ddGTP O
; O
' O
T O
' O
mix O
: O
6 O
. O
25MM O
dTTP O
, O
250yM O
ddTTP O
; O
as O
well O
as O
125MM O
of O
each O
of O
the O
three O
other O

dNTPs O
in O
each O
mix O
. O

Apart O
from O
the O
use O
of O
these O
modified O
mixes O
, O
no O
changes O
were O
made O
to O
the O
conventional O
sequencing O
procedure O
( O
20 O
) O
. O

Sequencing O
reactions O
were O
pooled O
and O
ethanol O
precipitated O
as O
appropriate O
. O

Precipitation O
in O
microtitre O
trays O
was O
carried O
out O
as O
follows O
: O
a O
mixture O
of O
3 O
. O
2M1 O
3M O
NaAc O
pH O
5 O
. O
0 O
and O
112Mi1 O
EtOH O
was O
dispensed O
to O
individual O
wells O
of O
a O
microtitre O
plate O
( O
Falcon O
3911 O
or O
Corning O
25855 O
) O
using O
an O
8 O
- O
channel O
pipettor O
. O

Each O
set O
of O
four O
reactions O
was O
added O
to O
the O
EtOH O
/ O
NaAc O
mixture O
, O
and O
the O
tray O
sealed O
using O
a O
Falcon O
3073 O
plate O
sealer O
. O

The O
samples O
were O
mixed O
by O
inversion O
and O
stored O
at O
- O
20 O
? O
C O
for O
30 O
minutes O
. O

The O
DNA O
was O
collected O
by O
a O
20 O
minute O
centrifugation O
at O
4 O
000 O
rpm O
in O
an O
IEC O
Centra O
3C O
centrifuge O
. O

The O
sealer O
was O
removed O
, O
and O
the O
plate O
inverted O
to O
discard O
the O
supernatant O
. O

After O
blotting O
the O
tray O
on O
tissue O
paper O
, O
200MI O
of O
95 O
% O
EtOH O
was O
added O
to O
each O
well O
. O

The O
plate O
was O
covered O
with O
a O
plastic O
lid O
and O
recentrifuged O
for O
2 O
minutes O
. O

The O
EtOH O
was O
discarded O
and O
the O
plate O
inverted O
for O
several O
minutes O
on O
tissue O
paper O
, O
then O
left O
for O
20 O
minutes O
to O
air O
dry O
. O

Precipitated O
samples O
were O
resuspended O
in O
6M1 O
deionized O
water O
by O
vortexing O
on O
an O
SMI O
multi O
- O
tube O
vortexer O
for O
1 O
minute O
. O

Samples O
were O
denatured O
and O
electrophoresed O
on O
6 O
% O
polyacrylamide O
buffer O
gradient O
gels O
as O
previously O
described O
( O
20 O
) O
. O

Following O
electrophoresis O
, O
the O
gel O
was O
transferred O
to O
a O
dry O
piece O
of O
Whatman O
3MM O
blotting O
paper O
, O
and O
placed O
on O
a O
second O
sheet O
of O
blotting O
paper O
supported O
on O
a O
glass O
plate O
and O
saturated O
in O
4 O
x O
SSC O
( O
SSC O
: O
150mM O
NaCl O
, O
l5mM O
trisodium O
citrate O
) O
. O

This O
sheet O
was O
wicked O
in O
a O
tray O
containing O
1 O
litre O
of O
4 O
x O
SSC O
. O

The O
DNA O
was O
transferred O
to O
a O
nylon O
membrane O
( O
Amersham O
Hybond O
N O
) O
by O
capillary O
blotting O
overnight O
( O
22 O
) O
. O

DNA O
was O
fixed O
to O
the O
membranes O
by O
U O
. O
V O
. O
crosslinking O
( O
23 O
) O
. O

Plex O
oligonucleotide O
probes O
were O
a O
kind O
gift O
of O
Dr O
. O
George O
Church O
. O

Probes O
were O
tailed O
at O
their O
3 O
' O
ends O
using O
[ O
a O
- O
32p O
] O
dCTP O
as O
previously O
described O
( O
3 O
) O
. O

For O
the O
preparation O
of O
digoxigenin O
( O
DIG O
) O
labelled O
probes O
, O
identical O
tailing O
reactions O
were O
carried O
out O
substituting O
I0pmols O
of O
DIG O
- O
II O
dUTP O
( O
Boehringer O
Mannheim O
) O
for O
[ O
a O
- O
32P O
] O
dCTP O
. O

Membranes O
were O
prehybridized O
for O
at O
least O
10 O
minutes O
in O
4 O
x O
SSC O
, O
5 O
x O
Denhardts O
' O
( O
0 O
. O
1 O
% O
( O
w O
/ O
v O
) O
each O
of O
BSA O
( O
heated O
at O
80 O
? O
C O
for O
30 O
minutes O
to O
inactivate O
any O
alkaline O
phosphatase O
activity O
) O
, O
Ficoll O
( O
Pharmacia O
) O
and O
polyvinylpyrrolidone O
) O
, O
0 O
. O
5 O
% O
( O
w O
/ O
v O
) O
SDS O
, O
5mM O
NaHPO4 O
( O
23 O
) O
. O

Hybridization O
was O
carried O
out O
in O
25 O
- O
50M1 O
of O
prehybridization O
buffer O
per O
cm2 O
of O
membrane O
. O

The O
probe O
concentration O
was O
approximately O
lnM O
. O

After O
lh O
at O
42 O
? O
C O
, O
unbound O
probe O
was O
removed O
by O
five O
1 O
minute O
washes O
at O
room O
temperature O
in O
1 O
x O
SSC O
, O
0 O
. O
5 O
% O
SDS O
( O
200MI O
/ O
cm2 O
membrane O
) O
. O

Radioactive O
blots O
were O
covered O
in O
Saran O
wrap O
and O
exposed O
to O
film O
immediately O
. O

Detection O
of O
DIG O
labelled O
probes O
used O
an O
anti O
- O
DIG O
antibodyalkaline O
phosphatase O
conjugate O
( O
Boehringer O
Mannheim O
) O
according O
to O
the O
manufacturer O
' O
s O
instructions O
, O
except O
that O
all O
volumes O
were O
reduced O
by O
70 O
% O
and O
the O
conjugate O
was O
used O
at O
a O
1 O
: O
10 O
000 O
dilution O
. O

Blots O
were O
developed O
in O
25M1 O
of O
100mM O
Tris O
. O
Cl O
pH9 O
. O
5 O
, O

mantane4 O
- O
methoxy4 O
( O
3 O
" O
- O
phosphoryloxy O
) O
phenyl O
- O
1 O
, O
2 O
- O
dioxetane O
) O
; O
Tropix O
) O
/ O
cm2 O
for O
30 O
minutes O
at O
37 O
? O
C O
, O
prior O
to O
exposure O
to O
film O
. O

Probes O
and O
dioxetane O
were O
stripped O
from O
the O
membranes O
by O
two O
10 O
minute O
washes O
at O
700C O
with O
0 O
. O
2 O
% O
SDS O
, O
2mM O
EDTA O
( O
200 O
, O
ul O
/ O
cm2 O
membrane O
) O
. O

The O
hybridization O
and O
washing O
procedures O
were O
carried O
out O
in O
plastic O
bags O
. O

However O
, O
washing O
steps O
have O
also O
been O
performed O
with O
gentle O
agitation O
in O
a O
perspex O
tub O
( O
43 O
x O
27 O
x O
15cm O
) O
mounted O
on O
a O
reciprocal O
shaker O
, O
with O
equivalent O
results O
. O

In O
the O
latter O
case O
a O
minimum O
wash O
volume O
of O
500mls O
was O
used O
. O

The O
use O
of O
a O
tub O
is O
more O
convenient O
for O
batch O
processing O
and O
should O
be O
straightforward O
to O
automate O
. O

RESULTS O

Autoradiograms O
revealed O
no O
difference O
in O
sequence O
quality O
when O
tagged O
primers O
were O
used O
instead O
of O
the O
17mer O
universal O
primer O
in O
conventional O
[ O
a O
- O
35S O
] O
dATP O
labelled O
sequencing O
reactions O
and O
in O
multiplex O
hybridization O
experiments O
using O
[ O
a O
- O
32P O
] O
dCTP O
- O
tailed O
probes O
( O
results O
not O
shown O
) O
. O

Experiments O
were O
then O
conducted O
to O
determine O
the O
feasibility O
of O
pooling O
the O
four O
base O
reactions O
for O
each O
clone O
and O
fractionating O
them O
in O
a O
single O
lane O
to O
obtain O
a O
superimposed O
but O
interpretable O
set O
of O
sequence O
ladders O
. O

The O
question O
addressed O
was O
whether O
or O
not O
difficulties O
in O
band O
registration O
might O
arise O
as O
a O
result O
of O
mobility O
differences O
between O
the O
different O
primer O
sequences O
and O
/ O
or O
distortion O
of O
the O
membrane O
between O
probings O
. O

It O
is O
relevant O
that O
an O
automated O
film O
reader O
employing O
an O
internal O
standard O
requires O
that O
the O
nylon O
membrane O
does O
not O
undergo O
significant O
distortions O
between O
probings O
( O
George O
Church O
, O
personal O
communication O
) O
. O

Clones O
were O
sequenced O
using O
the O
four O
tagged O
primers O
UEOlC O
, O
UPOIC O
, O
UE02C O
, O
and O
UP02C O
, O
one O
for O
each O
base O
reaction O
( O
Figure O
1 O
) O
. O

The O
A O
, O
C O
, O
G O
and O
T O
reactions O
for O
each O
clone O
were O
pooled O
, O
and O
processed O
as O
described O
above O
. O

A O
complete O
set O
of O
sequence O
autoradiograms O
was O
obtained O
from O
four O
consecutive O
rounds O
of O
probing O
with O
[ O
a O
- O
32P O
] O
dCTP O
- O
labelled O
oligonucleotides O
. O

Alignment O
of O
the O
films O
showed O
that O
sequence O
- O
specific O
mobility O
effects O
and O
distortion O
of O
the O
membrane O
between O
probings O
were O
sufficiently O
minor O
to O
allow O
accurate O
registration O
of O
the O
bands O
, O
and O
hence O
accurate O
reading O
of O
the O
sequence O
. O

At O
least O
200 O
nucleotides O
of O
sequence O
could O
be O
read O
accurately O
from O
a O
single O
clone O
by O
simply O
tracing O
the O
four O
sets O
of O
bands O
using O
different O
colours O
, O
overlaying O
the O
tracings O
, O
and O
reading O
the O
bands O
sequentially O
. O

In O
order O
to O
assess O
the O
practicality O
of O
reading O
the O
sequences O
by O
machine O
, O
the O
images O
were O
scanned O
to O
provide O
optical O
density O
profiles O
( O
Figure O
2 O
) O
. O

These O
profiles O
were O
overlaid O
, O
and O
were O
found O
to O
be O
sufficiently O
in O
register O
to O
allow O
accurate O
interpretation O
of O
the O
sequence O
for O
at O
least O
300 O
nucleotides O
. O

This O
was O
essentially O
the O
limit O
of O
resolution O
of O
the O
gel O
for O
accurate O
manual O
reading O
. O

In O
order O
to O
ensure O
that O
the O
relatively O
minor O
mobility O
differences O
observed O
between O
the O
four O
primers O
were O
not O
coincidental O
to O
the O
oligonucleotides O
used O
, O
a O
second O
set O
of O
four O
tagged O
M13 O
universal O
primers O
was O
synthesised O
, O
this O
time O
incorporating O
20mer O
sequences O
derived O
from O
the O
genome O
of O
murine B
herpesvirus O
- O
68 O
( O
UJOL14C O
, O
15C O
, O
16C O
, O
17C O
) O
. O

Sequencing O
reactions O
were O
performed O
using O
[ O
cx O
- O
35S O
] O
dATP O
to O
label O
the O
DNA O
directly O
. O

Various O
templates O
were O
sequenced O
, O
and O
in O
all O
cases O
correctly O
ordered O
sequence O
ladders O
were O
obtained O
following O
conventional O
electrophoresis O
in O
which O
the O
four O
reactions O
were O
run O
side O
- O
by O
- O
side O
( O
results O
not O
shown O
) O
. O

Initial O
hybridization O
experiments O
were O
conducted O
using O
[ O
f O
- O
32p O
] O

dCTP O
tailed O
oligonucleotide O
probes O
. O

However O
, O
the O
use O
of O

lOOmM O
NaCl O
, O
5OmM O
MgC12 O
, O
0 O
. O
15mM O
AMPPD O
( O
[ O
3 O
- O
( O
2 O
' O
- O
ada O

B O

C O

' O
Ordinary O
' O
Plex O
' O
4 O
- O
CJ3 O
4 O
- O
rn O
+ O

Primeir O
Primer O

" O
All O
" O
C O
" O
" O
G O
" O
" O
T O
" O

' O
Plex O
' O
' O
Ordinary O
' O
Pool O
and O

Vector O
Vector O
fractionate O

Resolve O
by O
sequential O

hybridizations O

Sequencing O
reactions O

SequencingreactIo O
product O

Sequencing O
reaction O
product O

- O
n O
; O

= O
= O

- O
= O

- O
n O
: O
= O

Figure O
1 O
. O
Approaches O
to O
enzymatic O
multiplex O
DNA O
sequencing O
. O
a O
) O
A O
set O
of O
sequence O
- O
tagged O
vectors O
can O
be O
used O
. O

The O
tag O
site O
is O
shown O
in O
red O
, O
and O
the O
insert O
to O
be O
sequenced O
in O
blue O
. O

However O
, O
the O
original O
plex O
vectors O
( O
3 O
) O
are O
plasmids O
, O
and O
therefore O
amenable O
only O
to O
dsDNA O
sequencing O
. O
Sets O
of O
bacteriophage O
M13 O
vectors O
have O
been O
constructed O
bearing O
either O
one O
( O
32 O
) O
or O
two O
[ O
Chee O
, O
unpublished O
] O
of O
the O
plex O
tag O
sites O
flanking O
the O
polylinker O
, O
which O
can O
be O
used O
for O
this O
approach O
. O

b O
) O
The O
strategy O
used O
in O
this O
paper O
. O

In O
this O
case O
the O
tag O
site O
is O
carried O
on O
the O
primer O
. O

c O
) O
If O
tagged O
primers O
are O
used O
, O
there O
is O
no O
practical O
impediment O
to O
performing O
each O
base O
reaction O
using O
a O
different O
primer O
, O
as O
depicted O
. O

The O
reactions O
can O
then O
be O
pooled O
in O
any O
combination O
desired O
. O

The O
configuration O
shown O
, O
in O
which O
the O
four O
reactions O
are O
electrophoresed O
in O
a O
single O
lane O
, O
is O
designed O
to O
facilitate O
accurate O
band O
registration O
and O
reading O
by O
an O
automatic O
film O
reader O
. O

In O
order O
to O
read O
the O
sequence O
manually O
, O
base O
reactions O
would O
be O
run O
side O
- O
by O
- O
side O
. O

The O
logistics O
of O
processing O
the O
reactions O
are O
essentially O
the O
same O
with O
either O
configuration O
; O
the O
same O
number O
of O
probings O
are O
required O
. O

Figure O
2 O
. O

Four O
overlaid O
one O
- O
dimensional O
optical O
density O
profiles O
for O
a O
single O
clone O
shown O
in O
two O
overlapping O
sections O
. O

The O
optical O
density O
profiles O
are O
unprocessed O
, O
except O
for O
a O
simple O
transform O
to O
correct O
for O
the O
relative O
displacement O
( O
translation O
and O
rotation O
) O
of O
the O
four O
images O
from O
which O
they O
are O
extracted O
. O

The O
profiles O
read O
5 O
' O
to O
3 O
' O
from O
right O
to O
left O
. O

Nucleotides O
positions O
66 O
to O
214 O
from O
the O
start O
of O
the O
universal O
priming O
site O
are O
shown O
. O

