How

valid

are

current

diagnostic

criteria

for

dental

erosion

In

principle

there

is

agreement

about

the

clinical

diagnostic

criteria

for

dental

erosion

basically

defined

as

cupping

and

grooving

of

the

occlusal/incisal

surfaces

shallow

defects

on

smooth

surfaces

located

coronal

from

the

enamel

cementum

junction

with

an

intact

cervical

enamel

rim

and

restorations

rising

above

the

adjacent

tooth

surface

This

lesion

characteristic

was

established

from

clinical

experience

and

from

observations

in

a

small

group

of

subjects

with

known

exposure

to

acids

rather

than

from

systematic

research

Their

prevalence

is

higher

in

risk

groups

for

dental

erosion

compared

to

subjects

not

particularly

exposed

to

acids

but

analytical

epidemiological

studies

on

random

or

cluster

samples

often

fail

to

find

a

relation

between

occurrence

or

severity

of

lesions

and

any

aetiological

factor

Besides

other

aspects

this

finding

might

be

due

to

lack

of

validity

with

respect

to

diagnostic

criteria

In

particular

cupping

and

grooving

might

be

an

effect

of

abrasion

as

well

as

of

erosion

and

their

value

for

the

specific

diagnosis

of

erosion

must

be

doubted

Knowledge

about

the

validity

of

current

diagnostic

criteria

of

different

forms

of

tooth

wear

is

incomplete

therefore

further

research

is

needed

The

process

of

diagnosis

and

current

criteria

for

dental

erosion

From

the

chemical

view

the

aetiology

of

dental

erosion

can

be

defined

as

the

chronic

exposure

of

the

teeth

to

extrinsic

or

intrinsic

acids

under

the

condition

that

the

oral

fluids

are

undersaturated

with

respect

to

tooth

mineral

-LSB-

-RSB-

Under

in

vitro

conditions

without

physical

impact

teeth

demineralise

centripetally

-LRB-

Fig.

-RRB-

a

feature

of

substance

loss

which

is

normally

not

observed

in

the

mouth

In

fact

the

multitudes

of

physical

and

chemical

assaults

occurring

during

a

lifetime

result

in

a

more

or

less

characteristic

pattern

of

tooth

wear

The

classification

of

wear

is

therefore

made

from

morphological

features

which

are

frequently

seen

clinically

The

tooth

morphology

as

apparent

after

eruption

is

the

idealised

status

deviations

of

which

if

not

caries

or

trauma

are

diagnosed

as

-LRB-

erosive

-RRB-

tooth

wear

Various

forms

of

wear

including

dental

erosion

are

listed

in

the

International

Classification

of

Diseases

-LSB-

-RSB-

thus

defining

them

as

a

disease

-LRB-

for

critical

discussion

of

this

notion

see

-LSB-

-RSB-

-RRB-

Fig.

