bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamate-binding affinity ofDrosophilametabotropic glutamate receptor is modulated by association with lipid rafts
Authors: Eroglu Çagla , Brügger Britta , Wieland Felix , Sinning Irmgard
Published: September 2003

Metabotropic glutamate receptors (mGluRs) are responsible for the effects of glutamate in slow synaptic transmission, and are implicated in the regulation of many processes in the CNS. Recently, we have reported the expression and purification of a mGluR fromDrosophila melanogaster(DmGluRA), a homologue of mammalian group II mGluRs. We have shown that ligand binding to reconstituted DmGluRA requires the presence of ergosterol in the liposomes [Eroglu, C., Cronet, P., Panneels, V., Beaufils, P. & Sinning, I. (2002)EMBO Rep.3, 491-496]. Here we demonstrate that the receptor exists in different affinity states for glutamate, depending on the membrane composition. The receptor is in a high-affinity state when associated with sterol-rich lipid microdomains (rafts), and in a low-affinity state out of rafts. Enrichment of the membranes with cholesterol shifts the receptor into the high-affinity state, and induces its association with rafts. The receptor was crosslinked to photocholesterol. Our data suggest that sterol-rich lipid rafts act as positive allosteric regulators of DmGluRA.
Fatty acid biosynthesis in
Mycobacterium tuberculosis
: Lateral gene transfer, adaptive evolution, and gene duplication
Authors: Kinsella Rhoda J. , Fitzpatrick David A. , Creevey Christopher J. , McInerney James O.
Published: September 2003

Mycobacterium tuberculosis
is a high GC Gram-positive member of
the actinobacteria. The mycobacterial cell wall is composed of a complex
assortment of lipids and is the interface between the bacterium and its
environment. The biosynthesis of fatty acids plays an essential role in the
formation of cell wall components, in particular mycolic acids, which have
been targeted by many of the drugs used to treat
M. tuberculosis
infection.
M. tuberculosis
has ≈250 genes involved in fatty acid
metabolism, a much higher proportion than in any other organism.
In
silico
methods have been used to compare the genome of
M.
tuberculosis
CDC1551 to a database of 58 complete bacterial genomes. The
resulting alignments were scanned for genes specifically involved in fatty
acid biosynthetic pathway I. Phylogenetic analysis of these alignments was
used to investigate horizontal gene transfer, gene duplication, and adaptive
evolution. It was found that of the eight gene families examined, five of the
phylogenies reconstructed suggest that the actinobacteria have a closer
relationship with the α-proteobacteria than expected. This is either due
to either an ancient transfer of genes or deep paralogy and subsequent
retention of the genes in unrelated lineages. Additionally, adaptive evolution
and gene duplication have been an influence in the evolution of the pathway.
This study provides a key insight into how
M. tuberculosis
has
developed its unique fatty acid synthetic abilities.
Conjugative coupling proteins interact with cognate and heterologous VirB10-like proteins while exhibiting specificity for cognate relaxosomes
Authors: Llosa Matxalen , Zunzunegui Sandra , de la Cruz Fernando
Published: September 2003

Conjugative coupling proteins (CPs) are proposed to play a role in connecting the relaxosome to a type IV secretion system (T4SS) during bacterial conjugation. Here we present biochemical and genetic evidence indicating that the prototype CP, TrwB, interacts with both relaxosome and type IV secretion components of plasmid R388. The cytoplasmic domain of TrwB immobilized in an affinity resin retained TrwC and TrwA proteins, the components of R388 relaxosome. By using the bacterial two-hybrid system, a strong interaction was detected between TrwB and TrwE, a core component of the conjugative T4SS. This interaction was lost when the transmembrane domains of either TrwB or TrwE were deleted, thus suggesting that it takes place within the membrane or periplasmic portions of both proteins. We have also analyzed the interactions with components of the related IncN plasmid pKM101. Its CP, TraJ, did not interact with TrwA, suggesting a highly specific interaction with the relaxosome. On the other side, CPs from three different conjugation systems were shown to interact with both their cognate TrwE-like component and the heterologous ones, suggesting that this interaction is less specific. Mating experiments among the three systems confirmed that relaxosome components need their cognate CP for transfer, whereas T4SSs are interchangeable. As a general rule, there is a correlation between the strength of the interaction seen by two-hybrid analysis and the efficiency of transfer.
Short- and long-range effects of
Sonic hedgehog
in limb development
Authors: Dillon Robert , Gadgil Chetan , Othmer Hans G.
Published: September 2003

The secreted protein Sonic hedgehog (Shh) and its transmembrane receptor Patched (Ptc) control a major signal transduction pathway in early vertebrate limb development. Ligand-free Ptc interacts with the transmembrane protein Smoothened (Smo) and blocks expression of Smo-controlled genes including
ptc
. Ligand-bound Ptc removes the block and leads to further expression of
ptc
, which in turn restricts the range of Shh transport. Currently it is not certain that Shh functions as a morphogen on the 300-μm scale of early chick limb development, because it has been difficult to determine how far different forms of Shh are transported. We develop a model to study the effects of two forms of Shh used experimentally and propose a mechanism for Shh signal transduction based on a two-state model for the Ptc-Smo interaction. Recent bead- and tissue-implant experiments can be explained by using this model without postulating different diffusivities for the two forms of Shh; a difference in other parameters such as the rate of release of Shh from the bead or transplant can explain the results equally well. The model also predicts that lower concentrations of Shh in a bead will produce a response similar to that after a tissue transplant. Our results provide an explanation for the counterintuitive experimental results and show that the same signal transduction mechanism can explain both short- and long-range Shh signaling. We conclude that Shh can function as a long-range morphogen.
Degradation of p57