The O
sequence O
is O
that O
of O
Bluescribe O
M13 O
+ O
( O
template O
DNA O
obtained O
by O
rescue O
with O
M13K07 O
helper O
phage O
( O
30 O
) O
) O
, O
and O
was O
determined O
using O
the O
primers O
UEOIC O
, O
UPOIC O
, O
UE02C O
, O
and O
UP02C O
for O
the O
T O
, O
C O
, O
G O
and O
A O

specific O
reactions O
respectively O
. O

Detection O
was O
by O
autoradiography O
following O
hybridization O
with O
[ O
a O
- O
32p O
] O
dCTP O
tailed O
plex O
probes O
. O

A O

Nucleic O
Acids O
Research O
, O
Vol O
. O

19 O
, O
No O
. O
12 O
3303 O

I O

3304 O
Nucleic O
Acids O
Research O
, O
Vol O
. O

19 O
, O
No O
. O
12 O

a O
) O

175 O
- O
. O
. O
. O

- O
w O

. O
m O
; O

b O
) O

182 O

a O
: O
. O
. O
. O
. O
. O
. O

F O
. O
VW O

an O

- O
w O

- O
1 O

S O
- O
K O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
~ O
i O

' O
F O
C O
C O
; O
A O
I1 O
' O
CG O
A O

Figure O
3 O
. O

Four O
separate O
base O
- O
specific O
reactions O
imaged O
from O
a O
single O
lane O
using O
chemiluminescent O
detection O
. O

The O
clones O
sequenced O
are O
: O
a O
) O
Bluescribe O
M13 O
+ O
( O
obtained O
by O
rescue O
with O
M13K07 O
helper O
phage O
( O
30 O
) O
) O
and O
b O
) O
an O
M13 O
recombinant O
clone O
prepared O
in O
a O
microtitre O
tray O
( O
21 O
) O
( O
a O
kind O
gift O
of O
Victoria O
Smith O
) O
. O

Nucleotide O
positions O
shown O
on O
the O
figure O
are O
numbered O
from O
the O
start O
of O
the O
universal O
priming O
site O
. O

The O
clones O
were O
sequenced O
using O
UEOlC O
, O
UPOIC O
, O
UE02C O
, O
and O
UP02C O
for O
the O
T O
, O
C O
, O
G O
and O
A O
specific O
reactions O
respectively O
. O

The O
blot O
was O
probed O
with O
corresponding O
DIG O
- O
11 O
- O
dUTP O
labelled O
oligonucleotides O
. O

radioactivity O
on O
the O
scale O
envisioned O
for O
a O
large O
sequencing O
project O
is O
undesirable O
for O
reasons O
of O
safety O
. O

The O
relatively O
long O
exposure O
times O
required O
( O
6 O
to O
24 O
hours O
) O
and O
the O
short O
half O
lives O
of O
the O
probes O
might O
also O
be O
inconvenient O
. O

It O
has O
been O
shown O
that O
a O
biotin O
/ O
streptavidin O
/ O
alkaline O
phosphatase O
based O
detection O
system O
used O
in O
conjunction O
with O
a O
chemiluminescent O
dioxetane O
substrate O
overcomes O
these O
disadvantages O
( O
24 O
, O
25 O
, O
26 O
) O
. O

We O
utilized O
a O
different O
bridging O
system O
with O
similar O
results O
. O

Digoxigenin O
( O
DIG O
) O
labelled O
oligonucleotide O
probes O
were O
detected O
using O
anti O
- O
DIG O
antibody O
- O
alkaline O
phosphatase O
conjugates O
and O
a O
chemiluminescent O
dioxetane O
substrate O
. O

Exposure O
times O
of O
10 O
to O
15 O
minutes O
were O
typically O
required O
, O
following O
a O
one O
hour O
preincubation O
period O
( O
Figure O
3 O
) O
. O

In O
our O
hands O
the O
DIG O
bridging O
system O
was O
similar O
in O
sensitivity O
to O
the O
streptavidin O
based O
system O
( O
24 O
) O
, O
and O
the O
practical O
lower O
limit O
of O
template O
ssDNA O
required O
in O
order O
to O
obtain O
an O
easily O
interpretable O
sequencing O
ladder O
was O
estimated O
to O
be O
in O
the O
range O
of O
20 O
to O
50fmols O
per O
reaction O
. O

However O
, O
the O
sensitivity O
of O
detection O
was O
limited O
only O
by O
enzymaticallytrigger O
background O
luminescence O
, O
and O
not O
by O
the O
level O
of O
signal O
obtained O
. O

The O
nonradioactive O
methods O
described O
have O
been O
used O
successfully O
in O
an O
8 O
- O
plex O
system O
. O

DISCUSSION O

Although O
the O
original O
multiplex O
protocol O
was O
based O
on O
a O
set O
of O
tagged O
vectors O
( O
3 O
) O
, O
tagged O
primers O
have O
also O
been O
used O
or O
proposed O
for O
various O
forms O
of O
multiplex O
DNA O
sequencing O
( O
George O
Church O
, O
personal O
communication O
; O
2 O
, O
27 O
) O
. O

For O
example O
, O
a O
proposal O
was O
recently O
put O
forward O
for O
multiplex O
sequencing O
using O
sequence O
- O
labelled O
primers O
and O
fluorophor O
- O
labelled O
probes O

( O
1 O
) O
, O
similar O
in O
principle O
to O
the O
methods O
used O
here O
. O

However O
, O
we O
use O
tagged O
primers O
and O
the O
superposition O
of O
the O
four O
sequencing O
reactions O
to O
address O
the O
problem O
of O
reading O
DNA O
sequence O
films O
; O
a O
part O
of O
this O
solution O
is O
to O
utilize O
M13 O
dideoxynucleotide O
sequencing O
, O
thereby O
improving O
the O
quality O
of O
the O
data O
to O
be O
analyzed O
. O

In O
addition O
, O
the O
proposal O
for O
fluorophor O
- O
labelled O
probes O
does O
not O
take O
into O
consideration O
any O
of O
the O
practical O
sequencing O
problems O
addressed O
here O
, O
and O
, O
in O
the O
version O
described O
, O
remains O
a O
promising O
but O
unproven O
scheme O
for O
large O
scale O
DNA O
sequencing O
. O

There O
are O
several O
advantages O
to O
tagging O
primers O
instead O
of O
vectors O
. O

Firstly O
, O
there O
is O
no O
need O
to O
prepare O
multiple O
libraries O
of O
clones O
in O
special O
vectors O
. O

This O
means O
that O
workers O
can O
use O
vector O
/ O
host O
combinations O
that O
yield O
good O
results O
in O
their O
hands O
, O
and O
an O
increased O
depth O
of O
multiplexing O
can O
easily O
be O
accomodated O
by O
synthesizing O
more O
primers O
. O

This O
should O
make O
multiplexing O
more O
accessible O
to O
workers O
undertaking O
smaller O
projects O
. O

A O
theoretical O
disadvantage O
of O
tagged O
primers O
is O
that O
the O
procedure O
can O
only O
be O
multiplexed O
following O
primer O
annealing O
( O
1 O
) O
, O
or O
following O
the O
sequencing O
reactions O
( O
this O
paper O
; O
in O
practice O
, O
pooling O
immediately O
after O
the O
annealing O
step O
might O
lead O
to O
increased O
backgrounds O
if O
one O
or O
more O
primers O
were O
present O
in O
excess O
over O
their O
template O
DNAs O
) O
. O

This O
is O
a O
relatively O
late O
stage O
. O

In O
the O
original O
procedure O
( O
3 O
) O
, O
clones O
were O
pooled O
prior O
to O
amplification O
by O
growth O
, O
an O
early O
step O
. O

However O
, O
we O
do O
not O
believe O
the O
sacrifice O
to O
be O
of O
practical O
importance O
when O
using O
phage O
vectors O
. O

In O
our O
experience O
, O
recombinant O
M13 O
phage O
have O
variable O
growth O
rates O
and O
the O
effects O
of O
competition O
are O
likely O
to O
severely O
limit O
the O
number O
of O
clones O
that O
can O
usefully O
be O
pooled O
for O
growth O
. O

In O
contrast O
, O
by O
growing O
clones O
individually O
, O
the O
depth O
of O
multiplexing O
is O
only O
really O
limited O
by O
probe O
sensitivity O
. O

We O
have O
not O
investigated O
the O
factors O
influencing O
variability O
in O
phage O
growth O
rates O
. O

It O
is O
worth O
noting O
that O
reliable O
protocols O
have O
been O
developed O
for O
growing O
large O
numbers O
of O
individual O
M13 O
clones O
and O
preparing O
high O
quality O
ssDNA O
templates O
in O
microtitre O
trays O
( O
28 O
, O
21 O
) O
. O

It O
is O
relatively O
simple O
to O
prepare O
manually O
two O
microtitre O
trays O
of O
ssDNA O
templates O
( O
192 O
clones O
) O
in O
a O
day O
. O

Sufficient O
clones O
can O
be O
prepared O
in O
a O
week O
to O
sequence O
a O
20kb O
fragment O
to O
a O
redundancy O
of O
10 O
( O
Victoria O
Smith O
, O
personal O
communication O
) O
. O

In O
this O
laboratory O
, O
ssDNA O
is O
now O
prepared O
with O
the O
aid O
of O
a O
commercially O
available O
robotic O
workstation O
( O
21 O
) O
. O

As O
sequencing O
reactions O
are O
also O
carried O
out O
in O
microtitre O
trays O
, O
manually O
or O
robotically O
( O
20 O
, O
29 O
) O
, O
the O
entire O
M13 O
- O
based O
dideoxynucleotide O
sequencing O
procedure O
is O
amenable O
to O
automation O
( O
29 O
) O
. O

For O
these O
reasons O
we O
see O
little O
practical O
advantage O
in O
pooling O
clones O
early O
. O

Finally O
, O
by O
not O
pooling O
clones O
early O
, O
the O
ability O
to O
easily O
retrieve O
individual O
clones O
is O
retained O
, O
which O
may O
facilitate O
directed O
sequencing O
later O
in O
a O
project O
should O
this O
become O
necessary O
. O

Multiplex O
DNA O
sequencing O
is O
currently O
limited O
by O
the O
lack O
of O
a O
robust O
computer O
program O
which O
can O
correct O
for O
the O
large O
variety O
of O
gel O
and O
sequencing O
artefacts O
that O
are O
normally O
encountered O
. O

The O
foundation O
of O
a O
film O
reading O
program O
is O
the O
ability O
to O
bring O
into O
register O
precisely O
vertical O
arrays O
of O
base O
- O
specific O
bands O
. O

This O
requires O
the O
ability O
to O
track O
lanes O
, O
correct O
for O
distortions O
, O
and O
order O
bands O
based O
on O
their O
relative O
spacing O
. O

A O
method O
of O
sequencing O
which O
has O
successfully O
overcome O
the O
problem O
of O
sequence O
reading O
uses O
real O
- O
time O
detection O
of O
fluorescently O
labelled O
DNA O
samples O
migrating O
through O
the O
gel O
( O
2 O
) O
. O

This O
system O
also O
utilizes O
the O
principle O
of O
running O
the O
four O
base O
reactions O
down O
the O
same O
lane O
( O
2 O
) O
. O

However O
, O
bands O
are O
detected O
at O
a O
fixed O
location O

in O
space O
, O
and O
their O
detection O
is O
separated O
in O
time O
. O

Hence O
the O

Nucleic O
Acids O
Research O
, O
Vol O
. O

19 O
, O
No O
. O
12 O
3305 O

problem O
of O
gel O
distortion O
is O
essentially O
avoided O
, O
although O
corrections O
for O
the O
different O
mobilities O
of O
the O
four O
dyes O
must O
be O
carried O
out O
. O

In O
contrast O
, O
we O
utilize O
the O
advantages O
of O
single O
lane O
electrophoresis O
to O
address O
the O
problem O
of O
superimposing O
four O
relatively O
large O
and O
complex O
two O
- O
dimensional O
images O
. O

Furthermore O
, O
by O
using O
sequence O
- O
tagged O
oligonucleotides O
which O
are O
detected O
by O
hybridization O
, O
a O
much O
greater O
depth O
of O
multiplexing O
can O
realistically O
be O
achieved O
than O
by O
real O
- O
time O
detection O
. O

The O
use O
of O
two O
- O
dimensional O
colour O
traces O
to O
depict O
the O
processed O
output O
of O
a O
film O
reader O
is O
consistent O
with O
the O
method O
of O
displaying O
fluorescence O
traces O
, O
and O
should O
facilitate O
the O
checking O
and O
editing O
of O
sequence O
databases O
in O
which O
both O
kinds O
of O
data O
have O
been O
entered O
. O

The O
sequence O
compilation O
programs O
used O
in O
this O
laboratory O
, O
which O
are O
already O
capable O
of O
handling O
large O
shotgun O
databases O
( O
8 O
, O
31 O
) O
, O
have O
recently O
undergone O
extensive O
improvements O
( O
Rodger O
Staden O
, O
personal O
communication O
) O
. O

There O
is O
now O
an O
interactive O
database O
editor O
which O
allows O
the O
graphical O
display O
of O
fluorescence O
traces O
, O
and O
it O
is O
envisaged O
that O
this O
feature O
could O
be O
extended O
to O
allow O
the O
handling O
of O
data O
from O
a O
film O
reader O
when O
a O
suitable O
machine O
is O
developed O
. O

ACKNOWLEDGEMENTS O

I O
am O
particularly O
grateful O
to O
George O
Church O
for O
thought O
- O
provoking O
discussions O
and O
gifts O
of O
vectors O
and O
oligonucleotide O
probes O
and O
to O
John O
Sulston O
for O
advice O
. O

I O
also O
thank O
Victoria O
Smith O
for O
the O
gift O
of O
DNA O
samples O
, O
Bart O
Barrell O
for O
long O
- O
term O
support O
, O
Tom O
O O
' O
Keefe O
of O
Milligen O
/ O
Biosearch O
for O
lessons O
in O
multiplexing O
and O
Amersham O
International O
for O
the O
optical O
density O
overlays O
shown O
in O
Figure O
2 O
. O

M O
. O
C O
. O

is O
supported O
by O
a O
fellowship O
from O
Applied O
Biosystems O
. O

12 O
. O

Hiratsuka O
, O
J O
. O
, O
Shimada O
, O
H O
. O
, O
Whittier O
, O
R O
. O
, O
Ishibashi O
, O
T O
. O
, O
Sakamoto O
, O
M O
. O
, O
Mori O
, O

M O
. O
, O
Kondo O
, O
C O
. O
, O
Honju O
, O
Y O
. O
, O
Sun O
, O
C O
. O

- O
R O
, O
Meng O
, O
B O
. O

- O
Y O
, O
Li O
, O
Y O
. O

- O
Q O
, O
Kanno O
, O
A O
. O
, O
Nisizawa O
, O
Y O
. O
, O
Hirai O
, O
A O
. O
, O
Shinozaki O
, O
K O
. O
and O
Sugiura O
, O
M O
. O

( O
1989 O
) O
Molecular O
and O
General O
Genetics O
, O
217 O
, O
185 O
- O
194 O
. O

13 O
. O

Komaromy O
, O
M O
. O
and O
Govan O
, O
H O
. O

( O
1984 O
) O
Nucleic O
Acids O
Research O
, O
12 O
, O
675 O
- O
678 O
. O

14 O
. O

Staden O
, O
R O
. O

( O
1984 O
) O
Nucleic O
Acids O
Research O
, O
12 O
, O
499 O
- O
503 O
. O

15 O
. O

Elder O
, O
J O
. O

K O
. O
, O
Green O
, O
D O
. O

K O
. O
and O
Southern O
, O
E O
. O

M O
. O

( O
1986 O
) O
Nucleic O
Acids O

Research O
, O
14 O
, O
417 O
- O
424 O
. O

16 O
. O

Maxam O
, O
A O
. O

M O
. O
and O
Gilbert O
, O
W O
. O

( O
1977 O
) O
Proceedings O
of O
the O
National O
Academy O

of O
Sciences O
, O
U O
. O
S O
. O
A O
. O
, O
74 O
, O
560 O
- O
564 O
. O

17 O
. O

Sanger O
, O
F O
. O
, O
Nicklen O
, O
S O
. O
and O
Coulson O
, O
A O
. O

R O
. O

( O
1977 O
) O
Proceedings O
of O
the O
National O

Academy O
of O
Sciences O
, O
U O
. O
S O
. O
A O
. O
, O
74 O
, O
5463 O
- O
5467 O
. O

