bluuebunny/crossref_metadata_2025 huggingface dataset:
Atomic layer deposition of hafnium oxide: A detailed reaction mechanism from first principles
Authors: Widjaja Yuniarto , Musgrave Charles B.
Published: July 2002

Atomic layer deposition (ALD) of hafnium oxide (HfO2) using HfCl4 and H2O as precursors is studied using density functional theory. The mechanism consists of two deposition half-reactions: (1) HfCl4 with Hf-OH sites, and (2) H2O with Hf-Cl sites. Both half-reactions exhibit stable intermediates with energies lower than those of the final products. We show that increasing the temperature reduces the stability of the complex. However, increasing temperature also increases the dissociation free-energy barrier, which in turn results in increased desorption of adsorbed precursors. Both half-reactions are qualitatively similar to the corresponding reactions of ZrO2 ALD using ZrCl4 and H2O.
Pt Schottky contacts to n-(Ga,Mn)N
Authors: Kim Jihyun , Ren F. , Thaler G. T. , Overberg M. E. , Abernathy C. R. , Pearton S. J. , Wilson R. G.
Published: July 2002

The Schottky barrier height of Pt contacts on n-(Ga,Mn)N (n∼3.5×1017 cm−3) thin films was obtained from current–voltage measurements as a function of temperature. The resulting values ranged from 0.82±0.04 eV at 25 °C to 0.79±0.06 eV at 100 °C with saturation current densities of 4.28×10−8 A cm−2 (25 °C) to 8.42×10−5 A cm−2 (100 °C), respectively. The barrier height at room temperature obtained from an activation energy plot was 0.91±0.06 eV. The reverse current magnitude was larger than predicted by thermionic emission alone, just as in n-GaN grown in a similar fashion on Al2O3 substrates. The measured barrier height for Pt on n-(Ga,Mn)N is lower than for the value reported on n-GaN(1.08 eV).
Distinct roles for jasmonate synthesis and action in the systemic wound response of tomato
Authors: Li Lei , Li Chuanyou , Lee Gyu In , Howe Gregg A.
Published: July 2002

Plant defense responses to wounding and herbivore attack are regulated by signal transduction pathways that operate both at the site of wounding and in undamaged distal leaves. Genetic analysis in tomato indicates that systemin and its precursor protein, prosystemin, are upstream components of a wound-induced, intercellular signaling pathway that involves both the biosynthesis and action of jasmonic acid (JA). To examine the role of JA in systemic signaling, reciprocal grafting experiments were used to analyze wound-induced expression of the
proteinase inhibitor II
gene in a JA biosynthetic mutant (
spr
-
2
) and a JA response mutant (
jai
-
1
). The results showed that
spr
-
2
plants are defective in the production, but not recognition, of a graft-transmissible wound signal. Conversely,
jai
-
1
plants are compromised in the recognition of this signal but not its production. It was also determined that a graft-transmissible signal produced in response to ectopic expression of prosystemin in rootstocks was recognized by
spr
-
2
but not by
jai
-
1
scions. Taken together, the results show that activation of the jasmonate biosynthetic pathway in response to wounding or (pro)systemin is required for the production of a long-distance signal whose recognition in distal leaves depends on jasmonate signaling. These findings suggest that JA, or a related compound derived from the octadecanoid pathway, may act as a transmissible wound signal.
Previously uncharacterized histone acetyltransferases implicated in mammalian spermatogenesis
Authors: Lahn Bruce T. , Tang Zhao Lan , Zhou Jianxin , Barndt Robert J. , Parvinen Martti , Allis C. David , Page David C.
Published: July 2002

During spermiogenesis (the maturation of spermatids into spermatozoa) in many vertebrate species, protamines replace histones to become the primary DNA-packaging protein.It has long been thought that this process is facilitated by the hyperacetylation of histone H4. However, the responsible histone acetyltransferase enzymes are yet to be identified.CDYis a human Y-chromosomal gene family expressed exclusively in the testis and implicated in male infertility. Its mouse homologCdyl, which is autosomal, is expressed abundantly in the testis. Proteins encoded byCDYand its homologs bear the “chromodomain,” a motif implicated in chromatin binding. Here, we show that (i) human CDY and mouse CDYL proteins exhibit histone acetyltransferase activityin vitro, with a strong preference for histone H4; (ii) expression of humanCDYand mouseCdylgenes during spermatogenesis correlates with the occurrence of H4 hyperacetylation; and (iii) CDY and CDYL proteins are localized to the nuclei of maturing spermatids where H4 hyperacetylation takes place. Taken together, these data link human CDY and mouse CDYL to the histone-to-protamine transition in mammalian spermiogenesis. This link offers a plausible mechanism to account for spermatogenic failure in patients bearing deletions of theCDYgenes.
Ab initio
protein structure prediction on a genomic scale: Application to the
Mycoplasma genitalium
genome
Authors: Kihara Daisuke , Zhang Yang , Lu Hui , Kolinski Andrzej , Skolnick Jeffrey
Published: July 2002

