bluuebunny/crossref_metadata_2025 huggingface dataset:
The plug domain of FepA, a TonB-dependent transport protein from
Escherichia coli
, binds its siderophore in the absence of the transmembrane barrel domain
Authors: Usher Ken C. , Özkan Engin , Gardner Kevin H. , Deisenhofer Johann
Published: July 2002

FepA, an outer membrane iron siderophore transporter from
Escherichia coli
, is composed of a 22-stranded membrane-spanning β barrel with a globular N-terminal “plug” domain of 148 residues that folds up inside the barrel and completely occludes the barrel's interior (1). We have overexpressed and purified this plug domain by itself and find that it behaves
in vitro
as a predominantly unfolded yet soluble protein, as determined by circular dichroism, thermal denaturation, and NMR studies. Despite its unfolded state, the isolated domain binds ferric enterobactin, the siderophore ligand of FepA, with an affinity of 5 μM, just 100-fold reduced from that of intact FepA. These findings argue against the hypothesis that the plug domain is pulled intact from the barrel during transport
in vivo
but may be consistent either with a model where the plug rearranges within the barrel to create a channel large enough to allow transport or with a model where the plug unfolds and comes out of the barrel.
Structure of the Sec23p/24p and Sec13p/31p complexes of COPII
Authors: Lederkremer Gerardo Z. , Cheng Yifan , Petre Benjamin M. , Vogan Erik , Springer Sebastian , Schekman Randy , Walz Thomas , Kirchhausen Tomas
Published: July 2002

COPII-coated vesicles carry proteins from the endoplasmic reticulum to the Golgi complex. This vesicular transport can be reconstituted by using three cytosolic components containing five proteins: the small GTPase Sar1p, the Sec23p/24p complex, and the Sec13p/Sec31p complex. We have used a combination of biochemistry and electron microscopy to investigate the molecular organization and structure of Sec23p/24p and Sec13p/31p complexes. The three-dimensional reconstruction of Sec23p/24p reveals that it has a bone-shaped structure, (17 nm in length), composed of two similar globular domains, one corresponding to Sec23p and the other to Sec24p. Sec13p/31p is a heterotetramer composed of two copies of Sec13p and two copies of Sec31p. It has an elongated shape, is 28–30 nm in length, and contains five consecutive globular domains linked by relatively flexible joints. Putting together the architecture of these Sec complexes with the interactions between their subunits and the appearance of the coat in COPII-coated vesicles, we present a model for COPII coat organization.
A simple model host for identifying Gram-positive virulence factors
Authors: Garsin Danielle A. , Sifri Costi D. , Mylonakis Eleftherios , Qin Xiang , Singh Kavindra V. , Murray Barbara E. , Calderwood Stephen B. , Ausubel Frederick M.
Published: July 2002

We demonstrate the use of the nematode
Caenorhabditis elegans
as a facile and inexpensive model host for several Gram-positive human bacterial pathogens.
Enterococcus faecalis, Streptococcus pneumoniae
, and
Staphylococcus aureus,
but not
Bacillus subtilis, Enterococcus faecium
, or
Streptococcus pyogenes
, kill adult
C. elegans.
Focusing our studies on the enterococcal species, we found that both
E. faecalis
and
E. faecium
kill
C. elegans
eggs and hatchlings, although only
E. faecalis
kills the adults. In the case of adults, a low inoculum of
E. faecalis
grows to a high titer in the
C. elegans
intestine, resulting in a persistent infection that cannot be eradicated by prolonged feeding on
E. faecium
. Interestingly, a high titer of
E. faecium
also accumulates in the nematode gut, but does not affect the longevity of the worms. Two
E. faecalis
virulence-related factors that play an important role in mammalian models of infection,
fsr
, a putative quorum-sensing system, and cytolysin, are also important for nematode killing. We exploit the apparent parallels between Gram-positive infection in simple and more complex organisms by using the nematode to identify an
E. faecalis
virulence factor, ScrB, which is relevant to mammalian pathogenesis.
Tonsillar memory B cells, latently infected with Epstein–Barr virus, express the restricted pattern of latent genes previously found only in Epstein–Barr virus-associated tumors
Authors: Babcock Gregory J. , Thorley-Lawson David A.
Published: July 2002

