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Biomedical subjects

H Wu

Publications and source records attributed to H Wu.

At least 433 records · Page 24Linked to original sources
PubMed1999

Theoretical and experimental tests of a chromosomal fingerprint for densely ionizing radiation based on F ratios calculated from stable and unstable chromosome aberrations.

In the present study, F ratios for both stable chromosome aberrations, i.e. ratios of translocations to pericentric inversions, and unstable aberrations, i.e. dicentrics and centric rings, were measured using fluorescence in situ hybridization. F ratios for stable aberrations measured after exposure to low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 1.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiation were 6.5 +/- 1.5, 4.7 +/- 1.6, 9.3 +/- 2.5 and 10.4 +/- 3.0, respectively. F ratios for unstable aberrations measured after low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiations were 6.5 +/- 1.6, 6.3 +/- 2.3 and 11.1 +/- 3.7, respectively. No significant difference between the F ratios for low- and high-LET radiation was found. Further tests on the models for calculation of the F ratio proposed by Brenner and Sachs (Radiat. Res. 140, 134-142, 1994) showed that the F ratio may not be straightforward as a practical fingerprint for densely ionizing radiation.

Cells, Cultured↗
PubMed1998

Sp1 and CREB mediate gastrin-dependent regulation of chromogranin A promoter activity in gastric carcinoma cells.

Chromogranin A (CgA) is a multifunctional acidic protein that in the stomach is expressed predominantly in enterochromaffin-like cells (ECL cells) where it is regulated by gastrin. In order to investigate the transcriptional response of the mouse CgA (mCgA) promoter to gastrin stimulation, we studied a 4.8-kilobase mCgA promoter-luciferase reporter gene construct in transiently transfected AGS-B cells. 5'-Deletion analysis and scanning mutagenesis of mCgA 5'-flanking DNA showed that a Sp1/Egr-1 site spanning -88 to -77 base pairs (bp) and a cyclic AMP-responsive element (CRE) at -71 to -64 bp are essential for gastrin-dependent mCgA transactivation. Gastrin stimulation increased cellular Sp1 protein levels and Sp1-binding to the mCgA -88 to -77 bp element, as well as binding of CREB to its consensus motif at -71 to -64 bp. Gastrin also stimulated CREB Ser-133 phosphorylation, and abundance of cellular CREB protein levels. Overexpression of either Sp1 or phosphorylated CREB transactivated the mCgA promoter dose dependently, while coexpression of both transcription factors resulted in an additive mCgA promoter response. mCgA -92 to -64 bp, comprising the Sp1/Egr-1 site and the CRE motif, conferred gastrin responsiveness to a heterologous thymidine kinase promoter system, and therefore functions as a "true" enhancer element. This report demonstrates that Sp1 and CREB mediate CCK-B/gastrin receptor-dependent gene regulation, and that the effect of gastrin on the CgA gene is brought about by cooperative action of both transcription factors.

Animals↗
PubMed1998

Estimation of HIV dynamic parameters.

Investigation of HIV viral dynamics is important for understanding the HIV pathogenesis and for development of treatment strategies. Perelson et al. demonstrated that simple viral dynamic models fit to data on viral load as measured by plasma HIV-RNA could produce estimates of rates of clearance of virus and of infected CD4+ T-lymphocytes. In this paper we extend the work of Perelson et al. by proposing models with less restrictive assumptions about drug activity. Our models take into account the fact that infectious and non-infectious virions are produced by infected T-cells both before and after the treatment. We also show that direct measurement of infectious virus load provides sufficient information for estimation of antiretroviral drug efficacy parameter. For characterizing viral dynamics of populations and estimation of dynamic parameters, we propose a hierarchical non-linear model. Compared to other methods such as the non-linear least square method used by Perelson et al., we show that the proposed approach has the following advantages: (i) it is more appropriate for modelling within-patient and between-patient variation and to characterize the population dynamics; (ii) it is flexible enough to deal with both rich and sparse individual data; (iii) it has more power to detect model misspecification; (iv) it allows incorporation of covariates for viral dynamic parameters; (v) it makes more efficient use of between-subject information to get better parameter estimates. We give two simulation examples to illustrate the proposed approach and its advantages. Finally, we discuss practical issues regarding the clinical trial design for viral dynamic studies.

Anti-HIV Agents↗
PubMed1998

Partial characterization of the calcium-releasing activity of porcine sperm cytosolic extracts.

Injection of sperm cytosolic extracts into mammalian eggs has been shown to elicit intracellular calcium ([Ca2+]i) oscillations that are similar in amplitude, duration, and frequency to those observed following fertilization. Thus, to characterize the Ca2+-release component(s) in porcine sperm cytosolic extracts, a combination of fractionation techniques was used. The fraction with Ca2+ releasing activity was precipitated by 50% saturating solutions of ammonium sulfate and Western blot analysis showed that the pellets contained glucosamine-6-phosphate deaminase (gpd)/oscillin, a protein which has been suggested to be the sperm's active component. Single and double isoelectrofocusing (IEF) of porcine sperm extracts generated fractions with different Ca2+-releasing activities. Fractions with maximal Ca2+-releasing activity did not contain material that was immunoreactive with antibodies against gpd/oscillin; adjacent fractions containing gpd/oscillin had no Ca2+-releasing activity. These findings were confirmed by IEF coupled with size exclusion chromatography on Superose 12 and with hydroxyapatite chromatography. These procedures predict an isoelectric point of our active component of 6.5-7.0 and a relative molecular weight ranging from 29 to 68 kDa. In summary, the data show that the Ca2+ release-inducing component(s) of porcine sperm extracts can be fractionated and that gpd/oscillin is not the pig sperm Ca2+ oscillogen.