An
ab initio
protein structure prediction procedure, TOUCHSTONE
,
was applied to all 85 small proteins of the
Mycoplasma genitalium
genome. TOUCHSTONE is based on a Monte Carlo refinement of a lattice model of proteins, which uses threading-based tertiary restraints. Such restraints are derived by extracting consensus contacts and local secondary structure from at least weakly scoring structures that, in some cases, can lack any global similarity to the sequence of interest. Selection of the native fold was done by using the convergence of the simulation from two different conformational search schemes and the lowest energy structure by a knowledge-based atomic-detailed potential. Among the 85 proteins, for 34 proteins with significant threading hits, the template structures were reasonably well reproduced. Of the remaining 51 proteins, 29 proteins converged to five or fewer clusters. In the test set, 84.8% of the proteins that converged to five or fewer clusters had a correct fold among the clusters. If this statistic is simply applied, 24 proteins (84.8% of the 29 proteins) may have correct folds. Thus, the topology of a total of 58 proteins probably has been correctly predicted. Based on these results,
ab initio
protein structure prediction is becoming a practical approach.
Rapid natural scene categorization in the near absence of attention
Authors: Li Fei Fei , VanRullen Rufin , Koch Christof , Perona Pietro
Published: July 2002

What can we see when we do not pay attention? It is well known that we can be “blind” even to major aspects of natural scenes when we attend elsewhere. The only tasks that do not need attention appear to be carried out in the early stages of the visual system. Contrary to this common belief, we report that subjects can rapidly detect animals or vehicles in briefly presented novel natural scenes while simultaneously performing another attentionally demanding task. By comparison, they are unable to discriminate large T's from L's, or bisected two-color disks from their mirror images under the same conditions. We conclude that some visual tasks associated with “high-level” cortical areas may proceed in the near absence of attention.
Identifying genes of agronomic importance in maize by screening microsatellites for evidence of selection during domestication
Authors: Vigouroux Y. , McMullen M. , Hittinger C. T. , Houchins K. , Schulz L. , Kresovich S. , Matsuoka Y. , Doebley J.
Published: July 2002

Crop species experienced strong selective pressure directed at genes controlling traits of agronomic importance during their domestication and subsequent episodes of selective breeding. Consequently, these genes are expected to exhibit the signature of selection. We screened 501 maize genes for the signature of selection using microsatellites or simple sequence repeats (SSRs). We applied the Ewens–Watterson test, which can reveal deviations from a neutral-equilibrium model, as well as two nonequilibrium tests that incorporate the domestication bottleneck. We investigated two classes of SSRs: those known to be polymorphic in maize (Class I) and those previously classified as monomorphic in maize (Class II). Fifteen SSRs exhibited some evidence for selection in maize and 10 showed evidence under stringent criteria. The genes containing nonneutral SSRs are candidates for agronomically important genes. Because demographic factors can bias our tests, further independent tests of these candidates are necessary. We applied such an additional test to one candidate, which encodes a MADS box transcriptional regulator, and confirmed that this gene experienced a selective sweep during maize domestication. Genomic scans for the signature of selection offer a means of identifying new genes of agronomic importance even when gene function and the phenotype of interest are unknown.
Characterization of an orphan G protein-coupled receptor localized in the dorsal root ganglia reveals adenine as a signaling molecule
Authors: Bender Eckhard , Buist Arjan , Jurzak Mirek , Langlois Xavier , Baggerman Geert , Verhasselt Peter , Ercken Martine , Guo Hong-Qing , Wintmolders Cindy , Van den Wyngaert Ilse , Van Oers Irma , Schoofs Liliane , Luyten Walter
Published: July 2002

The cloning of novel G protein-coupled receptors and the search for their natural ligands, a process called reverse pharmacology, is an excellent opportunity to discover novel hormones and neurotransmitters. Based on a degenerate primer approach we have cloned a G protein-coupled receptor whose mRNA expression profile indicates highest expression in the dorsal root ganglia, specifically in the subset of small neurons, suggesting a role in nociception. In addition, moderate expression was found in lung, hypothalamus, peripheral blood leukocytes, and ovaries. Guided by a receptor-activation bioassay, we identified adenine as the endogenous ligand, which activated the receptor potently and with high structural stringency. Therefore, we propose to name this receptor as the adenine receptor. Hormonal functions have already been demonstrated for adenine derivatives like 6-benzylaminopurine in plants and 1-methyladenine in lower animals. Here, we demonstrate that adenine functions as a signaling molecule in mammals. This finding adds a third family besides P1 and P2 receptors to the class of purinergic receptors.
The myelin basic protein gene is expressed in differentiated blood cell lineages and in hemopoietic progenitors
Authors: Marty M. C. , Alliot F. , Rutin J. , Fritz R. , Trisler D. , Pessac B.
Published: July 2002

Myelin basic proteins (MBP) are major constituents of the myelin sheath of oligodendrocytes and Schwann cells in the central nervous system and the peripheral nervous system, respectively. We previously showed that MBP-related transcripts are present in the bone marrow and the immune system. These mRNAs are transcribed from a region called 0′, consisting of three exons, located upstream of the classical MBP exons; these three exons belong to the long MBP gene otherwise called “Golli-MBP.” The most abundant of these mRNAs, now called HMBP (hemopoietic MBP), encompasses the sequence encoded by the region 0′ plus exon 1 and part of intron 1 of the classic MBP gene. Antisera to recombinant HMBP proteins are immunoreactive with proteins of about 26–28 kDa in brain, thymus, and spleen. This report demonstrates that HMBP proteins are present in the vast majority (>95%) of thymic T cells, which express the corresponding transcripts, as do mature T cells from lymph nodes and spleen. HMBP mRNAs and proteins are also manifest in the majority of spleen B lymphocytes and in B cell lines. In addition to lymphoid cells, HMBP proteins are in all types of myeloid lineage cells, i.e., macrophages, dendritic cells, and granulocytes, as well as in megakaryocytes and erythroblasts. Finally, HMBP proteins are present in CD34+bone marrow cells, and, furthermore, in highly proliferative cultures, these CD34+cells express HMBP RNAs and proteins. Thus, MBP gene products are present both in the nervous system and in the entire hemopoietic system.
Evolutionary genomics of
Salmonella
: Gene acquisitions revealed by microarray analysis
Authors: Porwollik Steffen , Wong Rita Mei-Yi , McClelland Michael
Published: July 2002