18 O
. O

Sanger O
, O
F O
. O
, O
Coulson O
, O
A O
. O

R O
. O
, O
Barrell O
, O
B O
. O

G O
. O
, O
Smith O
, O
A O
. O

J O
. O

H O
. O
and O
Roe O
, O
B O
. O

A O
. O

( O
1980 O
) O
Journal O
of O
Molecular O
Biology O
, O
143 O
, O
161 O
- O
178 O
. O

19 O
. O

Applied O
Biosystems O
User O
Bulletin O
( O
1987 O
) O
13 O
, O
11 O
- O
16 O
. O

20 O
. O

Bankier O
, O
A O
. O

T O
. O
, O
Weston O
, O
K O
. O

M O
. O
and O
Barrell O
, O
B O
. O

G O
. O

( O
1987 O
) O
Methods O
in O

Enzymology O
, O
155 O
, O
51 O
- O
93 O
. O

21 O
. O

Smith O
, O
V O
. O
, O
Brown O
, O
C O
. O

M O
. O
, O
Bankier O
, O
A O
. O

T O
. O
and O
Barrell O
, O
B O
. O

G O
. O

( O
1990 O
) O
DNA O

Sequence O
, O
1 O
, O
73 O
- O
78 O
. O

22 O
. O

Southern O
, O
E O
. O

M O
. O

( O
1975 O
) O
Journal O
of O
Molecular O
Biology O
, O
98 O
, O
503 O
- O
517 O
. O

23 O
. O

Church O
, O
G O
. O

M O
. O
and O
Gilbert O
, O
W O
. O

( O
1984 O
) O
Proceedings O
of O
the O
National O
Academy O

of O
Sciences O
, O
U O
. O
S O
. O
A O
. O
, O
81 O
, O
1991 O
- O
1995 O
. O

24 O
. O

Beck O
, O
S O
. O
, O
O O
' O
Keefe O
, O
T O
. O
, O
Coull O
, O
J O
. O

M O
. O
and O
Koster O
, O
H O
. O

( O
1989 O
) O
Nucleic O
Acids O

Research O
, O
17 O
, O
5115 O
- O
5123 O
. O

25 O
. O

Tizard O
, O
R O
. O
, O
Cate O
, O
R O
. O

L O
. O
, O
Ramachandran O
, O
K O
. O

L O
. O
, O
Wysk O
, O
M O
. O
, O
Voyta O
, O
J O
. O

C O
. O
, O

Murphy O
, O
0 O
. O

J O
. O
and O
Bronstein O
, O
I O
. O

( O
1990 O
) O
Proceedings O
of O
the O
National O
Academy O
of O
Sciences O
, O
U O
. O
S O
. O
A O
. O
, O
87 O
, O
4514 O
- O
4518 O
. O

26 O
. O

Beck O
, O
S O
. O
and O
Koster O
, O
H O
. O

( O
1990 O
) O
Analytical O
Chemistry O
, O
62 O
, O
2558 O
- O
2570 O
. O

27 O
. O

Jacobson O
, O
K O
. O

B O
. O
, O
Arlinghaus O
, O
H O
. O

F O
. O
, O
Schmitt O
, O
H O
. O

W O
. O
, O
Sachleben O
, O
R O
. O

A O
. O
, O

Brown O
, O
G O
. O

M O
. O
, O
Thonnard O
, O
N O
. O
, O
Sloop O
, O
F O
. O

V O
. O
, O
Foote O
, O
R O
. O

S O
. O
, O
Larimer O
, O
F O
. O

W O
. O
, O
Woychik O
, O
R O
. O

P O
. O
, O
England O
, O
M O
. O

W O
. O
, O
Burchett O
, O
K O
. O

L O
. O
and O
Jacobson O
, O
D O
. O

A O
. O

( O
1991 O
) O
Genomics O
, O
9 O
, O
51 O
- O
59 O
. O

28 O
. O

Eperon O
, O
I O
. O

C O
. O

( O
1986 O
) O
Analytical O
Biochemistry O
, O
56 O
, O
406 O
- O
412 O
. O

29 O
. O

Bankier O
, O
A O
. O

T O
. O
and O
Barrell O
, O
B O
. O

G O
. O

( O
1989 O
) O
In O
Howe O
, O
C O
. O

J O
. O
and O
Ward O
, O
E O
. O

S B
. I

( O
ed O
) O
, O
Nucleic O
acids O
sequencing O
: O
a O
practical O
approach O
. O

IRL O
Press O
, O
Oxford O
, O
Vol O
. O

1 O
, O
pp O
. O

37 O
- O
78 O
. O

30 O
. O

Vieira O
, O
J O
. O
and O
Messing O
, O
J O
. O

( O
1987 O
) O
Methods O
in O
Enzymology O
, O
153 O
, O
3 O
- O
11 O
. O

31 O
. O

Davison O
, O
A O
. O

DNA O
Sequence O
, O
in O
press O
. O

32 O
. O

Heller O
, O
C O
. O
, O
Radley O
, O
E O
. O
, O
Khurshid O
, O
F O
. O

A O
. O
and O
Beck O
, O
S O
. O

Gene O
, O
in O
press O
. O

REFERENCES O

1 O
. O

Yang O
, O
M O
. O

M O
. O
and O
Youvan O
, O
D O
. O

C O
. O

( O
1989 O
) O
Biotechnology O
, O
7 O
, O
576 O
- O
580 O
. O

2 O
. O

Smith O
, O
L O
. O

M O
. O
, O
Sanders O
, O
J O
. O

Z O
. O
, O
Kaiser O
, O
R O
. O

J O
. O
, O
Hughes O
, O
P O
. O
, O
Dodd O
, O
C O
. O
, O
Connell O
, O

C O
. O

R O
. O
, O
Heiner O
, O
C O
. O
, O
Kent O
, O
S O
. O

B O
. O

H O
. O
and O
Hood O
, O
L O
. O

E O
. O

( O
1986 O
) O
Nature O
, O
321 O
, O
674 O
- O
679 O
. O

3 O
. O

Church O
, O
G O
. O

M O
. O
and O
Kieffer O
- O
Higgins O
, O
S O
. O

( O
1988 O
) O
Science O
, O
240 O
, O
185 O
- O
188 O
. O

4 O
. O

Watson O
, O
J O
. O

D O
. O

( O
1990 O
) O
Science O
, O
248 O
, O
44 O
- O
49 O
. O

5 O
. O

Baer O
, O
R O
. O
, O
Bankier O
, O
A O
. O

T O
. O
, O
Biggin O
, O
M O
. O

D O
. O
, O
Deininger O
, O
P O
. O

L O
. O
, O
Farrell O
, O
P O
. O

J O
. O
, O

Gibson O
, O
T O
. O

J O
. O
, O
Hatfull O
, O
G O
. O
, O
Hudson O
, O
G O
. O

S O
. O
, O
Satchwell O
, O
S O
. O

C O
. O
, O
Seguin O
, O
C O
. O
, O
Tuffnell O
, O
P O
. O

S O
. O
and O
Barrell O
, O
B O
. O

G O
. O

( O
1984 O
) O
Nature O
, O
310 O
, O
207 O
- O
211 O
. O

6 O
. O

Davison O
, O
A O
. O

J O
. O
and O
Scott O
, O
J O
. O

E O
. O

( O
1986 O
) O
Journal O
of O
General O
Virology O
, O
67 O
, O

1759 O
- O
1816 O
. O

7 O
. O

McGeoch O
, O
D O
. O

J O
. O
, O
Dalrymple O
, O
M O
. O

A O
. O
, O
Davison O
, O
A O
. O

J O
. O
, O
Dolan O
, O
A O
. O
, O
Frame O
, O

M O
. O

C O
. O
, O
McNab O
, O
D O
. O
, O
Perry O
, O
L O
. O

J O
. O
, O
Scott O
, O
J O
. O

E O
. O
and O
Taylor O
, O
P O
. O

( O
1988 O
) O
Journal O
of O
General O
Virology O
, O
69 O
, O
1531 O
- O
1574 O
. O

8 O
. O

Chee O
, O
M O
. O

S O
. O
, O
Bankier O
, O
A O
. O

T O
. O
, O
Beck O
, O
S O
. O
, O
Bohni O
, O
R O
. O
, O
Brown O
, O
C O
. O

M O
. O
, O
Cerny O
, O

R O
. O
, O
Horsnell O
, O
T O
. O
, O
Hutchison O
III O
, O
C O
. O

A O
. O
, O
Kouzarides O
, O
T O
. O
, O
Martignetti O
, O
J O
. O

A O
. O
, O
Satchwell O
, O
S O
. O

C O
. O
, O
Tomlinson O
, O
P O
. O
, O
Weston O
, O
K O
. O

M O
. O
and O
Barrell O
, O
B O
. O

G O
. O

( O
1990 O
) O
Current O
Topics O
in O
Microbiology O
and O
Immunology O
, O
154 O
, O
125 O
- O
169 O
. O

9 O
. O

Goebel O
, O
S O
. O

J O
. O
, O
Johnson O
, O
G O
. O

P O
. O
, O
Perkus O
, O
M O
. O

E O
. O
, O
Davis O
, O
S O
. O

W O
. O
, O
Winslow O
, O
J O
. O

P O
. O
and O
Paoletti O
, O
E O
. O

( O
1990 O
) O
Virology O
, O
179 O
, O
247 O
- O
266 O
. O

10 O
. O

Ohyama O
, O
K O
. O
, O
Fukuzawa O
, O
H O
. O
, O
Kohchi O
, O
T O
. O
, O
Shirai O
, O
H O
. O
, O
Sano O
, O
T O
. O
, O
Sano O
, O
S O
. O
, O

Umesono O
, O
K O
. O
, O
Shiki O
, O
Y O
. O
, O
Takeuchi O
, O
M O
. O
, O
Chang O
, O
Z O
. O
, O
Aota O
, O
S O
. O

- O
I O
, O
Inokuchi O
, O
H O
. O
and O
Ozeki O
, O
H O
. O

( O
1986 O
) O
Nature O
, O
322 O
, O
572 O
- O
574 O
. O

11 O
. O

Shinozaki O
, O
K O
. O
, O
Ohme O
, O
M O
. O
, O
Tanaka O
, O
M O
. O
, O
Wakasugi O
, O
T O
. O
, O
Hayashida O
, O
N O
. O
, O

Matsubayashi O
, O
T O
. O
, O
Zaita O
, O
N O
. O
, O
Chunwongse O
, O
J O
. O
, O
Obokata O
, O
J O
. O
, O
YamaguchiShinozaki O
, O
K O
. O
, O
Ohto O
, O
C O
. O
, O
Torazawa O
, O
K O
. O
, O
Meng O
, O
B O
. O

Y O
. O
, O
Sugita O
, O
M O
. O
, O
Deno O
, O
H O
. O
, O
Kamogashira O
, O
T O
. O
, O
Yamada O
, O
K O
. O
, O
Kusuda O
, O
J O
. O
, O
Takaiwa O
, O
F O
. O
, O
Kato O
, O
A O
. O
, O
Tohdoh O
, O
N O
. O
, O
Shimada O
, O
H O
. O
and O
Sugiura O
, O
M O
. O

( O
1986 O
) O
EMBO O
Journal O
, O
5 O
, O
2043 O
- O
2049 O
. O

Mapping O
irradiation O
hybrids O
to O
cosmid O
and O
yeast B
artificial O
chromosome O
libraries O
by O
direct O
hybridization O
of O
Alu O
- O
PCR O
products O
. O

Abstract O

A O
direct O
hybridization O
protocol O
is O
described O
for O
screening O
cosmid O
and O
yeast B
artificial O
chromosome O
libraries O
with O
pools O
of O
Alu O
- O
PCR O
products O
from O
somatic O
cell O
or O
irradiation O
hybrids O
. O

This O
method O
eliminates O
purification O
, O
cloning O
and O
analysis O
of O
each O
individual O
Alu O
- O
PCR O
product O
before O
library O
screening O
. O

A O
series O
of O
human B
X O
chromosome O
irradiation O
hybrids O
were O
mapped O
by O
this O
method O
, O
using O
a O
cosmid O
reference O
library O
for O
comparisons O
between O
overlapping O
hybrids O
to O
identify O
interesting O
clones O
for O
further O
analysis O
. O
Images O

Nucleic O
Acids O
Research O
, O
Vol O
. O

19 O
, O
No O
. O
12 O
3315 O

Mapping O
irradiation O
hybrids O
to O
cosmid O
and O
yeast B
artificial O
chromosome O
libraries O
by O
direct O
hybridization O
of O
Alu O
- O
PCR O
products O

Anthony O
P O
. O
Monaco O
* O
, O
Veronica O
M O
. O
S O
. O
Lam2 O
, O
Gunther O
Zehetner O
, O
Gregory O
G O
. O
Lennon O
, O
Christal O
Douglas O
, O
Dean O
Nizetic O
, O
Peter O
N O
. O
Goodfellow1 O
and O
Hans O
Lehrach O

Genome O
Analysis O
Laboratory O
and O
' O
Molecular O
Human B
Genetics O
Laboratory O
, O
Imperial O
Cancer O
Research O
Fund O
, O
44 O
Lincoln O
' O
s O
Inn O
Fields O
, O
London O
WC2A O
3PX O
, O
UK O
and O
2Department O
of O
Biochemistry O
, O
Li O
Shu O
Fan O
Building O
, O
Sassoon O
Road O
, O
University O
of O
Hong B
Kong O
, O
Hong B
Kong O

Received O
March O
12 O
, O
1991 O
; O
Revised O
and O
Accepted O
May O
16 O
, O
1991 O

ABSTRACT O

A O
direct O
hybridization O
protocol O
is O
described O
for O
screening O
cosmid O
and O
yeast B
artificial O
chromosome O
libraries O
with O
pools O
of O
Alu O
- O
PCR O
products O
from O
somatic O
cell O
or O
irradiation O
hybrids O
. O

This O
method O
eliminates O
purification O
, O
cloning O
and O
analysis O
of O
each O
individual O
AluPCR O
product O
before O
library O
screening O
. O

A O
series O
of O
human B
X O
chromosome O
irradiation O
hybrids O
were O
mapped O
by O
this O
method O
, O
using O
a O
cosmid O
reference O
library O
for O
comparisons O
between O
overlapping O
hybrids O
to O
identify O
interesting O
clones O
for O
further O
analysis O
. O

INTRODUCTION O

The O
generation O
of O
human B
DNA O
probes O
specific O
for O
individual O
chromosomes O
and O
subregions O
of O
chromosomes O
has O
been O
advanced O
with O
Alu O
- O
sequence O
primed O
polymerase O
chain O
reaction O
amplification O
( O
Alu O
- O
PCR O
, O
1 O
- O
3 O
) O
. O

This O
method O
specifically O
amplifies O
sequences O
between O
Alu O
repeats O
from O
human B
DNA O
in O
somatic O
cell O
hybrids O
and O
yeast B
artificial O
chromosomes O
( O
YACs O
, O
4 O
) O
. O

Individual O
Alu O
- O
PCR O
products O
can O
be O
purified O
from O
agarose O
gels O
or O
ligated O
into O
plasmid O
vectors O
to O
screen O
for O
single O
copy O
sequences O
. O

Unique O
Alu O
- O
PCR O
products O
are O
then O
localized O
to O
certain O
chromosome O
regions O
using O
DNA O
blots O
of O
somatic O
cell O
hybrid O
panels O
. O

Once O
localized O
, O
Alu O
- O
PCR O
fragments O
can O
be O
screened O
against O
genomic O
libraries O
to O
isolate O
longer O
DNA O
fragments O
from O
the O
region O
of O
interest O
. O

