bluuebunny/crossref_metadata_2025 huggingface dataset:
Large-scale analysis of the human and mouse transcriptomes
Authors: Su Andrew I. , Cooke Michael P. , Ching Keith A. , Hakak Yaron , Walker John R. , Wiltshire Tim , Orth Anthony P. , Vega Raquel G. , Sapinoso Lisa M. , Moqrich Aziz , Patapoutian Ardem , Hampton Garret M. , Schultz Peter G. , Hogenesch John B.
Published: July 2002

High-throughput gene expression profiling has become an important tool for investigating transcriptional activity in a variety of biological samples. To date, the vast majority of these experiments have focused on specific biological processes and perturbations. Here, we have generated and analyzed gene expression from a set of samples spanning a broad range of biological conditions. Specifically, we profiled gene expression from 91 human and mouse samples across a diverse array of tissues, organs, and cell lines. Because these samples predominantly come from the normal physiological state in the human and mouse, this dataset represents a preliminary, but substantial, description of the normal mammalian transcriptome. We have used this dataset to illustrate methods of mining these data, and to reveal insights into molecular and physiological gene function, mechanisms of transcriptional regulation, disease etiology, and comparative genomics. Finally, to allow the scientific community to use this resource, we have built a free and publicly accessible website (
http://expression.gnf.org
) that integrates data visualization and curation of current gene annotations.
Optical imaging of
Renilla
luciferase reporter gene expression in living mice
Authors: Bhaumik S. , Gambhir S. S.
Published: July 2002

Imaging reporter gene expression in living subjects is a rapidly
evolving area of molecular imaging research. Studies have
validated the use of reporter genes with positron emission tomography
(PET), single photon emission computed tomography (SPECT), MRI,
fluorescence with wild-type and mutants of green fluorescent protein,
as well as bioluminescence using Firefly luciferase enzyme/protein
(FL). In the current study, we validate for the first time the ability
to image bioluminescence from
Renilla
luciferase
enzyme/protein (RL) by injecting the substrate coelenterazine in
living mice. A highly sensitive cooled charge-coupled device camera
provides images within a few minutes of photon counting. Cells,
transiently expressing the Rluc were imaged while located in the
peritoneum, s.c. layer, as well as in the liver and lungs of living
mice tail-vein injected with coelenterazine. Furthermore,
d
-luciferin (a substrate for FL) does not serve as a
substrate for RL, and coelenterazine does not serve as a substrate for
FL either in cell culture or in living mice. We also show that both
Rluc and Fluc expression can be imaged in the same living mouse and
that the kinetics of light production are distinct. The approaches
validated will have direct applications to various studies where two
molecular events need to be tracked, including cell trafficking of two
cell populations, two gene therapy vectors, and indirect monitoring of
two endogenous genes through the use of two reporter genes.
Visual arrestins in olfactory pathways of
Drosophila
and the malaria vector mosquito
Anopheles
gambiae
Authors: Merrill C. E. , Riesgo-Escovar J. , Pitts R. J. , Kafatos F. C. , Carlson J. R. , Zwiebel L. J.
Published: July 2002

Arrestins are important components for desensitization of G protein-coupled receptor cascades that mediate neurotransmission as well as olfactory and visual sensory reception. We have isolated
AgArr1
, an arrestin-encoding cDNA from the malaria vector mosquito,
Anopheles gambiae
, where olfaction is critical for vectorial capacity. Analysis of
AgArr1
expression revealed an overlap between chemosensory and photoreceptor neurons. Furthermore, an examination of previously identified arrestins from
Drosophila melanogaster
exposed similar bimodal expression, and
Drosophila
arrestin mutants demonstrate impaired electrophysiological responses to olfactory stimuli. Thus, we show that arrestins in
Drosophila
are required for normal olfactory physiology in addition to their previously described role in visual signaling. These findings suggest that individual arrestins function in both olfactory and visual pathways in Dipteran insects; these genes may prove useful in the design of control strategies that target olfactory-dependent behaviors of insect disease vectors.
Human macrophage activation programs induced by bacterial pathogens
Authors: Nau Gerard J. , Richmond Joan F. L. , Schlesinger Ann , Jennings Ezra G. , Lander Eric S. , Young Richard A.
Published: July 2002