The presence of homologues of
Salmonella
enterica
sv. Typhimurium LT2 genes was assessed in 22 other
Salmonella
including members of all seven subspecies and
Salmonella bongori
. Genomes were hybridized to a microarray of over 97% of the 4,596 annotated ORFs in the LT2 genome. A phylogenetic tree based on homologue content, relative to LT2, was largely concordant with previous studies using sequence information from several loci. Based on the topology of this tree, homologues of genes in LT2 acquired by various clades were predicted including 513 homologues acquired by the ancestor of all
Salmonella
, 111 acquired by
S. enterica
, 105 by diphasic
Salmonella
, and 216 by subspecies 1, most of which are of unknown function. Because this subspecies is responsible for almost all
Salmonella
infections of mammals and birds, these genes will be of particular interest for further mechanistic studies. Overall, a high level of gene gain, loss, or rapid divergence was predicted along all lineages. For example, at least 425 close homologues of LT2 genes may have been laterally transferred into
Salmonella
and then between
Salmonella
lineages.
Mutations in APC, Kirsten-ras, and p53—alternative genetic pathways to colorectal cancer
Authors: Smith Gillian , Carey Francis A. , Beattie Julie , Wilkie Murray J. V. , Lightfoot Tracy J. , Coxhead Jonathan , Garner R. Colin , Steele Robert J. C. , Wolf C. Roland
Published: July 2002

Colorectal cancer is one of the most significant causes of cancer death. A genetic model for colorectal cancer has been proposed in which the sequential accumulation of mutations in specific genes, including adenomatous polyposis coli (APC), Kirsten-ras (K-ras), and p53, drives the transition from healthy colonic epithelia through increasingly dysplastic adenoma to colorectal cancer. We have characterized tumor mutation spectra in a large cohort of colorectal cancer patients. In marked contrast to the predictions of the sequential model of mutation accumulation, only 6.6% of tumors were found to contain mutations in APC, K-ras, and p53, with 38.7% of tumors containing mutations in only one of these genes. The most common combination of mutations was p53 and APC (27.1%), whereas mutations in both p53 and K-ras were extremely rare. Statistical analysis (two-sided Fisher's exact test) confirmed that mutations in K-ras and p53 co-occurred less frequently than expected by chance (
P
< 0.01, Fisher's exact test). This finding suggests that these mutations lie on alternate pathways of colorectal tumor development. The heterogeneous pattern of tumor mutations in our patient cohort suggests that multiple alternative genetic pathways to colorectal cancer exist and that the widely accepted genetic model of cancer development is not representative of the majority of colorectal tumors.
Bucillamine, a thiol antioxidant, prevents transplantation-associated reperfusion injury
Authors: Amersi Farin , Nelson Sally K. , Shen Xiu Da , Kato Hirohisa , Melinek Judy , Kupiec-Weglinski Jerzy W. , Horwitz Lawrence D. , Busuttil Ronald W. , Horwitz Marcus A.
Published: July 2002

Ischemia/reperfusion (I/R) injury is a serious potential threat to outcomes in organ transplantation and other clinical arenas in which there is temporary interruption of blood flow. I/R is a frequent cause of primary failure in organ transplantation. We hypothesized that the antioxidant bucillamine, a potent sulfhydryl donor, would protect against I/R injury in high-risk organ transplants. Because livers subjected to prolonged ischemia and very fatty livers are highly susceptible to severe I/R injury, we studied the effect of bucillamine in three animal models of liver transplantation: two
ex vivo
models of isolated perfused livers, either normal or fatty rat livers, and an
in vivo
model of syngenic orthotopic liver transplants in rats. In all models, livers were deprived of oxygen for 24 h before either
ex vivo
reperfusion or transplantation. In the
ex vivo
models, bucillamine treatment significantly improved portal vein blood flow and bile production, preserved normal liver architecture, and significantly reduced liver enzyme release and indices of oxidative stress. Moreover, bucillamine treatment significantly increased levels of reduced glutathione in the liver and lowered levels of oxidized glutathione in both liver and blood. In rats subjected to liver transplants, bucillamine significantly enhanced survival and protected against hepatic injury. Possible mechanisms of this protection include prevention of excessive accumulation of toxic oxygen species, interruption of redox signaling in hepatocytes, and inhibition of macrophage activation. This study demonstrates the potential utility of bucillamine or other cysteine-derived thiol donors for improving outcomes in organ transplantation and other clinical settings involving I/R injury.
The aspartate-257 of presenilin 1 is indispensable for mouse development and production of β-amyloid peptides through β-catenin-independent mechanisms
Authors: Xia Xuefeng , Wang Pei , Sun Xiaoyan , Soriano Salvador , Shum Wan-Kyng , Yamaguchi Haruyasu , Trumbauer Myrna E. , Takashima Akihiko , Koo Edward H. , Zheng Hui
Published: July 2002