Epstein–Barr virus (EBV) establishes a life-long persistent
infection in most of the human population. In the peripheral blood, EBV
is restricted to memory B cells that are resting and express limited
genetic information. We have proposed that these memory cells are the
site of long-term persistent infection. We now show that memory cells
in the tonsil express the genes for EBV nuclear antigen 1 (EBNA1) (from
the Qp promoter), latent membrane protein 1 (LMP1), and LMP2a but do
not express EBNA2 or the EBNA3s. This pattern of latent gene expression
has only been seen previously in EBV-associated tumors such as
nasopharyngeal carcinoma, Hodgkin's disease (HD), and T/NK
lymphomas. Normal circulating memory B cells frequently reenter
secondary lymphoid tissue, where they receive signals essential for
their survival. Specifically they require signals from antigen-specific
T helper cells and from antigen itself. LMP1 and LMP2 are known to be
able to generate these signals in a ligand-independent fashion. We
suggest, therefore, that the transcription pattern we have found in
latently infected, tonsillar, memory B cells is used because it allows
for the expression of LMP1, LMP2a, and EBNA1 in the absence of the
immunogenic and growth-promoting EBNA2 and EBNA3 molecules. LMP1 and
LMP2a are produced to provide the surrogate rescue and survival signals
needed to allow latently infected memory cells to persist, and EBNA1 is
produced to allow replication of the viral episome.
Mapping adolescent brain change reveals dynamic wave of accelerated gray matter loss in very early-onset schizophrenia
Authors: Thompson Paul M. , Vidal Christine , Giedd Jay N. , Gochman Peter , Blumenthal Jonathan , Nicolson Robert , Toga Arthur W. , Rapoport Judith L.
Published: July 2002

Neurodevelopmental models for the pathology of schizophrenia propose both polygenetic and environmental risks, as well as early (pre/perinatal) and late (usually adolescent) developmental brain abnormalities. With the use of brain mapping algorithms, we detected striking anatomical profiles of accelerated gray matter loss in very early-onset schizophrenia; surprisingly, deficits moved in a dynamic pattern, enveloping increasing amounts of cortex throughout adolescence. Early-onset patients were rescanned prospectively with MRI, at 2-year intervals at three time points, to uncover the dynamics and timing of disease progression during adolescence. The earliest deficits were found in parietal brain regions, supporting visuospatial and associative thinking, where adult deficits are known to be mediated by environmental (nongenetic) factors. Over 5 years, these deficits progressed anteriorly into temporal lobes, engulfing sensorimotor and dorsolateral prefrontal cortices, and frontal eye fields. These emerging patterns correlated with psychotic symptom severity and mirrored the neuromotor, auditory, visual search, and frontal executive impairments in the disease. In temporal regions, gray matter loss was completely absent early in the disease but became pervasive later. Only the latest changes included dorsolateral prefrontal cortex and superior temporal gyri, deficit regions found consistently in adult studies. These emerging dynamic patterns were (i) controlled for medication and IQ effects, (ii) replicated in independent groups of males and females, and (iii) charted in individuals and groups. The resulting mapping strategy reveals a shifting pattern of tissue loss in schizophrenia. Aspects of the anatomy and dynamics of disease are uncovered, in a changing profile that implicates genetic and nongenetic patterns of deficits.
A structural basis for drug-induced long QT syndrome
Authors: Mitcheson John S. , Chen Jun , Lin Monica , Culberson Chris , Sanguinetti Michael C.
Published: July 2002

Mutations in the HERG K
+
channel gene cause inherited
long QT syndrome (LQT), a disorder of cardiac repolarization that
predisposes affected individuals to lethal arrhythmias [Curran,
M. E., Splawski, I., Timothy, K. W., Vincent, G. M.,
Green, E. D. & Keating, M. T. (1995)
Cell
80,
795–804]. Acquired LQT is far more common and is most often caused by
block of cardiac HERG K
+
channels by commonly used
medications [Roden, D. M., Lazzara, R., Rosen, M., Schwartz,
P. J., Towbin, J. & Vincent, G. M. (1996)
Circulation
94, 1996–2012]. It is unclear why so many
structurally diverse compounds block HERG channels, but this
undesirable side effect now is recognized as a major hurdle in the
development of new and safe drugs. Here we use alanine-scanning
mutagenesis to determine the structural basis for high-affinity drug
block of HERG channels by MK-499, a methanesulfonanilide antiarrhythmic
drug. The binding site, corroborated with homology modeling, is
comprised of amino acids located on the S6 transmembrane domain (G648,
Y652, and F656) and pore helix (T623 and V625) of the HERG channel
subunit that face the cavity of the channel. Other compounds that are
structurally unrelated to MK-499, but cause LQT, also were studied. The
antihistamine terfenadine and a gastrointestinal prokinetic drug,
cisapride, interact with Y652 and F656, but not with V625. The aromatic
residues of the S6 domain that interact with these drugs (Y652 and
F656) are unique to eag/erg K
+
channels. Other
voltage-gated K
+
(Kv) channels have Ile and Val (Ile) in
the equivalent positions. These findings suggest a possible structural
explanation for how so many commonly used medications block HERG but
not other Kv channels and should facilitate the rational design of
drugs devoid of HERG channel binding activity.
Normal kinetics of intestinal glucose absorption in the absence of GLUT2: Evidence for a transport pathway requiring glucose phosphorylation and transfer into the endoplasmic reticulum
Authors: Stümpel Frank , Burcelin Rémy , Jungermann Kurt , Thorens Bernard
Published: July 2002