Understanding the response of innate immune cells to pathogens may provide insights to host defenses and the tactics used by pathogens to circumvent these defenses. We used DNA microarrays to explore the responses of human macrophages to a variety of bacteria. Macrophages responded to a broad range of bacteria with a robust, shared pattern of gene expression. The shared response includes genes encoding receptors, signal transduction molecules, and transcription factors. This shared activation program transforms the macrophage into a cell primed to interact with its environment and to mount an immune response. Further study revealed that the activation program is induced by bacterial components that are Toll-like receptor agonists, including lipopolysaccharide, lipoteichoic acid, muramyl dipeptide, and heat shock proteins. Pathogen-specific responses were also apparent in the macrophage expression profiles. Analysis ofMycobacterium tuberculosis-specific responses revealed inhibition of interleukin-12 production, suggesting one means by which this organism survives host defenses. These results improve our understanding of macrophage defenses, provide insights into mechanisms of pathogenesis, and suggest targets for therapeutic intervention.
Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways
Authors: Duesbery N. S. , Resau J. , Webb C. P. , Koochekpour S. , Koo H.-M. , Leppla S. H. , Vande Woude G. F.
Published: July 2002

Lethal factor is a protease, one component of
Bacillus
anthracis
exotoxin, which cleaves many of the mitogen-activated
protein kinase kinases (MEKs). Given the importance of MEK signaling in
tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx)
on tumor cells. LeTx was very effective in inhibiting mitogen-activated
protein kinase activation in V12 H-ras-transformed NIH 3T3 cells.
In vitro
, treatment of transformed cells with LeTx
caused them to revert to a nontransformed morphology, and inhibited
their abilities to form colonies in soft agar and to invade Matrigel
without markedly affecting cell proliferation.
In vivo
,
LeTx inhibited growth of ras-transformed cells implanted in athymic
nude mice (in some cases causing tumor regression) at concentrations
that caused no apparent animal toxicity. Unexpectedly, LeTx also
greatly decreased tumor neovascularization. These results demonstrate
that LeTx potently inhibits ras-mediated tumor growth and is a
potential antitumor therapeutic.
Self-organization of self-assembled photonic materials into functional devices: Photo-switched conductors
Authors: Drain Charles Michael
Published: July 2002

Linear porphyrin arrays self-assembled by either hydrogen bonding or metal ion coordination self-organize into lipid bilayer membranes. The length of the transmembrane assemblies is determined both by the thermodynamics of the intermolecular interactions in the supermolecule and by the dimension and physical chemical properties of the bilayer. Thus, the size of the porphyrin assembly can self-adjust to the thickness of the bilayer. An aqueous electron acceptor is placed on one side of the membrane and an electron donor is placed on the opposite side. When illuminated with white light, substantial photocurrents are observed. Only the assembled structures give rise to the photocurrent, as no current is observed from any of the component molecules. The fabrication of this photogated molecular electronic conductor from simple molecular components exploits several levels of self-assembly and self-organization.
Preventing ventricular fibrillation by flattening cardiac restitution
Authors: Garfinkel Alan , Kim Young-Hoon , Voroshilovsky Olga , Qu Zhilin , Kil Jong R. , Lee Moon-Hyoung , Karagueuzian Hrayr S. , Weiss James N. , Chen Peng-Sheng
Published: July 2002

Ventricular fibrillation is the leading cause of sudden cardiac death. In fibrillation, fragmented electrical waves meander erratically through the heart muscle, creating disordered and ineffective contraction. Theoretical and computer studies, as well as recent experimental evidence, have suggested that fibrillation is created and sustained by the property of restitution of the cardiac action potential duration (that is, its dependence on the previous diastolic interval). The restitution hypothesis states that steeply sloped restitution curves create unstable wave propagation that results in wave break, the event that is necessary for fibrillation. Here we present experimental evidence supporting this idea. In particular, we identify the action of the drug bretylium as a prototype for the future development of effective restitution-based antifibrillatory agents. We show that bretylium acts in accord with the restitution hypothesis: by flattening restitution curves, it prevents wave break and thus prevents fibrillation. It even converts existing fibrillation, either to a periodic state (ventricular tachycardia, which is much more easily controlled) or to quiescent healthy tissue.
Horizontal transfer of oncogenes by uptake of apoptotic bodies
Authors: Bergsmedh Anna , Szeles Anna , Henriksson Marie , Bratt Anders , Folkman M. Judah , Spetz Anna-Lena , Holmgren Lars
Published: July 2002

Tumor formation involves the accumulation of a series of genetic alterations that are required for malignant growth. In most malignancies, genetic changes can be observed at the chromosomal level as losses or gains of whole or large portions of chromosomes.Here we provide evidence that tumor DNA may be horizontally transferred by the uptake of apoptotic bodies. Phagocytosis of apoptotic bodies derived from H-rasV12- and human c-myc-transfected rat fibroblasts resulted in loss of contact inhibitionin vitroand a tumorigenic phenotypein vivo. Fluorescencein situhybridization analysis revealed the presence of rat chromosomes or of rat and mouse fusion chromosomes in the nuclei of the recipient murine cells. The transferred DNA was propagated, provided that the transferred DNA conferred a selective advantage to the cell and that the phagocytotic host cell was p53-negative. These results suggest that lateral transfer of DNA between eukaryotic cells may result in aneuploidy and the accumulation of genetic changes that are necessary for tumor formation.
Monitoring the GAP catalyzed H-Ras GTPase reaction at atomic resolution in real time
Authors: Allin Christoph , Ahmadian Mohammad Reza , Wittinghofer Alfred , Gerwert Klaus
Published: July 2002