To differentiate multiple activities of presenilin 1 (PS1), we generated transgenic mice expressing two human
PS1
alleles: one with the aspartate to alanine mutation at residue 257 (hPS1D257A) that impairs the proteolytic activity of PS1, and the other deleting amino acids 340–371 of the hydrophilic loop sequence (hPS1Δcat) essential for β-catenin interaction. We show here that although hPS1Δcat is fully competent in rescuing the
PS1
-null lethal phenotype, hPS1D257A does not exhibit developmental activity. hPS1D257A also leads to the concurrent loss of the proteolytic processing of Notch and β-amyloid precursor protein (APP) and the generation of β-amyloid peptides (Aβ). Further, by measuring the levels of endogenous Aβ
X-40
and Aβ
X-42
in primary neuronal cultures, we confirmed the concept that PS1 is indispensable for the production of secreted Aβ.
PRKX, a phylogenetically and functionally distinct cAMP-dependent protein kinase, activates renal epithelial cell migration and morphogenesis
Authors: Li Xiaohong , Li Hsi-Ping , Amsler Kurt , Hyink Deborah , Wilson Patricia D. , Burrow Christopher R.
Published: July 2002

The human protein kinase X gene (
PRKX
) is a member of an ancient family of cAMP-dependent serine/threonine kinases here shown to be phylogenetically distinct from the classical
PKA
,
PKB
/
Akt
,
PKC
,
SGK
, and
PKG
gene families. Renal expression of the
PRKX
gene is developmentally regulated and restricted to the ureteric bud epithelium of the fetal metanephric kidney. Aberrant adult kidney expression of
PRKX
was found in autosomal dominant polycystic kidney disease. PRKX kinase expression markedly activated migration of cultured renal epithelial cells in the presence of cAMP; this effect was blocked by cell treatment with the PKA inhibitor H89 and was not observed in
PKA
-transfected cells. In addition, expression of PRKX kinase activated branching morphogenesis of Madin–Darby canine kidney cells in collagen gels even in the absence of cAMP and/or hepatocyte growth factor, an effect not seen with either PKA expression or expression of a mutant, kinase-inactivated PRKX. These results suggest that the PRKX kinase may regulate epithelial morphogenesis during mammalian kidney development. Because another member of the
PRKX
gene family (the
Dictyostelium discoideum
gene
KAPC
-
DICDI
) also plays a role in cellular migration, these studies suggest that regulation of morphogenesis may be a distinctive property of these genes that has been conserved in evolution that is not shared with
PKA
family genes.
Suppression and acceleration of cell elongation by integration of xyloglucans in pea stem segments
Authors: Takeda Takumi , Furuta Yuzo , Awano Tatsuya , Mizuno Koichi , Mitsuishi Yasushi , Hayashi Takahisa
Published: July 2002

Xyloglucan is a key polymer in the walls of growing plant cells. Using split pea stem segments and stem segments from which the epidermis had been peeled off, we demonstrate that the integration of xyloglucan mediated by the action of wall-bound xyloglucan endotransglycosylase suppressed cell elongation, whereas that of its fragment oligosaccharide accelerated it. Whole xyloglucan was incorporated into the cell wall and induced the rearrangement of cortical microtubules from transverse to longitudinal; in contrast, the oligosaccharide solubilized xyloglucan from the cell wall and maintained the microtubules in a transverse orientation. This paper proposes that xyloglucan metabolism controls the elongation of plant cells.
Human α-synuclein-harboring familial Parkinson's disease-linked Ala-53 → Thr mutation causes neurodegenerative disease with α-synuclein aggregation in transgenic mice
Authors: Lee Michael K. , Stirling Wanda , Xu Yanqun , Xu Xueying , Qui Dike , Mandir Allen S. , Dawson Ted M. , Copeland Neal G. , Jenkins Nancy A. , Price Don L.
Published: July 2002

Mutations in α-synuclein (α-Syn) cause Parkinson's disease (PD) in a small number of pedigrees with familial PD. Moreover, α-Syn accumulates as a major component of Lewy bodies and Lewy neurites, intraneuronal inclusions that are neuropathological hallmarks of PD. To better understand the pathogenic relationship between alterations in the biology of α-Syn and PD-associated neurodegeneration, we generated multiple lines of transgenic mice expressing high levels of either wild-type or familial PD-linked Ala-30 → Pro (A30P) or Ala-53 → Thr (A53T) human α-Syns. The mice expressing the A53T human α-Syn, but not wild-type or the A30P variants, develop adult-onset neurodegenerative disease with a progressive motoric dysfunction leading to death. Pathologically, affected mice exhibit neuronal abnormalities (in perikarya and neurites) including pathological accumulations of α-Syn and ubiquitin. Consistent with abnormal neuronal accumulation of α-Syn, brain regions with pathology exhibit increases in detergent-insoluble α-Syn and α-Syn aggregates. Our results demonstrate that the A53T mutant α-Syn causes significantly greater
in vivo
neurotoxicity as compared with other α-Syn variants. Further, α-Syn-dependent neurodegeneration is associated with abnormal accumulation of detergent-insoluble α-Syn.
An approach for global scanning of single nucleotide variations
Authors: Pan Xinghua , Weissman Sherman M.
Published: July 2002