As O
an O
alternative O
to O
this O
multistep O
process O
we O
have O
developed O
a O
hybridization O
protocol O
for O
screening O
of O
cosmid O
and O
YAC O
libraries O
directly O
with O
pools O
of O
Alu O
- O
PCR O
products O
. O

Two O
new O
human B
specific O
Alu O
primers O
were O
used O
to O
generate O
DNA O
probes O
from O
a O
series O
of O
irradiation O
- O
reduced O
hybrids O
containing O
multiple O
human B
X O
chromosome O
fragments O
of O
1 O
- O
2000 O
kb O
on O
a O
hamster B
chromosome O
background O
( O
5 O
; O
P O
. O
N O
. O
G O
. O
, O
unpublished O
) O
. O

The O
Alu O
- O
PCR O
products O
were O
hybridized O
as O
a O
pool O
of O
probes O
to O
X O
- O
specific O
cosmid O
and O
YAC O
libraries O
, O
after O

competitive O
reassociation O
with O
an O
excess O
of O
human B
DNA O
to O
both O
the O
library O
filters O
and O
radioactively O
labelled O
Alu O
- O
PCR O
products O
. O

Comparisons O
were O
made O
between O
clones O
identified O
by O
overlapping O
irradiation O
hybrids O
and O
single O
copy O
DNA O
probes O
hybridized O
to O
the O
cosmid O
and O
YAC O
libraries O
. O

METHODS O

Two O
human B
Alu O
sequence O
primers O
were O
generated O
which O
were O
shown O
to O
be O
human B
specific O
; O
3144 O
from O
the O
3 O
' O
end O
of O
Alu O
: O
5 O
' O
- O
GAGCGAGACTCCGTCTCAAA O
- O
3 O
' O
and O
2729 O
from O
the O
5 O
' O
end O
of O
Alu O
: O
5 O
' O
- O
GTGGATCACCTGAGGTCAGG O
- O
3 O
' O
. O

All O
PCR O
reactions O
were O
carried O
out O
with O
100 O
ng O
of O
hybrid O
DNA O
and O
0 O
. O
7 O
, O
^ O
g O
of O
a O
single O
Alu O
primer O
in O
100 O
d41 O
of O
0 O
. O
01 O
M O
Tris O
- O
HCl O
pH O
8 O
. O
3 O
, O
0 O
. O
0015 O
M O
MgCl2 O
, O
0 O
. O
05 O
M O
KCI O
, O
200 O
AtM O
each O
of O
dNTPs O
, O
10 O
% O
dimethlysulfoxide O
, O
and O
2 O
. O
5 O
units O
of O
Cetus O
Taq O
polymerase O
. O

Reactions O
were O
30 O
cycles O
of O
94 O
? O
C O
for O
2 O
min O
, O
57 O
? O
C O
for O
2 O
min O
, O
and O
74 O
? O
C O
for O
4 O
min O
followed O
by O
a O
final O
extension O
time O
at O
74 O
? O
C O
for O
9 O
min O
. O

Reactions O
products O
were O
analyzed O
on O
1 O
% O
agarose O
gels O
and O
shown O
to O
contain O
between O
five O
and O
twenty O
fragments O
, O
with O
sizes O
ranging O
from O
0 O
. O
1 O
to O
2 O
. O
0 O
kb O
. O

Chinese O
hamster I
DNA O
and O
no O
DNA O
PCR O
reactions O
were O
done O
to O
control O
for O
non O
- O
human B
products O
( O
data O
not O
shown O
) O
. O

Alu O
- O
PCR O
products O
were O
separated O
from O
Alu O
oligomers O
over O
Qiagen O
columns O
, O
and O
approximately O
50 O
- O
100 O
ng O
were O
labelled O
by O
random O
hexamer O
priming O
( O
6 O
) O
. O

The O
radioactively O
labelled O
pool O
of O
fragments O
was O
prehybridized O
with O
37 O
. O
5 O
, O
^ O
g O
of O
total O
human B
DNA O
and O
12 O
. O
5 O
tsg O
of O
hamster B
DNA O
immobilized O
on O
a O
cellulose O
support O
matrix O
, O
prepared O
as O
previously O
described O
( O
7 O
) O
. O

Reactions O
were O
at O
65 O
? O
C O
in O
1 O
ml O
of O
0 O
. O
75M O
NaCl O
, O
0 O
. O
05M O
sodium O
phosphate O
pH O
7 O
. O
2 O
, O
0 O
. O
005M O
EDTA O
, O
0 O
. O
1 O
% O
sodium O
dodecyl O
sulphate O
( O
SDS O
) O
, O
0 O
. O
5 O
mg O
/ O
ml O
heparin O
, O
and O
100 O
jig O
/ O
ml O
denatured O
salmon B
sperm O
DNA O
. O

The O
cellulose O
was O
pelleted O
and O
the O
supernatant O
boiled O
for O
2 O
min O
every O
12 O
- O
16 O
hours O
( O
three O
times O
in O
two O
days O
) O
. O

Cosmid O
and O
YAC O
library O
filters O
( O
Hybond O
N O
+ O
, O
Amersham O
) O
were O
prehybridized O
at O
42 O
? O
C O
for O
16 O
hours O
with O
100 O
jig O
/ O
ml O

* O
To O
whom O
correspondence O
should O
be O
addressed O
at O
Human B
Genetics O
Laboratory O
, O
Imperial O
Cancer O
Research O
Fund O
, O
Institute O
of O
Molecular O
Medicine O
, O
John O
Radcliffe O
Hospital O
, O
Headington O
, O
Oxford O
OX3 O
9DU O
, O
UK O

1991 O
Oxford O
University O
Press O

3316 O
Nucleic O
Acids O
Research O
, O
Vol O
. O

19 O
, O
No O
. O
12 O

Chromosome O
X O

. O
, O
. O
. O
, O
. O
, O
< O

_ O
. O
, O
. O

< O
. O
, O

* O
2 O
. O
' O
1 O
- O
7 O
- O
_ O

_ O
- O
_ O
1 O
. O

J O

, O
. O
1 O
. O
. O

- O
1 O
, O
_ O
i O
- O
_ O
1 O
. O

1 O

1 O
' O
4 O
- O
- O
1 O
+ O

1 O
1 O
L O
L O
. O

i O

. O
1 O
. O

_ O
1 O

1 O
1 O
s O
. O

1 O
i O

1 O
i O
; O
. O

- O
- O
r O
' O
F O
1 O
- O

1 O
- O
1 O
- O
l O

j O
i O
- O
_ O
1 O
. O

1 O

- O
_ O
1 O
. O
. O

s B
- O
S B
. I

i O
n O
- O
. O

. O
. O

: O
, O
1 O
' O
, O
. O

- O
1 O
_ O
E O
I O
i O
? O

, O
. O

. O
_ O
_ O
_ O

G O
- O

. O

* O
. O
- O
. O
, O
. O
, O
z O

_ O

_ O
, O

z O
; O
- O
[ O
X1 O

21 O
, O
In O

27 O
38 O
45 O
48 O
54 O
74 O
86 O

I O
1 O
I O

I O

I O

107 O

MD O

I O

: O
I O
I O

I O
I O

I O
I O

Figur O
2a O
and O
2b O
: O
Hybridization O
of O
Alu O
- O
PCR O
products O
generated O
with O
Alu O
primer O
3144 O
from O
irradiation O
hybrid O
48 O
to O
duplicate O
copies O
of O
22 O
x22cm O
filters O
containing O
9216 O
human B
X O
chromosome O
cosmids O
( O
8 O
) O
. O

2c O
: O
Hybridization O
of O
Alu O
- O
PCR O
products O
generated O
with O
Alu O
primer O
3144 O
from O
an O
independent O
hybrid O
( O
54 O
) O
to O
a O
third O
identical O
cosmid O
filter O
. O

Figure O
1 O
: O
A O
schematic O
diagram O
of O
the O
human B
X O
chromosome O
alongside O
the O
approximate O
cytogenetic O
location O
of O
fragments O
identified O
in O
nine O
irradiation O
hybrids O
( O
numbers O
across O
the O
top O
) O
. O

The O
human B
X O
fragments O
were O
identified O
by O
hybridization O
of O
27 O
known O
DNA O
markers O
( O
indicated O
by O
a O
black O
line O
; O
P O
. O
N O
. O
G O
, O
unpublished O
) O
or O
by O
cosmids O
in O
common O
with O
unique O
X O
chromosome O
probes O
in O
the O
reference O
library O
database O
( O
open O
boxes O
and O
Table O
1 O
) O
. O

The O
size O
of O
the O
lines O
and O
boxes O
relate O
to O
the O
best O
cytogenetic O
location O
of O
the O
probes O
used O
according O
to O
Human B
Gene O
Mapping O
10 O
. O
5 O
( O
14 O
) O
and O
does O
not O
indicate O
the O
physical O
extent O
of O
the O
irradiation O
hybrid O
fragments O
. O

denatured O
and O
sheared O
total O
human B
DNA O
in O
50 O
% O
formamide O
, O
4XSSC O
, O
0 O
. O
05 O
M O
sodium O
phosphate O
pH O
7 O
. O
2 O
, O
0 O
. O
001 O
M O
EDTA O
, O
10 O
% O
dextran O
sulphate O
, O
1 O
. O
0 O
% O
SDS O
, O
50 O
isg O
/ O
ml O
denatured O
salmon B
sperm O
DNA O
and O
OxDenhardt O
' O
s O
solution O
. O

The O
radioactively O
labelled O
Alu O
- O
PCR O
products O
were O
denatured O
and O
added O
to O
fresh O

hybridization O
solution O
without O
human B
DNA O
competitor O
at O
1 O
x O
106 O

cpm O
/ O
ml O
and O
hybridized O
at O
42 O
? O
C O
for O
16 O
hours O
. O

Filters O
were O
washed O
in O
0 O
. O
1 O
xSSC O
and O
1 O
. O
0 O
% O
SDS O
, O
twice O
at O
room O
temperature O
and O
twice O
at O
65 O
? O
C O
for O
30 O
min O
each O
and O
exposed O
to O
Kodak O
X O
- O
OMAT O
film O
for O
2 O
- O
3 O
days O
at O
- O
70 O
? O
C O
with O
an O
intensifying O
screen O
. O

For O
each O
hybridization O
, O
two O
sets O
of O
duplicate O
cosmid O
filters O
were O
used O
from O
the O
ICRF O
reference O
library O
system O
( O
8 O
) O
, O
each O
containing O
9216 O
flow O
- O
sorted O
human B
X O
chromosome O
cosmid O
clones O
or O
approximately O
2 O
chromosome O
equivalents O
on O
a O
22 O
x O
22 O
cm O
filter O
( O
9 O
) O
. O

The O
coordinates O
of O
signals O
positive O
on O
duplicate O
cosmid O
filters O
were O
entered O
into O
the O
reference O
library O
database O
( O
G O
. O
Z O
, O
unpublished O
) O
using O
a O
digitizing O
tablet O
. O

For O
the O
X O
chromosome O
specific O
YAC O
library O
( O
A O
. O
P O
. O
M O
. O
and O
H O
. O
L O
. O
, O
unpublished O
) O
, O
about O
420 O
YAC O
colonies O
were O
spotted O
manually O
onto O
filters O
from O
96 O
well O
microtiter O
dishes O
using O
a O
96 O
prong O
device O
. O

After O
growth O
on O
selective O
media O
for O
3 O
days O
, O
YAC O
filters O
were O
processed O
for O
hybridization O
as O
previously O
described O
( O
10 O
) O
. O

RESULTS O

A O
panel O
of O
195 O
X O
chromosome O
irradiation O
hybrids O
was O
constructed O
( O
50 O
, O
000 O
rads O
) O
and O
characterized O
by O
DNA O
hybridization O
using O
27 O
X O
chromosome O
markers O
and O
flourescence O
in O
situ O
hybridization O
using O
total O
human B
DNA O
as O
probe O
( O
Benham O
et O
al O
. O
, O
1989 O
; O
P O
. O
N O
. O
G O
. O
, O
unpublished O
) O
. O

This O
analysis O
indicated O
that O
the O
hybrids O
contained O
multiple O
small O
fragments O
( O
4 O
- O
10 O
fragments O
of O
1000 O
- O
5000 O
kb O
each O
) O
with O
a O
preferential O
retaining O
of O
centromere O
sequences O
( O
90 O
% O
) O
. O

From O
this O
panel O
, O
nine O
irradiation O
hybrids O
were O
chosen O
that O
contained O
less O
than O
five O
different O
regions O
by O
DNA O
probe O
hybridizations O
, O
mostly O
from O
the O
short O
arm O
of O
the O
X O
chromosome O
( O
Fig O
1 O
) O
. O

All O
nine O
hybrids O
were O
used O
in O
PCR O
reactions O
with O
3 O
' O
- O
Alu O
primer O
3144 O
and O
two O
were O
used O
with O
5 O
' O
- O
Alu O

Figure O
3 O
: O
Hybridization O
of O
Alu O
- O
PCR O
products O
generated O
with O
Alu O
primer O
3144 O
from O
irradiation O
hybrid O
54 O
to O
a O
filter O
containing O
420 O
YAC O
clones O
from O
the O
human B
X O
chromosome O
. O

The O
positive O
YAC O
was O
also O
identified O
in O
a O
separate O
hybridization O
with O
the O
DMD O
probe O
P20 O
( O
12 O
) O
. O

primer O
2729 O
. O

Example O
hybridizations O
to O
a O
human B
X O
chromosome O
cosmid O
filter O
in O
Fig O
2 O
shows O
the O
intensity O
and O
reliability O
of O
positive O
clones O
identified O
on O
duplicate O
filters O
with O
Alu O
- O
PCR O
products O
from O
the O
same O
irradiation O
hybrid O
( O
48 O
) O
and O
the O
independence O
of O
clones O
identified O
with O
Alu O
- O
PCR O
products O
from O
a O
different O
hybrid O
( O
54 O
) O
. O

Fig O
3 O
shows O
a O
single O
positive O
YAC O
clone O
after O
hybridization O
of O
Alu O
- O
PCR O
products O
from O
irradiation O
hybrid O
54 O
to O
a O
filter O
containing O
about O
420 O
YAC O
clones O
specific O
for O
the O
human B
X O
chromosome O
. O

The O
total O
number O
of O
cosmids O
identified O
with O
each O
pool O
of O
AluPCR O
products O
for O
each O
hybrid O
is O
shown O
in O
Table O
1 O
. O

From O
the O
average O
number O
of O
cosmids O
identified O
( O
24 O
) O
in O
four O
chromosome O
equivalents O
screened O
and O
the O
estimated O
average O
DNA O
content O
in O
each O
hybrid O
( O
3000 O
- O
15000 O
kb O
) O
, O
the O
Alu O
- O
PCR O
products O
generated O
by O
a O
single O
primer O
were O
calculated O
on O
average O
to O
be O
300 O
- O
1500 O
kb O
apart O
, O
similar O
to O
published O
estimates O
for O
this O
method O
( O
1 O
, O
2 O
) O
. O

Only O
3 O
- O
4 O
cosmids O
were O
identified O
in O
common O
using O
Alu O
- O
PCR O
products O
generated O
with O
either O
3 O
' O
or O
5 O
' O
Alu O
primers O
( O
3144 O
or O
2729 O
) O
from O
two O
hybrids O
( O
38 O
and O
45 O
) O
. O

This O
shows O
that O
separate O
products O
were O
amplified O
with O
the O
two O
Alu O
primers O
since O
they O
prime O
synthesis O
from O
opposite O
ends O
of O
the O
Alu O
consensus O
sequence O
and O
Alu O
sequences O
are O
oriented O
in O
the O
genome O
either O
head O
to O
head O
, O