Kip2

mediated by SCF
Skp2
-dependent ubiquitylation
Authors: Kamura Takumi , Hara Taichi , Kotoshiba Shuhei , Yada Masayoshi , Ishida Noriko , Imaki Hiroyuki , Hatakeyama Shigetsugu , Nakayama Keiko , Nakayama Keiichi I.
Published: September 2003

The abundance of the cyclin-dependent kinase (CDK) inhibitor
p57

Kip2

, an important regulator of cell cycle progression,
is thought to be controlled by the ubiquitin-proteasome pathway. The
Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCF
Skp2
has
now been shown to be responsible for regulating the cellular level of
p57

Kip2

by targeting it for ubiquitylation and
proteolysis. The elimination of p57

Kip2

was impaired in
Skp2
-/-
cells, resulting in abnormal accumulation of the
protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with
p57

Kip2
in vivo

. Overexpression of WT Skp2 promoted
degradation of p57

Kip2

, whereas expression of a dominant
negative mutant of Skp2 prolonged the half-life of
p57

Kip2

. Mutation of the threonine residue (Thr-310) of
human p57

Kip2

that is conserved between the COOH-terminal
QT domains of p57

Kip2

and p27

Kip1

prevented the effect of Skp2 on the stability of p57

Kip2

,
suggesting that phosphorylation at this site is required for
SCF
Skp2
-mediated ubiquitylation. Finally, the purified recombinant
SCF
Skp2
complex mediated p57

Kip2

ubiquitylation
in vitro
in a manner dependent on the presence of the cyclin E-CDK2
complex. These observations thus demonstrate that the SCF
Skp2
complex plays an important role in cell-cycle progression by determining the
abundance of p57

Kip2

and that of the related CDK inhibitor
p27

Kip1

.
S-adenosyl-
l
-methionine is an effector in the posttranscriptional autoregulation of the cystathionine γ-synthase gene in
Arabidopsis
Authors: Chiba Yukako , Sakurai Ryoko , Yoshino Michiko , Ominato Kimihiro , Ishikawa Mari , Onouchi Hitoshi , Naito Satoshi
Published: September 2003

Cystathionine γ-synthase, the first committed enzyme of methionine
biosynthesis in higher plants, is encoded by the
CGS1
gene in
Arabidopsis thaliana
. We have shown previously that the stability of
the
CGS1
mRNA is negatively regulated in response to methionine
application [Chiba, Y., Ishikawa, M., Kijima, F., Tyson, R. H., Kim, J.,
Yamamoto, A., Nambara, E., Leustek, T., Wallsgrove, R. M. & Naito, S.
(1999)
Science
286, 1371-1374]. To determine whether methionine
itself is the effector of the
CGS1
exon 1-mediated
posttranscriptional regulation, we carried out transfection experiments. The
results suggested that, rather than methionine,
S
-adenosyl-
l
-methionine (AdoMet), or one of its
metabolites, acts as the effector of this regulation. To further identify the
actual effector, we exploited the wheat germ
in vitro
translation
system. The effects of various metabolites and analogs of AdoMet were tested
by using RNA carrying a
CGS1
exon 1-reporter fusion. These tests
identified AdoMet as the effector of this regulation.
S
-adenosyl-
l
-ethionine, an analog of AdoMet, also had
effector activity.
A. thaliana mto1
mutants, which are deficient in
this regulation, showed a much reduced response to AdoMet
in vitro
,
with a leaky allele showing a less reduced response. RNA translated
in
vitro
in the presence of AdoMet contained a 5′-truncated RNA
species, similar to the one that we previously suggested was an
in
vivo
degradation intermediate of
CGS1
mRNA. Together, the
results show that the basic reactions of
CGS1
exon 1-mediated
posttranscriptional regulation occur in the wheat germ
in vitro
translation system, and that AdoMet acts as the effector.
Specific sequence changes in multiple transcript system
DYT3
are associated with X-linked dystonia parkinsonism
Authors: Nolte Dagmar , Niemann Stephan , Müller Ulrich
Published: September 2003

X-linked dystonia parkinsonism (XDP) is an X-linked recessive adult onset
movement disorder characterized by both dystonia and parkinsonism. We report
delineation of the disease gene within a 300-kb interval of Xq13.1 by allelic
association. Sequencing of this region in a patient revealed five
disease-specific single-nucleotide changes (here referred to as DSC) and a
48-bp deletion unique to XDP. One of the DSCs is located within an exon of a
not previously described multiple transcript system that is composed of at
least 16 exons. There is a minimum of three different transcription start
sites that encode four different transcripts. Two of these transcripts include
distal portions of the
TAF1
gene (TATA-box binding protein-associated
factor 1) and are alternatively spliced. Three exons overlap with
ING2
(a putative tumor suppressor) and with a homologue of
CIS4
(cytokine-inducible SH2 protein 4), both of which are encoded by
the opposite strand. Although all DSCs are located within this multiple
transcript system, only DSC3 lies within an exon. This exon is used by all
alternative transcripts making a pathogenic role of DSC3 in XDP likely. The
multiple transcript system is therefore referred to as
DYT3
(disease
locus in XDP).
Ian4 is required for mitochondrial integrity and T cell survival
Authors: Pandarpurkar Malini , Wilson-Fritch Leanne , Corvera Silvia , Markholst Helle , Hornum Lars , Greiner Dale L. , Mordes John P. , Rossini Aldo A. , Bortell Rita
Published: September 2003