Efficient global scanning of single nucleotide variations in DNA sequences between related, complex DNA samples remains a challenge. In the present article we present an approach to this problem. We have used immobilized thymidine DNA glycosylases to capture and enrich DNA fragments containing internal mismatched base pairs and separate these fragments as a pool from perfectly base-paired fragments as another pool. Enrichments of up to several hundredfold were obtained with one cycle of treatment, and all of the four groups of single nucleotide mismatches were fully covered by combining use of two thymine DNA glycosylases generated here. We have used a heterohybrid-orientating strategy for selective amplification of duplexes with one strand derived from each of two input DNA samples, which can also be used for selective amplification of duplexes with both strands derived from one of two input samples when desired. By combining these methods, the single nucleotide variations either between two DNA pools or within one DNA pool can be obtained in one process. This approach has been applied to the total cDNA from a human cell line and has several potential applications in mapping genetic variations, particularly global scanning of cDNA single nucleotide variations or polymorphisms, and finally high-throughput mapping of complex genetic traits.
An estrogen receptor repressor induces cataract formation in transgenic mice
Authors: Davis Vicki L. , Chan Chi-Chao , Schoen Timothy J. , Couse John F. , Chader Gerald J. , Korach Kenneth S.
Published: July 2002

Despite the high prevalence of age-related cataracts, there are currently no known therapies to delay or prevent their occurrence. Studies in humans and rodent models suggest that estrogen may provide protection against age-related cataracts. The discovery of ocular estrogen receptors (ERs) indicates that estrogen protection may result from direct interactions with its receptors in the eye, instead an indirect consequence from effects on another tissue. Studies in our transgenic mouse model validate the concept that estrogen is beneficial for the eye. These mice express ERΔ3, a dominant-negative form of ERα that inhibits ERα function. In the ERΔ3 transgenic mice, cortical cataracts spontaneously form in ERΔ3 females after puberty and progress with age. The cataracts initiate in the equatorial region of the lens where the epithelial cells differentiate into elongating fiber cells. Cataract formation can be prevented if the females are ovariectomized before, but not after, sexual maturity. Both male and female ERΔ3 mice develop cataracts after neonatal treatment with the potent estrogen diethylstilbestrol (DES). The incidence of spontaneous and DES-induced cataracts in ERΔ3 mice is 100%, yet these cataracts are absent from the wild-type mice. These data suggest that repression of estrogen action induces cortical cataract formation because estrogen is required to activate the ERΔ3 repressor. Evidence of DES-induced cataracts in the ERΔ3 males as well as the females suggests that estrogen is important in lens physiology in both sexes. The ERΔ3 mice provide a powerful model for assessing the role of estrogen in maintaining the transparency of the lens.
On the evolution of cells
Authors: Woese Carl R.
Published: July 2002

A theory for the evolution of cellular organization is presented. The model is based on the (data supported) conjecture that the dynamic of horizontal gene transfer (HGT) is primarily determined by the organization of the recipient cell. Aboriginal cell designs are taken to be simple and loosely organized enough that all cellular componentry can be altered and/or displaced through HGT, making HGT the principal driving force in early cellular evolution. Primitive cells did not carry a stable organismal genealogical trace. Primitive cellular evolution is basically communal. The high level of novelty required to evolve cell designs is a product of communal invention, of the universal HGT field, not intralineage variation. It is the community as a whole, the ecosystem, which evolves. The individual cell designs that evolved in this way are nevertheless fundamentally distinct, because the initial conditions in each case are somewhat different. As a cell design becomes more complex and interconnected a critical point is reached where a more integrated cellular organization emerges, and vertically generated novelty can and does assume greater importance. This critical point is called the “Darwinian Threshold” for the reasons given.
Three-dimensional structure of the amino-terminal domain of syntaxin 6, a SNAP-25 C homolog
Authors: Misura Kira M. S. , Bock Jason B. , Gonzalez Lino C. , Scheller Richard H. , Weis William I.
Published: July 2002

Soluble
N-
ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are required for intracellular membrane fusion, and are differentially localized throughout the cell. SNAREs on vesicle and target membranes contain “SNARE motifs” which interact to form a four-helix bundle that contributes to the fusion of two membranes. SNARE motif sequences fall into four classes, homologous to the neuronal proteins syntaxin 1a, VAMP 2, and the N- and C-terminal SNARE motifs of SNAP-25 (S25N and S25C), and it is thought that one member from each class interacts to form a SNARE complex. Many SNAREs also feature N-terminal domains believed to function in regulating SNARE complex assembly or other aspects of vesicle transport. Syntaxin 6 is a SNARE found primarily in endosomal transport vesicles and whose SNARE motif shows significant homology to both syntaxin 1a and S25C. The crystal structure of the syntaxin 6 N-terminal domain reveals strong structural similarity with the N-terminal domains of syntaxin family members syntaxin 1a, Sso1p, and Vam3p, despite a very low level of sequence similarity. The syntaxin 6 SNARE motif can substitute for S25C in
in vitro
binding experiments, supporting the classification of syntaxin 6 as an S25C family member. Secondary structure prediction of SNARE proteins shows that the N-terminal domains of many syntaxin, S25N, and S25C family members are likely to be similar to one another, but are distinct from those of VAMP family members, indicating that syntaxin, S25N, and S25C SNAREs may have shared a common ancestor.
Transcriptional response of
Saccharomyces cerevisiae
to DNA-damaging agents does not identify the genes that protect against these agents
Authors: Birrell Geoff W. , Brown James A. , Wu H. Irene , Giaever Guri , Chu Angela M. , Davis Ronald W. , Brown J. Martin
Published: July 2002

