bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and classification of p53-regulated genes
Authors: Yu Jian , Zhang Lin , Hwang Paul M. , Rago Carlo , Kinzler Kenneth W. , Vogelstein Bert
Published: July 2002

Sequence-specific transactivation by p53 is essential to its role as a tumor suppressor. A modified tetracycline-inducible system was established to search for transcripts that were activated soon after p53 induction. Among 9,954 unique transcripts identified by serial analysis of gene expression, 34 were increased more than 10-fold; 31 of these had not previously been known to be regulated by p53. The transcription patterns of these genes, as well as previously described p53-regulated genes, were evaluated and classified in a panel of widely studied colorectal cancer cell lines. “Class I” genes were uniformly induced by p53 in all cell lines; “class II” genes were induced in a subset of the lines; and “class III” genes were not induced in any of the lines. These genes were also distinguished by the timing of their induction, their induction by clinically relevant chemotherapeutic agents, the absolute requirement for p53 in this induction, and their inducibility by p73, a p53 homolog. The results revealed substantial heterogeneity in the transcriptional responses to p53, even in cells derived from a single epithelial cell type, and pave the way to a deeper understanding of p53 tumor suppressor action.
Abundant empty class II MHC molecules on the surface of immature dendritic cells
Authors: Santambrogio Laura , Sato Aaron K. , Fischer Falko R. , Dorf Martin E. , Stern Lawrence J.
Published: July 2002

A monoclonal antibody specific for the empty conformation of class II MHC molecules revealed the presence of abundant empty molecules on the surface of spleen- and bone marrow-derived dendritic cells (DC) among various types of antigen-presenting cells. The empty class II MHC molecules are developmentally regulated and expressed predominantly on immature DC. They can capture peptide antigens directly from the extracellular medium and present bound peptides to antigen-specific T lymphocytes. The ability of the empty cell-surface class II MHC proteins to bind peptides and present them to T cells without intracellular processing can serve to extend the spectrum of antigens able to be presented by DC, consistent with their role as sentinels in the immune system.
Origins of DNA-binding specificity: Role of protein contacts with the DNA backbone
Authors: Schildbach Joel F. , Karzai A. Wali , Raumann Brigitte E. , Sauer Robert T.
Published: July 2002

A central question in protein–DNA recognition is the origin of the specificity that permits binding to the correct site in the presence of excess, nonspecific DNA. In the P22 Arc repressor, the Phe-10 side chain is part of the hydrophobic core of the free protein but rotates out to pack against the sugar-phosphate backbone of the DNA in the repressor-operator complex. Characterization of a library of position 10 variants reveals that Phe is the only residue that results in fully active Arc. One class of mutants folds stably but binds operator with reduced affinity; another class is unstable. FV10, one member of the first class, binds operator DNA and nonoperator DNA almost equally well. The affinity differences between FV10 and wild type indicate that each Phe-10 side chain contributes 1.5–2.0 kcal to operator binding but less than 0.5 kcal/mol to nonoperator binding, demonstrating that contacts between Phe-10 and the operator DNA backbone contribute to binding specificity. This appears to be a direct contribution as the crystal structure of the FV10 dimer is similar to wild type and the Phe-10-DNA backbone interactions are the only contacts perturbed in the cocrystal structure of the FV10-operator complex.
Immunogenicity andin vitroprotective efficacy of a recombinant multistagePlasmodium falciparumcandidate vaccine
Authors: Shi Ya Ping , Hasnain Seyed E. , Sacci John B. , Holloway Brian P. , Fujioka Hisashi , Kumar Nirbhay , Wohlhueter Robert , Hoffman Stephen L. , Collins William E. , Lal Altaf A.
Published: July 2002

Compared with a single-stage antigen-based vaccine, a multistage and multivalentPlasmodium falciparumvaccine would be more efficacious by inducing “multiple layers” of immunity. We have constructed a synthetic gene that encodes for 12 B cell, 6 T cell proliferative, and 3 cytotoxic T lymphocyte epitopes derived from 9 stage-specificP. falciparumantigens corresponding to the sporozoite, liver, erythrocytic asexual, and sexual stages. The gene was expressed in the baculovirus system, and a 41-kDa antigen, termed CDC/NIIMALVAC-1, was purified. Immunization in rabbits with the purified protein in the presence of different adjuvants generated antibody responses that recognized vaccine antigen, linear peptides contained in the vaccine, and all stages ofP. falciparum.In vitroassays of protection revealed that the vaccine-elicited antibodies strongly inhibited sporozoite invasion of hepatoma cells and growth of blood-stage parasites in the presence of monocytes. These observations demonstrate that a multicomponent, multistage malaria vaccine can induce immune responses that inhibit parasite development at multiple stages. The rationale and approach used in the development of a multicomponentP. falciparumvaccine will be useful in the development of a multispecies human malaria vaccine and vaccines against other infectious diseases.
Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the β-subunit of the amiloride-sensitive epithelial sodium channel
Authors: Pradervand Sylvain , Barker Pierre M. , Wang Qing , Ernst Stephen A. , Beermann Friedrich , Grubb Barbara R. , Burnier Michel , Schmidt Andrea , Bindels Rene J. M. , Gatzy John T. , Rossier Bernard C. , Hummler Edith
Published: July 2002