Glucose is absorbed through the intestine by a transepithelial
transport system initiated at the apical membrane by the cotransporter
SGLT-1; intracellular glucose is then assumed to diffuse across the
basolateral membrane through GLUT2. Here, we evaluated the impact of
GLUT2
gene inactivation on this transepithelial
transport process. We report that the kinetics of transepithelial
glucose transport, as assessed in oral glucose tolerance tests, was
identical in the presence or absence of GLUT2; that the transport was
transcellular because it could be inhibited by the SGLT-1 inhibitor
phlorizin, and that it could not be explained by overexpression of
another known glucose transporter. By using an isolated intestine
perfusion system, we demonstrated that the rate of transepithelial
transport was similar in control and GLUT2
−/−
intestine
and that it was increased to the same extent by cAMP in both
situations. However, in the absence, but not in the presence, of GLUT2,
the transport was inhibited dose-dependently by the glucose-6-phosphate
translocase inhibitor S4048. Furthermore, whereas transport of
[
14
C]glucose proceeded with the same kinetics in control
and GLUT2
−/−
intestine,
[
14
C]3-
O
-methylglucose was transported in
intestine of control but not of mutant mice. Together our data
demonstrate the existence of a transepithelial glucose transport system
in GLUT2
−/−
intestine that requires glucose
phosphorylation and transfer of glucose-6-phosphate into the
endoplasmic reticulum. Glucose may then be released out of the cells by
a membrane traffic-based pathway similar to the one we previously
described in GLUT2-null hepatocytes.
Epstein–Barr virus nuclear antigen 1 activates transcription from episomal but not integrated DNA and does not alter lymphocyte growth
Authors: Kang Myung-Soo , Hung Siu Chun , Kieff Elliott
Published: July 2002

By binding to a cis-acting element
(
oriP
) in the Epstein–Barr virus (EBV) genome, EBV
nuclear antigen 1 (EBNA1) enables persistence and enhances
transcription from EBV episomes. To investigate whether EBNA1 also
directly affects cell gene transcription, we conditionally expressed a
Flag-tagged dominant negative EBNA1 (FDNE) in an EBV immortalized
lymphoblastoid cell line, in which the EBV genome is integrated into
cell DNA. FDNE induction inhibited expression from an EBNA1-dependent
oriP reporter plasmid by more than 90% in these cells but did not
affect expression from integrated EBV or oriP reporter DNA. FDNE
induction also did not alter expression of more than 1,800 cellular
mRNAs. Lymphoblastoid cell line growth under a variety of conditions
was unaffected by FDNE induction. Although Gal4-VP16 and EBNA1 strongly
activated and coactivated a Gal4-VP16- and oriP-dependent promoter that
was on an episome, only Gal4-VP16 activated the promoter when it was
integrated into chromosomal DNA. These data indicate that EBNA1 is
specifically deficient in activation of an integrated
oriP
enhancer and does not affect cell growth or gene
expression through an interaction with cognate chromosomal DNA.
HIV-1 Nef impairs MHC class II antigen presentation and surface expression
Authors: Stumptner-Cuvelette Pamela , Morchoisne Stephanie , Dugast Marc , Le Gall Sylvie , Raposo Graça , Schwartz Olivier , Benaroch Philippe
Published: July 2002

HIV-1-infected cells can avoid cytotoxic T lymphocyte killing by Nef-mediated down-regulation of surface MHC I. Here, we show that HIV-1 Nef inhibits MHC II restricted peptide presentation to specific T cells and thus may affect the induction of antiviral immune responses. Nef mediates this effect by reducing the surface level of mature (i.e., peptide-loaded) MHC II while increasing levels of immature MHC II, which are functionally incompetent because of their association with the invariant chain. Nef was the only HIV-1 gene product to possess this capacity, which was also observed in the context of the whole HIV-1 genome. Other proteins of the endocytic pathway were not affected by Nef expression, suggesting that Nef effects on MHC II did not result from a general alteration of the endocytic pathway. Response patterns to previously characterized mutations of Nef differed for Nef-induced modulation of mature and immature MHC II. Furthermore, the doses of Nef required to observe each of the two effects were clearly different, suggesting that Nef could affect MHC II peptide presentation through distinct mechanisms. Cooperation between those mechanisms may enable Nef to efficiently inhibit MHC II function.
p53 is a potential mediator of pregnancy and hormone-induced resistance to mammary carcinogenesis
Authors: Sivaraman Lakshmi , Conneely Orla M. , Medina Daniel , O'Malley Bert W.
Published: July 2002

Full-term pregnancy early in reproductive life is protective against breast cancer in women. Pregnancy also provides protection in animals against carcinogen-induced breast cancer, and this protection can be mimicked by using the hormones estrogen and progesterone. The molecular mechanisms that form the basis for this protective effect have not been elucidated. On the basis of our results, we propose a cell-fate hypothesis. At a critical period in adolescence the hormonal milieu of pregnancy affects the developmental fate of a subset of mammary epithelial cells and its progeny, which results in persistent differences in molecular pathways between the epithelial cells of hormone-treated and mature virgin mammary glands. These changes in turn dictate the proliferative response to carcinogen challenge and include a block in carcinogen-induced increase in mammary epithelial cell proliferation and an increased and sustained expression of nuclear p53 in the hormone-treated mammary gland. This hormone-induced nuclear p53 is transcriptionally active as evidenced by increased expression of mdm2 and p21 (CIP1/WAF1). Importantly, exposure to perphenazine, a compound that induces mammary gland differentiation but does not confer protection, does not induce p53 expression, indicating that p53 is not a differentiation marker. The proliferative block and induction of p53 are operative in both rats and mice, results that support the generality of the proposed hypothesis.
Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of β-sheets in Alzheimer's β-amyloid fibrils
Authors: Antzutkin Oleg N. , Balbach John J. , Leapman Richard D. , Rizzo Nancy W. , Reed Jennifer , Tycko Robert
Published: July 2002