The molecular reaction mechanism of the GTPase-activating protein (GAP)-catalyzed GTP hydrolysis by Ras was investigated by time resolved Fourier transform infrared (FTIR) difference spectroscopy using caged GTP (
P
3
-1-(2-nitro)phenylethyl guanosine 5′-
O
-triphosphate) as photolabile trigger. This approach provides the complete GTPase reaction pathway with time resolution of milliseconds at the atomic level. Up to now, one structural model of the GAP⋅Ras⋅GDP⋅AlF
x
transition state analog is known, which represents a “snap shot” along the reaction-pathway. As now revealed, binding of GAP to Ras⋅GTP shifts negative charge from the γ to β phosphate. Such a shift was already identified by FTIR in GTP because of Ras binding and is now shown to be enhanced by GAP binding. Because the charge distribution of the GAP⋅Ras⋅GTP complex thus resembles a more dissociative-like transition state and is more like that in GDP, the activation free energy is reduced. An intermediate is observed on the reaction pathway that appears when the bond between β and γ phosphate is cleaved. In the intermediate, the released P
i
is strongly bound to the protein and surprisingly shows bands typical of those seen for phosphorylated enzyme intermediates. All these results provide a mechanistic picture that is different from the intrinsic GTPase reaction of Ras. FTIR analysis reveals the release of P
i
from the protein complex as the rate-limiting step for the GAP-catalyzed reaction. The approach presented allows the study not only of single proteins but of protein–protein interactions without intrinsic chromophores, in the non-crystalline state, in real time at the atomic level.
Regulation of histone deacetylase 4 and 5 and transcriptional activity by 14-3- 3-dependent cellular localization
Authors: Grozinger Christina M. , Schreiber Stuart L.
Published: July 2002

Transcription is controlled in part by the dynamic acetylation and deacetylation of histone proteins. The latter process is mediated by histone deacetylases (HDACs). Previous analysis of the regulation of HDAC activity in transcription has focused primarily on the recruitment of HDAC proteins to specific promoters or chromosomal domains by association with DNA-binding proteins. To characterize the cellular function of the recently identified HDAC4 and HDAC5 proteins, complexes were isolated by immunoprecipitation. Both HDACs were found to interact with14-3-3 proteins at three phosphorylation sites. The association of 14-3-3 with HDAC4 and HDAC5 results in the sequestration of these proteins in the cytoplasm. Loss of this interaction allows HDAC4 and HDAC5 to translocate to the nucleus, interact with HDAC3, and repress gene expression. Regulation of the cellular localization of HDAC4 and HDAC5 by 14-3-3 represents a mechanism for controlling the transcriptional activity of these class II HDAC proteins.
Rapid mapping of protein functional epitopes by combinatorial alanine scanning
Authors: Weiss Gregory A. , Watanabe Colin K. , Zhong Alan , Goddard Audrey , Sidhu Sachdev S.
Published: July 2002

A combinatorial alanine-scanning strategy was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor. A phage-displayed protein library was constructed in which the 19 side chains were preferentially allowed to vary only as the wild type or alanine. The library pool was subjected to binding selections to isolate functional clones, and DNA sequencing was used to determine the alanine/wild-type ratio at each varied position. This ratio was used to calculate the effect of each alanine substitution as a change in free energy relative to that of wild type. Only seven side chains contribute significantly to the binding interaction, and these conserved residues form a compact cluster in the human growth hormone tertiary structure. The results were in excellent agreement with free energy data previously determined by conventional alanine-scanning mutagenesis and suggest that this technology should be useful for analyzing functional epitopes in proteins.
A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition
Authors: Bayfield Mark A. , Dahlberg Albert E. , Schulmeister Ulrike , Dorner Silke , Barta Andrea
Published: July 2002