The amiloride-sensitive epithelial sodium channel (ENaC) is a
heteromultimer of three homologous subunits (α-, β-, and
γ-subunits). To study the role of the β-subunit
in
vivo
, we analyzed mice in which the βENaC gene locus was
disrupted. These mice showed low levels of βENaC mRNA expression in
kidney (≈1%), lung (≈1%), and colon (≈4%). In homozygous
mutant βENaC mice, no βENaC protein could be detected with
immunofluorescent staining. At birth, there was a small delay in
lung-liquid clearance that paralleled diminished amiloride-sensitive
Na
+
absorption in tracheal explants. With normal salt
intake, these mice showed a normal growth rate. However,
in
vivo
, adult βENaC m/m mice exhibited a significantly
reduced ENaC activity in colon and elevated plasma aldosterone levels,
suggesting hypovolemia and pseudohypoaldosteronism type 1. This
phenotype was clinically silent, as βENaC m/m mice showed no weight
loss, normal plasma Na
+
and K
+
concentrations,
normal blood pressure, and a compensated metabolic acidosis. On
low-salt diets, βENaC-mutant mice developed clinical symptoms of an
acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and
decreased blood pressure), indicating that βENaC is required for
Na
+
conservation during salt deprivation.
Analysis of masked mutations in familial adenomatous polyposis
Authors: Laken Steven J. , Papadopoulos Nickolas , Petersen Gloria M. , Gruber Stephen B. , Hamilton Stanley R. , Giardiello Francis M. , Brensinger Jill D. , Vogelstein Bert , Kinzler Kenneth W.
Published: July 2002

Familial adenomatous polyposis (FAP) is an autosomal-dominant disease characterized by the development of hundreds of adenomatous polyps of the colorectum. Approximately 80% of FAP patients can be shown to have truncating mutations of the APC gene. To determine the cause of FAP in the other 20% of patients, MAMA (monoallelic mutation analysis) was used to independently examine the status of each of the two
APC
alleles. Seven of nine patients analyzed were found to have significantly reduced expression from one of their two alleles whereas two patients were found to have full-length expression from both alleles. We conclude that more than 95% of patients with FAP have inactivating mutations in
APC
and that a combination of MAMA and standard genetic tests will identify
APC
abnormalities in the vast majority of such patients. That no APC expression from the mutant allele is found in some FAP patients argues strongly against the requirement for dominant negative effects of
APC
mutations. The results also suggest that there may be at least one additional gene, besides
APC
, that can give rise to FAP.
Male gametic cell-specific gene expression in flowering plants
Authors: Xu Huiling , Swoboda Ines , Bhalla Prem L. , Singh Mohan B.
Published: July 2002

The role of the male gamete—the sperm cell—in the process of fertilization is to recognize, adhere to, and fuse with the female gamete. These highly specialized functions are expected to be controlled by activation of a unique set of genes. However, male gametic cells traditionally have been regarded as transcriptionally quiescent because of highly condensed chromatin and a very reduced amount of cytoplasm. Here, we provide evidence for male gamete-specific gene expression in flowering plants. We identified and characterized a gene,
LGC1
, which was shown to be expressed exclusively in the male gametic cells. The gene product of
LGC1
was localized at the surface of male gametic cells, suggesting a possible role in sperm–egg interactions. These findings represent an important step toward defining the molecular mechanisms of male gamete development and the cellular processes involved in fertilization of flowering plants.
A hierarchical approach to protein molecular evolution
Authors: Bogarad Leonard D. , Deem Michael W.
Published: July 2002

Biological diversity has evolved despite the essentially infinite complexity of protein sequence space. We present a hierarchical approach to the efficient searching of this space and quantify the evolutionary potential of our approach with Monte Carlo simulations. These simulations demonstrate that nonhomologous juxtaposition of encoded structure is the rate-limiting step in the production of new tertiary protein folds. Nonhomologous “swapping” of low-energy secondary structures increased the binding constant of a simulated protein by ≈107relative to base substitution alone. Applications of our approach include the generation of new protein folds and modeling the molecular evolution of disease.
Leishmania majorFriedlin chromosome 1 has an unusual distribution of protein-coding genes
Authors: Myler Peter J. , Audleman Lindsey , deVos Theo , Hixson Greg , Kiser Patti , Lemley Craig , Magness Charles , Rickel Erika , Sisk Ellen , Sunkin Susan , Swartzell Steven , Westlake Thomas , Bastien Patrick , Fu Guoliang , Ivens Alasdair , Stuart Kenneth
Published: July 2002