Senile plaques associated with Alzheimer's disease contain
deposits of fibrils formed by 39- to 43-residue β-amyloid peptides
with possible neurotoxic effects. X-ray diffraction measurements on
oriented fibril bundles have indicated an extended β-sheet structure
for Alzheimer's β-amyloid fibrils and other amyloid fibrils, but the
supramolecular organization of the β-sheets and other structural
details are not well established because of the intrinsically
noncrystalline, insoluble nature of amyloid fibrils. Here we report
solid-state NMR measurements, using a multiple quantum (MQ)
13
C NMR technique, that probe the β-sheet organization in
fibrils formed by the full-length, 40-residue β-amyloid peptide
(Aβ
1–40
). Although an antiparallel β-sheet
organization often is assumed and is invoked in recent structural
models for full-length β-amyloid fibrils, the MQNMR data indicate an
in-register, parallel organization. This work provides site-specific,
atomic-level structural constraints on full-length β-amyloid fibrils
and applies MQNMR to a significant problem in structural biology.
Cell-type specificity of short-range transcriptional repressors
Authors: Ryu Jae-Ryeon , Olson L. Karl , Arnosti David N.
Published: July 2002

Transcriptional repressors can be classified as short- or long-range, according to their range of activity. Functional analysis of identified short-range repressors has been carried out largely in transgenic
Drosophila
, but it is not known whether general properties of short-range repressors are evident in other types of assays. To study short-range transcriptional repressors in cultured cells, we created chimeric tetracycline repressors based on
Drosophila
transcriptional repressors Giant,
Drosophila
C-terminal-binding protein (dCtBP), and Knirps. We find that Giant and dCtBP are efficient repressors in
Drosophila
and mammalian cells, whereas Knirps is active only in insect cells. The restricted activity of Knirps, in contrast to that of Giant, suggests that not all short-range repressors possess identical activities, consistent with recent findings showing that short-range repressors act through multiple pathways. The mammalian repressor Kid is more effective than either Giant or dCtBP in mammalian cells but is inactive in
Drosophila
cells. These results indicate that species-specific factors are important for the function of the Knirps and Kid repressors. Giant and dCtBP repress reporter genes in a variety of contexts, including genes that were introduced by transient transfection, carried on episomal elements, or stably integrated. This broad activity indicates that the context of the target gene is not critical for the ability of short-range repressors to block transcription, in contrast to other repressors that act only on stably integrated genes.
The Zα domain of the editing enzyme dsRNA adenosine deaminase binds left-handed Z-RNA as well as Z-DNA
Authors: Brown Bernard A. , Lowenhaupt Ky , Wilbert Christina M. , Hanlon Eugene B. , Rich Alexander
Published: July 2002

The Zα domain of human double-stranded RNA adenosine deaminase 1
binds specifically to left-handed Z-DNA and stabilizes the
Z-conformation. Here we report spectroscopic and analytical results
that demonstrate that Zα can also stabilize the left-handed
Z-conformation in double-stranded RNA. Zα induces a slow transition
from the right-handed A-conformation to the Z-form in duplex
r(CG)
6
, with an activation energy of 38 kcal
mol
−1
. We conclude that Z-RNA as well as Z-DNA can be
accommodated in the tailored binding site of Zα. The specific binding
of Z-RNA by Zα may be involved in targeting double-stranded RNA
adenosine deaminase 1 for a role in hypermutation of RNA viruses.
S100A1: A regulator of myocardial contractility
Authors: Most Patrick , Bernotat Juliane , Ehlermann Philipp , Pleger Sven T. , Reppel Michael , Börries Melanie , Niroomand Ferraydoon , Pieske Burkert , Janssen Paul M. L. , Eschenhagen Thomas , Karczewski Peter , Smith Godfrey L. , Koch Walter J. , Katus Hugo A. , Remppis Andrew
Published: July 2002