The ribosome is a dynamic particle that undergoes many structural changes during translation. We show through chemical probing with dimethyl sulfate (DMS) that conformational changes occur at several nucleotides in the peptidyl transferase center upon alterations in pH, temperature, and monovalent ion concentration, consistent with observations made by Elson and coworkers over 30 years ago. Moreover, we have found that the pH-dependent DMS reactivity of A2451 in the center of the 23S rRNA peptidyl transferase region, ascribed to a perturbed pKa of this base, occurs only in inactive 50S and 70S ribosomes. The degree of DMS reactivity of this base in the inactive ribosomes depends on both the identity and amount of monovalent ion present. Furthermore, G2447, a residue proposed to be critical for the hypothesized pKa perturbation, is not essential for the conditional DMS reactivity at A2451. Given that the pH-dependent change in DMS reactivity at A2451 occurs only in inactive ribosomes, and that this DMS reactivity can increase with increasing salt (independently of pH), we conclude that this observation cannot be used as supporting evidence for a recently proposed model of acid/base catalyzed ribosomal transpeptidation.
Okazaki fragment processing: Modulation of the strand displacement activity of DNA polymerase δ by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1
Authors: Maga Giovanni , Villani Giuseppe , Tillement Vanessa , Stucki Manuel , Locatelli Giada A. , Frouin Isabelle , Spadari Silvio , Hübscher Ulrich
Published: July 2002

DNA polymerase (pol) δ is essential for both leading and lagging
strand DNA synthesis during chromosomal replication in
eukaryotes. Pol δ has been implicated in the Okazaki fragment
maturation process for the extension of the newly synthesized fragment
and for the displacement of the RNA/DNA segment of the preexisting
downstream fragment generating an intermediate flap structure that is
the target for the Dna2 and flap endonuclease-1 (Fen 1) endonucleases.
Using a single-stranded minicircular template with an annealed
RNA/DNA primer, we could measure strand displacement by pol δ
coupled to DNA synthesis. Our results suggested that pol δ alone can
displace up to 72 nucleotides while synthesizing through a
double-stranded DNA region in a distributive manner. Proliferating cell
nuclear antigen (PCNA) reduced the template dissociation rate of pol
δ, thus increasing the processivity of both synthesis and strand
displacement, whereas replication protein A (RP-A) limited the size of
the displaced fragment down to 20–30 nucleotides, by generating a
“locked” flap DNA structure, which was a substrate for processing
of the displaced fragment by Fen 1 into a ligatable product. Our data
support a model for Okazaki fragment processing where the strand
displacement activity of DNA polymerase δ is modulated by the
concerted action of PCNA, RP-A and Fen 1.
Human V delta 2+ gamma delta T-cell tolerance to foreign antigens of Toxoplasma gondii.
Authors: Hara T , Ohashi S , Yamashita Y , Abe T , Hisaeda H , Himeno K , Good R A , Takeshita K
Published: July 2002

Little is known about the mechanisms involved in human gammadelta T-cell tolerance to self or to foreign antigens. Patients with congenital toxoplasmosis offer a unique opportunity to examine Vdelta2+ gammadelta T-cell tolerance. Analysis of gammadelta T cells in patients with congenital toxoplasmosis revealed evidence for anergy of these cells with or without clonal Vdelta2+ gammadelta T-cell expansion in the acute phase of the Toxoplasma infection. T cells in general were unresponsive and did not proliferate upon exposure to mitogens or to Toxoplasma lysate antigens or in response to live Toxoplasma-infected cells when the congenitally infected infants were 1 month of age, and they exhibited selective anergy to Toxoplasma lysate antigens and live Toxoplasma-infected cells when the infants were aged 5 months. During the chronic phase of congenital toxoplasmosis in the patients who were more than I year of age, the repertoires of the gammadelta T-cell receptors were found to be within normal ranges. In addition, in the chronic phase, the gammadelta T cells proliferated and secreted gamma-interferon in response to exposure to live Toxoplasmia-infected cells. By contrast, alphabeta T cells remained anergic. Vdelta2+ gammadelta T cells have been considered to undergo extrathymic maturation and thus to be subject to development of peripheral tolerance. Our findings indicate that Vdelta2+ gammadelta T-cell tolerance was lost in these infected infants earlier than alphabeta T-cell tolerance. These findings suggest that gammadelta T cells play a role in protection against Toxoplasma gondii in the chronic phase when congenitally infected children are more than 1 year of age, especially in those in whom alphabeta T cells continue to exhibit deficits in specific immune responses to Toxoplasma antigens.
Microfabricated structures for integrated DNA analysis.
Authors: Burns M A , Mastrangelo C H , Sammarco T S , Man F P , Webster J R , Johnsons B N , Foerster B , Jones D , Fields Y , Kaiser A R , Burke D T
Published: July 2002