of

the

continuous

exposure

of

a

human

third

molar

to

citric

acid

The

amorphous

centripetal

tissue

loss

is

obvious

-LRB-

a

unaffected

tooth

b

tissue

loss

after

c

and

d

h

immersion

time

-RRB-

Looking

at

what

diagnosis

is

one

will

find

mostly

definitions

like

identification

of

disease

from

signs

or

symptoms

implicating

that

the

physician

or

scientist

reads

off

from

the

patient

thus

detecting

the

disease

Health

and

disease

however

is

not

a

given

condition

but

is

constituted

by

the

theoretical

concepts

and

the

discursive

practice

the

physician

or

scientist

is

subjected

to

-LSB-

-RSB-

That

is

to

say

rather

than

reading

off

the

disease

from

the

patient

a

pattern

of

criteria

is

projected

onto

the

diagnosed

subject

which

determines

the

diagnostic

procedure

and

outcome

Following

this

approach

the

diagnostic

process

in

a

first

step

is

a

theoretical

concept

ordering

signs

and

symptoms

to

diseases

which

takes

place

within

the

configurative

power

of

the

discourses

In

whatever

discourse

the

physician

acts

the

act

of

clinical

diagnosis

in

a

second

step

is

classification

The

conclusion

reached

through

this

process

is

called

a

diagnosis

From

this

background

it

becomes

obvious

that

the

diagnostic

process

must

be

accompanied

by

a

recurrent

reconsideration

of

its

theoretical

concepts

and

the

reevaluation

of

the

criteria

used

The

latter

is

what

the

paper

aims

to

achieve

The

diagnosis

of

tooth

wear

in

general

and

erosion

in

particular

is

made

from

its

lesion

characteristics

from

the

results

of

nutritional

medical

and

occupational

analysis

and

from

dietary

records

The

diagnostic

process

can

be

more

differentiated

with

the

individual

patient

whereas

in

field

trials

it

is

restricted

to

the

classification

of

lesion

shape

Throughout

the

literature

there

is

more

or

less

consensus

about

the

aetiology

of

the

various

forms

of

tooth

wear

the

clinical

criteria

for

dental

erosion

and

its

differential

diagnosis

-LSB-

-RSB-

The

early

signs

of

erosive

tooth

wear

appear

as

changes

of

the

optical

properties

of

enamel

resulting

in

a

smooth

silky

shining

glazed

surface

When

the

tissue

loss

continues

changes

in

the

original

morphology

occur

On

smooth

surfaces

convex

areas

flatten

or

concavities

develop

the

width

of

which

clearly

exceeds

the

depth

Lesions

are

located

coronal

from

the

enamel

cementum

junction

-LRB-

CEJ

-RRB-

with

an

intact

enamel

rim

along

the

gingival

margin

On

occlusal

and

incisal

surfaces

rounding

and

cupping

of

the

cusps

and

grooving

of

the

incisal

edges

occur

and

restorations

may

rise

above

the

level

of

the

adjacent

tooth

surfaces

In

advanced

cases

the

whole

occlusal

morphology

disappears

The

validity

of

these

clinical

criteria

for

erosion

however

has

not

been

under

critical

consideration

Validity

in

general

terms

means

the

degree

to

which

a

measurement

measures

what

it

purports

to

measure

For

the

issue

addressed

here

criterion

validity

is

the

relevant

term

meaning

the

extent

to

which

the

measurement

correlates

with

an

external

criterion

under

study

A

further

aspect

of

validity

is

concurrent

validity

Concurrent

validity

means

to

which

extent

the

measurement

and

the

criterion

refer

to

the

same

time

-LSB-

-RSB-

In

the

case

of

erosion

this

would

address

the

question

if

-LRB-

a

-RRB-

the

diagnostic

criteria

reflect

lesions

being

an

effect

of

an

exposure

to

acids

and

-LRB-

b

-RRB-

if

the

presence

of

characteristic

signs

is

concurrent

with

an

acid

exposure

The

implementation

of

current

diagnostic

criteria

Considering

how

the

diagnostic

criteria

for

erosion

were

established

the

literature

reveals

that

it

was

individual

clinical

experience

and

case

reports

rather

than

systematic

research

One

of

the

first

publications

on

the

characteristics

of

acid

induced

tissue

loss

was

in

from

Robinson

-LSB-

-RSB-

and

in

from

Stafne

and

Lovestedt

-LSB-

-RSB-

Robinson

whilst

being

in

doubt

about

the

aetiology

of

dental

erosion

described

the

lesions

as

located

on

smooth

surfaces

and

as

wedge

shaped

cup

shaped

disk

shaped

irregular

in

form

L

or

U-shaped

or

simply

as

small

round

depressions

or

larger

surface

lesions

It

is

not

clear

how

these

descriptions

were

established

but

Robinson

referred

to

a

work

from

McClure

and

Ruzicka

-LSB-

-RSB-

describing

the

morphology

of

lesions

of

rats

teeth

after

being

fed

with

lactate

and

citrate

drinking

fluid

Stafne

and

Lovestedt

presented

their

observations

in

subjects

with

known

acid

exposure

amongst

them

patients

with

frequent

consumption

of

lemon

juice

They

did

not

give

a

concrete

description

of

lesion

shape

but

attributed

hypersensitivity

absence

of

stain

and

defects

with

rounded

margins

as

effect

of

the

action

of

the

acids

Their

most

important

sign

of

diagnostic

value

was

the

presence

of

fillings

projecting

above

the

surface

of

the

tooth

In

their

publication

a

number

of

clinical

images

were

included

presenting

lesions

clearly

matching

current

erosion

criteria

More

than

twenty

years

later

in

it

was

Pindborg

-LSB-

-RSB-

who

gave

the

often

cited

definition

of

erosion

as

being

superficial

loss

of

dental

hard

tissue

by

a

chemical

process

which

does

not

involve

bacteria

He

described

the

clinical

signs

of

chemically-induced

tissue

loss

as

usually

located

to

the

gingival

third

of

the

facial

surfaces

possibly

also

located

at

proximal

surfaces

lesions

to

appear

shallow

disc-shaped

smooth

polished

or

scooped

out

In

contrast

to

abrasion

he

attributed

erosive

lesions

to

be

located

evenly

on

the

left

and

right

side

It

appears

noteworthy

that

Pindborg

as

well

as

Robinson

ascribed

cupping

of

the

cusps

loss

of

the

occlusal

morphology

or

loss

of

crown

height

and

incisal

grooving

to

attrition

which

in

their

publications

was

defined

as

result

of

mastication

It

is

probably

Eccles

and

Jenkins

-LSB-

-RSB-

who

were

the

first

to

give

a

detailed

and

systematic

description

of

the

lesion

characteristics

and

also

suggested

a

system

for

classification

The

basis

of

the

development

of

the

clinical

criteria

was

a

sample

of

patients

seen

in

the

dental

hospital

over

a

period

of

years

-LSB-

-RSB-

All

cases

were

thoroughly

documented

with

respect

to

their

medical

and

dietary

history

that

is

it

was

a

group

of

subjects

with

known

exposure

to

intrinsic

or

dietary

acids

The

findings

derived

from

this

group

of

subjects

-LRB-

Table

-RRB-

have

been

retained

nearly

unchanged

until

now

The

main

aspects

are

in

general

terms

loss

of

surface

contour

shallow

concavities

on

smooth

surfaces

cupping

and

grooving

on

occlusal/incisal

surfaces

and

restorations

rising

above

the

level

of

the

adjacent

tooth

surface

Table

criteria

for

dental

erosion

as

outlined

by

Eccles

and

Jenkins

-LSB-

-RSB-

and

Eccles

-LSB-

-RSB-

Diagnostic

criteria

for

dental

erosionInitialAbsence

of

developmental

ridges

of

the

enamel

smooth

glazed

surfaceAdvanced

Facial/oral

surfacesConcavities

whose

breadth

greatly

exceeds

their

depthLesion

ovoid

or

crescentic

in

outline

concave

in

cross

section

orLesion

entirely

in

the

crown

irregular

in

outline

punched

out

appearance

Occlusal/incisal

surfacesSurfaces

appear

flattened

depression

of

the

cusps

-LRB-

cupping

-RRB-

and

on

the

incisal

edges

-LRB-

grooving

-RRB-

edges

of

restorations

raising

above

the

level

of

the

adjacent

tooth

surface

Conclusions

from

epidemiological

studies

using

current

diagnostic

criteria

Current

criteria

derived

from

a

relatively

small

sample

have

been

applied

in

case

reports

-LSB-

-RSB-

and

studies

with

risk

groups

-LSB-

-RSB-

Indeed

when

used

in

subjects

with

known

or

strongly

assumed

exposure

to

acids

most

studies

revealed

a

higher

prevalence

of

lesions

in

the

exposed

groups

compared

to

the

control

groups

-LRB-

Table

-RRB-

Table

of

lesions

in

risk

groups

deriving

from

the

use

of

current

diagnostic

criteria

for

dental

erosion

IndexGroup

sizePrevalence

risk

groupPrevalence

control

groupIntrinsic

acid

exposureMeurman

et

al.

-LSB-

-RSB-

reflux

diseaseEccles

and

Jenkins

indexn

No

control

groupRytömaa

et

al.

-LSB-

-RSB-

eating

disordersEccles

and

Jenkins

indexn

Öhrn

et

al.

-LSB-

-RSB-

eating

disordersLussi

indexn

Minor

less

severeIncisal/occlusalIncisal/occlusalGrade

Grade

Grade

Grade

BuccalBuccalGrade

Grade

Grade

Grade

PalatalPalatalGrade

Grade

Grade

Grade

Extrinsic

acid

exposureLinkosalo

and

Markkanen

-LSB-

-RSB-

vegetariansEccles

and

Jenkins

indexn

Wiktorsson

et

al.

-LSB-

-RSB-

wine

tastersEccles

and

Jenkins

indexn

No

control

groupGanss

et

al.

-LSB-

-RSB-

raw

food

dietLussi

indexn

These

observations

support

the

finding

that

subjects

with

continuous

exposure

to

acids

have

a

higher

rate

of

lesions

with

a

specific

characterisation

This

is

however

not

enough

support

for

the

assumption

that

vice

versa

subjects

presenting

with

such

defects

must

be

exposed

to

acids

Analytical

epidemiological

studies

on

random

or

cluster

samples

attempted

to

relate

the

occurrence

of

lesions

with

any

of

the

known

aetiological

factors

for

erosion

but

only

few

studies

of

this

type

have

been

published

Most

of

them

include

children

or

adolescents

but

there

is

lack

of

studies

on

older

population

groups

Furthermore

off

the

few

studies

some

used

the

Tooth

Wear

Index

-LRB-

TWI

-LSB-

-RSB-

-RRB-

which

is

not

designed

to

assess

dental

erosion

specifically

The

overall

findings

-LRB-

Table

-RRB-

are

controversial

since

some

authors

found

no

or

only

a

partial

relation

between

aetiological

factors

and

the

occurrence

or

severity

of

lesions

-LSB-

-RSB-

whereas

others

revealed

strong

relationships

-LSB-

-RSB-

In

addition

a

relation

to

the

intake

of

yoghurt

or

other

foodstuff

which

certainly

has

no

erosive

potential

was

mentioned

-LSB-

-RSB-

Table

epidemiological

studies

attempting

to

relate

the

occurrence

of

-LRB-

erosive

-RRB-

wear

to

aetiological

factors

Index

group

size

age

and

prevalenceConclusionJärvinen

et

al.