S100A1, a Ca
2+
binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been found to colocalize with the sarcoplasmic reticulum (SR) and the contractile filaments in cardiac tissue. Because S100A1 is known to modulate SR Ca
2+
handling in skeletal muscle, we sought to investigate the specific role of S100A1 in the regulation of myocardial contractility. To address this issue, we investigated contractile properties of adult cardiomyocytes as well as of engineered heart tissue after S100A1 adenoviral gene transfer. S100A1 gene transfer resulted in a significant increase of unloaded shortening and isometric contraction in isolated cardiomyocytes and engineered heart tissues, respectively. Analysis of intracellular Ca
2+
cycling in S100A1-overexpressing cardiomyocytes revealed a significant increase in cytosolic Ca
2+
transients, whereas in functional studies on saponin-permeabilized adult cardiomyocytes, the addition of S100A1 protein significantly enhanced SR Ca
2+
uptake. Moreover, in Triton-skinned ventricular trabeculae, S100A1 protein significantly decreased myofibrillar Ca
2+
sensitivity ([EC
50%
]) and Ca
2+
cooperativity, whereas maximal isometric force remained unchanged. Our data suggest that S100A1 effects are cAMP independent because cellular cAMP levels and protein kinase A-dependent phosphorylation of phospholamban were not altered, and carbachol failed to suppress S100A1 actions. These results show that S100A1 overexpression enhances cardiac contractile performance and establish the concept of S100A1 as a regulator of myocardial contractility. S100A1 thus improves cardiac contractile performance both by regulating SR Ca
2+
handling and myofibrillar Ca
2+
responsiveness.
DNA supercoiling allows enhancer action over a large distance
Authors: Liu Ye , Bondarenko Vladimir , Ninfa Alexander , Studitsky Vasily M.
Published: July 2002

Enhancers are regulatory DNA elements that can activate their
genomic targets over a large distance. The mechanism of enhancer
action over large distance is unknown. Activation of the
glnAp2
promoter by NtrC-dependent enhancer in
Escherichia coli
was analyzed by using a purified system
supporting multiple-round transcription
in vitro
. The
data suggest that DNA supercoiling is an essential requirement for
enhancer action over a large distance (2,500 bp) but not over a short
distance (110 bp). DNA supercoiling facilitates functional
enhancer–promoter communication over a large distance, probably by
bringing the enhancer and promoter into close proximity.
Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in Escherichia coli.
Authors: Jones P G , Mitta M , Kim Y , Jiang W , Inouye M
Published: July 2002

A 70-kDa protein was specifically induced in Escherichia coli when the culture temperature was shifted from 37 to 15 degrees C. The protein was identified to be the product of the deaD gene (reassigned csdA) encoding a DEAD-box protein. Furthermore, after the shift from 37 to 15 degrees C, CsdA was exclusively localized in the ribosomal fraction and became a major ribosomal-associated protein in cells grown at 15 degrees C. The csdA deletion significantly impaired cell growth and the synthesis of a number of proteins, specifically the derepression of heat-shock proteins, at low temperature. Purified CsdA was found to unwind double-stranded RNA in the absence of ATP. Therefore, the requirement for CsdA in derepression of heat-shock protein synthesis is a cold shock-induced function possibly mediated by destabilization of secondary structures previously identified in the rpoH mRNA.
Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity.
Authors: Przedborski S , Jackson-Lewis V , Yokoyama R , Shibata T , Dawson V L , Dawson T M
Published: July 2002

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson disease (PD). To study the role of NO radical in MPTP-induced neurotoxicity, we injected MPTP into mice in which nitric oxide synthase (NOS) was inhibited by 7-nitroindazole (7-NI) in a time- and dose-dependent fashion. 7-NI dramatically protected MPTP-injected mice against indices of severe injury to the nigrostriatal dopaminergic pathway, including reduction in striatal dopamine contents, decreases in numbers of nigral tyrosine hydroxylase-positive neurons, and numerous silver-stained degenerating nigral neurons. The resistance of 7-NI-injected mice to MPTP is not due to alterations in striatal pharmacokinetics or content of 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP. To study specifically the role of neuronal NOS (nNOS), MPTP was administered to mutant mice lacking the nNOS gene. Mutant mice are significantly more resistant to MPTP-induced neurotoxicity compared with wild-type littermates. These results indicate that neuronally derived NO mediates, in part, MPTP-induced neurotoxicity. The similarity between the MPTP model and PD raises the possibility that NO may play a significant role in the etiology of PD.
Culture as shared cognitive representations.
Authors: Romney A K , Boyd J P , Moore C C , Batchelder W H , Brazill T J
Published: July 2002

Culture consists of shared cognitive representations in the minds of individuals. This paper investigates the extent to which English speakers share the "same" semantic structure of English kinship terms. The semantic structure is defined as the arrangement of the terms relative to each other as represented in a metric space in which items judged more similar are placed closer to each other than items judged as less similar. The cognitive representation of the semantic structure, residing in the mind of an individual, is measured by judged similarity tasks involving comparisons among terms. Using six independent measurements, from each of 122 individuals, correspondence analysis represents the data in a common multidimensional spatial representation. Judged by a variety of statistical procedures, the individuals in our sample share virtually identical cognitive representations of the semantic structure of kinship terms. This model of culture accounts for 70-90% of the total variability in these data. We argue that our findings on kinship should generalize to all semantic domains--e.g., animals, emotions, etc. The investigation of semantic domains is important because they may reside in localized functional units in the brain, because they relate to a variety of cognitive processes, and because they have the potential to provide methods for diagnosing individual breakdowns in the structure of cognitive representations typical of such ailments as Alzheimer disease.
Escherichia coli exhibits negative chemotaxis in gradients of hydrogen peroxide, hypochlorite, and N-chlorotaurine: products of the respiratory burst of phagocytic cells.
Authors: Benov L , Fridovich I
Published: July 2002