Leishmaniaare evolutionarily ancient protozoans (Kinetoplastidae) and important human pathogens that cause a spectrum of diseases ranging from the asymptomatic to the lethal. TheLeishmaniagenome is relatively small [≈34 megabases (Mb)], lacks substantial repetitive DNA, and is distributed among 36 chromosomes pairs ranging in size from 0.3 Mb to 2.5 Mb, making it a useful candidate for complete genome sequence determination. We report here the nucleotide sequence of the smallest chromosome, chr1. The sequence of chr1 has a 257-kilobase region that is densely packed with 79 protein-coding genes. This region is flanked by telomeric and subtelomeric repetitive elements that vary in number and content among the chr1 homologs, resulting in an ≈27.5-kilobase size difference. Strikingly, the first 29 genes are all encoded on one DNA strand, whereas the remaining 50 genes are encoded on the opposite strand. Based on the gene density of chr1, we predict a total of ≈9,800 genes inLeishmania, of which 40% may encode unknown proteins.
Regulation of T cell receptor (TCR) β gene expression by CD3 complex signaling in immature thymocytes: Implications for TCRβ allelic exclusion
Authors: Biro Judit , Würch Andreas , Potocnik Alexandre J. , Falk Ingrid , Mossmann Horst , Eichmann Klaus
Published: July 2002

During αβ thymocyte development, clonotype-independent CD3 complexes are expressed at the cell surface before the pre-T cell receptor (TCR). Signaling through clonotype-independent CD3 complexes is required for expression of rearranged TCRβ genes. On expression of a TCRβ polypeptide chain, the pre-TCR is assembled, and TCRβ locus allelic exclusion is established. We investigated the putative contribution of clonotype-independent CD3 complex signaling to TCRβ locus allelic exclusion in mice single-deficient or double-deficient for CD3ζ/η and/or p56
lck
. These mice display defects in the expression of endogenous TCRβ genes in immature thymocytes, proportional to the severity of CD3 complex malfunction. Exclusion of endogenous TCRβ VDJ (variable, diversity, joining) rearrangements by a functional TCRβ transgene was severely compromised in the single-deficient and double-deficient mutant mice. In contrast to wild-type mice, most of the CD25
+
double-negative (DN) thymocytes of the mutant mice failed to express the TCRβ transgene, suggesting defective expression of the TCRβ transgene similar to endogenous TCRβ genes. In the mutant mice, a proportion of CD25
+
DN thymocytes that failed to express the transgene expressed endogenous TCRβ polypeptide chains. Many double-positive cells of the mutant mice coexpressed endogenous and transgenic TCRβ chains or more than one endogenous TCRβ chain. The data suggest that signaling through clonotype-independent CD3 complexes may contribute to allelic exclusion of the TCRβ locus by inducing the expression of rearranged TCRβ genes in CD25
+
DN thymocytes.
Optimal prediction and the Klein–Gordon equation
Authors: Hald Ole H.
Published: July 2002

The method of optimal prediction is applied to calculate the future means of solutions to the Klein–Gordon equation. It is shown that, in an appropriate probability space, the difference between the average of all solutions that satisfy certain constraints at time
t
= 0 and the average computed by an approximate method is small with high probability.
Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [
PSI
+
] of
Saccharomyces cerevisiae
Authors: Eaglestone Simon S. , Ruddock Lloyd W. , Cox Brian S. , Tuite Mick F.
Published: July 2002

The cytoplasmic heritable determinant [
PSI
+
] of the yeast
Saccharomyces cerevisiae
reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [
PSI
+
] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [
PSI
+
] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are “cured” of [
PSI
+
] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [
PSI
+
] determinant is not derived from the direct dissolution of self-replicating [
PSI
+
] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [
PSI
+
] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [
PSI
+
] element is diluted from a culture, after the total inhibition of prion replication by GuHCl.
Molecular mechanism for silencing virally transduced genes involves histone deacetylation and chromatin condensation
Authors: Chen Wen Yong , Townes Tim M.
Published: July 2002

Virally transduced genes are often silenced after integration into the host genome. Chromatin immunoprecipitation and nuclease sensitivity experiments now demonstrate that silencing of the transgene is characterized by deacetylation of histone H4 lysines and chromatin condensation. Trichostatin A treatment results in dramatic reactivation of gene expression that is preceded by histone acetylation and chromatin decondensation. Analysis of individual histone H4 lysines demonstrate that chromatin domain opening is coincident with rapid acetylation of histone H4 K5, K12, and K16 and that maintenance of the open domain is correlated with acetylation of histone H4 K8. Removal of trichostatin A results in rapid deacetylation of histone H4 K8, chromatin condensation, and transcription silencing. The results suggest that deacetylation of histone H4 lysines and coincident chromatin condensation are critically involved in the silencing of virally transduced genes.
Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling
Authors: Russell Kerry Strong , Haynes M. Page , Sinha Diviya , Clerisme Emmanuelle , Bender Jeffrey R.
Published: July 2002