-LSB-

-RSB-

Eccles

Jenkins

indexCitrus

fruits

odds

ratio

-LRB-

OR

-RRB-

n

eachSoft

drinks

OR

et

al.

-LSB-

-RSB-

Lussi

indexSignificant

relation

to

the

consumption

of

fruit

acidic

drinks

yoghurt

vomitingn

and

at

least

and

resp

with

any

erosionBartlett

et

al.

-LSB-

-RSB-

TWI

-LRB-

Smith

and

Knight

-RRB-

No

significant

relation

to

drinks

or

other

acidic

foodn

relation

to

heart

had

wear

in

enamel

on

more

than

teethJaeggi

et

al.

-LSB-

-RSB-

Lussi

indexNo

relation

to

any

aetiological

factorn

at

least

with

erosionAl-Dlaigan

et

al.

-LSB-

-RSB-

TWI

-LRB-

Smith

and

Knight

-RRB-

Significant

relation

to

drinks

and

fruit

but

also

to

milk

yoghurt

and

beern

low

moderate

severe

lesionsAl-Majed

at

al.

-LSB-

-RSB-

TWI

-LRB-

Smith

and

Knight

-RRB-

modified

for

erosionNo

association

to

erosive

drinks

for

the

total

samplen

association

to

frequency

of

drinks

at

night

and

duration

of

drinks

retained

in

the

mouth

only

in

advanced

cases

-LRB-

n

-RRB-

with

erosionMathew

et

al.

-LSB-

-RSB-

Lussi

indexNo

relation

to

the

intake

of

sport

drinksn

with

erosionVan

Rijkom

et

al.

-LSB-

-RSB-

Modified

Lussi

indexNo

relation

to

acidic

drinks

and

fruitsn

with

visible

smooth

wearArnadottir

et

al.

-LSB-

-RSB-

Modified

Lussi

indexNo

significant

association

to

risk

factorsn

grade

grade

grade

et

al.

-LSB-

-RSB-

TWI

-LRB-

Smith

and

Knight

-RRB-

modified

for

erosionNo

significant

association

with

dietary

factorsn

relationship

with

gastro-oesophageal

and

with

any

erosion

on

buccal

and

palatal

surfaces

of

the

incisors

and

first

permanent

molars

resp.Dugmore

and

Rock

-LSB-

-RSB-

TWI

-LRB-

Smith

and

Knight

-RRB-

modified

for

erosionDrinking

fizzy

pop

odds

ratio

depending

on

amount

and

frequencyn

relation

to

eating

apples

citrus

with

erosionMilosevic

et

al.

-LSB-

-RSB-

TWI

-LRB-

Smith

and

Knight

-RRB-

on

labial

and

lingual

surfaces

in

front

teeth

occlusal

surfaces

of

first

molarsNo

association

to

apples

fresh

orangesn

association

-LRB-

OR

-RRB-

to

yoghurt

grapefruit

salad

dressing

vinegar

fruit

juice

fizzy

association

to

herbal/lemon

tea

-LRB-

OR

-RRB-

with

exposed

dentine

There

are

many

points

of

discussion

for

explanation

At

first

-LRB-

erosive

-RRB-

wear

is

the

effect

of

various

concurrent

or

past

chemical

exposures

from

different

sources

the

variety

of

which

can

hardly

be

covered

by

simple

questionnaires

and

especially

in

younger

people

the

dietary

intake

and

lifestyle

might

be

open

to

variations

Furthermore

questionnaires

may

reflect

only

in

part

the

dietary

habits

due

to

interpretation

by

the

responder

or

seasonal

changes

in

daily

life

habits

When

the

young

age

of

the

groups

studied

is

considered

the

determinants

for

dental

erosion

may

not

have

acted

for

long

enough

and

in

addition

there

might

be

considerable

differences

in

the

individual

susceptibility

to

erosive

demineralisation

-LSB-

-RSB-

It

must

however

also

be

taken

into

consideration

that

current

diagnostic

criteria

might

not

be

valid

enough

to

really

reflect

the

effect

of

a

chronic

acid

exposure

Taking

into

account

that

tooth

wear

is

the

result

of

various

factors

the

occlusal

and

incisal

surfaces

of

teeth

are

not

only

exposed

to

acids

but

are

particularly

prone

to

physical

impacts

from

mastication

From

this

background

the

finding

that

many

studies

using

erosion

indices

reveal

that

occlusal

lesions

are

the

most

prevalent

and

that

it

is

the

first

lower

molar

which

is

most

affected

-LSB-

-RSB-

is

of

particular

importance

Occlusal

surfaces

might

be

the

only

location

exhibiting

a

relation

between

lesion

severity

and

prevalence

and

age

-LSB-

-RSB-

thus

indicating

a

significant

contribution

of

abrasion

from

mastication

Furthermore

particularly

the

teeth

from

ancient

remains

exhibit

defects

being

strikingly

of

the

same

shape

as

those

attributed

to

erosion

today

-LRB-

Fig.

-RRB-

Fig.

Occlusal

aspect

of

a

subject

living

on

a

raw

food

diet

with

multiple

acid

impacts

and

a

medieval

subject

b

with

an

assumed

abrasive

diet

-LRB-

images

a

b

and

c

samples

from

-LSB-

-RSB-

-RRB-

Occlusal/incisal

defects

in

a

subject

with

chronic

vomiting

d

and

in

a

medieval

subject

c

The

shape

of

lesions

from

predominantly

erosive

and

predominantly

abrasive

aetiology

is

strikingly

similar

Comparative

studies

on

lesion

characteristics

of

wear

Attempts

have

been

made

to

characterise

the

shape

of

wear

resulting

from

abrasion

and

erosion

-LSB-

-RSB-

A

comparison

of

subjects

with

known

exposure

to

a

non-acidic

but

coarse

diet

and

subjects

with

an

acidic

but

refined

diet

using

silicon

impressions

revealed

that

in

the

former

the

scooped

out

dentine

was

significantly

shallower

than

in

the

latter

Furthermore

the

deepest

lesion

region

in

teeth

from

subjects

with

abrasive

diet

was

located

at

the

functional

cusps

whereas

in

teeth

from

subjects

with

an

erosive

diet

the

deepest

region

of

the

defects

was

located

more

centrally

The

authors

concluded

that

in

cases

when

the

dentine

is

severely

scooped

out

the

causative

agent

is

most

likely

of

erosive

origin

whereas

scooping

tends

to

be

shallower

when

wear

is

caused

by

abrasion

The

conclusion

of

the

study

was

to

suggest

a

quantitative

diagnostic

procedure

by

calculating

the

depth/breadth

ratio

for

clinical

differential

diagnosis

-LSB-

-RSB-

Another

approach

was

to

compare

the

shape

of

defects

occurring

in

subjects

with

substantially

different

nutrition

patterns

-LSB-

-RSB-

The

study

included

three

groups

of

age

matched

individuals

randomly

selected

contemporary

subjects

medieval

remains

and

a

group

of

subjects

living

on

raw

food

diet

From

the

latter

group

extensive

information

was

available

on

their

intake

of

acid

food

qualifying

their

nutrition

as

rather

erosive

For

the

medieval

group

an

abrasive

nutrition

was

assumed

from

knowledge

about

the

general

nature

of

the

early

medieval

diet

As

to

the

smooth

surfaces

the

most

interesting

finding

was

that

in

the

medieval

group

no

lesions

were

observed

-LRB-

Table

-RRB-

Even

in

cases

with

severe

wear

the

lingual

and

buccal

aspects

of

the

teeth

appeared

undisturbed

and

in

many

cases

even

developmental

ridges

were

present

The

lack

of

cervical

defects

is

also

reported

by

Aubry

et

al.