Escherichia coli can respond to gradients of specific compounds, moving up gradients of attractants and down gradients of repellents. Stimulated phagocytic leukocytes produce H2O2, OCl-, and N-chlorotaurine in a response termed the respiratory burst. E. coli is actively repelled by these compounds. Catalase in the suspending medium eliminated the effect of H2O2. Repulsion by H2O2 could be demonstrated with 1 microM H2O2, which is far below the level that caused overt toxicity. Strains with defects in the biosynthesis of glutathione or lacking hydroperoxidases I and II retained this response to H2O2, and 2.0 mM CN- did not interfere with it. Mutants with defects in any one of the four known methyl-accepting chemotaxis proteins also retained the ability to respond to H2O2, but a "gutted" mutant that was deleted for all four methyl-accepting chemotaxis proteins, as well as for CheA, CheW, CheR, CheB, CheY, and CheZ, did not respond to H2O2. Hypochlorite and N-chlorotaurine were also strongly repellent. Chemotaxis down gradients of H2O2, OCl-, and N-chlorotaurine may contribute to the survival of commensal or pathogenic microorganisms.
Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity.
Authors: Southworth M W , Kong H , Kucera R B , Ware J , Jannasch H W , Perler F B
Published: July 2002

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.
Behavior predicts genes structure in a wild primate group.
Authors: Altmann J , Alberts S C , Haines S A , Dubach J , Muruthi P , Coote T , Geffen E , Cheesman D J , Mututua R S , Saiyalel S N , Wayne R K , Lacy R C , Bruford M W
Published: July 2002

The predictability of genetic structure from social structure and differential mating success was tested in wild baboons. Baboon populations are subdivided into cohesive social groups that include multiple adults of both sexes. As in many mammals, males are the dispersing sex. Social structure and behavior successfully predicted molecular genetic measures of relatedness and variance in reproductive success. In the first quantitative test of the priority-of-access model among wild primates, the reproductive priority of dominant males was confirmed by molecular genetic analysis. However, the resultant high short-term variance in reproductive success did not translate into equally high long-term variance because male dominance status was unstable. An important consequence of high but unstable short-term variance is that age cohorts will tend to be paternal sibships and social groups will be genetically substructured by age.
Conformational transitions monitored for single molecules in solution.
Authors: Edman L , Mets U , Rigler R
Published: July 2002

Phenomena that can be observed for a large number of molecules may not be understood if it is not possible to observe the events on the single-molecule level. We measured the fluorescence lifetimes of individual tetramethylrhodamine molecules, linked to an 18-mer deoxyribonucleotide sequence specific for M13 DNA, by time-resolved, single-photon counting in a confocal fluorescence microscope during Brownian motion in solution. When many molecules were observed, a biexponential fluorescence decay was observed with equal amplitudes. However, on the single-molecule level, the fraction of one of the amplitudes spanned from 0 to unity for a collection of single-molecule detections. Further analysis by fluorescence correlation spectroscopy made on many molecules revealed a process that obeys a stretched exponential relaxation law. These facts, combined with previous evidence of the quenching effect of guanosine on rhodamines, indicate that the tetramethylrhodamine molecule senses conformational transitions as it associates and dissociates to a guanosine-rich area. Thus, our results reveal conformational transitions in a single molecule in solution under conditions that are relevant for biological processes.
Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system.
Authors: Hagedorn M , Hsu E W , Pilatus U , Wildt D E , Rall W R , Blackband S J
Published: July 2002

Successful cryopreservation of most multicompartmental biological systems has not been achieved. One prerequisite for success is quantitative information on cryoprotectant permeation into and amongst the compartments. This report describes direct measurements of cryoprotectant permeation into a multicompartmental system using chemical shift selective magnetic resonance (MR) microscopy and MR spectroscopy. We used the developing zebrafish embryo as a model for studying these complex systems because these embryos are composed of two membrane-limited compartments: (i) a large yolk (surrounded by the yolk syncytial layer) and (ii) differentiating blastoderm cells (each surrounded by a plasma membrane). MR images of the spatial distribution of three cryoprotectants (dimethyl sulfoxide, propylene glycol, and methanol) demonstrated that methanol permeated the entire embryo within 15 min. In contrast, the other cryoprotectants exhibited little or no permeation over 2.5 h. MR spectroscopy and microinjections of cryoprotectants into the yolk inferred that the yolk syncytial layer plays a critical role in limiting the permeation of some cryoprotectants throughout the embryo. This study demonstrates the power of MR technology combined with micromanipulation for elucidating key physiological factors in cryobiology.
Structure of the catalytic fragment of poly(AD-ribose) polymerase from chicken.
Authors: Ruf A , Mennissier de Murcia J , de Murcia G , Schulz G E
Published: July 2002