Estrogen induces both rapid and delayed effects on the cardiovascular system. The early effects take place within minutes (e.g., changes in vasomotor tone) and are mediated through rapid intracellular signaling pathways; whereas the delayed effects (e.g., remodeling or lipid alterations) require hours to days to occur and require transcriptional effects with subsequent modulation of protein expression. To study the acute effects of 17β-estradiol (E2) treatment on vascular function, we have investigated the rapid (on the order of minutes) effects of E2 treatment on intracellular signaling in human endothelial cells (EC). Our previous data have shown that E2 induces rapid release of NO from and activation of guanylate cyclase in human EC. In this study, we demonstrate that E2 also activates mitogen-activated protein kinase (extracellular signal-related kinase) signaling within minutes in EC. We hypothesized that this effect might be mediated by estrogen receptors (ER) localized to the cell surface. Our data show that membrane-impermeant forms of E2 also activate EC mitogen-activated protein kinase as well as stimulate cGMP production and NO release. The ER antagonist ICI 182,780 blocks this effect. Using confocal microscopy and flow cytometric analysis, we demonstrate that EC contain surface binding sites for E2, detectable by cell-impermeant ligand binding and equally with an anti-ERα antibody. Immunoreactive bands of 66 and 45 kDa are detectable with an anti-ERα mAb in human EC, and their individual presence correlates functionally with E2-stimulated genomic and rapid nongenomic responses, respectively. Membrane ERs may provide key molecular switches in these novel, rapid signaling pathways induced by E2 in EC.
Optimal prediction of stiff oscillatory mechanics
Authors: Kast Anton P.
Published: July 2002

We consider many-body problems in classical mechanics where a wide range of time scales limits what can be computed. We apply the method of optimal prediction to obtain equations that are easier to solve numerically. We demonstrate by examples that optimal prediction can reduce the amount of computation needed to obtain a solution by several orders of magnitude.
Identification of phylogenetic footprints in primate tumor necrosis factor-α promoters
Authors: Leung Jessica Y. , McKenzie F. Ellis , Uglialoro Adele M. , Flores-Villanueva Pedro O. , Sorkin Barbara C. , Yunis Edmond J. , Hartl Daniel L. , Goldfeld Anne E.
Published: July 2002

The human tumor necrosis factor-α (TNF-α) gene encodes a pleiotropic cytokine that plays a critical role in basic immunologic processes. To investigate the TNF-α regulatory region in the primate lineage, we isolated TNF-α promoters from representative great apes, Old World monkeys, and New World monkeys. We demonstrate that there is a nonuniform distribution of fixed human differences in the TNF-α promoter. We define a “fixed human difference” as a site that is not polymorphic in humans, but which differs in at least one of the seven primate sequences examined. Furthermore, we identify two human TNF-α promoter single nucleotide polymorphisms that are putative ancestral polymorphisms, because each of the human polymorphic nucleotides was found at the identical site in at least one of the other primate sequences. Strikingly, the largest conserved region among the primate species, a 69-nt “phylogenetic footprint,” corresponds to a region of the human TNF-α promoter that forms the transcriptionally active nucleoprotein–DNA complex, essential for gene regulation. By contrast, other regions of the TNF-α promoter, which exhibit a high density of variable sites, are nonessential for gene expression, indicating that distinct TNF-α promoter regions have been subjected to different evolutionary constraints depending on their function. TNF-α is the first case in which a promoter region dissected by functional analyses can be correlated with nucleotide polymorphism and variability in primate lineages. The results suggest that patterns of polymorphism and divergence are likely to be useful in identifying candidate regions important for gene regulation in other immune-response genes.
Inhibition of sonic hedgehog autoprocessing in cultured mammalian cells by sterol deprivation
Authors: Guy R. Kip
Published: July 2002

Sonic hedgehog (Shh) is a signaling molecule that is important for defining patterning in the developing vertebrate central nervous system. After translation, Shh autoproteolyzes and covalently attaches cholesterol to the newly formed carboxyl terminus, a modification crucial for normal Shh signaling. Presented here is evidence that acute severe sterol deprivation in cultured Chinese hamster ovary cells expressing mouse
Shh
(
mShh
) inhibits autoprocessing of the protein. These conditions allowed the first detailed kinetic analysis of mShh autoprocessing and turnover rates revealing that cells rapidly degrade both precursor and mature mShh regardless of sterol content and sterol deprivation increases the rate of precursor degradation. Inhibition of mShh autoprocessing also allowed the determination of the subcellular localization of mShh precursor which accumulates in a pre-medial Golgi intracellular compartment. Finally, the precursor form of mShh that results from autoprocessing inhibition appears to accumulate as an amide rather than a stable thioester.
Placental expression of the nonclassical MHC class I molecule Mamu-AG at implantation in the rhesus monkey
Authors: Slukvin I. I. , Lunn D. P. , Watkins D. I. , Golos T. G.
Published: July 2002