-LSB-

-RSB-

and

Kaidonis

-LRB-

in

this

issue

-RRB-

and

also

inherently

supported

by

the

fact

that

the

overwhelming

number

of

anthropological

studies

on

tooth

wear

only

deal

with

occlusal

and

proximal

wear

and

that

the

indices

used

there

do

not

include

criteria

for

cervical

wear

-LSB-

-RSB-

There

are

however

few

studies

reporting

wear

on

lingual

surfaces

in

archaeological

remains

-LSB-

-RSB-

and

there

is

one

publication

discussing

regurgitation

as

a

possible

aetiological

factor

-LSB-

-RSB-

In

this

study

adult

pre-Conquest

British

skulls

were

examined

using

the

TWI

Indeed

buccal

and

lingual

wear

was

observed

to

the

same

degree

as

in

a

contemporary

comparison

group

but

cervical

wear

did

not

develop

beyond

criterion

in

most

individuals

In

of

the

skulls

however

a

cervical

wear

to

TWI

score

of

occurred

No

information

was

given

about

the

shape

of

the

lesions

and

from

the

images

included

it

appears

that

their

characteristics

would

not

match

the

current

criteria

for

dental

erosion

Table

of

lesions

of

defined

shape

in

three

groups

-LRB-

n

each

-RRB-

of

subjects

with

substantially

different

nutrition

patterns

-LSB-

-RSB-

Abrasive

diet

-LRB-

medieval

group

-RRB-

Acidic

diet

-LRB-

raw

food

group

-RRB-

Average

western

diet

Incisal/occlusal

n.s.

Molars/premolarsShallow

cupping

-LRB-

mm

-RRB-

p

Deep

cupping

-LRB-

mm

-RRB-

p

Smooth

surfaces

-LRB-

all

teeth

-RRB-

Concavity

coronal

to

the

p

p

In

contrast

to

the

findings

in

the

medieval

group

shallow

defects

located

coronal

from

the

CEJ

were

considerably

prevalent

in

the

raw

food

group

-LRB-

Table

Fig.

-RRB-

whereas

in

the

western

diet

group

the

prevalence

of

this

kind

of

lesions

was

much

lower

and

corresponds

to

its

general

prevalence

in

Germany

-LSB-

-RSB-

Fig.

Buccal

aspect

of

teeth

with

significant

loss

of

crown

height

but

without

any

lesion

in

a

medieval

remain

-LSB-

-RSB-

with

severe

generalised

occlusal

wear

c.

b

Occlusal

defects

in

a

subject

living

on

a

raw

food

diet

with

a

high

intake

of

acidic

food

-LSB-

-RSB-

The

shape

of

the

occlusal

lesions

is

similar

to

c

but

combined

with

shallow

lesions

with

intact

cervical

rim

lesions

d

Same

subject

with

Fig.

with

an

initial

buccal

lesion

As

to

the

occlusal/incisal

surfaces

grooving

and

cupping

was

common

in

all

groups

even

though

most

often

in

the

medieval

group

followed

by

the

raw

food

group

and

the

western

diet

group

The

conclusion

from

this

study

was

that

shallow

defects

on

smooth

surfaces

might

be

a

valid

criterion

for

dental

erosion

whereas

cupping

and

especially

incisal

grooving

was

common

in

all

groups

and

therefore

not

valid

for

a

differential

diagnosis

The

similarity

of

occlusal/incisal

defects

in

all

groups

might

be

explained

from

a

tribological

view

-LSB-

-RSB-

Wear

as

a

result

of

abrasion

occurs

as

three

body

wear

that

means

the

intervention

of

an

abrasive

slurry

or

bolus

During

mastication

the

abrasive

tends

to

hollow

out

softer

surface

regions

Cupping

or

grooving

can

occur

when

deeper

enamel

regions

with

lower

microhardness

-LSB-

-RSB-

are

exposed

or

when

the

dentine

is

reached

In

the

case

of

erosion

tissue

loss

is

on

one

hand

caused

by

direct

dissolution

of

mineral

but

also

due

to

an

increased

susceptibility

of

acid

softened

surfaces

to

physical

wear

In

these

cases

also

a

non

or

less

abrasive

bolus

could

cause

similar

defects

when

acting

on

acid

weakened

surfaces

Hence

the

occlusal/incisal

substance

loss

observed

in

individuals

prone

to

dietary

acids

may

be

explained

as

pronounced

abrasion/demastication

of

acid

softened

surfaces

Therefore

it

is

questionable

if

the

occlusal

morphological

criteria

used

for

the

diagnosis

of

occlusal

erosion

per

se

are

valid

Even

though

there

are

often

striking

similarities

between

lesions

of

predominantly

abrasive

or

predominantly

erosive

origin

there

is

on

the

other

hand

often

also

considerable

variation

in

lesion

shape

in

cases

of

erosion

The

morphology

of

occlusal

lesions

in

subjects

with

known

exposure

to

acids

varies

from

deep

hollowing

out

of

the

cusps

to

totally

amorphous

loss

of

the

occlusal

structure

even

in

subjects

with

a

similar

dietary

history

-LRB-

Fig.