Apoptosis is a regulated cell death program controlled by extrinsic and intrinsic signaling pathways. The intrinsic pathway involves stress signals that activate pro-apoptotic members of the Bcl-2 family, inducing permeabilization of mitochondria and release of apoptogenic factors. These proteins localize to the outer mitochondrial membrane. Ian4, a mitochondrial outer membrane protein with GTP-binding activity, is normally present in thymocytes, T cells, and B cells. We and others have recently discovered that a mutation in the ratIan4gene results in severe T cell lymphopenia that is associated with the expression of autoimmune diabetes. The mechanism by which Ian4 controls T cell homeostasis is unknown. Here we show that the absence of Ian4 in T cells causes mitochondrial dysfunction, increased mitochondrial levels of stress-inducible chaperonins and a leucine-rich protein, and T cell-specific spontaneous apoptosis. T cell activation and caspase 8 inhibition both prevented apoptosis, whereas transfection of T cells with Ian4-specific small interfering RNA recapitulated the apoptotic phenotype. The findings establish Ian4 as a tissue-specific regulator of mitochondrial integrity.
Chaperone-mediated
in vitro
assembly of Polyomavirus capsids
Authors: Chromy Laura R. , Pipas James M. , Garcea Robert L.
Published: September 2003

The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic
ability to self-assemble
in vitro
into polymorphic capsid-like
structures on addition of calcium. In contrast, polyomavirus assembly
in
vivo
is rigorously controlled, such that virions of uniform size are
formed only in the cell nucleus. During viral infection, the 72 kDa cellular
chaperone heat shock cognate protein (hsc70) binds VP1 posttranslation and
colocalizes with VP1 to the nucleus, thereby suggesting a role for ≈70-kDa
heat shock protein (hsp70) family chaperones in regulating the quality and
location of capsid assembly. We found that, after expression of recombinant
VP1 in
Escherichia coli
, the prokaryotic hsp70 chaperone DnaK
copurified with the VP1 C-terminal domain that links pentamers in an assembled
capsid. When stably bound to VP1, DnaK inhibited
in vitro
assembly
induced by calcium. However, in the presence of ATP, the hsp70 chaperone
system comprised of DnaK, DnaJ, and GrpE assembled VP1 into uniform capsids
without requiring calcium. Chaperone-mediated assembly was similarly catalyzed
by the eukaryotic hsc70 protein, in combination with the J-domain function of
the simian virus 40 large T-antigen protein. Thus, polyomavirus capsid
assembly can be recapitulated with high-fidelity
in vitro
using
either prokaryotic or eukaryotic hsp70 chaperone systems, thereby supporting a
role for cellular chaperones in the
in vivo
regulation of virion
assembly.
Expression deconvolution: A reinterpretation of DNA microarray data reveals dynamic changes in cell populations
Authors: Lu Peng , Nakorchevskiy Aleksey , Marcotte Edward M.
Published: September 2003

Cells grow in dynamically evolving populations, yet this aspect of
experiments often goes unmeasured. A method is proposed for measuring the
population dynamics of cells on the basis of their mRNA expression patterns.
The population's expression pattern is modeled as the linear combination of
mRNA expression from pure samples of cells, allowing reconstruction of the
relative proportions of pure cell types in the population. Application of the
method, termed expression deconvolution, to yeast grown under varying
conditions reveals the population dynamics of the cells during the cell cycle,
during the arrest of cells induced by DNA damage and the release of arrest in
a cell cycle checkpoint mutant, during sporulation, and following
environmental stress. Using expression deconvolution, cell cycle defects are
detected and temporally ordered in 146 yeast deletion mutants; six of these
defects are independently experimentally validated. Expression deconvolution
allows a reinterpretation of the cell cycle dynamics underlying all previous
microarray experiments and can be more generally applied to study most forms
of cell population dynamics.
Synaptopodin-deficient mice lack a spine apparatus and show deficits in synaptic plasticity
Authors: Deller Thomas , Korte Martin , Chabanis Sophie , Drakew Alexander , Schwegler Herbert , Stefani Giulia Good , Zuniga Aimee , Schwarz Karin , Bonhoeffer Tobias , Zeller Rolf , Frotscher Michael , Mundel Peter
Published: September 2003

The spine apparatus is a cellular organelle that is present in many dendritic spines of excitatory neurons in the mammalian forebrain. Despite its discovery >40 years ago, the function of the spine apparatus is still unknown although calcium buffering functions as well as roles in synaptic plasticity have been proposed. We have recently shown that the 100-kDa protein synaptopodin is associated with the spine apparatus. Here, we now report that mice homozygous for a targeted deletion of thesynaptopodingene completely lack spine apparatuses. Interestingly, this absence of the spine apparatus is accompanied by a reduction in hippocampal long-term potentiation (LTP) in the CA1 region of the hippocampus and by an impairment of spatial learning in the radial arm maze test. This genetic analysis points to a role of the spine apparatus in synaptic plasticity.
Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease
Authors: Wang Xin , Zhu Shan , Drozda Martin , Zhang Wenhua , Stavrovskaya Irina G. , Cattaneo Elena , Ferrante Robert J. , Kristal Bruce S. , Friedlander Robert M.
Published: September 2003