During human implantation trophoblasts mediate attachment of the embryo to the uterine epithelium and invade and reorganize vessels of the maternal endometrium to initiate blood flow to the intervillous space. Expression of the nonclassical MHC class I molecule HLA-G by invading trophoblasts may play a central role in their protection from recognition by the maternal immune system; however, the ontogeny of trophoblast HLA-G expression during the earliest stages of implantation is difficult to evaluate in human pregnancy. We previously identified a novel nonclassical MHC class I molecule, Mamu-AG, which is expressed in the rhesus monkey placenta and shares many unique characteristics of HLA-G. Immunocytochemical analysis with a Mamu-AG-specific mAb and locus-specificin situhybridization of rhesus implantation sites 7–12 days after embryo attachment (days 14–19 of pregnancy) demonstrated that Mamu-AG molecules are expressed predominantly in cytotrophoblasts invading the maternal vessels and endometrium, whereas syncytiotrophoblasts covering trophoblastic lacunae or newly formed chorionic villi remained largely Mamu-AG-negative. By day 36 of pregnancy, Mamu-AG glycoprotein also was expressed in villous syncytiotrophoblasts, and accumulation of Mamu-AG glycoprotein was noted at the border between maternal decidua and fetal trophoblasts. The ontogeny of a nonclassical MHC class I molecule at the implantation site supports the hypothesis that its expression is important for the establishment of maternal-fetal immune tolerance.
NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein
Authors: Yuvaniyama Pramvadee , Agar Jeffrey N. , Cash Valerie L. , Johnson Michael K. , Dean Dennis R.
Published: July 2002

The NifS and NifU proteins from
Azotobacter vinelandii
are required for the full activation of nitrogenase. NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S]
2+,+
cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with NifS and that a second, transient [2Fe-2S] cluster can be assembled within NifU
in vitro
when incubated in the presence of ferric ion,
l
-cysteine, and catalytic amounts of NifS. Approximately one transient [2Fe-2S] cluster is assembled per homodimer. The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU. Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.
Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells
Authors: Schuldiner Maya , Yanuka Ofra , Itskovitz-Eldor Joseph , Melton Douglas A. , Benvenisty Nissim
Published: July 2002

Human embryonic stem (ES) cells are pluripotent cells derived from
the inner cell mass of
in vitro
fertilized human
blastocysts. We examined the potential of eight growth factors [basic
fibroblast growth factor (bFGF), transforming growth factor β1
(TGF-β1), activin-A, bone morphogenic protein 4 (BMP-4), hepatocyte
growth factor (HGF), epidermal growth factor (EGF), β nerve growth
factor (βNGF), and retinoic acid] to direct the differentiation of
human ES-derived cells
in vitro
. We show that human ES
cells that have initiated development as aggregates (embryoid bodies)
express a receptor for each of these factors, and that their effects
are evident by differentiation into cells with different epithelial or
mesenchymal morphologies. Differentiation of the cells was assayed by
expression of 24 cell-specific molecular markers that cover all
embryonic germ layers and 11 different tissues. Each growth factor has
a unique effect that may result from directed differentiation and/or
cell selection, and we can divide the overall effects of the factors
into three categories: growth factors (Activin-A and TGFβ1) that
mainly induce mesodermal cells; factors (retinoic acid, EGF, BMP-4, and
bFGF) that activate ectodermal and mesodermal markers; and factors (NGF
and HGF) that allow differentiation into the three embryonic germ
layers, including endoderm. None of the growth factors directs
differentiation exclusively to one cell type. This analysis sets the
stage for directing differentiation of human ES cells in culture and
indicates that multiple human cell types may be enriched
in
vitro
by specific factors.
Statistics of lateral geniculate nucleus (LGN) activity determine the segregation of ON/OFF subfields for simple cells in visual cortex
Authors: Lee Ann B. , Blais Brian , Shouval Harel Z. , Cooper Leon N
Published: July 2002

The receptive fields for simple cells in visual cortex show a
strong preference for edges of a particular orientation and display
adjacent excitatory and inhibitory subfields. These subfields are
projections from ON-center and OFF-center lateral geniculate nucleus
cells, respectively. Here we present a single-cell model using ON and
OFF channels, a natural scene environment, and synaptic modification
according to the Bienenstock, Cooper, and Munro (BCM) theory. Our
results indicate that lateral geniculate nucleus cells must act
predominantly in the linear region around the level of spontaneous
activity, to lead to the observed segregation of ON/OFF subfields.
Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays
Authors: Tenenbaum Scott A. , Carson Craig C. , Lager Patrick J. , Keene Jack D.
Published: July 2002

Genomic array technologies provide a means for profiling global
changes in gene expression under a variety of conditions. However, it
has been difficult to assess whether transcriptional or
posttranscriptional regulation is responsible for these changes.
Additionally, fluctuations in gene expression in a single cell type
within a complex tissue like a tumor may be masked by overlapping
profiles of all cell types in the population. In this paper, we
describe the use of cDNA arrays to identify subsets of mRNAs contained
in endogenous messenger ribonucleoprotein complexes (mRNPs)
that are cell type specific. We identified mRNA subsets from P19
embryonal carcinoma stem cells by using mRNA-binding proteins HuB,
eIF-4E, and PABP that are known to play a role in translation. The mRNA
profiles associated with each of these mRNPs were unique and
represented gene clusters that differed from total cellular RNA.
Additionally, the composition of mRNAs detected in HuB–mRNP complexes
changed dramatically after induction of neuronal differentiation with
retinoic acid. We suggest that the association of structurally related
mRNAs into mRNP complexes is dynamic and may help regulate
posttranscriptional events such as mRNA turnover and translation.
Recovering proteins specifically associated with mRNP complexes to
identify and profile endogenously clustered mRNAs should
provide insight into structural and functional relationships among gene
transcripts and/or their protein products. We have termed this
approach to functional genomics
ribonomic
s and suggest
that it will provide a useful paradigm for organizing genomic
information in a biologically relevant manner.
Toward a protein–protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins
Authors: Ito Takashi , Tashiro Kosuke , Muta Shigeru , Ozawa Ritsuko , Chiba Tomoko , Nishizawa Mayumi , Yamamoto Kiyoshi , Kuhara Satoru , Sakaki Yoshiyuki
Published: July 2002