The crystal structures of the catalytic fragment of chicken poly(ADP-ribose) polymerase [NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] with and without a nicotinamide-analogue inhibitor have been elucidated. Because this enzyme is involved in the regulation of DNA repair, its inhibitors are of interest for cancer therapy. The inhibitor shows the nicotinamide site and also suggests the adenosine site. The enzyme is structurally related to bacterial ADP-ribosylating toxins but contains an additional alpha-helical domain that is suggested to relay the activation signal issued on binding to damaged DNA.
Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis.
Authors: Sharma A K , Pangburn M K
Published: July 2002

Human complement factor H controls spontaneous activation of complement in plasma and appears to play a role in distinguishing host cells from activators of the alternative pathway of complement. In both mice and humans, the protein is composed of 20 homologous short consensus repeat (SCR) domains. The size of the protein suggests that portions of the structure outside the known C3b binding site (SCR 1-4) possess a significant biological role. We have expressed the full-length cDNA of factor H in the baculovirus system and have shown the recombinant protein to be fully active. Mutants of this full-length protein have now been prepared, purified, and examined for cofactor activity and binding to C3b and heparin. The results demonstrate (i) that factor H has at least three sites that bind C3b, (ii) that one of these sites is located in SCR domains 1-4, as has been shown by others, (iii) that a second site exists in the domain 6-10 region, (iv) that a third site resides in the SCR 16-20 region, and (v) that two heparin binding sites exist in factor H, one near SCR 13 and another in the SCR 6-10 region. Functional assays demonstrated that only the first C3b site located in SCR 1-4 expresses factor I cofactor activity. Mutant proteins lacking any one of the three C3b binding sites exhibited 6- to 8-fold reductions in affinity for C3b on sheep erythrocytes, indicating that all three sites contribute to the control of complement activation on erythrocytes. The identification of multiple functionally distinct sites on factor H clarifies many of the heretofore unexplainable behaviors of this protein, including the heterogeneous binding of factor H to surface-bound C3b, the effects of trypsin cleavage, and the differential control of complement activation on activators and nonactivators of the alternative pathway of complement.
Activation of memory circuits during cue-elicited cocaine craving.
Authors: Grant S , London E D , Newlin D B , Villemagne V L , Liu X , Contoreggi C , Phillips R L , Kimes A S , Margolin A
Published: July 2002

Evidence accumulated over more than 45 years has indicated that environmental stimuli can induce craving for drugs of abuse in individuals who have addictive disorders. However, the brain mechanisms that subserve such craving have not been elucidated. Here a positron emission tomographic study shows increased glucose metabolism in cortical and limbic regions implicated in several forms of memory when human volunteers who abuse cocaine are exposed to drug-related stimuli. Correlations of metabolic increases in the dorsolateral prefrontal cortex, medial temporal lobe (amygdala), and cerebellum with self-reports of craving suggest that a distributed neural network, which integrates emotional and cognitive aspects of memory, links environmental cues with cocaine craving.
Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica.
Authors: Mujer C V , Andrews D L , Manhart J R , Pierce S K , Rumpho M E
Published: July 2002

The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic theory of eukaryote origins.
Light-mediated retinoic acid production.
Authors: McCaffery P , Mey J , Dräger U C
Published: July 2002

Retinoids serve two main functions in biology: retinaldehyde forms the chromophore bound to opsins, and retinoic acid (RA) is the activating ligand of transcription factors. These two functions are linked in the vertebrate eye: we describe here that illumination of the retina results in an increase in RA synthesis, as detected with a RA bioassay and by HPLC. The synthesis is mediated by retinaldehyde dehydrogenases which convert some of the chromophore all-trans retinaldehyde, released from bleached rhodopsin, into RA. As the eye contains high levels of retinaldehyde dehydrogenases, and as the oxidation of retinaldehyde is an irreversible reaction, RA production has to be considered an unavoidable by-product of light. Through RA synthesis, light can thus directly influence gene transcription in the eye, which provides a plausible mechanism for light effects that cannot be explained by electric activity. Whereas the function of retinaldehyde as chromophore is conserved from bacteria to mammals, RA-mediated transcription is fully evolved only in vertebrates. Invertebrates differ from vertebrates in the mechanism of chromophore regeneration: while in the invertebrate visual cycle the chromophore remains bound, it is released as free all-trans retinaldehyde from illuminated vertebrate rhodopsin. RA synthesis occurring as corollary of dark regeneration in the vertebrate visual cycle may have given rise to the expansion of RA-mediated transcriptional regulation.
Induction of pheromone production in a moth by topical application of a pseudopeptide mimic of a pheromonotropic neuropeptide.
Authors: Abernathy R L , Teal P E , Meredith J A , Nachman R J
Published: July 2002