A O

B O

C O

El O

I0 O

Nucleic O
Acids O
Research O
, O
Vol O
. O

19 O
, O
No O
. O
12 O
3317 O

Table O
1 O
. O

Cosmids O
identified O
by O
hybrids O
and O
unique O
probes O

hybrids O
21 O
27 O
38 O
38 O
45 O
45 O
48 O
54 O
74 O
86 O
107 O
unique O

primer O
3144 O
3144 O
3144 O
2729 O
3144 O
2729 O
3144 O
3144 O
3144 O
33144 O
3144 O
probes O

21 O
3144 O
59 O
1 O
27 O
3144 O
7 O
14 O
2 O
38 O
3144 O
3 O
1 O
28 O
2 O
38 O
2729 O
0 O
0 O
4 O
25 O
0 O
45 O
3144 O
13 O
2 O
2 O
0 O
32 O
3 O
45 O
2729 O
1 O
0 O
0 O
0 O
3 O
31 O
0 O
48 O
3144 O
10 O
2 O
4 O
0 O
2 O
0 O
28 O
3 O
54 O
3144 O
11 O
3 O
0 O
0 O
5 O
0 O
5 O
30 O
2 O
74 O
3144 O
5 O
0 O
0 O
0 O
4 O
0 O
5 O
12 O
20 O
3 O
86 O
3144 O
3 O
1 O
0 O
0 O
4 O
1 O
3 O
4 O
3 O
17 O
0 O
107 O
3144 O
1 O
0 O
1 O
1 O
0 O
0 O
3 O
0 O
0 O
0 O
13 O
0 O

tail O
to O
tail O
or O
head O
to O
tail O
relative O
to O
each O
other O
. O

Therefore O
, O
by O
using O
the O
3 O
' O
- O
and O
5 O
' O
- O
Alu O
primers O
in O
separate O
PCR O
reactions O
with O
the O
same O
hybrid O
DNA O
, O
the O
total O
number O
of O
products O
and O
cosmid O
clones O
identified O
was O
almost O
doubled O
. O

Table O
1 O
also O
indicates O
how O
many O
cosmids O
were O
identified O
by O
Alu O
- O
PCR O
products O
from O
other O
hybrids O
, O
and O
16 O
cosmids O
previously O
identified O
with O
unique O
DNA O
probes O
in O
the O
reference O
library O
database O
. O

As O
can O
be O
seen O
in O
Fig O
1 O
and O
previous O
irradiation O
hybrid O
analysis O
, O
there O
is O
a O
preferential O
retention O
of O
centromere O
sequences O
( O
2 O
, O
11 O
) O
. O

However O
, O
there O
were O
no O
cosmids O
identified O
in O
common O
from O
all O
the O
hybrids O
positive O
with O
centromere O
sequences O
. O

This O
is O
probably O
due O
to O
a O
paucity O
of O
Alu O
repeats O
in O
the O
correct O
orientation O
in O
alphoid O
centromere O
sequences O
and O
thus O
few O
or O
no O
Alu O
- O
PCR O
products O
would O
be O
amplified O
from O
the O
centromere O
. O

Cosmids O
identified O
in O
common O
by O
several O
irradiation O
hybrids O
( O
Table O
1 O
) O
were O
most O
likely O
from O
regions O
of O
overlap O
outside O
the O
centromere O
area O
as O
shown O
by O
the O
previous O
DNA O
probe O
characterization O
( O
Fig O
1 O
) O
. O

The O
overlap O
regions O
between O
hybrids O
were O
also O
seen O
by O
16 O
cosmids O
( O
Table O
1 O
, O
Fig O
1 O
) O
that O
were O
hybridization O
targets O
of O
unique O
DNA O
markers O
in O
the O
reference O
library O
database O
that O
mapped O
in O
independent O
experiments O
to O
the O
overlap O
region O
. O

At O
least O
for O
several O
cosmids O
this O
showed O
that O
the O
Alu O
- O
PCR O
products O
identified O
target O
cosmids O
that O
were O
definitely O
from O
the O
expected O
region O
contained O
in O
the O
hybrids O
. O

For O
example O
, O
hybrids O
21 O
and O
54 O
were O
both O
shown O
to O
contain O
part O
of O
the O
Duchenne O
muscular O
dystrophy O
( O
DMD O
) O
locus O
( O
Fig O
1 O
; O
P O
. O
N O
. O
G O
. O
, O
unpublished O
) O
and O
had O
11 O
cosmids O
in O
common O
, O
including O
one O
identified O
by O
the O
probe O
P20 O
from O
the O
deletion O
hotspot O
region O
of O
the O
DMD O
gene O
( O
12 O
) O
. O

In O
Fig O
3 O
the O
hybridization O
of O
Alu O
- O
PCR O
products O
from O
hybrid O
54 O
identified O
a O
YAC O
clone O
which O
was O
also O
positive O
for O
the O
DMD O
probe O
P20 O
( O
data O
not O
shown O
) O
. O

This O
method O
also O
identified O
fragments O
in O
the O
hybrids O
that O
were O
not O
seen O
in O
the O
initial O
DNA O
characterization O
( O
Fig O
1 O
and O
Table O
1 O
) O
. O

Since O
the O
27 O
DNA O
probes O
were O
not O
close O
enough O
to O
each O
other O
along O
the O
chromosome O
to O
detect O
all O
possible O
hybrid O
fragments O
( O
1000 O
- O
5000 O
kb O
) O
, O
many O
regions O
would O
have O
been O
untested O
. O

For O
example O
, O
AluPCR O
products O
from O
hybrids O
45 O
and O
48 O
identified O
several O
cosmids O
, O
also O
seen O
by O
the O
cDNA O
for O
chronic O
granulomatous O
disease O
gene O
( O
CYBB O
, O
13 O
) O
in O
Xp2 O
1 O
. O
1 O
, O
although O
this O
region O
was O
not O
tested O
in O
the O
original O
hybrid O
characterization O
. O

DISCUSSION O

The O
direct O
hybridization O
of O
Alu O
- O
PCR O
products O
from O
somatic O
cell O
hybrids O
to O
genomic O
libraries O
can O
bypass O
gel O
purification O
or O
ligation O
of O
fragments O
into O
plasmid O
vectors O
and O
individual O
analysis O
for O
single O
copy O
sequences O
. O

Hybridization O
of O
Alu O
- O
PCR O
products O
as O

a O
pool O
to O
ordered O
array O
libraries O
such O
as O
the O
flow O
- O
sorted O
X O
chromosome O
cosmid O
library O
( O
9 O
) O
, O
allows O
the O
direct O
comparison O
of O
overlapping O
hybrids O
to O
pinpoint O
cosmids O
most O
likely O
to O
be O
from O
the O
region O
of O
interest O
. O

In O
conjunction O
with O
the O
reference O
library O
database O
( O
G O
. O
Z O
, O
unpublished O
) O
with O
183 O
X O
chromosome O
probe O
hybridization O
entries O
, O
cosmids O
identified O
with O
both O
Alu O
- O
PCR O
products O
and O
uniquely O
mapped O
X O
probes O
immediately O
map O
them O
to O
the O
region O
of O
interest O
and O
proove O
that O
the O
method O
has O
worked O
. O

Similar O
hybridization O
experiments O
using O
Alu O
- O
PCR O
products O
from O
four O
overlapping O
irradiation O
hybrids O
identified O
four O
cosmids O
in O
common O
that O
mapped O
to O
the O
region O
of O
overlap O
by O
independent O
experiments O
( O
F O
. O
Muscatelli O
, O
A O
. O
P O
. O
M O
. O
, O
P O
. O
N O
. O
G O
. O
, O
H O
. O
L O
. O
and O
M O
. O
Fontes O
, O
in O
preparation O
) O
. O

Since O
only O
27 O
probes O
from O
the O
X O
chromosome O
were O
used O
to O
initially O
characterize O
the O
hybrids O
and O
the O
length O
of O
individual O
human B
fragments O
in O
the O
irradiation O
hybrids O
is O
about O
1000 O
- O
5000 O
kb O
, O
many O
regions O
could O
have O
been O
undetected O
in O
the O
original O
analysis O
. O

The O
direct O
hybridization O
of O
Alu O
- O
PCR O
products O
to O
the O
cosmid O
reference O
library O
detected O
such O
fragments O
since O
they O
identified O
cosmids O
in O
common O
with O
uniquely O
mapped O
probes O
in O
regions O
not O
tested O
originally O
( O
Table O
1 O
and O
Fig O
1 O
) O
. O

This O
should O
prove O
to O
be O
a O
sensitive O
and O
efficient O
method O
to O
determine O
content O
and O
overlap O
of O
irradiation O
hybrids O
in O
conjunction O
with O
DNA O
blot O
hybridization O
. O

However O
, O
since O
the O
exact O
length O
of O
the O
human B
DNA O
fragments O
for O
each O
hybrid O
and O
the O
spacing O
of O
Alu O
- O
PCR O
products O
along O
the O
chromosome O
is O
not O
known O
, O
it O
is O
difficult O
to O
directly O
correlate O
the O
number O
of O
target O
cosmids O
to O
the O
DNA O
content O
of O
the O
hybrids O
. O

The O
direct O
hybridization O
of O
Alu O
- O
PCR O
products O
from O
irradiation O
or O
somatic O
cell O
hybrids O
to O
total O
genomic O
YAC O
libraries O
will O
be O
especially O
useful O
to O
construct O
long O
range O
YAC O
contigs O
from O
specific O
subregions O
of O
chromosomes O
. O

The O
dissection O
of O
a O
total O
genomic O
YAC O
library O
by O
this O
method O
may O
be O
more O
efficient O
than O
generating O
chromosome O
specific O
YAC O
libraries O
from O
somatic O
cell O
hybrids O
( O
usually O
a O
haploid O
human B
chromosome O
on O
a O
diploid O
or O
greater O
rodent B
background O
) O
or O
flow O
- O
sorted O
chromosomes O
, O
because O
of O
the O
low O
transformation O
efficiency O
of O
yeast B
. O

ACKNOWLEDGEMENTS O

We O
would O
like O
to O
thank O
Gert O
- O
Jan O
Van O
Ommen O
for O
the O
probe O
P20 O
and O
Stuart O
Orkin O
for O
the O
CYBB O
cDNA O
probe O
. O

A O
. O
P O
. O
M O
is O
supported O
in O
part O
by O
a O
research O
fellowship O
from O
the O
Muscular O
Dystrophy O
Association O
of O
America O
. O

V O
. O
M O
. O
S O
. O
L O
. O

is O
supported O
in O
part O
by O
the O
Medical O
Research O
Grant O
of O
the O
University O
of O
Hong O
Kong O
. O

Reference O
library O
filters O
and O
cosmids O
identified O
by O
Alu O
- O
PCR O
products O
can O
be O
requested O
from O
the O
Reference O
Library O
Database O
, O
ICRF O
, O
44 O
Lincoln O
' O
s O
Inn O
Fields O
, O
London O
WC2A O
3PX O
, O
U O
. O
K O
. O

3318 O
Nucleic O
Acids O
Research O
, O
Vol O
. O

19 O
, O
No O
. O
12 O

REFERENCES O

1 O
. O

Nelson O
, O
D O
. O
L O
. O
, O
Ledbetter O
, O
S O
. O
A O
. O
, O
Corbo O
, O
L O
. O
, O
Victoria O
, O
M O
. O
F O
. O
, O
Ramirez O
- O
Solis O
, O

R O
. O
, O
Webster O
, O
T O
. O
D O
. O
, O
Ledbetter O
, O
D O
. O
H O
. O
, O
and O
Caskey O
, O
C O
. O
T O
. O

( O
1989 O
) O
Proc O
. O

Natl O
. O

Acad O
. O

Sci O
. O

USA O
, O
86 O
: O
6686 O
- O
6690 O
. O

2 O
. O

Ledbetter O
, O
S O
. O
A O
. O
, O
Nelson O
, O
D O
. O
L O
. O
, O
Warren O
, O
S O
. O
T O
. O
, O
and O
Ledbetter O
, O
D O
. O
H O
. O

( O
1990 O
) O

Genomics O
, O
6 O
: O
475 O
- O
481 O
. O

3 O
. O

Cotter O
, O
F O
. O
E O
. O
, O
Hampton O
, O
G O
. O
M O
. O
, O
Nasipuri O
, O
S O
. O
, O
Bodmer O
, O
W O
. O
F O
. O
, O
and O
Young O

B O
. O
D O
. O

( O
1990 O
) O
Genomics O
, O
7 O
: O
257 O
- O
263 O
. O

4 O
. O

Burke O
, O
D O
. O
T O
. O
, O
Carle O
, O
G O
. O
F O
. O
, O
and O
Olson O
, O
M O
. O
V O
. O

( O
1987 O
) O
Science O
, O
236 O
: O
806 O
- O
812 O
. O

5 O
. O

Benham O
, O
F O
. O
, O
Hart O
, O
K O
. O
, O
Crolla O
, O
J O
. O
, O
Bobrow O
, O
M O
. O
, O
Francavilla O
, O
M O
. O
, O
and O

Goodfellow O
, O
P O
. O
N O
. O

( O
1989 O
) O
Genomics O
, O
4 O
: O
509 O
- O
517 O
. O

6 O
. O

Feinberg O
, O
A O
. O
P O
. O
, O
and O
Vogelstein O
, O
B O
. O

( O
1984 O
) O
Anal O
. O

Biochem O
. O
, O
137 O
: O
266 O
- O
267 O
. O

7 O
. O

Hochgeschwender O
, O
U O
. O
, O
Sutciffe O
, O
J O
. O
G O
. O
, O
and O
Brennan O
, O
M O
. O
B O
. O

( O
1989 O
) O
Proc O
. O

Natl O
. O

Acad O
. O

Sci O
. O

USA O
, O
86 O
: O
8482 O
- O
8486 O
. O

8 O
. O

Lehrach O
, O
H O
. O
, O
Drmanac O
, O
R O
. O
, O
Hoheisel O
, O
J O
. O
, O
Larin O
, O
Z O
. O
, O
Lennon O
, O
G O
. O
, O
Monaco O
, O

A O
. O
P O
. O
, O
Nizetic O
, O
D O
. O
, O
Zehetner O
, O
G O
. O
, O
and O
Poustka O
, O
A O
. O

( O
1990 O
) O
In O
Davies O
, O
K O
. O
E O
. O

& O
Tilghman O
, O
S O
. O
M O
. O

( O
eds O
. O
) O
, O
Genome O
Analysis O
Volume O
1 O
: O
Genetic O
and O
Physical O
Mapping O
. O

Cold O
Spring O
Harbor O
Laboratory O
Press O
, O
Cold O
Spring O
Harbor O
, O
pp O
. O

39 O
- O
81 O
. O

9 O
. O

Nizetic O
, O
D O
. O
, O
Zehetner O
, O
G O
. O
, O
Monaco O
, O
A O
. O
P O
. O
, O
Gellen O
, O
L O
. O
, O
Young O
, O
B O
. O
D O
. O
, O
and O

Lehrach O
, O
H O
. O

( O
1991 O
) O
Proc O
. O

Natl O
. O

Acad O
. O

Sci O
. O

USA O
, O
88 O
: O
3233 O
- O
3237 O
. O

10 O
. O

Larin O
, O
Z O
. O
and O
Lehrach O
, O
H O
. O

( O
1990 O
) O
Genet O
. O

Res O
. O

Camb O
. O
, O
56 O
: O
203 O
- O
208 O
. O

11 O
. O

Cox O
, O
D O
, O
R O
. O
, O
Pritchard O
, O
C O
. O
A O
. O
, O
Uglum O
, O
E O
. O
, O
Casher O
, O
D O
. O
, O
Koborl O
. O

J O
. O
and O
Myers O
, O

R O
. O
M O
. O

( O
1989 O
) O
Genomics O
, O
4 O
: O
397 O
- O
407 O
. O

12 O
. O

Wapenaar O
, O
M O
. O
C O
. O
, O
Kievits O
, O
T O
. O
, O
Hart O
, O
K O
. O
A O
. O
, O
Abbs O
S O
. O
, O
Blonden O
, O
L O
. O
A O
. O
J O
. O
, O

denDunnen O
, O
J O
. O
T O
. O
, O
Grootscholten O
, O
P O
. O
M O
. O
, O
Bakker O
, O
E O
. O
, O
Verellen O
- O
Dumoulin O
, O
C O
. O
, O
Bobrow O
, O
M O
. O
, O
vanOmmen O
, O
G O
. O
J O
. O
B O
. O
, O
and O
Pearson O
, O
P O
. O
L O
. O