Protein–protein interactions play pivotal roles in various aspects
of the structural and functional organization of the cell, and their
complete description is indispensable to thorough understanding of the
cell. As an approach toward this goal, here we report a comprehensive
system to examine two-hybrid interactions in all of the possible
combinations between proteins of
Saccharomyces
cerevisiae
. We cloned all of the yeast ORFs individually as a
DNA-binding domain fusion (“bait”) in a
MAT
a
strain and as an activation domain fusion (“prey”) in a
MAT
α strain, and subsequently divided them into pools,
each containing 96 clones. These bait and prey clone pools were
systematically mated with each other, and the transformants were
subjected to strict selection for the activation of three reporter
genes followed by sequence tagging. Our initial examination of
≈4 × 10
6
different combinations, constituting
≈10% of the total to be tested, has revealed 183 independent
two-hybrid interactions, more than half of which are entirely novel.
Notably, the obtained binary data allow us to extract more complex
interaction networks, including the one that may explain a currently
unsolved mechanism for the connection between distinct steps of
vesicular transport. The approach described here thus will provide many
leads for integration of various cellular functions and serve as a
major driving force in the completion of the protein–protein
interaction map.
HIV-1 envelope induces activation of caspase-3 and cleavage of focal adhesion kinase in primary human CD4+T cells
Authors: Cicala Claudia , Arthos James , Rubbert Andrea , Selig Sara , Wildt Kathryn , Cohen Oren J. , Fauci Anthony S.
Published: July 2002

Binding of HIV type 1 (HIV-1) envelope glycoproteins to the surface of a CD4+T cell transduces intracellular signals through the primary envelope receptor, CD4, and a coreceptor, either CCR5 or CXCR4. Furthermore, envelope–CD4+cell interactions increase rates of apoptosis in peripheral blood mononuclear cells (PBMCs). We demonstrate that in primary T lymphocytes, recombinant HIV-1 envelope proteins induce the activation of caspase-3 and caspase-6, which belong to a family of cysteine proteases that, upon activation, promote programmed cell death. Envelope-mediated activation of caspase-3 and caspase-6 depended on envelope–CD4 receptor interactions; CCR5-utilizing as well as CXCR4-utilizing envelopes elicited this response. Focal adhesion kinase (FAK) is a substrate of both caspase-3 and caspase-6, and inactivation of FAK by these caspases promotes apoptosis. Envelope treatment of lymphocytes led to the cleavage of FAK in a manner consistent with caspase-mediated cleavage.
Brown dwarfs: At last filling the gap between stars and planets
Authors: Zuckerman Ben
Published: July 2002

Until the mid-1990s a person could not point to any celestial
object and say with assurance that “here is a brown dwarf.” Now
dozens are known, and the study of brown dwarfs has come of age,
touching upon major issues in astrophysics, including the nature of
dark matter, the properties of substellar objects, and the origin of
binary stars and planetary systems.
Shotgun sequencing of the human transcriptome with ORF expressed sequence tags
Authors: Dias Neto Emmanuel , Garcia Correa Ricardo , Verjovski-Almeida Sergio , Briones Marcelo R. S. , Nagai Maria Aparecida , da Silva Wilson , Zago Marco Antonio , Bordin Silvana , Costa Fernando Ferreira , Goldman Gustavo Henrique , Carvalho Alex F. , Matsukuma Adriana , Baia Gilson S. , Simpson David H. , Brunstein Adriana , de Oliveira Paulo S. L. , Bucher Philipp , Jongeneel C. Victor , O'Hare Michael J. , Soares Fernando , Brentani Ricardo R. , Reis Luis F. L. , de Souza Sandro J. , Simpson Andrew J. G.
Published: July 2002

Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription–PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed sequence tag (EST) database consisting of central, generally coding regions of expressed genes. Such a database would add significant value to existing public EST databases, which consist mostly of sequences derived from the extremities of cDNAs, and facilitate the construction of contigs of transcript sequences. We tested our predictions, creating a database of 10,000 sequences from human breast tumors. The data confirmed the central distribution of the sequences, the significant normalization of the sequence population, the frequent extension of contigs composed of existing human ESTs, and the identification of a series of potentially important homologues of known genes. This approach should make a significant contribution to the early identification of important human genes, the deciphering of the draft human genome sequence currently being compiled, and the shotgun sequencing of the human transcriptome.
Human herpesvirus 8 infections in the Amsterdam Cohort Studies (1984–1997): Analysis of seroconversions to ORF65 and ORF73
Authors: Goudsmit Jaap , Renwick Neil , Dukers Nicole H. T. M. , Coutinho Roel A. , Heisterkamp Siem , Bakker Margreet , Schulz Thomas F. , Cornelissen Marion , Weverling Gerrit J.
Published: July 2002