Minocycline is broadly protective in neurologic disease models featuring
cell death and is being evaluated in clinical trials. We previously
demonstrated that minocycline-mediated protection against caspase-dependent
cell death related to its ability to prevent mitochondrial cytochrome
c
release. These results do not explain whether or how minocycline
protects against caspase-independent cell death. Furthermore, there is no
information on whether Smac/Diablo or apoptosis-inducing factor might play a
role in chronic neurodegeneration. In a striatal cell model of Huntington's
disease and in R6/2 mice, we demonstrate the association of cell death/disease
progression with the recruitment of mitochondrial caspase-independent
(apoptosis-inducing factor) and caspase-dependent (Smac/Diablo and cytochrome
c
) triggers. We show that minocycline is a drug that directly
inhibits both caspase-independent and -dependent mitochondrial cell death
pathways. Furthermore, this report demonstrates recruitment of Smac/Diablo and
apoptosis-inducing factor in chronic neurodegeneration. Our results further
delineate the mechanism by which minocycline mediates its remarkably broad
neuroprotective effects.
Mpi recombinase globally modulates the surface architecture of a human commensal bacterium
Authors: Coyne Michael J. , Weinacht Katja G. , Krinos Corinna M. , Comstock Laurie E.
Published: September 2003

The mammalian gut represents a complex and diverse ecosystem, consisting of
unique interactions between the host and microbial residents. Bacterial
surfaces serve as an interface that promotes and responds to this dynamic
exchange, a process essential to the biology of both symbionts. The human
intestinal microorganism,
Bacteroides fragilis
, is able to
extensively modulate its surface. Analysis of the
B. fragilis
genomic
sequence, together with genetic conservation analyses, cross-species cloning
experiments, and mutational studies, revealed that this organism utilizes an
endogenous DNA inversion factor to globally modulate the expression of its
surface structures. This DNA invertase is necessary for the inversion of at
least 13 regions located throughout the genome, including the promoter regions
for seven of the capsular polysaccharide biosynthesis loci, an accessory
polysaccharide biosynthesis locus, and five other regions containing consensus
promoter sequences. Bacterial DNA invertases of the serine site-specific
recombinase family are typically encoded by imported elements such as phage
and plasmids, and act locally on a single region of the imported element. In
contrast, the conservation and unique global regulatory nature of the process
in
B. fragilis
suggest an evolutionarily ancient mechanism for
surface adaptation to the changing intestinal milieu during commensalism.
Gating currents associated with intramembrane charge displacement in HERG potassium channels
Authors: Piper David R. , Varghese Anthony , Sanguinetti Michael C. , Tristani-Firouzi Martin
Published: September 2003

HERG(human ether-a-go-go-related gene) encodes a delayed rectifier K+channel vital to normal repolarization of cardiac action potentials. Attenuation of repolarizing K+current caused by mutations in HERG or channel block by common medications prolongs ventricular action potentials and increases the risk of arrhythmia and sudden death. The critical role of HERG in maintenance of normal cardiac electrical activity derives from its unusual gating properties. Opposite to other voltage-gated K+channels, the rate of HERG channel inactivation is faster than activation and appears to be intrinsically voltage dependent. To investigate voltage sensor movement associated with slow activation and fast inactivation, we characterized HERG gating currents. When the cut-open oocyte voltage clamp technique was used, membrane depolarization elicited gating current with fast and slow components that differed 100-fold in their kinetics. Unlike previously studied voltage-gated K+channels, the bulk of charge movement in HERG was protracted, consistent with the slow rate of ionic current activation. Despite similar kinetic features, fast inactivation was not derived from the fast gating component. Analysis of an inactivation-deficient mutant HERG channel and a Markov kinetic model suggest that HERG inactivation is coupled to activation.
Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy
Authors: Urbanek Konrad , Quaini Federico , Tasca Giordano , Torella Daniele , Castaldo Clotilde , Nadal-Ginard Bernardo , Leri Annarosa , Kajstura Jan , Quaini Eugenio , Anversa Piero
Published: September 2003

It is generally believed that increase in adult contractile cardiac mass
can be accomplished only by hypertrophy of existing myocytes. Documentation of
myocardial regeneration in acute stress has challenged this dogma and led to
the proposition that myocyte renewal is fundamental to cardiac homeostasis.
Here we report that in human aortic stenosis, increased cardiac mass results
from a combination of myocyte hypertrophy and hyperplasia. Intense new myocyte
formation results from the differentiation of stem-like cells committed to the
myocyte lineage. These cells express stem cell markers and telomerase. Their
number increased >13-fold in aortic stenosis. The finding of cell clusters
with stem cells making the transition to cardiogenic and myocyte precursors,
as well as very primitive myocytes that turn into terminally differentiated
myocytes, provides a link between cardiac stem cells and myocyte
differentiation. Growth and differentiation of these primitive cells was
markedly enhanced in hypertrophy, consistent with activation of a restricted
number of stem cells that, through symmetrical cell division, generate
asynchronously differentiating progeny. These clusters strongly support the
existence of cardiac stem cells that amplify and commit to the myocyte lineage
in response to increased workload. Their presence is consistent with the
notion that myocyte hyperplasia significantly contributes to cardiac
hypertrophy and accounts for the subpopulation of cycling myocytes.
A tenth
atp
gene and the conserved
atpI
gene of a
Bacillus atp
operon have a role in Mg
2+
uptake
Authors: Hicks David B. , Wang ZhenXiong , Wei Yi , Kent Rebecca , Guffanti Arthur A. , Banciu Horia , Bechhofer David H. , Krulwich Terry A.
Published: September 2003