Photolithographic micromachining of silicon is a candidate technology for the construction of high-throughput DNA analysis devices. However, the development of complex silicon microfabricated systems has been hindered in part by the lack of a simple, versatile pumping method for integrating individual components. Here we describe a surface-tension-based pump able to move discrete nanoliter drops through enclosed channels using only local heating. This thermocapillary pump can accurately mix, measure, and divide drops by simple electronic control. In addition, we have constructed thermal-cycling chambers, gel electrophoresis channels, and radiolabeled DNA detectors that are compatible with the fabrication of thermocapillary pump channels. Since all of the components are made by conventional photolithographic techniques, they can be assembled into more complex integrated systems. The combination of pump and components into self-contained miniaturized devices may provide significant improvements in DNA analysis speed, portability, and cost. The potential of microfabricated systems lies in the low unit cost of silicon-based construction and in the efficient sample handling afforded by component integration.
Stimulation of new bone formation by direct transfer of osteogenic plasmid genes.
Authors: Fang J , Zhu Y Y , Smiley E , Bonadio J , Rouleau J P , Goldstein S A , McCauley L K , Davidson B L , Roessler B J
Published: July 2002

Degradable matrices containing expression plasmid DNA [gene-activated matrices (GAMs)] were implanted into segmental gaps created in the adult rat femur. Implantation of GAMs containing beta-galactosidase or luciferase plasmids led to DNA uptake and functional enzyme expression by repair cells (granulation tissue) growing into the gap. Implantation of a GAM containing either a bone morphogenetic protein-4 plasmid or a plasmid coding for a fragment of parathyroid hormone (amino acids 1-34) resulted in a biological response of new bone filling the gap. Finally, implantation of a two-plasmid GAM encoding bone morphogenetic protein-4 and the parathyroid hormone fragment, which act synergistically in vitro, caused new bone to form faster than with either factor alone. These studies demonstrate for the first time that repair cells (fibroblasts) in bone can be genetically manipulated in vivo. While serving as a useful tool to study the biology of repair fibroblasts and the wound healing response, the GAM technology may also have wide therapeutic utility.
Chemical basis of courtship in a beetle (Neopyrochroa flabellata): Cantharidin as "nuptial gift".
Authors: Eisner T , Smedley S R , Young D K , Eisner M , Roach B , Meinwald J
Published: July 2002

The amount of cantharidin (Spanish fly) that the Neopyrochroa flabellata male presents to the female as a glandular offering during courtship represents only a small fraction of the total cantharidin the male accumulates systemically following ingestion of the compound. A major fraction of the acquired cantharidin is stored by the male in the large accessory glands of the reproductive system. At mating, the male transfers this supply, presumably as part of the sperm package, to the spermatheca of the female. The female in turn allocates the gift to the eggs. Eggs endowed with cantharidin proved relatively invulnerable to attack by a predaceous beetle larva (Coleomegilla maculata).
Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development.
Authors: Schmidt J V , Su G H , Reddy J K , Simon M C , Bradfield C A
Published: July 2002

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain insight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murine Ahr gene by homologous recombination. Ahr-/- mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. The Ahr-/- phenotype is most severe between 0-3 weeks of age and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedullary hematopoiesis, and portal hypercellularity with thickening and fibrosis.
The structure of bovine F1-ATPase complexed with the peptide antibiotic efrapeptin.
Authors: Abrahams J P , Buchanan S K , Van Raaij M J , Fearnley I M , Leslie A G , Walker J E
Published: July 2002

In the previously determined structure of mitochondrial F1-ATPase determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. AMP-PNP and ADP are bound to subunits beta TP and beta DP, respectively, and the third beta-subunit (beta E) has no bound nucleotide. The efrapeptins are a closely related family of modified linear peptides containing 15 amino acids that inhibit both ATP synthesis and hydrolysis by binding to the F1 catalytic domain of F1F0-ATP synthase. In crystals of F1-ATPase grown in the presence of both nucleotides and inhibitor, efrapeptin is bound to a unique site in the central cavity of the enzyme. Its binding is associated with small structural changes in side chains of F1-ATPase around the binding pocket. Efrapeptin makes hydrophobic contacts with the alpha-helical structure in the gamma-subunit, which traverses the cavity, and with subunit beta E and the two adjacent alpha-subunits. Two intermolecular hydrogen bonds could also form. Intramolecular hydrogen bonds probably help to stabilize efrapeptin's two domains (residues 1-6 and 9-15, respectively), which are connected by a flexible region (beta Ala-7 and Gly-8). Efrapeptin appears to inhibit F1-ATPase by blocking the conversion of subunit beta E to a nucleotide binding conformation, as would be required by an enzyme mechanism involving cyclic interconversion of catalytic sites.
The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.
Authors: White C E , Hunter M J , Meininger D P , Garrod S , Komives E A
Published: July 2002

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.
Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3
Authors: Yang Wen-Ming , Inouye Carla , Zeng Yingying , Bearss David , Seto Edward
Published: July 2002