We have shown previously that human herpesvirus 8 (HHV8) seroconversion for antibodies to the latency-associated nuclear antigen encoded by ORF73 and/or the lytic capsid antigen (vp19) encoded by ORF65 is associated with orogenital contact and is strongly linked to the development of Kaposi's sarcoma among HIV-infected individuals in the Amsterdam Cohort Studies. Here, we investigate the relationship between seroconversion to these antigens and primary HHV8 infection. Between 1984 and 1997, 215 HHV8 seroconversions to ORF73 (106 cases or 49%) and/or to ORF65 (159 cases or 74%) were recorded in the cohort of homosexual men. The HHV8 seroconversion rate among HIV-infected homosexual men (6.2 per 100 person years) was consistently higher than among HIV-uninfected men (2.6 per 100 person years). In HIV-infected but not in uninfected individuals, seroconversion to ORF73/latency-associated nuclear antigen precedes that to ORF65/vp19. Antibody levels to both ORF65- and ORF73-encoded antigens were higher in HIV-infected than in HIV-uninfected men, and among HIV-seropositives, antibody levels to ORF65/vp19 rise even higher with declining CD4 cell counts and peak with Kaposi's sarcoma development, suggesting continuing and increasing viral replication. In 10.3% of HHV8 seroconversions, transient serum viremia could be demonstrated before or at seroconversion. Together with the previously reported link between unprotected orogenital sex and HHV8 seroconversion, our observations suggest that HHV8 seroconversions result from primary infections.
Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution
Authors: Yarbrough Daniel , Wachter Rebekka M. , Kallio Karen , Matz Mikhail V. , Remington S. James
Published: July 2002

The crystal structure of DsRed, a red fluorescent protein
from a corallimorpharian, has been determined at 2.0-Å
resolution by multiple-wavelength anomalous dispersion and
crystallographic refinement. Crystals of the
selenomethionine-substituted protein have space group P2
1
and contain a tetramer with 222 noncrystallographic symmetry in the
asymmetric unit. The refined model has satisfactory stereochemistry and
a final crystallographic
R
factor of 0.162. The protein,
which forms an obligatory tetramer in solution and in the crystal, is a
squat rectangular prism comprising four protomers whose fold is
extremely similar to that of the
Aequorea victoria
green
fluorescent protein despite low (≈23%) amino acid sequence homology.
The monomer consists of an 11-stranded β barrel with a coaxial helix.
The chromophores, formed from the primary sequence -Gln-Tyr-Gly-
(residues 66–68), are arranged in a ≈27 × 34-Å rectangular
array in two approximately antiparallel pairs. The geometry at the α
carbon of Gln-66 (refined without stereochemical restraints) is
consistent with an sp
2
hybridized center, in accord with
the proposal that red fluorescence is because of an additional
oxidation step that forms an acylimine extension to the chromophore
[Gross, L. A., Baird, G. S., Hoffman, R. C., Baldridge,
K. K. & Tsien, R. Y. (2000)
Proc. Natl. Acad. Sci.
USA
87, 11990–11995]. The carbonyl oxygen of Phe-65 is almost
90° out of the plane of the chromophore, consistent with theoretical
calculations suggesting that this is the minimum energy conformation of
this moiety despite the conjugation of this group with the rest of the
chromophore.
Simultaneous observation of the O—O and Fe—O
2
stretching modes in oxyhemoglobins
Authors: Das Tapan Kanti , Couture Manon , Ouellet Yannick , Guertin Michel , Rousseau Denis L.
Published: July 2002

Understanding of the chemical nature of the dioxygen moiety
of oxyhemoglobin is crucial for elucidation of its physiological
function. In the present work, direct Raman spectroscopic observation
of both the Fe—O
2
and O—O stretching modes
unambiguously establishes the vibrational characteristics of the
oxygen-bound heme moiety in the hemoglobins of
Chlamydomonas
eugametos
and
Synechocystis
PCC6803. In addition
to providing the resonance Raman assignment of the O—O stretching
mode (1136 cm
−1
for
Chlamydomonas
, 1133
cm
−1
for
Synechocystis
) in an oxyhemoglobin
with an iron-porphyrin, this study also reports unusually low
frequencies for the Fe—O
2
stretching modes (554
cm
−1
). The effect of strong hydrogen bonding to the bound
oxygen is confirmed by changes in the frequency of the
Fe—O
2
stretching mode on mutation of distal residues.
These findings suggest an enzymatic function rather than an oxygen
transport role for these hemoglobins.
A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG
Authors: Rumfelt Lynn L. , Avila David , Diaz Marilyn , Bartl Simona , McKinney E. Churchill , Flajnik Martin F.
Published: July 2002