The
atp
operon of alkaliphilic
Bacillus pseudofirmus
OF4,
as in most prokaryotes, contains the eight structural genes for the F-ATPase
(ATP synthase), which are preceded by an
atpI
gene that encodes a
membrane protein of unknown function. A tenth gene,
atpZ
, has been
found in this operon, which is upstream of and overlapping with
atpI
.
Most
Bacillus
species, and some other bacteria, possess
atpZ
homologues. AtpZ is predicted to be a membrane protein with a hairpin
topology, and was detected by Western analyses. Deletion of
atpZ,
atpI
, or
atpZI
from
B. pseudofirmus
OF4 led to a
requirement for a greatly increased concentration of Mg
2+
for
growth at pH 7.5. Either
atpZ, atpI
, or
atpZI
complemented
the similar phenotype of a triple mutant of
Salmonella typhimurium
(MM281), which is deficient in Mg
2+
uptake.
atpZ
and
atpI
, separately and together, increased the
Mg
2+
-sensitive
45
Ca
2+
uptake by vesicles of
an
Escherichia coli
mutant that is defective in Ca
2+
and
Na
+
efflux. We hypothesize that AtpZ and AtpI, as homooligomers,
and perhaps as heterooligomers, are Mg
2+
transporter,
Ca
2+
transporter, or channel proteins. Such proteins could provide
Mg
2+
, which is required by ATP synthase, and also support charge
compensation, when the enzyme is functioning in the hydrolytic direction;
e.g., during cytoplasmic pH regulation.
Estimating the distribution of fitness effects from DNA sequence data: Implications for the molecular clock
Authors: Piganeau Gwenaël , Eyre-Walker Adam
Published: September 2003

We present a method for estimating the distribution of fitness effects of
new amino acid mutations when those mutations can be assumed to be slightly
advantageous, slightly deleterious, or strongly deleterious. We apply the
method to mitochondrial data from several different species. In the majority
of the data sets, the shape of the distribution is approximately exponential.
Our results provide an estimate of the distribution of fitness effects of
weakly selected mutations and provide a possible explanation for why the
molecular clock is fairly constant across taxa and time.
Can energy landscape roughness of proteins and RNA be measured by using mechanical unfolding experiments?
Authors: Hyeon Changbong , Thirumalai D.
Published: September 2003

By considering temperature effects on the mechanical unfolding rates of
proteins and RNA, whose energy landscape is rugged, the question posed in the
title is answered in the affirmative. Adopting a theory by Zwanzig [Zwanzig,
R. (1988)
Proc. Natl. Acad. Sci. USA
85, 2029-2030], we show that,
because of roughness characterized by an energy scale ε, the unfolding
rate at constant force is retarded. Similarly, in nonequilibrium experiments
done at constant loading rates, the most probable unfolding force increases
because of energy landscape roughness. The effects are dramatic at low
temperatures. Our analysis suggests that, by using temperature as a variable
in mechanical unfolding experiments of proteins and RNA, the ruggedness energy
scale ε, can be directly measured.
ACE2
is required for daughter cell-specific G
1
delay in
Saccharomyces cerevisiae
Authors: Laabs Tracy L. , Markwardt David D. , Slattery Matthew G. , Newcomb Laura L. , Stillman David J. , Heideman Warren
Published: September 2003

Saccharomyces cerevisiae
cells reproduce by budding to yield a
mother cell and a smaller daughter cell. Although both mother and daughter
begin G
1
simultaneously, the mother cell progresses through
G
1
more rapidly. Daughter cell G
1
delay has long been
thought to be due to a requirement for attaining a certain critical cell size
before passing the commitment point in the cell cycle known as START. We
present an alternative model in which the daughter cell-specific Ace2
transcription factor delays G
1
in daughter cells. Deletion of
ACE2
produces daughter cells that proceed through G
1
at
the same rate as mother cells, whereas a mutant Ace2 protein that is not
restricted to daughter cells delays G
1
equally in both mothers and
daughters. The differential in G
1
length between mothers and
daughters requires the Cln3 G
1
cyclin, and
CLN3
-GFP
reporter expression is reduced in daughters in an
ACE2
-dependent
manner. Specific daughter delay elements in the
CLN3
promoter are
required for normal daughter G
1
delay, and these elements bind to
an unidentified 127-kDa protein. This DNA-binding activity is enhanced by
deletion of
ACE2
. These results support a model in which daughter
cell G
1
delay is determined not by cell size but by an intrinsic
property of the daughter cell generated by asymmetric cell division.
Homez
, a homeobox leucine zipper gene specific to the vertebrate lineage
Authors: Bayarsaihan Dashzeveg , Enkhmandakh Badam , Makeyev Aleksandr , Greally John M. , Leckman James F. , Ruddle Frank H.
Published: September 2003

This work describes a vertebrate homeobox gene, designated
Homez
(
homeo
domain leucine
z
ipper-encoding gene), that encodes a
protein with an unusual structural organization. There are several regions
within Homez, including three atypical homeodomains, two leucine zipper-like
motifs, and an acidic domain. The gene is ubiquitously expressed in human and
murine tissues, although the expression pattern is more restricted during
mouse development. Genomic analysis revealed that human and mouse genes are
located at 14q11.2 and 14C, respectively, and are composed of two exons. The
zebrafish and pufferfish homologs share high similarity to mammalian
sequences, particularly within the homeodomain sequences. Based on homology of
homeodomains and on the similarity in overall protein structure, we deliniate
Homez and members of ZHX family of zinc finger homeodomain factors as a subset
within the superfamily of homeobox-containing proteins. The type and
composition of homeodomains in the Homez subfamily are vertebrate-specific.
Phylogenetic analysis indicates that
Homez
lineage was separated from
related genes >400 million years ago before separation of ray- and
lobe-finned fishes. We apply a duplication-degeneration-complementation model
to explain how this family of genes has evolved.
Hierarchical model of gene regulation by transforming growth factor β
Authors: Yang Yaw-Ching , Piek Ester , Zavadil Jiri , Liang Dan , Xie Donglu , Heyer Joerg , Pavlidis Paul , Kucherlapati Raju , Roberts Anita B. , Böttinger Erwin P.
Published: September 2003