The recent completion of the deletion of all of the nonessential genes in budding yeast has provided a powerful new way of determining those genes that affect the sensitivity of this organism to cytotoxic agents. We have used this system to test the hypothesis that genes whose transcription is increased after DNA damage are important for the survival to that damage. We used a pool of 4,627 diploid strains each with homozygous deletion of a nonessential gene to identify those genes that are important for the survival of yeast to four DNA-damaging agents: ionizing radiation, UV radiation, and exposure to cisplatin or to hydrogen peroxide. In addition we measured the transcriptional response of the wild-type parental strain to the same DNA-damaging agents. We found no relationship between the genes necessary for survival to the DNA-damaging agents and those genes whose transcription is increased after exposure. These data show that few, if any, of the genes involved in repairing the DNA lesions produced in this study, including double-strand breaks, pyrimidine dimers, single-strand breaks, base damage, and DNA cross-links, are induced in response to toxic doses of the agents that produce these lesions. This finding suggests that the enzymes necessary for the repair of these lesions are at sufficient levels within the cell. The data also suggest that the nature of the lesions produced by DNA-damaging agents cannot easily be deduced from gene expression profiling.
Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC- restricted T cell receptor-like specificity
Authors: Denkberg Galit , Cohen Cyril J. , Lev Avital , Chames Patrick , Hoogenboom Hennie R. , Reiter Yoram
Published: July 2002

Specificity in the cellular immune system is controlled and regulated by the T cell antigen receptor (TCR), which specifically recognizes peptide/major histocompatibility complex (MHC) molecules. In recent years many cancer-associated MHC-restricted peptides have been isolated and because of their highly restricted fine specificity, they are desirable targets for novel approaches in immunotherapy. Antibodies that would recognize tumor-associated MHC–peptide complexes with the same specificity as the TCR would be valuable reagents for studying antigen presentation by tumor cells, for visualizing MHC–peptide complexes on cells, and eventually for monitoring the expression of specific complexes during immunotherapy. To generate molecules with such a unique fine specificity, we selected a large nonimmune repertoire of phage Fab antibodies on recombinant HLA-A2 complexed with three common antigenic T cell, HLA-A2-restricted epitopes derived from the melanoma differentiation antigen gp100. We were able to isolate a surprisingly large panel of human recombinant Fab antibodies that exhibit a characteristic TCR-like binding specificity to each of the three gp100-derived epitopes, yet unlike TCRs, they did so with an affinity in the nanomolar range. These TCR-like antibodies recognize the native MHC–peptide complex expressed on the surface of antigen-presenting cells. Moreover, they can detect the specific MHC–peptide complexes on the surface of melanoma tumor cells. These results demonstrate the ability to isolate high-affinity human recombinant antibodies with the antigen-specific, MHC-restricted specificity of T cells, and this ability was demonstrated for three different epitopes of the same melanoma-derived antigen.
Interactions between Eph kinases and ephrins provide a mechanism to support platelet aggregation once cell-to-cell contact has occurred
Authors: Prevost Nicolas , Woulfe Donna , Tanaka Takako , Brass Lawrence F.
Published: July 2002

Eph kinases are receptor tyrosine kinases whose ligands, the ephrins, are also expressed on the surface of cells. Interactions between Eph kinases and ephrins on adjacent cells play a central role in neuronal patterning and vasculogenesis. Here we examine the expression of ephrins and Eph kinases on human blood platelets and explore their role in the formation of the hemostatic plug. The results show that human platelets express EphA4 and EphB1, and the ligand, ephrinB1. Forced clustering of EphA4 or ephrinB1 led to cytoskeletal reorganization, adhesion to fibrinogen, and α-granule secretion. Clustering of ephrinB1 also caused activation of the Ras family member, Rap1B. In platelets that had been activated by ADP and allowed to aggregate, EphA4 formed complexes with two tyrosine kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Blockade of Eph/ephrin interactions prevented the formation of these complexes and caused platelet aggregation at low ADP concentrations to become more readily reversible. We propose that when sustained contacts between platelets have occurred in response to agonists such as collagen, ADP, and thrombin, the binding of ephrins to Eph kinases on adjacent platelets provides a mechanism to perpetuate signaling and promote stable platelet aggregation.
Increased competition may promote species coexistence
Authors: Vandermeer J. , Evans M. A. , Foster P. , Höök T. , Reiskind M. , Wund M.
Published: July 2002