-RRB-

Fig.

tissue

loss

from

erosive

aetiology

can

also

be

of

strikingly

different

shape

either

presenting

as

deeply

hollowed

out

lesions

-LRB-

a

subject

with

raw

food

diet

-LSB-

-RSB-

b

subject

with

excessive

consumption

of

orange

juice

-RRB-

or

as

amorphous

generalised

tissue

loss

affecting

the

entire

surface

-LRB-

c

d

subjects

with

excessive

consumption

of

erosive

drinks

-RRB-

An

interesting

feature

is

seen

in

an

adolescent

with

a

history

of

severe

anterior

open

bite

with

only

the

molars

being

in

function

e

Substance

loss

occurred

from

excessive

consumption

of

a

cola

type

drink

In

the

premolars

dentine

is

proud

of

the

surface

f

Hollowing

out

the

entire

occlusal

surface

with

enamel

remnants

in

the

centre

aetiology

is

the

excessive

consumption

of

sport

drinks

Conclusion

The

validity

of

current

diagnostic

criteria

for

dental

erosion

has

not

been

systematically

studied

even

though

there

is

consensus

about

their

definition

The

following

conclusions

can

be

drawn

Grooving

of

incisal

surfaces

is

a

common

phenomenon

and

possibly

the

effect

of

any

physical

or

chemical

impact

It

should

be

considered

to

abandon

grooving

of

anterior

teeth

and

canines

as

a

clinical

criterion

for

dental

erosion.Shallow

defects

located

coronal

from

the

CEJ

may

predominantly

occur

as

effect

of

chronic

acid

exposure

and

might

be

pathogonomic

for

dental

erosion

This

assumption

is

supported

by

the

finding

that

these

types

of

lesions

are

not

present

in

ancient

remains

even

in

cases

of

severe

wear.Cupping

of

cusps

is

the

most

uncertain

criterion

because

it

can

be

an

effect

of

abrasion

as

well

as

of

erosion

In

industrialised

countries

abrasion

is

not

expected

to

be

a

significant

factor

in

young

people

Cupping

occurring

at

younger

ages

can

therefore

be

an

effect

of

erosion

At

older

ages

however

physical

and

chemical

impacts

add

up

increasingly

and

cupping

will

therefore

be

of

little

diagnostic

value

in

adults

These

conclusions

are

drawn

from

very

few

studies

therefore

systematic

research

on

this

issue

is

needed

Nevertheless

there

is

enough

support

for

a

criticism

of

current

diagnostic

criteria

particularly

in

the

light

of

the

development

of

a

new

index

O O O O O B O O B O O O O O O O O O O O B O O B 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 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 B 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 O B O O O O B O O O O O O O O O O O O O O O O O B O O O O O O B O B O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O B O O B O O B O O O O O O O O O B 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 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 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 O O O O O O B 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 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 B 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 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 B 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 O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B 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 B O O O O O O O O O O O O O B O O O O O O B 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 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 B O O B O O O B 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 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 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 B O O O B O B O O O O O O O O O O B 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 B O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O 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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 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 B O O B O O O O O O O O O O O O O O O B 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 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 B O B 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 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 O O O O O O O O O O O B O O B 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O B 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 O O O O O O O O O O O B 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 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 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O B 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 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 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 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 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 B 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 B 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 O O O O O O O O O O O O O O O O B 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 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 B 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 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 O O O O O O O O O O B 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 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 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 O O O O O O O O B 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O B 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 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 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 O O B O O B 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 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 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 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 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 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 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 B O O O O O O O O O O O O O O O O O O O O O B 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 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 B 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 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 B O O O B O O B 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 B 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 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 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 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 B O O O B O O B 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 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 B 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 B 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 B 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 O O O O O O O O O O B O O O O O O O O O O O O


Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4 -- 5 days . In our previous study , we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase -LRB- gs -RRB- as one of the most abundantly expressed genes during this process . In the present study , we show that the full-length sequence of E. japonensis glutamine synthetase -LRB- EjGS -RRB- , which is the first reported annelid glutamine synthetase , is highly similar to other known class II glutamine synthetases . EjGS shows a 61 -- 71 % overall amino acid sequence identity with its counterparts in various other animal species , including Drosophila and mouse . We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation . gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema . Furthermore , in the elongated blastema , gs expression was detectable also in the presumptive regions of the brain , ventral nerve cord , and stomodeum . In the fully formed intact head , gs expression was also evident in the prostomium , brain , the anterior end of the ventral nerve cord , the epithelium of buccal and pharyngeal cavities , the pharyngeal pad , and in the esophageal appendages . In intact E. japonensis tails , gs expression was found in the growth zone in actively growing worms but not in full-grown individuals . In the nonblastemal regions of regenerating fragments and in intact worms , gs expression was also detected in the nephridia , chloragocytes , gut epithelium , epidermis , spermatids , and oocytes . These results suggest that EjGS may play roles in regeneration , nerve function , cell proliferation , nitrogenous waste excretion , macromolecule synthesis , and gametogenesis . Introduction Some types of annelids exhibit remarkable regenerative abilities that are comparable to planarians . However , the regeneration mechanisms are thought to be quite different between planarians and annelids . Whereas planarians regenerate via totipotent stem cells -LRB- neoblasts -RRB- that are widely distributed throughout their bodies -LRB- Reddien and Alvarado 2004 -RRB- , annelid regeneration is thought to occur mainly through the dedifferentiation and subsequent redifferentiation of cells without any contribution from totipotent stem cells -LRB- Thouveny and Tassava 1998 -RRB- . In this respect , annelids regenerate in a manner similar to that of amphibians -LRB- Carlson 1998 -RRB- but on a much larger scale . The elucidation of the annelid regeneration mechanisms is thus expected to provide valuable information that may allow us in the future to explore strategies to enhance the regenerative capabilities in vertebrates . In our recent study , we have proposed the oligochaete annelid Enchytraeus japonensis as a new model system for regeneration studies -LRB- Myohara et al. 1999 -RRB- . E. japonensis has striking features that are highly advantageous in this regard : -LRB- 1 -RRB- It reproduces asexually by dividing its body into several fragments , which then regenerate a complete individual within 4 -- 5 days , -LRB- 2 -RRB- artificially amputated fragments can also regenerate new individuals in the same manner , and -LRB- 3 -RRB- both asexual and sexual reproduction can be artificially induced in the laboratory -LRB- Myohara et al. 1999 -RRB- . As an initial strategy toward the elucidation of the molecular mechanisms underlying annelid regeneration , we previously performed complementary deoxyribonucleic acid -LRB- cDNA -RRB- subtraction cloning using regenerating fragments of E. japonensis and intact animals -LRB- Myohara et al. 2006 -RRB- . Filter array screening subsequently revealed that about 38 % of the forward-subtracted cDNA clones we isolated contained genes that were upregulated during regeneration . These clones hybridized with probes derived from regenerating fragments but not with probes from intact worms . Of these clones , 279 were then sequenced and found to contain 165 different sequences -LRB- 79 known and 86 unknown -RRB- . Among these 279 regeneration-upregulated clones , as many as 13 -LRB- 4.7 % -RRB- were found to encode glutamine synthetase , a ubiquitous and key enzyme in nitrogen metabolism -LRB- Kumada et al. 1993 -RRB- . Because of its abundance in our subtracted cDNA library , we cloned the full-length sequence of E. japonensis glutamine synthetase -LRB- EjGS -RRB- . By reverse transcriptase -- polymerase chain reaction -LRB- RT-PCR -RRB- , we subsequently revealed that the expression levels of the glutamine synthetase gene -LRB- gs -RRB- were consistently high during the regenerative process , with a gradual decline seen only in the very late stages , and our preliminary analysis by whole mount in situ hybridization further revealed strong gs expression in the blastemal tissues of the regenerating fragments -LRB- Myohara et al. 2006 -RRB- . As these previous results suggested an important role for gs in annelid regeneration , in the present study , we have examined the detailed expression pattern of gs in regenerating fragments and intact worms and considered the possible roles of this enzyme in the biology of E. japonensis . This is the first detailed report of the sequence and expression of gs in annelids . Materials and methods Animals E. japonensis worms were reared at 24 °C in 1.1 % -LRB- w/v -RRB- plain agar medium in disposable Petri dishes and were fed with rolled oats as previously described -LRB- Myohara 2004 -RRB- . Under these conditions , the worms grow continuously to about 10 mm long , consisting of 50 -- 60 segments , and reproduces asexually by fragmentation every 2 weeks . To induce sexual reproduction , starved worms were cultured in wet leaf mold and refed with rolled oats as previously described -LRB- Myohara 2004 -RRB- . Molecular analysis The full-length cDNA of EjGS -LRB- accession number AB109095 -RRB- has been isolated in our previous study -LRB- Myohara et al. 2006 -RRB- . Multiple alignments of the entire amino acid sequences of the glutamine synthetase proteins from representative species , which were obtained through GenBank/EMBL/DDBJ Data Bank and chosen to cover a wide range of phylogenic groups , were performed using ClustalW software -LRB- Thompson et al 1994 -RRB- . To avoid errors resulting from mixing of possible pseudogenes , only data originating from full-length messenger ribonucleic acid -LRB- mRNA -RRB- sequences were used . In situ hybridization Both sense and antisense digoxigenin -LRB- DIG -RRB- - labeled RNA probes were synthesized using the DIG RNA labeling kit -LRB- SP6/T7 , Roche -RRB- as previously described -LRB- Myohara et al. 2006 -RRB- . Whole-mount in situ hybridizations were also performed as previously described -LRB- Myohara et al. 2006 -RRB- with some modifications to enhance the signals . Briefly , hybridization was carried out overnight with 1 μg/ml of gs DIG-labeled RNA probe at 55 °C . An anti-DIG alkaline phosphatase -LRB- AP -RRB- conjugate antibody -LRB- Roche -RRB- was preabsorbed by incubation for 2.5 h with fixed and rehydrated worms and used at a dilution of 1:2,000 . In situ hybridization of paraffin sections -LRB- 6 μm -RRB- was carried out according to Hoshino et al. -LRB- 1999 -RRB- and Abe et al. -LRB- 2003 -RRB- with a few modifications . Briefly , sections were hybridized with DIG-labeled probes at a concentration of 100 ng/ml in probe diluent -LRB- Genostaff , Tokyo , Japan -RRB- at 60 °C for 16 h. Washes were then carried out using 5 × Hybri-wash -LRB- Genostaff -RRB- , and an anti-DIG AP conjugate antibody -LRB- Roche Applied Science , Tokyo , Japan -RRB- was then applied at a 1:1,000 dilution in Tris-buffered saline with 0.1 % Tween 20 for 2 h . After an overnight coloring reaction with nitro blue tetrazolium/5-bromo -4-chloro-3-indolyl-phosphate -LRB- Roche -RRB- , the sections were counterstained with a Kernechtrot stain solution -LRB- Muto Pure Chemicals , Tokyo , Japan -RRB- , dehydrated , and mounted in Malinol -LRB- Muto Pure Chemicals -RRB- . Results and discussion Comparisons between E. japonensis glutamine synthetase and other known glutamine synthetases A full-length 2,717-bp cDNA encoding gs was previously isolated from regenerating E. japonensis in our laboratory -LRB- Myohara et al. 2006 -RRB- . The sequence of this gene was found to contain a putative open reading frame of 360 amino acids with a calculated molecular mass of 40,509 Da . Because this was the first report in annelids of a full-length sequence for gs , we performed a database search in the present study to determine whether similarities existed with the gs genes from other organisms . BLASTP analysis revealed that the deduced glutamine synthetase protein in E. japonensis -LRB- EjGS -RRB- was highly similar to known class II glutamine synthetases along its entire amino acid sequence -LRB- Fig. 1 -RRB- , which is well conserved across species -LRB- Kumada et al. 1993 -RRB- . Within the EjGS sequence , we identified a beta-grasp -LRB- catalytic -RRB- and a noncatalytic domain , five conserved regions that are common to both prokaryotes and eukaryotes , ligands for Mn2 + ions , and an adenosine triphosphate-binding site -LRB- Pesole et al. 1991 -RRB- . An amino acid alignment between EjGS and the gs proteins from five invertebrates , sea anemone -LRB- Cnidaria -RRB- , C. elegans -LRB- Nematoda -RRB- , Pacific oyster -LRB- Mollusca -RRB- , fruit fly -LRB- Arthropoda -RRB- , sea urchin -LRB- Echinodermata -RRB- , and four vertebrates -LRB- zebrafish , clawed frog , chicken , and mouse -RRB- showed that the overall amino acid identities between EjGS and its counterparts in various other animal species were within a range of 61 -- 71 % -LRB- Fig. 1 -RRB- . Fig. 1Amino acid alignment of E. japonensis glutamine synthetase -LRB- EjGS -RRB- with representative GSs from various other species . The species and protein ID of the GSs listed here are as follows : Enchytraeus japonensis -LRB- BAE93509 .1 -RRB- , sea anemone Aiptasia pallida -LRB- AAR36878 .1 -RRB- , nematode Caenorhabditis elegans -LRB- NP_001041010.1 -RRB- , Pacific oyster Crassostea gigas -LRB- CAD90162 .1 -RRB- , fruit fly Drosophila melanogaster -LRB- AAZ41780 .1 -RRB- , sea urchin Paracentrotus lividus -LRB- AAC41562 .1 -RRB- , zebrafish Danio rerio -LRB- AAH66735 .1 -RRB- , clawed frog Xenopus laevis -LRB- AAH46681 .1 -RRB- , chicken Gallus gallus -LRB- AAA48783 .1 -RRB- , and mouse Mus musculus -LRB- AAH15086 .1 -RRB- . Amino acid residues are indicated in blue when greater than or equal to 70 % of these residues are identical in the ten representative species . The beta grasp domain , catalytic domain , and the regions that are conserved in prokaryotes and eukaryotes are underlined by broken single lines , broken double lines , and thick single lines , respectively . Ligands for Mn2 + ions are indicated by arrowheads . The ATP-binding sites are boxed . The protein length and overall amino acid identities with EjGS are indicated at the end of each sequence Expression of the gs gene in the blastema of regenerating E. japonensis By whole-mount in situ hybridization analysis , we detected strong gs expression in the blastemal region in regenerating fragments of E. japonensis -LRB- Fig. 2a -RRB- . This expression was detectable from the early stages of the regenerative process in the cell layer covering the wound -LRB- Fig. 2b , k -RRB- and was found to persist in the epidermal cells during the formation and elongation of the blastema -LRB- Fig. 2c , d -RRB- . There was also a notable concentration of this transcript in the region corresponding to the prostomium anlagen in the anterior blastema -LRB- Fig. 2e -RRB- and in the growth zone in the posterior blastema -LRB- Fig. 2l -RRB- . In situ hybridization analyses with paraffin-embedded sections additionally revealed that the gs expression pattern was mainly restricted to the epidermal cells in the early blastema -LRB- Fig. 2h -RRB- but did occur also in the inside tissues of the elongated blastema , where it was detected in the presumptive regions of the brain , ventral nerve cord , and buccal cavity -LRB- stomodeum -RRB- , in addition to the prostomium and epidermal cells -LRB- Fig. 2i -RRB- . Fig. 2Glutamine synthetase mRNA expression during regeneration in E. japonensis . Transcripts were detected using whole-mount in situ hybridization -LRB- a -- g and k -- o -RRB- or in situ hybridization analysis of paraffin-embedded sections -LRB- h -- j -RRB- using an antisense gs riboprobe , with the exception of the lower specimen shown in a . The anterior is to the left , and the ventral side is down in each image . Arrowheads indicate the amputated sites . a Whole-body images of regenerating fragments at about 1 day after amputation with the anterior blastema and an intact tail . The lower specimen was hybridized with a sense probe . b -- f Lateral views of anterior blastemas at about 1 -LRB- b -RRB- , 1.5 -LRB- c -RRB- , 2 -LRB- d -RRB- , 2.5 -LRB- e -RRB- , and 4 days -LRB- f -RRB- after amputation . g Lateral view of an intact head . h -- i Sagittal sections of anterior blastemas at about 1.5 -LRB- h -RRB- and 2 days -LRB- i -RRB- after amputation . j Sagittal section of an intact head . k -- m Lateral views of posterior blastemas at about 0.5 -LRB- k -RRB- , 1 -LRB- l -RRB- , and 1.5 days -LRB- m -RRB- after amputation . n -- o Ventral views of intact tails in an actively growing -LRB- n -RRB- and a full-grown individual -LRB- o -RRB- . ba Brain anlage , br brain , bc buccal cavity , ch chloragocyte , ea esophageal appendage , ep epidermal cell , es esophagus , gl gut lumen , gz growth zone , m mouth , pc pharyngeal cavity , pp pharyngeal pad , pr prostomium , py pygidium , st stomodeum , va ventral nerve cord anlage , vn ventral nerve cord . Scale bars , 50 μm In the later stages of E. japonensis regeneration , by which time segmentation and organogenesis have almost completed , we observed that the gs expression levels in the blastemal regions had become reduced -LRB- Fig. 2f , m -RRB- . In the fully formed intact head , however , the expression of this gene was detectable in the prostomium , brain , anterior end of the ventral nerve cord , epithelial cells lining the buccal and pharyngeal cavities , pharyngeal pad , and esophageal appendages -LRB- Fig. 2g , j ; for E. japonensis morphology , see Schmelz et al. 2000 -RRB- . In intact tails , gs expression was also evident in the growth zone in actively growing worms -LRB- Fig. 2n -RRB- but was absent in full-grown individuals in which the growth zone had become indistinguishable -LRB- Fig. 2o -RRB- . These observations are in agreement with the RT-PCR data included in our previous report , which showed that the expression levels of gs were maintained at high levels during the early stages of regeneration , decreased temporarily in the later stages of this process , and were upregulated again in the fully formed intact worms -LRB- Myohara et al. 2006 -RRB- . Expression of the gs gene in nonblastemal regions In accordance with the abovementioned RT-PCR results , we found by in situ hybridization analysis in the present study that gs expression was not restricted to the blastemal regions -LRB- Figs. 2a and 3a -RRB- but that less intense but clear expression could be observed also in the nephridia -LRB- Fig. 3a , b -RRB- , chloragocytes -LRB- Figs. 2a , 3a -- c -RRB- , gut epithelium -LRB- Fig. 3c -RRB- , epidermis of the nonblastemal regions of regenerating fragments -LRB- Fig. 3a -RRB- , and also in intact worms -LRB- Fig. 3d , e -RRB- . Nephridia are the excretory organs of oligochaete annelids and the chloragocytes -LRB- chloragogen cells -RRB- are specialized peritoneal cells of the intestine that participate in the metabolism , storage , and excretion of glycogen and lipids , thus resembling the vertebrate liver in terms of function -LRB- Jamieson 1981 ; Edwards and Bohlen 1996 -RRB- . The gs expression levels in these tissues were observed to be variable among the specimens examined and also among regions within the same specimen -LRB- Figs. 2a and 3c -RRB- . We were unable to find any correlation between this pattern and a specific gut region or specific condition of our specimens , although an uneven distribution of glutamine synthetase has been reported previously in the mammalian and fish digestive tracts -LRB- James et al. 1998 ; Mommsen et al. 2003 -RRB- . Intense gs expression was also found in the spermatids in the seminal vesicles and in oocytes in the ovisacs in sexually mature worms -LRB- Fig. 3e -RRB- . Fig. 3Glutamine synthetase mRNA expression in nonblastemal tissues . Transcripts were detected by in situ hybridization on paraffin-embedded sections -LRB- a -- c and e -RRB- or by whole-mount in situ hybridization -LRB- d -RRB- using an antisense gs riboprobe , except for the right-hand specimen in a . The anterior is to the left , and the ventral side is down in each image . a Sagittal sections of regenerating fragments at about 0.5 days after amputation . The right-hand specimen was hybridized with a sense probe . Arrowheads indicate the amputated sites . b -- c Sagittal sections of intact worms . d Whole-mount in situ hybridization analysis of an intact worm showing gs expression in the epidermal cells . e Sagittal section of a sexually mature worm . ch Chloragocyte , ep epidermal cell , ge gut epithelium , gl gut lumen , ne nephridium , oo oocyte , sc spermatocyte , st spermatid , sp sperm . Scale bars , 50 μm General comparisons between the distribution of glutamine synthetase in different organisms Glutamine synthetase is found in a wide range of organisms , including microorganisms , plants , and animals and is known to be expressed in a tissue-specific and developmentally controlled manner . In murine organs , glutamine synthetase activity is highest in the liver , followed in descending order by the epididymis , testes , uterine tube , stomach , kidney , brain , and adipose tissues -LRB- van Straaten et al. 2006 -RRB- . High glutamine synthetase expression in the brain , kidney , and liver has also been reported in fish -LRB- Murray et al. 2003 -RRB- . In Xenopus , gs transcripts have been detected in the ovary , kidney , eye , muscle , and embryo but not in the brain , heart , liver , stomach , or testis -LRB- Hatada et al. 1995 -RRB- . In the case of invertebrates , little information is currently available regarding the expression profile of this enzyme and is restricted to arthropods ; gs expression has been reported in mosquito midgut epithelium , ovary , and fat body -LRB- Smartt et al. 2001 -RRB- and in crustacean neural tissues -LRB- Linser et al. 1997 ; Allodi et al. 2006 ; Sullivan et al. 2007 -RRB- . Our present study , therefore , provides novel information regarding the expression of this enzyme in nonarthropod invertebrates , and our results are in general agreement with those of previous reports ; that is , in full-grown E. japonensis , gs is expressed in -LRB- 1 -RRB- chloragogen tissues , which correspond to the vertebrate liver in terms of functionality -LRB- Jamieson 1981 -RRB- , -LRB- 2 -RRB- nephridia , which are the annelid counterparts of the kidney and uterine tube in vertebrates -LRB- Jamieson 1981 -RRB- , -LRB- 3 -RRB- the brain and ventral nerve cord , -LRB- 4 -RRB- the prostomium where large numbers of sensory cells including photoreceptor cells are present -LRB- Jamieson 1981 ; Edwards and Bohlen 1996 -RRB- , -LRB- 5 -RRB- the epithelium of the digestive tract , and -LRB- 6 -RRB- spermatids and oocytes . Possible functions of glutamine synthetase in E. japonensis Regeneration The possible involvement of glutamine synthetase in the regeneration of damaged tissue has been reported in only a few cases . These include the observed increase in gs expression after peripheral motor nerve