( O
1988 O
) O
Genomics O
, O
2 O
: O
101 O
- O
108 O
. O

13 O
. O

Royer O
- O
Pokora O
, O
B O
. O
, O
Kunkel O
, O
L O
. O
M O
. O
, O
Monaco O
, O
A O
. O
P O
. O
, O
Goff O
, O
S O
. O
C O
. O
, O
Newburger O
, O

P O
. O
E O
. O
, O
Baehner O
, O
R O
. O
L O
. O
, O
Cole O
F O
. O
S O
. O
, O
Curnette O
J O
. O
T O
. O
, O
and O
Orkin O
, O
S O
. O
A O
. O

( O
1986 O
) O
Nature O
, O
322 O
: O
32 O
- O
38 O
. O

14 O
. O

Davies O
, O
K O
. O
E O
. O
, O
Mandel O
, O
J O
. O
L O
. O
, O
Monaco O
, O
A O
. O
P O
. O
, O
Nussbaum O
, O
R O
. O
L O
. O
and O
Willard O
, O

H O
. O
F O
. O

( O
1990 O
) O
Cytogenet O
. O

Cell O
Genet O
. O

55 O
: O
254 O
- O
313 O
. O

The O
Caenorhabditis B
elegans I
genome O
contains O
monomorphic O
minisatellites O
and O
simple O
sequences O
. O

Abstract O

Many O
species O
have O
been O
shown O
to O
contain O
tandemly O
repeated O
short O
sequence O
DNA O
known O
as O
minisatellites O
and O
simple O
sequence O
motifs O
. O

Due O
to O
allelic O
variation O
in O
the O
copy O
number O
of O
the O
repeat O
unit O
these O
loci O
are O
usually O
highly O
polymorphic O
. O

Here O
we O
demonstrate O
the O
presence O
of O
sequences O
in O
the O
genome O
of O
the O
nematode B
Caenorhabditis B
elegans I
which O
are O
homologous O
to O
two O
sets O
of O
short O
sequence O
DNA O
. O

However O
, O
when O
two O
independent O
strains O
were O
compared O
no O
polymorphism O
for O
these O
sequences O
could O
be O
detected O
. O
Images O

Volume O
17 O
Number O
23 O
1989 O
Nucleic O
Acids O
Research O

The O
Caenorhabds O
elegans I
genome O
contains O
monomorphic O
minisatellites O
and O
simple O
sequences O
Andrd O
G O
. O
Uitterlinden O
, O
P O
. O
Eline O
Slagboom O
, O
Thomas O
E O
. O
Johnsonl O
and O
Jan O
Vijg O

Department O
of O
Molecular O
Biology O
, O
TNO O
Institute O
for O
Experimental O
Gerontology O
, O
PO O
Box O
5815 O
, O
2280 O
HV O
Rijswijk O
, O
The O
Netherlands O
and O
lInstitute O
for O
Behavioral O
Genetics O
, O
University O
of O
Colorado O
, O
Boulder O
, O
CO O
80309 O
, O
USA O

Received O
October O
27 O
, O
1989 O
; O
Accepted O
November O
3 O
, O
1989 O

ABSTRACT O

Many O
species O
have O
been O
shown O
to O
contain O
tandemly O
repeated O
short O
sequence O
DNA O
kinown O
as O
minisatellites O
and O
simple O
sequence O
motifs O
. O

Due O
to O
allelic O
variation O
in O
the O
copy O
number O
of O
the O
repeat O
unit O
these O
loci O
are O
usually O
highly O
polymorphic O
. O

Here O
we O
demonstrate O
the O
presence O
of O
sequences O
in O
the O
genome O
of O
the O
nematode B
Caenorhabditis B
elegans I
which O
are O
homologous O
to O
two O
sets O
of O
short O
sequence O
DNA O
. O

However O
, O
when O
two O
independent O
strains O
were O
compared O
no O
polymorphism O
for O
these O
sequences O
could O
be O
detected O
. O

INTRODUCTION O

The O
genome O
of O
many O
species O
, O
including O
lower O
organisms O
, O
contain O
minisatellite O
sequences O
and O
so O
- O
called O
simple O
sequence O
motifs O
( O
1 O
, O
2 O
, O
3 O
) O
. O

Due O
to O
extensive O
variation O
in O
the O
number O
of O
repeat O
units O
, O
many O
of O
these O
loci O
have O
been O
shown O
useful O
as O
polymorphic O
markers O
, O
e O
. O
g O
. O
for O
genetic O
linkage O
studies O
and O
identification O
purposes O
. O

Here O
we O
demonstrate O
that O
the O
genome O
of O
the O
nematode B
Caenorhabditis B
elegans I
contains O
sequences O
that O
are O
homologous O
to O
the O
minisatellite O
core O
sequence O
33 O
. O
6 O
( O
1 O
) O
and O
to O
the O
simple O
sequence O
motif O
( O
AGC O
) O
n O
. O

Surprisingly O
, O
these O
sequences O
did O
not O
display O
polymorphism O
when O
two O
independent O
C B
. I
elegans I
strains O
, O
Bristol O
and O
Bergerac O
, O
were O
compared O
. O

MATERIALS O
AND O
METHODS O
Genomic O
DNA O
from O
nematodes B

Genomic O
DNA O
was O
isolated O
according O
to O
standard O
procedures O
from O
the O
two O
strains O
Bergerac O
( O
BO O
) O
and O
Bristol O
( O
strain O
N2 O
) O
. O

These O
strains O
are O
derived O
from O
two O
different O
individual O
worms O
isolated O
in O
France O
and O
England O
, O
respectively O
. O

Southern O
blotting O

Genomic O
DNA O
digests O
( O
5 O
Ag O
) O
were O
separated O
in O
a O
1 O
% O
agarose O
gel O
in O
1 O
x O
TAE O
( O
40 O
mM O
Tris O
. O
HCl O
, O
pH O
7 O
. O
4 O
/ O
20 O
mM O
sodium O
acetate O
/ O
I O
mM O
NaEDTA O
) O
by O
electrophoresis O
at O
75 O
V O
for O
16 O
h O
. O

Separation O
patterns O
were O
transferred O
to O
Zetaprobe O
membrane O
( O
BIORAD O
) O
in O
0 O
. O
4 O
N O
NaOH O
, O
0 O
. O
6 O
M O
NaCl O
in O
a O
vacu O
- O
blot O
apparatus O
( O
LKB O
) O
according O
to O
the O
manufacturer O
' O
s O
instructions O
. O

The O
minisatellite O
and O
simple O
sequence O
probes O
used O
were O
prepared O
essentially O
as O
described O
( O
5 O
) O
. O

The O
Tcl O
probe O
is O
a O
plasmid O
containing O
an O
insert O
of O
the O
transposable O
element O
Tcl O
( O
4 O
) O
. O

All O
probes O
were O
labelled O
by O
random O
- O
priming O
. O

Hybridization O
was O
performed O
for O
12 O
h O
in O
7 O
% O
SDS O
, O
0 O
. O
5 O
M O
phosphate O
buffer O
, O
1 O
mM O
Na2EDTA O
at O
650C O
. O

Blots O
were O
washed O
twice O
in O
2 O
. O
5 O
x O
SSC O
at O
650C O
and O
exposed O
to O
Kodak O
XAR O
- O
5 O
film O
with O
intensifying O
screens O
. O

Exposure O
times O
are O
indicated O
in O
Fig O
. O

1 O
. O

( O
IRL O
Press O

Nucleic O
Acids O
Research O

Volume O
17 O
Number O
23 O
1989 O

9527 O

Nucleic O
Acids O
Research O

C B
. I
elegans I

N O
0 O
$ O
8 O

_ O
_ O

: O
, O
s O
, O
. O
. O
e O
: O
* O
. O

, O
~ O
: O
: O
: O
: O
: O

. O
4i O

. O
t O
_ O
: O
- O
: O
: O
? O
J O
? O
. O
: O

i O
- O

. O
a O

E O
? O
: O
< O
. O

: O
: O
y O
: O

* O
01 O

4 O
? O
j O
* O

. O
- O
I O

kb O
- O
27 O

- O
9A O
- O

: O
. O
: O
: O
. O
. O
. O
. O
. O
. O
. O

Mi O
. O
. O
. O
. O

- O
4v3 O

IC O
. O
3 O

If O
. O
0r O

MW O

! O
5 O
c6 O

I O

" O
R O
1d O
~ O
! O

; O
, O

Figure O
1 O
. O

Southern O
hybridization O
analysis O
of O
Hae O
III O
, O
Rsa O
I O
and O
Eco O
RI O
digested O
genomic O
DNA O
isolated O
from O
the O
two O
C B
. I
elegans I
strains O
Bristol O
( O
N O
) O
and O
Bergerac O
( O
B O
) O
. O

The O
probes O
used O
and O
the O
exposure O
times O
of O
the O
autoradiograph O
are O
indicated O
below O
the O
figure O
. O

kb O
= O
kilo O
basepairs O
. O

9528 O

* O
: O
. O
. O
? O
. O
; O
. O

. O
* O
. O

i O
. O
. O
. O

: O
" O
. O

ANN O
& O

' O
ROW O
: O
. O
lwmwww O
. O

. O
Ammkw O
. O
l O

4w O
, O

9 O

_ O
i O
ob O

Ill O
a O

Nucleic O
Acids O
Research O

RESULTS O

In O
Figure O
1 O
the O
hybridization O
patterns O
are O
shown O
of O
C B
. I
elegans I
genomic O
DNA O
digested O
with O
Hae O
II O
, O
Rsa O
I O
and O
Eco O
RI O
, O
and O
subsequently O
hybridized O
to O
minisatellite O
core O
probe O
33 O
. O
6 O
, O
the O
simple O
sequence O
probe O
( O
AGC O
) O
n O
and O
Tcl O
. O

The O
latter O
probe O
contains O
a O
member O
of O
the O
Tcl O
family O
of O
transposable O
elements O
which O
is O
present O
in O
different O
copy O
numbers O
in O
the O
two O
different O
strains O
( O
4 O
) O
. O

The O
hybridization O
patterns O
of O
probes O
33 O
. O
6 O
and O
( O
AGC O
) O
n O
obtained O
after O
Hae O
Im O
and O
Rsa O
I O
digestion O
and O
the O
differences O
in O
intensities O
of O
the O
hybridizing O
bands O
is O
reminiscent O
of O
DNA O
- O
fingerprint O
patterns O
obtained O
with O
these O
probes O
in O
other O
species O
( O
1 O
, O
2 O
, O
3 O
) O
. O

However O
, O
identical O
hybridization O
patterns O
for O
the O
two O
strains O
with O
the O
three O
enzymes O
tested O
indicated O
that O
in O
C B
. I
elegans I
these O
sequences O
are O
monomorphic O
( O
Fig O
. O
1 O
) O
. O

Based O
on O
the O
number O
of O
hybridizing O
bands O
we O
estimate O
the O
C B
. I
elegans I
genome O
to O
contain O
about O
30 O
33 O
. O
6 O
homologous O
loci O
and O
about O
20 O
( O
AGC O
) O
n O
homologous O
loci O
. O

Rehybridization O
of O
the O
same O
blot O
with O
a O
Tcl O
probe O
revealed O
extensive O
RFLPs O
due O
to O
the O
different O
copy O
number O
of O
the O
transposon O
in O
the O
two O
strains O
, O
a O
phenomenon O
which O
has O
been O
described O
previously O
by O
others O
( O
4 O
) O
. O

DISCUSSION O

The O
findings O
presented O
in O
this O
paper O
indicate O
that O
polymorphism O
of O
minisatellite O
sequences O
and O
simple O
sequence O
motifs O
, O
is O
not O
a O
general O
phenomenon O
in O
animal O
species O
. O

So O
far O
, O
only O
some O
species O
of O
whales O
have O
displayed O
similar O
high O
levels O
of O
monomorphism O
( O
6 O
) O
. O

In O
other O
species O
thus O
far O
tested O
both O
minisatellites O
and O
simple O
sequences O
display O
high O
to O
very O
high O
levels O
of O
polymorphism O
. O

It O
should O
be O
noted O
, O
however O
, O
that O
at O
least O
in O
humans B
, O
a O
substantial O
part O
of O
the O
minisatellites O
detected O
by O
core O
probes O
also O
displays O
high O
levels O
of O
monomorphism O
as O
analysed O
by O
cloning O
( O
1 O
) O
or O
by O
two O
- O
dimensional O
DNA O
fingerprinting O
( O
5 O
) O
. O

The O
fact O
that O
nematodes O
are O
hermaphrodites O
, O
and O
thus O
inbred O
, O
might O
be O
a O
contributing O
factor O
to O
the O
observed O
lack O
of O
polymorphism O
. O

However O
, O
the O
high O
levels O
of O
polymorphism O
detected O
by O
the O
Tcl O
probe O
do O
not O
indicate O
a O
general O
absence O
of O
events O
causing O
genetic O
variation O
. O

Indeed O
, O
Eide O
and O
Anderson O
( O
7 O
) O
showed O
that O
tandemly O
repeated O
duplications O
in O
the O
unc O
- O
54 O
gene O
of O
C B
. I
elegans I
revert O
at O
high O
frequencies O
. O

Since O
in O
all O
cases O
the O
revertants O
had O
the O
normal O
genomic O
configuration O
this O
suggests O
that O
unequal O
crossing O
- O
over O
does O
occur O
in O
the O
nematode O
. O

A O
more O
likely O
explanation O
for O
the O
monomorphic O
nature O
of O
the O
sequences O
detected O
with O
33 O
. O
6 O
and O
( O
AGC O
) O
n O
in O
C B
. I
elegans I
is O
selection O
against O
sequence O
variants O
at O
these O
loci O
. O

This O
might O
be O
the O
result O
of O
the O
presence O
of O
a O
particular O
subset O
of O
minisatellites O
and O
/ O
or O
simple O
sequences O
at O
sites O
in O
the O
genome O
of O
this O
organism O
, O
e O
. O
g O
. O
in O
coding O
sequences O
, O
in O
which O
variation O
in O
copy O
number O
of O
repeat O
units O
cannot O
be O
tolerated O
. O

An O
example O
of O
such O
a O
coding O
sequence O
could O
be O
the O
High O
Mobility O
Group O
proteins O
which O
usually O
have O
stretches O
of O
identical O
( O
acidic O
) O
amino O
acids O
( O
2 O
, O
8 O
) O
. O

An O
interesting O
observation O
in O
this O
respect O
is O
the O
demonstration O
of O
the O
absence O
of O
any O
protein O
polymorphisms O
in O
electrophoretic O
comparisons O
for O
24 O
different O
enzymes O
between O
the O
Bristol O
and O
Bergerac O
strains O
( O
9 O
) O
. O

An O
important O
step O
in O
understanding O
this O
phenomenon O
will O
therefore O
be O
the O
isolation O
and O
analysis O
of O
individual O
homologous O
minisatellite O
and O
simple O
sequence O
loci O
from O
a O
genomic O
library O
of O
C B
. I
elegans I
. O

REFERENCES O

1 O
. O

Jeffreys O
, O
A O
. O
J O
. O
, O
Wilson O
, O
V O
. O
, O
and O
Thein O
, O
S O
. O
L O
. O

( O
1985 O
) O
Nature O
314 O
, O
67 O
- O
73 O
. O

2 O
. O

Tautz O
, O
D O
. O
, O
Trick O
M O
. O
, O
and O
Dover O
, O
G O
. O
A O
. O

( O
1986 O
) O
Nature O
322 O
, O
652 O
- O
656 O
. O

3 O
. O

Rogstad O
, O
S O
. O
H O
. O
, O
Herwaldt O
, O
B O
. O
L O
. O
, O
Schlesinger O
, O
P O
. O
H O
. O
, O
and O
Krogstad O
, O
D O
. O
J O
. O