It is a mainstay of community ecology that local exclusion of species will result if competitive pressures become too large. The pattern of exclusion may be complicated, but the qualitative orthodoxy has changed little since the pioneering work of Lotka, Volterra, and Gause—no two species can occupy the same niche. Stated in a more precise form, the higher the intensity of interspecific competition in an assemblage of species, the fewer the number of species that can coexist in perpetuity. We suggest that this orthodoxy results from “linear” thinking, and that if the classical equations are formulated more realistically with attendant nonlinearities, the orthodoxy breaks down and higher levels of competition may actually increase the likelihood that species will avoid competitive exclusion. Furthermore, this increased probability of coexistence at higher levels of competition is accompanied by characteristic dynamic patterns: (
i
) at lower levels of competition, after all extinction events have occurred, remaining species follow irregular chaotic patterns; (
ii
) at higher levels of competition, when most species coexist, all species are entrained in a single large limit cycle; (
iii
) the transient behavior appears to correspond to a special case of chaos, uniform phase chaotic amplitude.
Visualization of maltose uptake in living yeast cells by fluorescent nanosensors
Authors: Fehr Marcus , Frommer Wolf B. , Lalonde Sylvie
Published: July 2002

Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring. Currently, methods are not available for
in vivo
metabolite imaging at cellular or subcellular levels. Limited information derives from methods requiring fixation or fractionation of tissue (1, 2). We thus developed a flexible strategy for designing protein-based nanosensors for a wide spectrum of solutes, allowing analysis of changes in solute concentration in living cells. We made use of bacterial periplasmic binding proteins (PBPs), where we show that, on binding of the substrate, PBPs transform their hinge–bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins. By using the maltose-binding protein as a prototype, nanosensors were constructed allowing
in vitro
determination of FRET changes in a concentration-dependent fashion. For physiological applications, mutants with different binding affinities were generated, allowing dynamic
in vivo
imaging of the increase in cytosolic maltose concentration in single yeast cells. Control sensors allow the exclusion of the effect from other cellular or environmental parameters on ratio imaging. Thus the myriad of PBPs recognizing a wide spectrum of different substrates is suitable for FRET-based
in vivo
detection, providing numerous scientific, medical, and environmental applications.
Assessing the role of Ih channels in synaptic transmission and mossy fiber LTP
Authors: Chevaleyre Vivien , Castillo Pablo E.
Published: July 2002

Hyperpolarization-activated nonselective cation channels (Ih channels) play an important role in the control of membrane excitability and rhythmic neuronal activity. The functional relevance of presynaptic Ih channels in regulating synaptic function, however, is not well established. Recently, it has been proposed [Mellor, J., Nicoll, R. A. & Schmitz, D. (2002)
Science
295, 143–147] that presynaptic Ih channels are necessary for hippocampal mossy fiber long-term potentiation (LTP). This observation challenges an alternative model that suggests presynaptic forms of LTP are caused by a direct modification of the transmitter release machinery. Here, we assess the role of Ih in hippocampal mossy fiber LTP as well as cerebellar parallel fiber LTP, forms of potentiation that share common mechanisms. Our results show that after Ih blockade neither mossy fiber LTP nor parallel fiber LTP are affected. Furthermore, Ih does not significantly modify basal excitatory synaptic transmission in the hippocampus, whereas the organic Ih blockers ZD7288 and DK-AH 269 induce a large Ih-independent depression of synaptic transmission. In summary, our results indicate that Ih-mediated persistent changes in presynaptic excitability do not underlie presynaptic forms of LTP.
VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial cells
Authors: Shay-Salit Ayelet , Shushy Moran , Wolfovitz Efrat , Yahav Hava , Breviario Ferruccio , Dejana Elisabetta , Resnick Nitzan
Published: July 2002

Blood-flow interactions with the vascular endothelium represents a specialized example of mechanical regulation of cell function that has important physiological and pathophysiological cardiovascular consequences. Yet, the mechanisms of mechanostransduction are not understood fully. This study shows that shear stress induces a rapid induction as well as nuclear translocation of the vascular endothelial growth factor (VEGF) receptor 2 and promotes the binding of the VEGF receptor 2 and the adherens junction molecules, VE-cadherin and β-catenin, to the endothelial cytoskeleton. These changes are accompanied by the formation of a complex containing the VEGF receptor 2–VE-cadherin–β-catenin. In endothelial cells lacking VE-cadherin, shear stress did not augment nuclear translocation of the VEGF receptor 2 and phosphorylation of Akt1 and P38 as well as transcriptional induction of a reporter gene regulated by a shear stress-responsive promoter. These results suggest that VEGF receptor 2 and the adherens junction act as shear-stress cotransducers, mediating the transduction of shear-stress signals into vascular endothelial cells.
R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1
Authors: Hu Guang , Wensel Theodore G.
Published: July 2002

The regulator of G protein signaling (RGS)-9-1⋅G
β5
complex forms the GTPase accelerating protein for G
αt
in vertebrate photoreceptors. Although the complex is soluble when expressed
in vitro
, extraction of the endogenous protein from membranes requires detergents. The detergent extracts contain a complex of RGS9-1, G
β5
, G
αt
, and a 25-kDa phosphoprotein, R9AP (
R
GS
9
-1-
A
nchor
P
rotein). R9AP is encoded by one intronless gene in both human and mouse. Full or partial cDNA or genomic clones were obtained from mice, cattle, human, zebrafish, and
Xenopus
laevis
. R9AP mRNA was detected only in the retina, and the protein only in photoreceptors. R9AP binds to the N-terminal domain of RGS9-1, and anchors it to the disk membrane via a C-terminal transmembrane helix.
Global repression of exotoxin synthesis by staphylococcal superantigens
Authors: Vojtov Nikola , Ross Hope F. , Novick Richard P.
Published: July 2002