Transforming growth factor βs (TGF-βs) regulate key aspects of
embryonic development and major human diseases. Although Smad2, Smad3, and
extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases
(MAPKs) have been proposed as key mediators in TGF-β signaling, their
functional specificities and interactivity in controlling transcriptional
programs in different cell types and (patho)physiological contexts are not
known. We investigated expression profiles of genes controlled by TGF-β
in fibroblasts with ablations of Smad2, Smad3, and ERK MAPK. Our results
suggest that Smad3 is the essential mediator of TGF-β signaling and
directly activates genes encoding regulators of transcription and signal
transducers through Smad3/Smad4 DNA-binding motif repeats that are
characteristic for immediate-early target genes of TGF-β but absent in
intermediate target genes. In contrast, Smad2 and ERK predominantly
transmodulated regulation of both immediate-early and intermediate genes by
TGF-β/Smad3. These results suggest a previously uncharacterized
hierarchical model of gene regulation by TGF-β in which TGF-β causes
direct activation by Smad3 of cascades of regulators of transcription and
signaling that are transmodulated by Smad2 and/or ERK.
The yeasts Rho1p and Pkc1p regulate the transport of chitin synthase III (Chs3p) from internal stores to the plasma membrane
Authors: Valdivia Raphael H. , Schekman Randy
Published: September 2003

During cell stress,
Saccharomyces cerevisiae
increases the
synthesis of chitin and glucans to strengthen and repair the cell wall. In
this study, we show that under conditions of cell stress, the steady-state
localization of chitin synthase III (Chs3p) shifts from internal stores
(chitosomes) to the plasma membrane (PM). This redistribution occurs rapidly
and requires the activators of the cell wall stress response signaling
pathway, the G protein Rho1p, and the protein kinase Pkc1p, but not the cell
integrity response mitogen-activated protein kinase cascade. Furthermore,
expression of activated forms of Rho1p or Pkc1p, in the absence of cell
stress, is sufficient to redistribute Chs3p to the PM. In cells deficient for
both the clathrin adaptor complex 1 and Chs6p, where Chs3p is transported to
the PM by an alternative bypass pathway, cell wall stress did not cause
mobilization of Chs3p, suggesting that Rho1p/Pkc1p regulate Chs3p exit from
the trans-Golgi network. The mobilization of an intracellular reservoir of
Chs3p presents a novel opportunity to investigate the genetic basis of
regulated vesicular traffic.
DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus
Authors: Hall Roy A. , Nisbet Debra J. , Pham Kim B. , Pyke Alyssa T. , Smith Greg A. , Khromykh Alexander A.
Published: September 2003

A plasmid DNA directing transcription of the infectious full-length RNA genome of Kunjin (KUN) virusin vivofrom a mammalian expression promoter was used to vaccinate mice intramuscularly. The KUN viral cDNA encoded in the plasmid contained the mutation in the NS1 protein (Pro-250 to Leu) previously shown to attenuate KUN virus in weanling mice. KUN virus was isolated from the blood of immunized mice 3-4 days after DNA inoculation, demonstrating that infectious RNA was being transcribedin vivo; however, no symptoms of virus-induced disease were observed. By 19 days postimmunization, neutralizing antibody was detected in the serum of immunized animals. On challenge with lethal doses of the virulent New York strain of West Nile (WN) or wild-type KUN virus intracerebrally or intraperitoneally, mice immunized with as little as 0.1-1 μg of KUN plasmid DNA were solidly protected against disease. This finding correlated with neutralization datain vitroshowing that serum from KUN DNA-immunized mice neutralized KUN and WN viruses with similar efficiencies. The results demonstrate that delivery of an attenuated but replicating KUN virus via a plasmid DNA vector may provide an effective vaccination strategy against virulent strains of WN virus.
Amphetamine or cocaine limits the ability of later experience to promote structural plasticity in the neocortex and nucleus accumbens
Authors: Kolb Bryan , Gorny Grazyna , Li Yilin , Samaha Anne-Noël , Robinson Terry E.
Published: September 2003

Drugs of abuse and many other kinds of experiences share the ability to
alter the morphology of neuronal dendrites and spines, the primary site of
excitatory synapses in the brain. We hypothesized, therefore, that exposure to
psychostimulant drugs might influence later experience-dependent structural
plasticity. We tested this hypothesis by treating rats repeatedly with
amphetamine or cocaine and then housing them in either a complex environment
or standard laboratory cages for 3-3.5 mo. The brains were processed for
Golgi-Cox staining, and the number of dendritic branches and the density of
dendritic spines on medium spiny neurons in the nucleus accumbens and
pyramidal cells in the parietal cortex were quantified. On most measures,
prior treatment with amphetamine or cocaine interfered with the ability of
experience in a complex environment to increase dendritic arborization and
spine density. We conclude that in some brain regions, repeated exposure to
psychomotor-stimulant drugs limits the ability of later experience to produce
this form of synaptic plasticity, which may contribute to the persistent
behavioral and cognitive deficits associated with drug abuse.
Rapamycin causes down-regulation of CCR5 and accumulation of anti-HIV β-chemokines: An approach to suppress R5 strains of HIV-1
Authors: Heredia A. , Amoroso A. , Davis C. , Le N. , Reardon E. , Dominique J. K. , Klingebiel E. , Gallo R. C. , Redfield R. R.
Published: September 2003