An amphiphilic analog of Locusta myotropin II (Lom-MT-II), Glu-Gly-Asp-Phe-Thr-Pro-Arg-Leu-amide, was synthesized by addition of 6-phenylhexanoic acid (6-Pha) linked through alanine to the amino terminus. This pseudopeptide, [6-Pha-Ala0]Lom-MT-II, was found to have pheromonotropic activity equivalent to pheromone biosynthesis activating neuropeptide when injected into females of Heliothis virescens. Topical application of [6-Pha-Ala0]Lom-MT-II or Helicoverpa zea-pheromone biosynthesis activating neuropeptide (PBAN), dissolved in dimethyl sulfoxide, to the descaled abdomen of females induced production of pheromone, although more Hez-PBAN than [6-Pha-Ala0]Lom-MT-II was required to obtain significant production of pheromone. Application of [6-Pha-Ala0]Lom-MT-II, dissolved in water, to the abdomen induced production of pheromone, but neither Hez-PBAN nor Lom-MT-II dissolved in water stimulated production of significant amounts of pheromone. Dose- and time-response studies indicated that application of the amphiphilic mimetic in water induced pheromone production in as little as 15 min after application and that the effects were maintained for prolonged periods. These findings show that amphiphilic pseudopeptide mimics of insect neuropeptides will penetrate the insect cuticle when applied topically in water and induce an endogenous response.
Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus
Candida albicans
Authors: Leberer Ekkehard , Harcus Doreen , Broadbent Ian D. , Clark Karen L. , Dignard Daniel , Ziegelbauer Karl , Schmidt Axel , Gow Neil A. R. , Brown Alistair J. P. , Thomas David Y.
Published: July 2002

The
CST20
gene of
Candida
albicans
was cloned by functional complementation of a deletion
of the
STE20
gene in
Saccharomyces
cerevisiae
.
CST20
encodes a homolog of the
Ste20p/p65
PAK
family of protein kinases. Colonies of
C. albicans
cells deleted for
CST20
revealed defects in the lateral formation of mycelia on synthetic solid
“Spider” media. However, hyphal development was not impaired in
some other media. A similar phenotype was caused by deletion of
HST7
, encoding a functional homolog of the
S.
cerevisiae
Ste7p protein kinase. Overexpression of
HST7
partially complemented the deletion of
CST20
. Cells deleted for
CST20
were less virulent in a mouse model for systemic candidiasis. Our
results suggest that more than one signaling pathway can trigger hyphal
development in
C. albicans
, one of which has a protein
kinase cascade that is analogous to the mating response pathway in
S. cerevisiae
and might have become adapted to the
control of mycelial formation in asexual
C. albicans
.
Levels of the growth-associated protein GAP-43 are selectively increased in association cortices in schizophrenia
Authors: Perrone-Bizzozero Nora I. , Sower Angela C. , Bird Edward D. , Benowitz Larry I. , Ivins Kathryn J. , Neve Rachael L.
Published: July 2002

The pathophysiology of schizophrenia may involve
perturbations of synaptic organization during development. The presence
of cytoarchitectural abnormalities that may reflect such perturbations
in the brains of patients with this disorder has been well-documented.
Yet the mechanistic basis for these features of the disorder is still
unknown. We hypothesized that altered regulation of the neuronal
growth-associated protein GAP-43, a membrane phosphoprotein found at
high levels in the developing brain, may play a role in the alterations
in brain structure and function observed in schizophrenia. In the
mature human brain, GAP-43 remains enriched primarily in association
cortices and in the hippocampus, and it has been suggested that this
protein marks circuits involved in the acquisition, processing,
and/or storage of new information. Because these processes are known
to be altered in schizophrenia, we proposed that GAP-43 levels might be
altered in this disorder. Quantitative immunoblots revealed that the
expression of GAP-43 is increased preferentially in the visual
association and frontal cortices of schizophrenic patients, and that
these changes are not present in other neuropsychiatric conditions
requiring similar treatments. Examination of the levels of additional
markers in the brain revealed that the levels of the synaptic vesicle
protein synaptophysin are reduced in the same areas, but that the
abundance of the astrocytic marker of neurodegeneration, the glial
fibrillary acidic protein, is unchanged.
In situ
hybridization histochemistry was used to show that the laminar pattern
of GAP-43 expression appears unaltered in schizophrenia. We propose
that schizophrenia is associated with a perturbed organization of
synaptic connections in distinct cortical associative areas of the
human brain, and that increased levels of GAP-43 are one manifestation
of this dysfunctional organization.
Five-fold symmetry in crystalline quasicrystal lattices
Authors: Caspar Donald L. D. , Fontano Eric
Published: July 2002

To demonstrate that crystallographic methods can be applied to index and interpret diffraction patterns from well-ordered quasicrystals that display non-crystallographic 5-fold symmetry, we have characterized the properties of a series of periodic two-dimensional lattices built from pentagons, called Fibonacci pentilings, which resemble aperiodic Penrose tilings. The computed diffraction patterns from periodic pentilings with moderate size unit cells show decagonal symmetry and are virtually indistinguishable from that of the infinite aperiodic pentiling. We identify the vertices and centers of the pentagons forming the pentiling with the positions of transition metal atoms projected on the plane perpendicular to the decagonal axis of quasicrystals whose structure is related to crystalline η phase alloys. The characteristic length scale of the pentiling lattices, evident from the Patterson (autocorrelation) function, is ∼τ2times the pentagon edge length, where τ is the golden ratio. Within this distance there are a finite number of local atomic motifs whose structure can be crystallographically refined against the experimentally measured diffraction data.