VirulentStaphylococcus aureusstrains typically produce and secrete large quantities of many extracellular proteins involved in pathogenesis. Such strains cause the classical staphylococcal lesion—local tissue destruction and aggressive inflammation accompanied by the massive influx of polymorphonuclear leukocytes, leading to the formation of pus. Most strains causing toxic shock syndrome, however, produce and secrete very small quantities of most exoproteins although they elaborate high levels of toxic shock syndrome toxin-1 (TSST-1). These strains cause local infections that are remarkably apurulent although potentially fatal owing to the superantigen. We have analyzed this disparity and have found that TSST-1 itself is a negative global regulator of exoprotein gene transcription. TSST-1 not only represses most exoprotein genes but determines its own high expression level by autorepression. We report also that a second superantigen, enterotoxin B, has similar regulatory properties.
dELL is an essential RNA polymerase II elongation factor with a general role in development
Authors: Eissenberg Joel C. , Ma Jiyan , Gerber Mark A. , Christensen Alan , Kennison James A. , Shilatifard Ali
Published: July 2002

Several eukaryotic proteins increase RNA polymerase II (Pol II) transcription rates
in vitro
. The relative contributions of these factors to gene expression
in vivo
is unknown. The ELL family of proteins promote Pol II elongation
in vitro
, and the
Drosophila
ELL homolog (dELL) is associated with Pol II at sites of transcription
in vivo
. The purpose of this study was to test whether an ELL family protein is required for gene expression
in vivo
. We show that dELL is encoded by the
Suppressor of Triplo-lethal
locus [
Su(Tpl)
]. We have characterized seven distinct mutant alleles of
Su(Tpl)
and show that a dELL transgene rescues recessive lethality of
Su(Tpl)
.
Su(Tpl)
mutations cause abnormal embryonic segmentation and dominantly modify expression of diverse genes during development. These data show that an ELL family elongation factor is essential, acts broadly in development, and is not functionally redundant to other elongation factors
in vivo
.
Vanadate-catalyzed photocleavage of the signature motif of an ATP-binding cassette (ABC) transporter
Authors: Fetsch Erin E. , Davidson Amy L.
Published: July 2002

The maltose transport complex of
Escherichia coli
, a member of the ATP-binding cassette (ABC) superfamily, is made up of two nucleotide-binding subunits, MalK
2
, which hydrolyze ATP with positive cooperativity, and two transmembrane subunits, MalF and MalG. The ABC family is defined in part by the canonical signature motif LSGGQ whose exact function remains controversial. Taking advantage of the dual function of vanadate as a transition state analogue and as a photoactive chemical, we demonstrate that vanadate catalyzes the UV-dependent cleavage of the polypeptide backbone at both the LSGGQ motif and the nucleotide-binding, or Walker A, motif when it is trapped in the nucleotide-binding site of the bacterial maltose transporter. This highly specific cleavage pattern indicates that residues in both motifs are immediately adjacent to ATP during hydrolysis, and are therefore likely to participate directly in ATP-binding and/or hydrolysis. Because the LSGGQ motif is too distant from the nucleotide in the structure of an ABC monomer for cleavage to occur, these data support a model in which the LSGGQ motif contacts the nucleotide across the interface of a MalK dimer, as seen in the crystal structure of Rad50. This architecture provides a basis for the cooperativity observed in the nucleotide-binding domains of ABC transporters and a function for this highly conserved family signature motif.
Identification of two cerebral malaria resistance loci using an inbred wild-derived mouse strain
Authors: Bagot Sébastien , Campino Susana , Penha-Gonçalves Carlos , Pied Sylviane , Cazenave Pierre-André , Holmberg Dan
Published: July 2002

Malaria is a complex infectious disease in which the host/parasite interaction is strongly influenced by host genetic factors. The consequences of plasmodial infections range from asymptomatic to severe complications like the neurological syndrome cerebral malaria induced byPlasmodium falciparumin humans andPlasmodium bergheiANKA in rodents. Mice infected withP. bergheiANKA show marked differences in disease manifestation and either die from experimental cerebral malaria (ECM) or from hemolytic anemia caused by hyperparasitemia (HP). A majority of laboratory mouse strains so far investigated are susceptible to ECM; however, a number of wild-derived inbred strains show resistance. To evaluate the genetic basis of this difference, we crossed a uniquely ECM-resistant, wild-derived inbred strain (WLA) with an ECM susceptible laboratory strain (C57BL/6J). All of the (WLA × C57BL/6J) F1and 97% of the F2progeny displayed ECM resistance similar to the WLA strain. To screen for loci contributing to ECM resistance, we analyzed a cohort of mice backcrossed to the C57BL/6J parental strain. A genome wide screening of this cohort provided significant linkage of ECM resistance to marker loci in two genetic regions on chromosome 1 (χ2= 18.98,P= 1.3 × 10−5) and on chromosome 11 (χ2= 16.51,P= 4.8 × 10−5), being designatedBerr1andBerr2, respectively. These data provide the first evidence of loci associated with resistance to murine cerebral malaria, which may have important implications for the search for genetic factors controlling cerebral malaria in humans.