Propagation of R5 strains of HIV-1 on CD4 lymphocytes and macrophages requires expression of the CCR5 coreceptor on the cell surface. Individuals lacking CCR5 (CCR5Δ32 homozygous genotype) are phenotypically normal and resistant to infection with HIV-1. CCR5 expression on lymphocytes depends on signaling through the IL-2 receptor. By FACS analysis we demonstrate that rapamycin (RAPA), a drug that disrupts IL-2 receptor signaling, reduces CCR5 surface expression on T cells at concentrations as low as 1 nM. In addition, lower concentrations of RAPA (0.01 nM) were sufficient to reduce CCR5 surface expression on maturing monocytes. PCR analysis on peripheral blood mononuclear cells (PBMCs) showed that RAPA interfered with CCR5 expression at the transcriptional level. Reduced expression of CCR5 on PBMCs cultured in the presence of RAPA was associated with increased extracellular levels of macrophage inflammatory protein (MIP)-1α and MIP-1β. In infectivity assays, RAPA suppressed the replication of R5 strains of HIV-1 both in PBMC and macrophage cultures. In total PBMC cultures, RAPA-mediated inhibition of CCR5-using strains of HIV-1 occurred at 0.01 nM, a concentration of drug that is ∼103times lower than therapeutic through levels of drug in renal transplant recipients. In addition, RAPA enhanced the antiviral activity of the CCR5 antagonist TAK-779. These results suggest that low concentrations of RAPA may have a role in both the treatment and prevention of HIV-1 infection.
Hydrogen atoms in proteins: Positions and dynamics
Authors: Engler Niklas , Ostermann Andreas , Niimura Nobuo , Parak Fritz G.
Published: September 2003

Hydrogen atoms constitute about half of the atoms in proteins. Thus they
contribute to the complex energy landscape of proteins [Frauenfelder, H.,
Sligar, S. G. & Wolynes, P. G. (1991)
Science
254, 1598-1603].
Neutron crystal structure analysis was used to study the positions and
mean-square displacements of hydrogen in myoglobin. A test of the reliability
of calculated hydrogen atom coordinates by a comparison with our experimental
results has been carried out. The result shows that >70% of the coordinates
for hydrogen atoms that have a degree of freedom is predicted worse than 0.2
Å. It is shown that the mean-square displacements of the hydrogen atoms
obtained from the Debye-Waller factor can be divided into three classes. A
comparison with the dynamic mean-square displacements calculated from the
elastic intensities obtained from incoherent neutron scattering [Doster, W.,
Cusack, S. & Petry, W. (1989)
Nature
337, 754-756] shows that
mainly the side-chain hydrogen atoms contribute to dynamic displacements on a
time scale faster than 100 ps.
A mouse model of Rubinstein-Taybi syndrome: Defective long-term memory is ameliorated by inhibitors of phosphodiesterase 4
Authors: Bourtchouladze Rusiko , Lidge Regina , Catapano Ray , Stanley Jennifer , Gossweiler Scott , Romashko Darlene , Scott Rod , Tully Tim
Published: September 2003

Mice carrying a truncated form of cAMP-responsive element binding protein
(CREB)-binding protein (CBP) show several developmental abnormalities similar
to patients with Rubinstein-Taybi syndrome (RTS). RTS patients suffer from
mental retardation, whereas long-term memory formation is defective in mutant
CBP mice. A critical role for cAMP signaling during CREB-dependent long-term
memory formation appears to be evolutionarily conserved. From this
observation, we reasoned that drugs that modulate CREB function by enhancing
cAMP signaling might yield an effective treatment for the memory defect(s) of
CBP
+/−
mice. To this end, we designed a cell-based drug
screen and discovered inhibitors of phosphodiesterase 4 (PDE4) to be
particularly effective enhancers of CREB function. We extend previous
behavioral observations by showing that CBP
+/−
mutants have
impaired long-term memory but normal learning and short-term memory in an
object recognition task. We demonstrate that the prototypical PDE4 inhibitor,
rolipram, and a novel one (HT0712) abolish the long-term memory defect of
CBP
+/−
mice. Importantly, the genetic lesion in CBP acts
specifically to shift the dose sensitivity for HT0712 to enhance memory
formation, which conveys molecular specificity on the drug's mechanism of
action. Our results suggest that PDE4 inhibitors may be used to treat the
cognitive dysfunction of RTS patients.
Cuticular hydrocarbons mediate discrimination of reproductives and nonreproductives in the ant
Myrmecia gulosa
Authors: Dietemann Vincent , Peeters Christian , Liebig Jürgen , Thivet Virginie , Hölldobler Bert
Published: September 2003

In many species of social insects, the cuticular hydrocarbons of adults
vary with both colony identity and individual physiology (oogenesis). Such
variations have been shown in some ants and social wasps to function in
nestmate recognition, but as yet there is no demonstration of their use by
workers to recognize egg layers. We report that in the ant
Myrmecia
gulosa
, workers can discriminate queens and fertile workers from
infertile individuals based on distinctive blends of long-chained hydrocarbons
present both on the cuticle and in the postpharyngeal gland. The purified
hydrocarbon fraction of cuticular extracts from queens elicited high interest
in workers, unlike the nonhydrocarbon fraction. However, both fractions were
necessary to trigger a response of maximal intensity. In contrast, extracts of
mandibular and Dufour glands from queens or infertile workers were not treated
differentially by workers. We suggest that cuticular hydrocarbons function as
pheromones allowing for recognition of the queen as well as egg-laying
workers.
Participation of the S4 voltage sensor in the Mg
2+
-dependent activation of large conductance (BK) K
+
channels
Authors: Hu Lei , Shi Jingyi , Ma Zhongming , Krishnamoorthy Gayathri , Sieling Fred , Zhang Guangping , Horrigan Frank T. , Cui Jianmin
Published: September 2003