( O
1989 O
) O
Nucleic O
Acids O
Res O
. O

17 O
, O
9 O
, O
3610 O
. O

4 O
. O

Emmons O
, O
S O
. O
W O
. O
, O
Yesner O
, O
L O
. O
, O
Ruan O
, O
K O
. O
and O
Katzenberg O
, O
D O
. O

( O
1983 O
) O
Cell O
32 O
, O
55 O
- O
65 O
. O

9529 O

Nucleic O
Acids O
Research O

5 O
. O

Uitterlinden O
, O
A O
. O
G O
. O
, O
Slagboom O
, O
P O
. O
E O
. O
, O
Knook O
, O
D O
. O
L O
. O
, O
and O
Vijg O
, O
J O
. O

( O
1989 O
) O
Proc O
. O

Natl O
. O

Acad O
. O

USA O
86 O
, O
2742 O
- O
2746 O
. O

6 O
. O

Rus O
Hoelzel O
, O
A O
. O
, O
and O
Amos O
, O
W O
. O

( O
1988 O
) O
Nature O
333 O
, O
305 O
. O

7 O
. O

Eide O
, O
D O
. O
, O
and O
Anderson O
, O
P O
. O

( O
1985 O
) O
Genetics O
109 O
, O
67 O
- O
79 O
. O

8 O
. O

Pentecost O
, O
B O
. O
T O
. O
, O
Wright O
, O
J O
. O
M O
. O
, O
and O
Dixon O
, O
G O
. O
H O
. O

( O
1985 O
) O
Nucleic O
Acids O
Res O
. O

13 O
, O
4871 O
- O
4888 O
. O

9 O
. O

Butler O
et O
al O
. O

( O
1981 O
) O
J O
. O

Molec O
. O

Evolution O
18 O
, O
18 O
- O
23 O
. O

9530 O

This O
article O
, O
submitted O
on O
disc O
, O
has O
been O
automatically O

converted O
into O
this O
typeset O
format O
by O
the O
publisher O
. O

How O
can O
Health O
Behavior O
Theory O
be O
made O
more O
useful O
for O
intervention O
research O
? O

Abstract O

Background O

The O
present O
paper O
expresses O
the O
author O
' O
s O
views O
about O
the O
practical O
utility O
of O
Health O
Behavior O
Theory O
for O
health O
behavior O
intervention O
research O
. O

The O
views O
are O
skeptical O
and O
perhaps O
even O
a O
bit O
exaggerated O
. O

They O
are O
, O
however O
, O
also O
based O
on O
20 O
- O
plus O
years O
of O
in O
- O
the O
- O
trenches O
research O
focused O
on O
improving O
health O
behavior O
practice O
through O
research O
. O

Discussion O

The O
author O
' O
s O
research O
has O
been O
theoretically O
driven O
and O
has O
involved O
measurement O
of O
varying O
variables O
considered O
to O
be O
important O
theoretical O
mediators O
and O
moderators O
of O
health O
behavior O
. O

Regretfully O
, O
much O
of O
this O
work O
has O
found O
these O
variables O
wanting O
in O
basic O
scientific O
merit O
. O

Health O
Behavior O
Theory O
as O
we O
have O
known O
it O
over O
the O
last O
25 O
years O
or O
so O
has O
been O
dominated O
by O
conceptualizations O
of O
behavior O
change O
processes O
that O
highlight O
cognitive O
decision O
- O
making O
. O

Although O
much O
of O
health O
behavior O
practice O
targets O
what O
people B
do O
rather O
than O
what O
they O
think O
, O
the O
logic O
of O
focusing O
on O
thoughts O
is O
that O
what O
people B
think O
about O
is O
the O
key O
to O
what O
they O
will O
do O
in O
the O
future O
, O
and O
that O
interventions O
that O
can O
measure O
and O
harness O
those O
processes O
will O
succeed O
to O
a O
greater O
extent O
than O
those O
that O
do O
not O
. O

Unfortunately O
, O
in O
the O
author O
' O
s O
experience O
, O
the O
premise O
of O
cognitive O
theories O
has O
fallen O
short O
empirically O
in O
a O
number O
of O
ways O
. O

The O
cognitive O
schemata O
favored O
by O
most O
health O
behavior O
theories O
are O
difficult O
to O
measure O
, O
they O
do O
not O
predict O
behavioral O
outcomes O
very O
well O
, O
there O
is O
little O
evidence O
that O
they O
cause O
behavior O
, O
and O
they O
are O
hard O
to O
change O
directly O
. O

Summary O

It O
is O
suggested O
that O
health O
behavior O
researchers O
reconsider O
their O
use O
of O
these O
theories O
in O
favor O
of O
models O
whose O
variables O
are O
more O
accessible O
to O
observation O
and O
experimental O
manipulation O
and O
that O
most O
importantly O
have O
strong O
empirical O
support O
. O

Background O

The O
author O
has O
been O
conducting O
research O
on O
behavioral O
treatment O
of O
obesity O
for O
about O
25 O
years O
. O

During O
that O
time O
, O
the O
dominant O
conceptual O
models O
guiding O
intervention O
development O
have O
been O
cognitive O
behavior O
models O
that O
have O
their O
origin O
in O
psychological O
theory O
. O

Those O
most O
often O
cited O
include O
the O
Health O
Belief O
Model O
[ O
1 O
] O
, O
Protection O
Motivation O
Theory O
[ O
2 O
] O
, O
Subjective O
Expected O
Utility O
Theory O
[ O
3 O
] O
, O
the O
Theory O
of O
Reasoned O
Action O
[ O
4 O
] O
, O
Social O
Cognitive O
Theory O
[ O
5 O
] O
, O
and O
the O
Transtheoretical O
Model O
[ O
6 O
] O
. O

All O
of O
these O
theories O
are O
concerned O
with O
how O
people B
make O
behavioral O
choices O
and O
the O
general O
idea O
is O
that O
people B
decide O
what O
to O
do O
based O
on O
the O
extent O
to O
which O
they O
expect O
that O
their O
choices O
will O
produce O
results O
that O
they O
value O
. O

Much O
of O
the O
content O
of O
the O
theories O
is O
concerned O
with O
factors O
that O
may O
affect O
value O
/ O
expectancy O
calculations O
. O

As O
summarized O
by O
Weinstein O
in O
a O
comparative O
review O
of O
four O
social O
psychological O
theories O
[ O
7 O
] O
, O
variables O
thought O
to O
influence O
value O
/ O
expectancy O
judgments O
include O
such O
factors O
as O
perceived O
rewards O
of O
current O
behavior O
, O
self O
- O
efficacy O
, O
normative O
beliefs O
, O
motivation O
, O
and O
the O
perceived O
consequences O
of O
not O
changing O
behavior O
. O

Weinstein O
' O
s O
summary O
is O
illustrative O
of O
the O
fact O
that O
Health O
Behavior O
Theory O
has O
tended O
to O
be O
particularly O
interested O
in O
understanding O
people B
' O
s O
motivation O
to O
change O
behavior O
rather O
than O
ability O
to O
change O
. O

Moreover O
, O
motivation O
is O
thought O
to O
be O
the O
result O
of O
a O
relatively O
complex O
, O
but O
logical O
, O
interpretation O
of O
large O
quantities O
of O
information O
about O
self O
and O
environment O
. O

The O
theories O
that O
Weinstein O
reviewed O
deal O
almost O
exclusively O
with O
behavioral O
decision O
processes O
in O
people B
' O
s O
minds O
. O

They O
have O
few O
if O
any O
terms O
relating O
to O
how O
information O
gets O
into O
peoples B
minds O
or O
how O
subsets O
of O
it O
receive O
more O
or O
less O
attention O
. O

Broader O
health O
behavior O
theories O
such O
as O
Social O
Cognitive O
Theory O
or O
the O
Transtheoretical O
model O
have O
addressed O
issues O
and O
variables O
outside O
the O
person B
to O
a O
greater O
extent O
, O
but O
the O
fundamental O
interest O
in O
and O
belief O
in O
psychological O
variables O
as O
the O
key O
force O
in O
determining O
health O
behavior O
remains O
. O

The O
implications O
of O
the O
focus O
of O
health O
behavior O
theory O
on O
psychological O
determinants O
of O
behavioral O
decision O
- O
making O
for O
my O
own O
research O
area O
of O
interest O
, O
obesity O
treatment O
, O
are O
several O
. O

One O
is O
the O
inclusion O
of O
measures O
of O
psychological O
characteristics O
in O
most O
research O
protocols O
( O
e O
. O
g O
. O
, O
assessment O
of O
behavioral O
intentions O
, O
self O
- O
efficacy O
, O
perception O
of O
barriers O
to O
change O
, O
perception O
of O
social O
support O
, O
and O
outcome O
expectations O
) O
. O

A O
second O
is O
the O
inclusion O
of O
treatment O
elements O
that O
specifically O
target O
psychological O
perceptions O
and O
processes O
independent O
of O
the O
diet O
and O
physical O
activity O
behaviors O
that O
actually O
produce O
weight O
change O
( O
e O
. O
g O
. O
, O
how O
to O
deal O
with O
emotional O
eating O
, O
how O
to O
deal O
with O
the O
frustration O
of O
lapses O
and O
relapses O
, O
and O
how O
to O
talk O
to O
yourself O
to O
increase O
self O
- O
motivation O
) O
. O

A O
third O
is O
the O
belief O
that O
psychological O
reactions O
to O
treatment O
experiences O
themselves O
are O
very O
important O
and O
deserve O
independent O
attention O
. O

Common O
behavioral O
prescriptions O
for O
weight O
- O
loss O
goals O
and O
frequency O
of O
self O
- O
weighing O
are O
exemplary O
( O
i O
. O
e O
. O
, O
recommending O
infrequent O
weighing O
to O
prevent O
discouraging O
feedback O
about O
progress O
and O
encouraging O
smaller O
and O
thus O
" O
more O
attainable O
" O
behavior O
and O
weight O
- O
loss O
goals O
in O
the O
belief O
that O
they O
will O
be O
more O
motivating O
) O
. O

The O
problem O
with O
the O
emphasis O
on O
cognitive O
variables O
in O
weight O
- O
control O
research O
is O
that O
they O
have O
so O
far O
failed O
to O
meet O
fundamental O
scientific O
criteria O
for O
empirical O
verification O
. O

Thus O
, O
they O
also O
have O
not O
led O
to O
a O
better O
understanding O
of O
the O
weight O
- O
loss O
process O
, O
have O
not O
improved O
our O
ability O
to O
predict O
weight O
- O
loss O
outcomes O
, O
and O
have O
not O
led O
to O
improvement O
in O
treatment O
methods O
. O

In O
some O
cases O
it O
is O
even O
arguable O
that O
they O
have O
made O
treatment O
worse O
. O

I O
will O
illustrate O
these O
problems O
with O
results O
from O
my O
own O
research O
. O

Discussion O

Like O
most O
behavioral O
researchers O
in O
the O
obesity O
area O
, O
I O
have O
attempted O
to O
measure O
elements O
of O
health O
behavior O
theory O
in O
every O
obesity O
intervention O
project O
I O
have O
ever O
conducted O
. O

I O
have O
assessed O
weight O
- O
loss O
goals O
, O
behavioral O
and O
weight O
- O
loss O
self O
- O
efficacy O
, O
psychological O
well O
- O
being O
, O
perceived O
barriers O
to O
diet O
and O
physical O
activity O
change O
, O
stages O
- O
of O
- O
change O
, O
and O
perceived O
social O
support O
. O

How O
well O
have O
empirical O
examinations O
of O
these O
factors O
fared O
as O
predictors O
of O
success O
in O
weight O
control O
? O

Self O
- O
efficacy O

We O
have O
examined O
the O
predictive O
value O
of O
self O
- O
efficacy O
assessments O
in O
several O
of O
our O
studies O
and O
describe O
the O
results O
from O
three O
of O
these O
here O
in O
more O
detail O
[ O
8 O
- O
10 O
] O
. O

In O
the O
first O
study O
, O
self O
- O
efficacy O
was O
assessed O
at O
baseline O
, O
posttreatment O
, O
and O
one O
year O
later O
in O
85 O
men B
participating O
in O
a O
15 O
- O
week O
weight O
- O
loss O
program O
[ O
8 O
] O
. O

The O
self O
- O
efficacy O
instrument O
had O
subscales O
for O
emotional O
states O
( O
e O
. O
g O
. O
, O
anxiety O
) O
and O
situations O
( O
e O
. O
g O
. O
, O
eating O
away O
from O
home O
) O
. O

Higher O
baseline O
self O
- O
efficacy O
on O
both O
subscales O
was O
associated O
with O
greater O
weight O
loss O
in O
treatment O
and O
at O
1 O
- O
and O
2 O
- O
year O
follow O
- O
up O
. O

Emotional O
self O
- O
efficacy O
at O
posttreatment O
did O
not O
predict O
weight O
loss O
at O
1 O
- O
or O
2 O
- O
year O
follow O
- O
up O
. O

Situational O
self O
- O
efficacy O
at O
posttreatment O
predicted O
weight O
loss O
at O
1 O
- O
year O
but O
not O
2 O
- O
year O
follow O
- O
up O
. O

The O
second O
study O
examined O
mood O
and O
situational O
self O
- O
efficacy O
in O
55 O
men B
and O
58 O
women B
before O
and O
after O
a O
16 O
- O
week O
weight O
- O
loss O
treatment O
with O
a O
1 O
- O
year O
follow O
- O
up O
[ O
9 O
] O
. O

Women B
had O
lower O
pretreatment O
self O
- O
efficacy O
than O
men B
. O

Self O
- O
efficacy O
was O
predictive O
of O
weight O
loss O
and O
maintenance O
in O
men B
but O
not O
in O
women B
. O

Change O
in O
self O
- O
efficacy O
over O
time O
was O
positively O
related O
to O
weight O
change O
in O
women B
but O
not O
in O
men B
. O

The O
third O
study O
examined O
predictors O
of O
weight O
change O
over O
a O
2 O
- O
year O
period O
in O
460 O
men B
and O
1172 O
women B
who O
received O
a O
low O
- O
intensity O
weight O
- O
loss O
intervention O
delivered O
through O
their O
HMO O
[ O
10 O
] O
. O

The O
self O
- O
efficacy O
measure O
was O
the O
WEL O
questionnaire O
. O

Men B
again O
were O
found O
to O
have O
higher O
baseline O
self O
- O
efficacy O
than O
women B
. O

Self O
- O
efficacy O
did O
not O
predict O
weight O
change O
in O
men B
but O
was O
positively O
, O
though O
weakly O
, O
related O
to O
weight O
change O
at O
6 O
months O
only O
in O
women B
. O

Our O
overall O
conclusion O
from O
the O
analyses O
described O
above O
, O
as O
well O
as O
others O
not O
pursued O
in O
as O
great O
detail O
, O
is O
that O
self O
- O
efficacy O
is O
a O
weak O
predictor O
of O
weight O
loss O
and O
is O
inconsistent O
across O
study O
populations O
and O
gender O
. O

It O
tends O
to O
increase O
with O
weight O
loss O
. O

However O
, O
treatment O
- O
induced O
increases O
in O
efficacy O
are O
not O
predictive O
of O
longer O
- O
term O
weight O
- O
loss O
success O
. O

Barriers O
to O
Adherence O

We O
have O
also O
attempted O
to O
measure O
barriers O
to O
adherence O
to O
weight O
- O
control O
behaviors O
in O
many O
of O
our O
studies O
[ O
11 O
- O
14 O
] O
. O

The O
instruments O
used O
for O
this O
have O
typically O
been O
formatted O
similarly O
to O
efficacy O
questionnaires O
in O
that O
people B
are O
asked O
to O
indicate O
how O
difficult O
they O
find O
situational O
, O
knowledge O
, O
and O
motivational O
challenges O
to O
achieving O
diet O
and O
exercise O
changes O
. O