YY1 is a mammalian zinc-finger transcription factor with unusual
structural and functional features. It has been implicated as a
positive and a negative regulatory factor that binds to the CCATNTT
consensus DNA element located in promoters of many cellular and viral
genes. A mammalian cDNA that encodes a YY1-binding protein and
possesses sequence homology with the yeast transcriptional factor RPD3
has been identified. A Gal4 DNA binding domain–mammalian RPD3 fusion
protein strongly represses transcription from a promoter containing
Gal4 binding sites. Association between YY1 and mammalian RPD3 requires
a glycine-rich region on YY1. Mutations in this region abolish the
interaction with mammalian RPD3 and eliminate transcriptional
repression by YY1. These data suggest that YY1 negatively regulates
transcription by tethering RPD3 to DNA as a cofactor and that this
transcriptional mechanism is highly conserved from yeast to human.
Increased sensitivity to apoptotic stimuli in c-abl-deficient progenitor B-cell lines
Authors: Dorsch Marion , Goff Stephen P.
Published: July 2002

The protooncogene c-ablencodes a nonreceptor tyrosine kinase whose cellular function is unknown. To study the possible involvement of c-Abl in proliferation, differentiation, and cell cycle regulation of early B cells, long-term lymphoid bone marrow cultures were established from c-abl-deficient mice and their wild-type littermates. Interleukin 7-dependent progenitor B-cell clones and lines expressing B220 and CD43 could be generated from both mutant and wild-type mice. The mutant and wild-type lines displayed no difference in their proliferative capacity as measured by thymidine incorporation in response to various concentrations of interleukin 7. Similarly, c-abldeficiency did not interfere with the ability of mutant clones to differentiate into surface IgM-positive cellsin vitro. Analysis of cultures after growth factor deprivation, however, revealed a strikingly accelerated rate of cell death in c-ablmutant cells, due to apoptosis as confirmed by terminal deoxynucleotidyltransferase-mediated UTP nick end labeling analysis. Furthermore, a greater susceptibility to apoptotic cell death in c-ablmutant cells was also observed after glucocorticoid treatment. These results suggest that mutant c-Abl renders the B-cell progenitors more sensitive to apoptosis, and may account for some of the phenotypes observed in c-abl-deficient animals.
Distinct short-term plasticity at two excitatory synapses in the hippocampus
Authors: Salin Paul A. , Scanziani Massimo , Malenka Robert C. , Nicoll Roger A.
Published: July 2002

A single mossy fiber input contains several release sites and is
located on the proximal portion of the apical dendrite of CA3 neurons.
It is, therefore, well suited to exert a strong influence on pyramidal
cell excitability. Accordingly, the mossy fiber synapse has been
referred to as a detonator or teacher synapse in autoassociative
network models of the hippocampus. The very low firing rates of granule
cells [Jung, M. W. & McNaughton, B. L. (1993)
Hippocampus
3, 165–182], which give rise to the mossy
fibers, raise the question of how the mossy fiber synapse temporally
integrates synaptic activity. We have therefore addressed the frequency
dependence of mossy fiber transmission and compared it to
associational/commissural synapses in the CA3 region of the
hippocampus. Paired pulse facilitation had a similar time course, but
was 2-fold greater for mossy fiber synapses. Frequency facilitation,
during which repetitive stimulation causes a reversible growth in
synaptic transmission, was markedly different at the two synapses. At
associational/commissural synapses facilitation occurred only at
frequencies greater than once every 10 s and reached a magnitude
of about 125% of control. At mossy fiber synapses, facilitation
occurred at frequencies as low as once every 40 s and reached a
magnitude of 6-fold. Frequency facilitation was dependent on a rise in
intraterminal Ca
2+
and activation of
Ca
2+
/calmodulin-dependent kinase II, and was greatly
reduced at synapses expressing mossy fiber long-term potentiation.
These results indicate that the mossy fiber synapse is able to
integrate granule cell spiking activity over a broad range of
frequencies, and this dynamic range is substantially reduced by
long-term potentiation.
How the brain keeps the eyes still
Authors: Seung H. S.
Published: July 2002

The brain can hold the eyes still because it stores a memory of eye
position. The brain’s memory of horizontal eye position appears to be
represented by persistent neural activity in a network known as the
neural integrator, which is localized in the brainstem and cerebellum.
Existing experimental data are reinterpreted as evidence for an
“attractor hypothesis” that the persistent patterns of activity
observed in this network form an attractive line of fixed points in its
state space. Line attractor dynamics can be produced in linear or
nonlinear neural networks by learning mechanisms that precisely tune
positive feedback.
The role of symmetry in fundamental physics
Authors: Gross David J.
Published: July 2002

The role of symmetry in fundamental physics is reviewed.
Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity.
Authors: Paulsen I T , Brown M H , Littlejohn T G , Mitchell B A , Skurray R A
Published: July 2002