The S4 transmembrane segment is the primary voltage sensor in voltage-dependent ion channels. Its movement in response to changes in membrane potential leads to the opening of the activation gate, which is formed by a separate structural component, the S6 segment. Here we show in voltage-, Ca
2+
-, and Mg
2+
-dependent, large conductance K
+
channels that the S4 segment participates not only in voltage- but also Mg
2+
-dependent activation. Mutations in S4 and the S4-S5 linker alter voltage-dependent activation and have little or no effect on activation by micromolar Ca
2+
. However, a subset of these mutations in the C-terminal half of S4 and in the S4-S5 linker either reduce or abolish the Mg
2+
sensitivity of channel gating. Cysteine residues substituted into positions R210 and R213, marking the boundary between S4 mutations that alter Mg
2+
sensitivity and those that do not, are accessible to a modifying reagent [sodium (2-sulfonatoethyl)methane-thiosulfonate] (MTSES) from the extracellular and intracellular side of the membrane, respectively, at -80 mV. This implies that interactions between S4 and a cytoplasmic domain may be involved in Mg
2+
-dependent activation. These results indicate that the voltage sensor is critical for Mg
2+
-dependent activation and the coupling between the voltage sensor and channel gate is a converging point for voltage- and Mg
2+
-dependent activation pathways.
Alzheimer's disease-affected brain: Presence of oligomeric Aβ ligands (ADDLs) suggests a molecular basis for reversible memory loss
Authors: Gong Yuesong , Chang Lei , Viola Kirsten L. , Lacor Pascale N. , Lambert Mary P , Finch Caleb E. , Krafft Grant A. , Klein William L.
Published: September 2003

A molecular basis for memory failure in Alzheimer's disease (AD) has been recently hypothesized, in which a significant role is attributed to small, soluble oligomers of amyloid β-peptide (Aβ). Aβ oligomeric ligands (also known as ADDLs) are known to be potent inhibitors of hippocampal long-term potentiation, which is a paradigm for synaptic plasticity, and have been linked to synapse loss and reversible memory failure in transgenic mouse AD models. If such oligomers were to build up in human brain, their neurological impact could provide the missing link that accounts for the poor correlation between AD dementia and amyloid plaques. This article, using antibodies raised against synthetic Aβ oligomers, verifies the predicted accumulation of soluble oligomers in AD frontal cortex. Oligomers in AD reach levels up to 70-fold over control brains. Brain-derived and synthetic oligomers show structural equivalence with respect to mass, isoelectric point, and recognition by conformation-sensitive antibodies. Both oligomers, moreover, exhibit the same striking patterns of attachment to cultured hippocampal neurons, binding on dendrite surfaces in small clusters with ligand-like specificity. Binding assays using solubilized membranes show oligomers to be high-affinity ligands for a small number of nonabundant proteins. Current results confirm the prediction that soluble oligomeric Aβ ligands are intrinsic to AD pathology, and validate their use in new approaches to therapeutic AD drugs and vaccines.
Activation of CB
2
cannabinoid receptors by AM1241 inhibits experimental neuropathic pain: Pain inhibition by receptors not present in the CNS
Authors: Ibrahim Mohab M. , Deng Hongfeng , Zvonok Alexander , Cockayne Debra A. , Kwan Joyce , Mata Heriberto P. , Vanderah Todd W. , Lai Josephine , Porreca Frank , Makriyannis Alexandros , Malan T. Philip
Published: September 2003

We designed AM1241, a selective CB
2
cannabinoid receptor
agonist, and used it to test the hypothesis that CB
2
receptor
activation would reverse the sensory hypersensitivity observed in neuropathic
pain states. AM1241 exhibits high affinity and selectivity for CB
2
receptors. It also exhibits high potency
in vivo
. AM1241
dose-dependently reversed tactile and thermal hypersensitivity produced by
ligation of the L5 and L6 spinal nerves in rats. These effects were
selectively antagonized by a CB
2
but not by a CB
1
receptor antagonist, suggesting that they were produced by actions of AM1241
at CB
2
receptors. AM1241 was also active in blocking spinal nerve
ligation-induced tactile and thermal hypersensitivity in mice lacking
CB
1
receptors (CB
1
-/-
mice), confirming that
AM1241 reverses sensory hypersensitivity independent of actions at
CB
1
receptors. These findings demonstrate a mechanism leading to
the inhibition of pain, one that targets receptors localized exclusively
outside the CNS. Further, they suggest the potential use of CB
2
receptor-selective agonists for treatment of human neuropathic pain, a
condition currently without consistently effective therapies. CB
2
receptor-selective agonist medications are predicted to be without the CNS
side effects that limit the effectiveness of currently available
medications.
Self-assembly of synthetic peptides control conformation and optical properties of a zwitterionic polythiophene derivative
Authors: Nilsson K. Peter R. , Rydberg Johan , Baltzer Lars , Inganäs Olle
Published: September 2003

The optical transitions of a chiral, three-substituted polythiophene with
an amino acid function can be tuned by interactions with synthetic peptides.
The addition of a positively charged peptide with a random-coil formation will
force the polymer to adopt a nonplanar conformation, and the intensity of the
emitted light is increased and blue-shifted. After the addition of a
negatively charged peptide with a random-coil conformation, the backbone of
the polymer adopts a planar conformation and an aggregation of the polymer
chains occurs, seen as a red shift and a decrease of the intensity of the
emitted light. By adding the positively charged peptide designed to form a
four-helix bundle with the negatively charged peptide, the polymer aggregates
are disrupted and the intensity of the emitted light is increased because of
separation of the polymer chains. This technique could be used as a platform
for making novel sensors and biomolecular switches.