injury in mouse -LRB- Toki et al. 1998 -RRB- and also in Xenopus tail regeneration in which gs expression was found to be upregulated and could be detected in the terminal vesicle of the regenerating spiral cord -LRB- Tazaki et al. 2005 -RRB- . Our present results , together with our previously reported findings , strongly suggest the involvement of glutamine synthetase also in annelid regeneration . In the previous study , we have shown that glutamine synthetase is one of the most abundantly expressed upregulated genes during regeneration in E. japonensis -LRB- Myohara et al. 2006 -RRB- , and in the present study , our detailed expression analysis by in situ hybridization has revealed strong gs expression in the blastemal regions of regenerating E. japonensis . It has been reported previously in Xenopus that embryos abundantly express glutamine synthetase in the developing nervous system during the gastrula and neurula stages -LRB- Hatada et al. 1995 -RRB- . In accordance with this finding , we observed in the present analyses that gs is expressed in the presumptive regions of the brain and ventral nerve cord anlagen in the blastema during E. japonensis regeneration . This further suggests the involvement of glutamine synthetase in annelid neural regeneration . Nerve function Glutamine synthetase has been shown to play an important role in glutamate transmitter degradation in the nervous system -LRB- Derouiche and Frotscher 1991 -RRB- and is used as a specific glial cell marker in the neural retina -LRB- Vardimon et al. 1993 -RRB- . In intact E. japonensis , the expression of this enzyme was evident in the brain and ventral nerve cord , suggesting that it has a nervous system function . Its expression in the prostomium may also be attributable to the distribution of numerous sensory cells , probably including photoreceptor cells , in this region -LRB- Jamieson 1981 ; Edwards and Bohlen 1996 -RRB- . Cell proliferation Glutamine synthetase is the only enzyme that can synthesize glutamine , which provides nitrogen for the synthesis of amino acids , purines , pyrimidines , and other compounds required in many critical cellular events including proliferation -LRB- Meister 1980 -RRB- . In the rat intestinal epithelium , high concentrations of glutamine synthetase protein and mRNA have been found in the highly proliferative crypt region -LRB- Roig et al. 1995 -RRB- . Hence , the high gs expression levels that we observed in blastemal cells and in the growth zone in actively growing worms could be explained , at least in part , by a high demand for glutamine during the intense cell proliferation that would be ongoing in these areas . Detoxification Glutamine synthetase is also important for the detoxification of excess glutamate and ammonia by converting them to glutamine , which is nontoxic and can be stored in tissues or circulate throughout the body without causing any harm -LRB- Meister 1980 -RRB- . A comparison between the cellular concentrations of glutamine synthetase in various murine organs in a previous report has also revealed that the highest concentrations of this enzyme positively correlate with the cells that have detoxifying functions -LRB- van Straaten et al. 2006 -RRB- . The gs expression that we observed in the nephridia and chloragocytes in E. japonensis herein could therefore be ascribed to the involvement of these tissues in excretion of nitrogenous waste in oligochaete annelids -LRB- Jamieson 1981 -RRB- . Macromolecule synthesis Glutamine is known to be required for the synthesis of certain macromolecules that are present in mucus , such as the hexosamines that lubricate and protect the gut mucus -LRB- Weiss et al. 1999 -RRB- . The mucus secreted by epidermal cells covering the earthworm body is also essential for locomotion , gas exchange , and protection in these animals -LRB- Jamieson 1981 -RRB- , and hexosamines have been found to be a component of their cuticles -LRB- Muir and Lee 1970 -RRB- . We speculate therefore that the gs transcripts in the E. japonensis epidermal cells have a role in mucus and/or cuticle synthesis in intact and regenerating individuals . In addition , the gs expression found in epithelial cells of the buccal and pharyngeal cavities may be attributed to cuticle synthesis in these tissues -LRB- Jamieson 1981 -RRB- . Gametogenesis Glutamine synthetase expression in the ovary has been reported in Xenopus -LRB- Hatada et al. 1995 -RRB- and in the yellow fever mosquito -LRB- Smartt et al. 2001 -RRB- . In oligochaete annelids , the testes and ovaries are small organs located in the ventral part of the genital segments -LRB- Edwards and Bohlen 1996 -RRB- . The spermatogonia that are formed in the annelid testes pass into the seminal vesicles , which contain male cells at all stages of development . Oogonia in annelids are formed in the ovaries and divide to form oocytes , which are shed from the ovaries into the ovisacs . In E. japonensis , gs transcripts were detectable in spermatids in the seminal vesicles and in oocytes in the ovisacs but not in the testes or ovaries , suggesting that glutamine synthetase may have a role in the later stage of gametogenesis . In summary , in the present study , we have characterized an annelid gs gene and shown that it is expressed in a tissue-specific manner in regenerating and intact E. japonensis . To further elucidate the actual functions of this gene , additional studies including RNAi gene knockdown experiments will be needed in the future . Ongoing investigations in our laboratory of other regeneration-associated upregulated genes , several of which are novel -LRB- Myohara et al. 2006 -RRB- , should also shed important new light on the network of biological pathways involved in the regenerative processes in E. japonensis .

B I B I O O B O O O B I B I O O O O B I 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 B O O B I 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 B I O O O O O O O O O O B I 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 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 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 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 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 O O O B 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 B 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 B 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 O O O O O O O O O O O B 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 B I B I O O O O O O B 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 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 O O O O O O O B 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 O O O O B 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 O O O O O O O O O O O O O B I 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 B I O O O O O O O O O O O O O O O O O O O O O O O O B I 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 O O O O O O O O O O O O O O O O O O O O B 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 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 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 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 B I 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 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 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 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 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 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 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 O O O O O O O O O O O O O B I 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 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 O O O O O O B I 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 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 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 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 B I 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 B I 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 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 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 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 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 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 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 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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 O O O O O O O O O O O O O O O O O B 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 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 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 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 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 O O O O O O O O O O O O O O O O O O B I 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 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 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 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 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 B I O O O B I 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 B I 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 B I 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 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 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 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 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 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 B I O O O B O O O O B I O O B 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 B 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 B I O O O B 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glutamine synthetase
gene expression
regeneration
oligochaete annelid
enchytraeus japonensis