The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.
Stable triple helices formed by oligonucleotide N3'-->P5' phosphoramidates inhibit transcription elongation.
Authors: Escudé C , Giovannangeli C , Sun J S , Lloyd D H , Chen J K , Gryaznov S M , Garestier T , Hélène C
Published: July 2002

Oligonucleotide analogs with N3'-->P5' phosphoramidate linkages bind to the major groove of double-helical DNA at specific oligopurine.oligopyrimidine sequences. These triple-helical complexes are much more stable than those formed by oligonucleotides with natural phosphodiester linkages. Oligonucleotide phosphoramidates containing thymine and cytosine or thymine, cytosine, and guanine bind strongly to the polypurine tract of human immunodeficiency virus proviral DNA under physiological conditions. Site-specific cleavage by the Dra I restriction enzyme at the 5' end of the polypurine sequence was inhibited by triplex formation. A eukaryotic transcription assay was used to investigate the effect of oligophosphoramidate binding to the polypurine tract sequence on transcription of the type 1 human immunodeficiency virus nef gene under the control of a cytomegalovirus promoter. An efficient arrest of RNA polymerase II was observed at the specific triplex site at submicromolar concentrations.
Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice
Authors: Bhatia Mickie , Wang Jean C. Y. , Kapp Ursula , Bonnet Dominique , Dick John E.
Published: July 2002

The purification of primitive human hematopoietic stem cells has been impaired by the absence of repopulation assays. By using a stringent two-step strategy involving depletion of lineage-positive cells followed by fluorescence-activated cell sorting, we have purified a cell population that is highly enriched for cells capable of multilineage repopulation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) recipients. TheseSCID-repopulatingcells (SRCs) were exclusively found in a cell fraction that expressed high levels of CD34 and no CD38. Through limiting dilution analysis using Poisson statistics, we calculated a frequency of 1 SRC in 617 CD34+CD38−cells. The highly purified SRC were capable of extensive proliferation in NOD/SCID mice. Mice transplanted with 1 SRC (at limiting cell doses) were able to produce approximately 400,000 progeny 6 weeks after the transplant. Detailed flow cytometric analysis of the marrow of highly engrafted mice demonstrated both lymphoid and myeloid differentiation, as well as the retention of a significant fraction of CD34+CD38−cells. These highly purified fractions should be useful for identification of the cellular and molecular mechanisms that regulate primitive human hematopoietic cells. Moreover, the ability to detect and purify primitive cells provides a means to develop conditions for maintaining and/or expanding these cells duringin vitroculture.
X-ray structures of a hydrolytic antibody and of complexes elucidate catalytic pathway from substrate binding and transition state stabilization through water attack and product release
Authors: Gigant Benoît , Charbonnier Jean-Baptiste , Eshhar Zelig , Green Bernard S. , Knossow Marcel
Published: July 2002

The x-ray structures of the unliganded esterase-like catalytic antibody D2.3 and its complexes with a substrate analogue and with one of the reaction products are analyzed. Together with the structure of the phosphonate transition state analogue hapten complex, these crystal structures provide a complete description of the reaction pathway. At alkaline pH, D2.3 acts by preferential stabilization of the negatively charged oxyanion intermediate of the reaction that results from hydroxide attack on the substrate. A tyrosine residue plays a crucial role in catalysis: it activates the ester substrate and, together with an asparagine, it stabilizes the oxyanion intermediate. A canal allows facile diffusion of water molecules to the reaction center that is deeply buried in the structure. Residues bordering this canal provide targets for mutagenesis to introduce a general base in the vicinity of the reaction center.
Characteristics of the deep ocean carbon system during the past 150,000 years: ΣCO
2
distributions, deep water flow patterns, and abrupt climate change
Authors: Boyle Edward A.
Published: July 2002

Studies of carbon isotopes and cadmium in bottom-dwelling
foraminifera from ocean sediment cores have advanced our knowledge of
ocean chemical distributions during the late Pleistocene. Last Glacial
Maximum data are consistent with a persistent high-ΣCO
2
state for eastern Pacific deep water. Both tracers indicate that the
mid-depth North and tropical Atlantic Ocean almost always has lower
ΣCO
2
levels than those in the Pacific. Upper waters of
the Last Glacial Maximum Atlantic are more ΣCO
2
-depleted
and deep waters are ΣCO
2
-enriched compared with the
waters of the present. In the northern Indian Ocean, δ
13
C
and Cd data are consistent with upper water ΣCO
2
depletion relative to the present. There is no evident proximate source
of this ΣCO
2
-depleted water, so I suggest that
ΣCO
2
-depleted North Atlantic intermediate/deep water
turns northward around the southern tip of Africa and moves toward the
equator as a western boundary current. At long periods (>15,000
years), Milankovitch cycle variability is evident in paleochemical time
series. But rapid millennial-scale variability can be seen in cores
from high accumulation rate series. Atlantic deep water chemical
properties are seen to change in as little as a few hundred years or
less. An extraordinary new 52.7-m-long core from the Bermuda Rise
contains a faithful record of climate variability with century-scale
resolution. Sediment composition can be linked in detail with the
isotope stage 3 interstadials recorded in Greenland ice cores. This new
record shows at least 12 major climate fluctuations within marine
isotope stage 5 (about 70,000–130,000 years before the present).
Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them
Authors: Beresford Paul J. , Kam Chih-Min , Powers James C. , Lieberman Judy
Published: July 2002