The O
findings O
in O
these O
studies O
have O
been O
quite O
consistent O
. O

Baseline O
assessments O
of O
perceived O
barriers O
to O
behavior O
change O
are O
not O
predictive O
of O
weight O
change O
. O

Weight O
loss O
is O
associated O
with O
reported O
decreases O
in O
perceived O
barriers O
. O

Treatment O
- O
induced O
change O
in O
perceived O
barriers O
are O
not O
predictive O
of O
future O
weight O
change O
. O

In O
other O
words O
, O
barrier O
perceptions O
as O
we O
have O
measured O
them O
do O
not O
appear O
to O
have O
pragmatic O
significance O
. O

Weight O
Goals O

Goal O
- O
setting O
has O
long O
been O
of O
interest O
to O
health O
behavior O
theory O
and O
in O
recent O
years O
has O
attracted O
attention O
in O
weight O
- O
loss O
research O
when O
it O
was O
realized O
that O
most O
people B
who O
enter O
weight O
- O
loss O
treatments O
want O
to O
lose O
a O
lot O
more O
weight O
than O
is O
realistic O
given O
the O
potency O
of O
current O
weight O
- O
loss O
methodologies O
[ O
15 O
] O
. O

When O
asked O
to O
describe O
weight O
losses O
they O
deem O
to O
represent O
" O
dream O
, O
happy O
, O
acceptable O
, O
and O
disappointing O
, O
" O
many O
individuals O
in O
treatment O
fail O
to O
reach O
even O
" O
disappointing O
" O
weight O
losses O
even O
though O
in O
objective O
medical O
terms O
the O
results O
are O
positive O
. O

Based O
on O
the O
argument O
that O
failure O
to O
reach O
gratifying O
weight O
- O
loss O
goals O
leads O
to O
psychological O
distress O
that O
lowers O
weight O
self O
- O
efficacy O
and O
undermines O
weight O
- O
loss O
efforts O
, O
it O
has O
become O
popular O
to O
recommend O
counseling O
in O
weight O
- O
loss O
treatments O
specifically O
targeting O
the O
lowering O
of O
weight O
- O
loss O
goals O
. O

The O
theoretical O
argument O
is O
that O
excessive O
outcome O
expectations O
undermine O
behavioral O
efforts O
. O

We O
have O
now O
completed O
three O
sets O
of O
formal O
analyses O
examining O
whether O
weight O
goals O
are O
predictive O
of O
weight O
- O
loss O
success O
. O

In O
one O
of O
these O
analyses O
the O
relationship O
between O
weight O
- O
loss O
goals O
, O
weight O
- O
loss O
goal O
attainment O
, O
and O
long O
- O
term O
( O
30 O
months O
) O
weight O
- O
loss O
attainment O
and O
psychological O
well O
- O
being O
were O
assessed O
in O
69 O
men B
and O
61 O
women B
participating O
in O
an O
intensive O
behavioral O
treatment O
program O
[ O
16 O
] O
. O

Results O
indicated O
that O
weight O
- O
loss O
goals O
were O
unrealistically O
high O
on O
average O
and O
that O
lower O
goals O
were O
more O
likely O
to O
be O
reached O
. O

Nevertheless O
, O
weight O
- O
loss O
goals O
did O
not O
predict O
either O
short O
- O
or O
long O
- O
term O
weight O
losses O
and O
were O
not O
associated O
with O
elevated O
psychological O
distress O
. O

Two O
more O
recent O
analyses O
we O
have O
conducted O
looking O
at O
weight O
- O
loss O
goals O
as O
predictors O
of O
success O
have O
produced O
similar O
results O
[ O
Linde O
JA O
, O
Jeffery O
RW O
, O
Levy O
RL O
, O
Pronk O
NP O
and O
Boyle O
RG O
, O
unpublished O
data O
[ O
17 O
] O
] O
. O

Weight O
- O
loss O
goals O
either O
did O
not O
predict O
weight O
loss O
at O
all O
or O
were O
slightly O
positively O
related O
to O
weight O
- O
loss O
success O
. O

Perceived O
Social O
Support O

Perceived O
social O
support O
is O
another O
psychological O
factor O
thought O
to O
influence O
health O
behavior O
decision O
- O
making O
. O

We O
have O
measured O
social O
support O
in O
a O
variety O
of O
ways O
in O
our O
studies O
, O
ranging O
from O
single O
- O
item O
questions O
to O
multipaged O
assessments O
attempting O
to O
differentiate O
among O
informational O
, O
instrumental O
, O
and O
emotional O
support O
. O

The O
results O
, O
unfortunately O
, O
have O
closely O
paralleled O
those O
we O
have O
seen O
with O
other O
assessments O
of O
barriers O
to O
adherence O
. O

Assessments O
of O
social O
support O
prior O
to O
treatment O
do O
not O
predict O
weight O
loss O
. O

Average O
reports O
of O
social O
support O
tend O
to O
parallel O
weight O
loss O
itself O
. O

When O
people B
lose O
weight O
they O
report O
more O
social O
support O
. O

When O
they O
regain O
, O
they O
report O
less O
. O

In O
other O
words O
, O
perceptions O
of O
social O
support O
are O
not O
predictive O
of O
success O
in O
weight O
- O
loss O
treatments O
. O

Frequency O
Weight O
Self O
- O
monitoring O

Self O
- O
monitoring O
of O
health O
behavior O
is O
incorporated O
into O
many O
health O
behavior O
theories O
, O
usually O
as O
part O
of O
a O
person B
' O
s O
assessment O
of O
achieved O
outcomes O
. O

Although O
self O
- O
monitoring O
is O
usually O
considered O
a O
positive O
element O
in O
the O
adoption O
of O
health O
behavior O
, O
in O
obesity O
treatment O
frequent O
self O
- O
monitoring O
of O
weight O
has O
tended O
to O
be O
down O
- O
played O
or O
even O
discouraged O
on O
the O
grounds O
that O
disappointing O
results O
( O
i O
. O
e O
. O
, O
less O
than O
desired O
weight O
change O
) O
may O
undermine O
motivation O
. O

This O
is O
another O
example O
in O
which O
health O
behavior O
theory O
may O
have O
indirectly O
led O
to O
incorrect O
treatment O
recommendations O
. O

In O
weight O
- O
loss O
treatments O
, O
active O
discouragement O
of O
frequent O
self O
- O
observation O
of O
weight O
has O
become O
popular O
based O
on O
the O
premise O
that O
more O
frequent O
weighting O
will O
cause O
psychological O
stress O
and O
lower O
self O
- O
efficacy O
. O

Recently O
, O
we O
have O
examined O
the O
relationship O
between O
frequency O
of O
self O
- O
weighing O
and O
body O
weight O
in O
both O
clinical O
and O
population O
samples O
and O
have O
found O
, O
somewhat O
to O
our O
surprise O
, O
that O
frequency O
of O
self O
- O
weighing O
is O
one O
of O
the O
strongest O
single O
predictors O
of O
body O
weight O
cross O
- O
sectionally O
, O
and O
change O
in O
the O
frequency O
of O
self O
- O
weighing O
is O
one O
of O
the O
strongest O
predictors O
of O
weight O
change O
[ O
Linde O
JA O
, O
Jeffery O
RW O
and O
French O
SA O
, O
unpublished O
data O
] O
. O

The O
direction O
of O
predictions O
, O
however O
, O
is O
opposite O
that O
derived O
from O
theory O
. O

People B
who O
weigh O
themselves O
more O
weigh O
less O
and O
are O
more O
successful O
in O
losing O
weight O
. O

Stage O
- O
of O
- O
Change O

A O
final O
failure O
of O
current O
health O
behavior O
theory O
to O
prove O
useful O
in O
weight O
- O
control O
research O
is O
a O
recent O
examination O
of O
the O
relationship O
between O
a O
stage O
- O
of O
- O
change O
measure O
adopted O
from O
Prochaska O
and O
short O
- O
and O
long O
- O
term O
weight O
loss O
[ O
18 O
] O
. O

Categories O
of O
precontemplation O
, O
contemplation O
, O
preparation O
, O
and O
action O
were O
defined O
based O
on O
questions O
about O
weight O
- O
loss O
intentions O
and O
recent O
weight O
- O
loss O
attempts O
. O

Despite O
a O
large O
sample O
size O
, O
excellent O
follow O
- O
up O
rates O
, O
and O
well O
- O
measured O
objective O
outcomes O
, O
we O
were O
unable O
to O
demonstrate O
that O
staging O
algorithms O
recommended O
by O
proponents O
of O
the O
Transtheoretical O
Model O
could O
predict O
weight O
- O
loss O
outcomes O
. O

Experimental O
Modification O
of O
Expectations O

Our O
most O
recent O
effort O
to O
utilize O
health O
behavior O
theory O
in O
obesity O
intervention O
research O
is O
a O
study O
that O
attempted O
to O
examine O
the O
effectiveness O
of O
experimentally O
- O
induced O
outcome O
expectancies O
on O
weight O
loss O
[ O
Finch O
EA O
, O
Linde O
JA O
, O
Jeffery O
RW O
, O
Rothman O
AJ O
and O
King O
CM O
, O
unpublished O
data O
] O
. O

Obese O
men B
and O
women B
participated O
in O
an O
8 O
- O
week O
weight O
- O
loss O
program O
with O
18 O
- O
month O
follow O
- O
up O
in O
which O
they O
were O
assigned O
to O
one O
of O
two O
expectancy O
groups O
. O

The O
optimistic O
group O
was O
told O
that O
focusing O
exclusively O
on O
the O
positive O
benefits O
of O
weight O
loss O
would O
be O
valuable O
in O
ensuring O
that O
they O
remained O
motivated O
in O
their O
weight O
- O
loss O
efforts O
and O
was O
given O
assignments O
during O
weekly O
group O
sessions O
and O
homework O
between O
sessions O
to O
reinforce O
this O
optimistic O
mindset O
. O

A O
" O
balanced O
" O
expectancy O
group O
received O
the O
instructions O
that O
focusing O
on O
both O
the O
positive O
and O
negative O
aspects O
of O
weight O
loss O
, O
a O
balanced O
approach O
, O
would O
be O
most O
conducive O
to O
maintaining O
weight O
- O
loss O
motivation O
. O

This O
group O
also O
received O
assignments O
to O
reinforce O
their O
message O
. O

Results O
of O
this O
study O
indicated O
that O
the O
expectation O
induction O
was O
successful O
initially O
but O
difficult O
to O
maintain O
in O
the O
face O
of O
real O
weight O
- O
loss O
experience O
. O

We O
were O
also O
unable O
to O
show O
that O
experimentally O
- O
induced O
expectations O
influenced O
weight O
- O
loss O
success O
. O

Summary O
and O
Conclusion O

To O
summarize O
the O
findings O
described O
above O
, O
I O
have O
had O
considerable O
difficulty O
over O
the O
last O
25 O
years O
in O
confirming O
that O
the O
psychosocial O
variables O
favored O
by O
health O
behavior O
theory O
are O
of O
much O
value O
for O
obesity O
intervention O
research O
. O

They O
do O
not O
predict O
weight O
loss O
well O
, O
either O
as O
mediators O
or O
moderators O
. O

There O
is O
little O
evidence O
to O
support O
the O
idea O
that O
targeting O
them O
for O
intervention O
improves O
weight O
- O
loss O
outcomes O
. O

It O
is O
, O
of O
course O
, O
arguable O
that O
the O
weak O
findings O
relating O
to O
health O
behavior O
theory O
variables O
are O
due O
in O
large O
part O
to O
methodological O
weaknesses O
, O
either O
in O
measurement O
tools O
and O
/ O
or O
their O
frequency O
of O
measurement O
. O

I O
would O
argue O
, O
however O
, O
that O
25 O
years O
is O
long O
enough O
to O
wait O
for O
improved O
methods O
and O
that O
it O
is O
time O
to O
look O
elsewhere O
for O
variables O
that O
better O
predict O
weight O
- O
change O
outcomes O
and O
that O
, O
therefore O
, O
may O
form O
a O
better O
basis O
for O
improving O
future O
treatments O
. O

Implication O
for O
Weight O
- O
Loss O
Treatment O

Given O
the O
lack O
of O
success O
finding O
support O
for O
cognitive O
mediators O
of O
behavior O
change O
in O
weight O
loss O
, O
one O
might O
surmise O
that O
progress O
in O
improving O
weight O
- O
loss O
interventions O
over O
the O
last O
20 O
years O
must O
have O
been O
dreary O
indeed O
. O

Somewhat O
surprisingly O
, O
however O
, O
that O
is O
not O
the O
case O
. O

In O
fact O
, O
the O
short O
- O
term O
( O
6 O
to O
12 O
months O
) O
success O
of O
weight O
- O
loss O
treatments O
has O
approximately O
doubled O
over O
that O
time O
and O
several O
variables O
have O
been O
identified O
that O
reliably O
enhance O
treatment O
outcomes O
. O

It O
has O
been O
clearly O
shown O
experimentally O
that O
increasing O
treatment O
length O
[ O
19 O
] O
, O
prescribing O
low O
- O
energy O
intakes O
[ O
20 O
] O
, O
prescribing O
high O
- O
energy O
expenditure O
[ O
21 O
] O
, O
using O
a O
deposit O
contract O
and O
group O
- O
based O
reward O
systems O
[ O
22 O
] O
, O
and O
simplifying O
adherence O
to O
diet O
through O
meal O
substitutes O
[ O
23 O
] O
and O
exercise O
by O
providing O
exercise O
equipment O
[ O
24 O
] O
all O
improve O
initial O
weight O
loss O
. O

From O
a O
theoretical O
perspective O
, O
however O
, O
one O
thing O
is O
noteworthy O
about O
these O
successful O
innovations O
. O

Although O
not O
incompatible O
with O
health O
behavior O
theory O
, O
none O
of O
them O
are O
specifically O
derived O
from O
cognitive O
decision O
- O
making O
models O
. O

Indeed O
, O
health O
behavior O
theory O
does O
not O
include O
variables O
like O
these O
in O
its O
models O
. O

Where O
Do O
We O
Go O
From O
Here O
? O

The O
argument O
above O
about O
the O
practical O
limitations O
of O
many O
popular O
theories O
of O
health O
behavior O
is O
not O
meant O
to O
be O
a O
call O
to O
abandon O
theory O
. O

Behavior O
scientists O
have O
amassed O
much O
useful O
information O
about O
the O
principles O
underlying O
human B
behavior O
that O
should O
be O
valuable O
for O
health O
behavior O
interventions O
. O

Much O
is O
known O
about O
human B
perception O
, O
learning O
, O
motivation O
, O
and O
responsiveness O
to O
environmental O
opportunities O
and O
contingencies O
. O

Health O
behavior O
intervention O
lies O
at O
the O
interface O
between O
people B
and O
their O
environment O
. O

Interventionists O
change O
aspects O
of O
the O
environment O
( O
cues O
, O
information O
, O
behavioral O
contingencies O
) O
with O
the O
intention O
of O
producing O
changes O
in O
how O
people B
behave O
. O

What O
is O
needed O
to O
advance O
health O
behavior O
intervention O
is O
theory O
that O
addresses O
relationships O
between O
modifiable O
aspects O
of O
the O
environment O
and O
behavior O
. O

There O
is O
no O
doubt O
that O
cognitive O
processes O
are O
involved O
in O
these O
relationships O
. O

However O
, O
the O
extent O
to O
which O
current O
theories O
capture O
this O
is O
questionable O
. O

Data O
now O
available O
suggest O
that O
easily O
obtainable O
information O
about O
people B
' O
s O
cognitive O
processes O
adds O
little O
to O
our O
ability O
to O
predict O
the O
results O
of O
interventions O
. O

Thus O
, O
it O
may O
be O
wise O
to O
pay O
more O
attention O
to O
applied O
theories O
like O
classical O
behavior O
theory O
[ O
25 O
] O
, O
communications O
theory O
[ O
26 O
] O
, O
and O
learning O
theory O
[ O
27 O
] O
than O
to O
those O
coming O
out O
of O
the O
social O
cognitive O
traditions O
. O

Competing O
interests O

None O
declared O
. O