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM1gj, from the nurse sharkGinglymostoma cirratumthat is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells (“germline-joined”). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H1gjin primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H1gjchain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM1gj. Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.
Croyances de contrôle et rendement scolaire chez des élèves francophones du primaire au Québec
Authors: Bouffard Thérèse , Bordeleau Luc
Published: July 2002

Des recherches en éducation faites aux États‐Unis et en Europe suggèrent que les croyances de contrôle sont un facteur qui permet de mieux comprendre les variations individuelles des élèves dans la qualité de leur engagement cognitif dans les divers apprentissages à réaliser et dans les rendements scolaires qu'ils obtiennent. Les trois objectifs poursuivis dans cette recherche faite auprès de 780 élèves de la quatrième à la sixième année du primaire sont d'examiner: (1) la manière dont les diverses dimensions des croyances de contrôle sont reliées entre elles, (2) l'importance que leur accordent les élèves et la manière dont elles évoluent de la quatrième à la sixième année du primaire, et (3) l'impact de ces croyances sur le rendement scolaire des élèves. Les résultats montrent que les relations entre les facteurs composant les croyances de contrôle ne different pas d'un niveau scolaire à l'autre. En outre, les élèves des trois niveaux s'entendent quant à l'importance relative des différents facteurs en regard de la réussite scolaire. Enfin, alors que le jugement des élèves sur la valeur des différents facteurs dans la réussite est peu lié à leur rendement scolaire, leurs croyances dans la capacité d'accéder à ces facteurs expliquent 21% de la variance observée dans le rendement scolaire. La discussion met en relation les résultats obtenus dans cette recherche avec ceux obtenus dans des études similaires réalisées aux États‐Unis et en Europe.
Consciousness from the Viewpoint of the Structural‐functional Relationships of the Brain
Authors: Ito Masao
Published: July 2002

The brain of vertebrates consists of brainstem and spinal cord conducting reflexes, compound movements, and innate behaviour, and the cerebral neocortex generating sensorimotor function and association function. These five major functions are assisted by four regulatory systems: limbic system, basal ganglia, cerebellum and sleep‐wakefulness brainstem centres. Consciousness contains three different levels, i.e., wakefulness, awareness and self‐consciousness. Wakefulness is a fundamental brain function regulated by the brainstem wakefulness centres. Awareness represents integration of diverse sensory signals, largely in the neocortex, for the perception of what is going on in the external world. Consciousness in humans is directed to the self so that an individual is aware of what is going on in his or her internal world, i.e., the mind, and seems to be inherent to the association cortex, in particular the frontal lobe. Freud's id emerges from the hypothalamus and limbic system, whereas the ego involves both the sensorimotor and association cortices. The super‐ego is likely to be embodied in part of the association cortex. When movement and thought are conceived as a control system function, instruction for control corresponds to the will which initiates these actions and which represents a positive aspect of consciousness. Though consciousness is related to brain structures in these ways, a crucial question remains as to how we subjectively experience will, affect or self‐consciousness as a consequence of neuronal activities in the brain structures.
Introduction to the Symposium on Drug Improvement of Memory
Authors: Rosenzweig Mark R.
Published: July 2002

This paper introduces a symposium in which three main speakers discussed the prospect that drugs may improve cognitive functioning, including memory. Even though the participants agreed that presently available drugs can alleviate memory disorders only to a limited extent and do not increase ability of well‐functioning individuals, it is important to consider social implications of drugs that might increase learning ability and intelligence. It is also important to consider, as alternatives to drugs, behavioural approaches to improving abilities that do not share possible drawbacks of pharmacological treatments.
Semantic Contribution to Immediate Serial Recall Using an Unlimited Set of Items: Evidence for a Multi‐level Capacity View of Short‐term Memory
Authors: Caza Nicole , Belleville Sylvie
Published: July 2002

The purpose of this study was to assess the respective roles of lexical and semantic levels of representations in immediate serial recall, by testing participants with items that varied on both these dimensions. Contrary to most studies where a small fixed set of words are repeated over trials, the current items were tested once by sampling them from an unlimited set of items without replacement. Participants recalled three classes of words under articulatory suppression: nonwords, function words, and content words. Results indicated an advantage for function words over nonwords, confirming a specifically lexical contribution to immediate serial recall. Additionally, content words were more frequently recalled than function words, confirming a semantic contribution. These results imply that non‐phonological factors influence immediate serial recall and are consistent with a multi‐level capacity view of short‐term memory.
Networking Psychology in Finland: New Collaboration Between Research and Profession
Authors: Niemi Pekka , Hämäläinen Heikki
Published: July 2002

A firmly structured collaborative scheme was created in Finland during the 1990s, involving all psychology departments and national scientific as well as professional societies. The experiences have been very rewarding. This article reports the main events, crucial strategies and decisions, and evaluates the reasons for success and the dangers to be avoided in such a project.