The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death. Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes. We expressed human granzyme A in bacteria as a proenzyme capable ofin vitroactivation by enterokinase. The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin. An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates. Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment. PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack. PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.
Subtle atomic group discrimination in the RNA minor groove
Authors: Frugier Magali , Schimmel Paul
Published: July 2002

As a problem in molecular recognition and for drug discovery, great interest has developed around the possibility that RNA structures could be discriminated by peptides and other small molecules. Although small peptides have been shown to have the capacity to discriminate specific bulges and loops in RNA molecules, discrimination of double helical regions by a peptide binder has not been reported. Indeed, the most accessible part of an RNA helix is the minor groove, and fundamental stereochemical considerations have suggested that discrimination of at least some base pairs would be difficult in the minor groove. Here we report the design and isolation of a peptide binder that manifests the most subtle kind of discrimination of base pair differences in the RNA minor groove. Functional discrimination of a single atomic group is demonstrated as well as the difference between two different angular orientations of the same group. This report of RNA helix discrimination by a peptide binder suggests a richer potential for RNA minor groove recognition than previously thought.
cAMP receptor protein–cAMP plays a crucial role in glucose–lactose diauxie by activating the major glucose transporter gene in
Escherichia coli
Authors: Kimata Keiko , Takahashi Hideyuki , Inada Toshifumi , Postma Pieter , Aiba Hiroji
Published: July 2002

The inhibition of β-galactosidase expression in a medium containing both glucose and lactose is a typical example of the glucose effect in
Escherichia coli.
We studied the glucose effect in the
lacL8UV5
promoter mutant, which is independent of cAMP and cAMP receptor protein (CRP). A strong inhibition of β-galactosidase expression by glucose and a diauxic growth were observed when the
lacL8UV5
cells were grown on a glucose–lactose medium. The addition of isopropyl β-
d
-thiogalactoside to the culture medium eliminated the glucose effect. Disruption of the
crr
gene or overproduction of LacY also eliminated the glucose effect. These results are fully consistent with our previous finding that the glucose effect in wild-type cells growing in a glucose–lactose medium is not due to the reduction of CRP–cAMP levels but is due to the inducer exclusion. We found that the glucose effect in the
lacL8UV5
cells was no longer observed when either the
crp
or the
cya
gene was disrupted. Evidence suggested that CRP–cAMP may not enhance directly the
lac
repressor action
in vivo
. Northern blot analysis revealed that the mRNA for
ptsG,
a major glucose transporter gene
,
was markedly reduced in a Δ
crp
or Δ
cya
background. The constitutive expression of the
ptsG
gene by the introduction of a multicopy plasmid restored the glucose effect in Δ
cya
or Δ
crp
cells. We conclude that CRP–cAMP plays a crucial role in inducer exclusion, which is responsible for the glucose–lactose diauxie, by activating the expression of the
ptsG
gene.
Targeted deletion of all isoforms of the
trkC
gene suggests the use of alternate receptors by its ligand neurotrophin-3 in neuronal development and implicates
trkC
in normal cardiogenesis
Authors: Tessarollo Lino , Tsoulfas Pantelis , Donovan Michael J. , Palko Mary Ellen , Blair-Flynn Janet , Hempstead Barbara L. , Parada Luis F.
Published: July 2002

We have generated null mutant mice that lack expression of all isoforms encoded by the
trkC
locus. These mice display a behavioral phenotype characterized by a loss of proprioceptive neurons. Neuronal counts of sensory ganglia in the
trkC
mutant mice reveal less severe losses than those in
NT-3
null mutant mice, strongly suggesting that
NT-3
,
in vivo
, may signal through receptors other than
trkC
. Mice lacking either
NT-3
or all
trkC
receptor isoforms die in the early postnatal period. Histological examination of
trkC
-deficient mice reveals severe cardiac defects such as atrial and ventricular septal defects, and valvular defects including pulmonic stenosis. Formation of these structures during development is dependent on cardiac neural crest function. The similarities in cardiac defects observed in the
trkC
and
NT-3
null mutant mice indicate that the
trkC
receptor mediates most
NT-3
effects on the cardiac neural crest.