Aldose-Ketose Isomerases↗
PubMed1998

Cardiac myocytes express mRNA for ten RGS proteins: changes in RGS mRNA expression in ventricular myocytes and cultured atria.

Regulators of G-protein signalling (RGS) are recently identified proteins that shorten the lifetime of the activated G protein. We now show that rat cardiac myocytes express mRNA for at least 10 RGS. The mRNA for RGS-r is barely detectable in rat ventricles, but increases more than 20-fold during the 60- to 90-min process of isolating ventricular myocytes, and after 90 min of culture of atrial pieces in medium with Ca2+. Both in myocytes and in atria, the rise in RGS-r is transient. The mRNA for cardiac RGS5, but not RGS-r, is developmentally regulated. These studies suggest that rapid regulation of RGS levels may be a new mechanism that governs how signals are transmitted across the cardiac cell membrane.

Aging↗
PubMed1998

Sequencing regular and labeled oligonucleotides using enzymatic digestion and ionspray mass spectrometry.

A method using a combination of enzymatic digestion and ionspray mass spectrometry (MS) was developed for sequencing oligodeoxynucleotides (ODNs) containing more than 20 bases. Phosphodiesterase (PDE) digestion of ODNs produced truncated ODNs whose molecular weights (MWs) were determined by ionspray MS. It was demonstrated that reconstruction of MW spectra over a large MW range produced easy-to-read sequence ladders similar to those obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Sample and enzyme cleanup, digestion control, and MW reconstruction were found to be crucial factors. For regular ODNs, both 5'- and 3'-PDE digestions are needed for complete sequencing. Late in the time course of PDE digestions, 5'-nucleoside monophosphates were found to produce artifactual peaks in the reconstructed MW spectra, and a table correlating base compositions and MS ions was compiled to handle such situations. For labeled ODNs, it is necessary to use collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) for complete sequence determination. Sequencing of regular 22-mer and labeled 18-mer ODNs was demonstrated in this work.

Animals↗
PubMed1998

Disruption of primary mesenchyme cell patterning by misregulated ectodermal expression of SpMsx in sea urchin embryos.

The patterning of the mesoderm of the sea urchin embryo is a classical paradigm of epithelial mesenchymal interactions in organogenesis, yet little is known of its molecular basis. Here we address the role of the homeobox gene, SpMsx, a member of the highly conserved Msx gene family, in this process. Msx genes have been shown to function in the dorsoventral patterning of the central nervous system in Drosophila and in a variety epithelial-mesenchymal interactions in vertebrates. We showed previously that the SpMsx gene is expressed during embryogenesis in a complex and dynamic pattern consistent with roles in the development of subpopulations of endoderm, mesoderm, and oral ectoderm. To perturb this pattern of expression and thus probe the function of SpMsx, we injected SpMsx mRNA into single-cell zygotes and monitored development morphologically and with a series of territory-specific molecular markers. RT-PCR analysis revealed that injected SpMsx transcripts persisted at least until the gastrula stage in amounts comparable to endogenous levels. Injected embryos exhibited deficiencies in the organization of primary and secondary mesenchyme cells within the blastocoelic cavity, as well as abnormalities in spicule number and shape. Defects in the endoderm were also common, including reduced or absent archenterons. Micromere transplantation experiments revealed that the defects in skeletogenic mesenchyme patterning were non-cell autonomous, consistent with findings that cell-cell interactions between ectoderm and the progenitors of the skeletogenic mesenchyme, the primary mesenchyme cells (PMCs), are important both for PMC guidance and spicule morphogenesis. Our data, taken together with observations in other organisms on the role of Msx genes in embryonic signaling processes, particularly involving the BMP pathway, suggest that SpMsx may be a part of the mechanism by which the ectoderm influences both the arrangement of primary mesenchyme cells within the blastocoel and the shapes of the skeletal rods.

Animals↗
PubMed1998

Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein.

A 429 aa theoretical intein is encoded in the dnaB gene (DNA helicase) of the cyanobacterium Synechocystis sp. strain PCC6803. This intein is shown to be capable of protein splicing with or without its native exteins when tested in E. coli cells. A centrally located 275 amino acid sequence (residues 107-381) of this intein can be deleted without loss of the protein splicing activity, resulting in a functional mini-intein of 154 aa in size. Efficient in vivo protein trans-splicing was observed when this mini-intein was split into a 106 aa N-terminal fragment containing intein motifs A and B, and a 48 aa C-terminal fragment containing intein motifs F and G. These results indicate that the N- and C-terminal regions of the Ssp DnaB intein, whether covalently linked with each other or not, can come together through non-covalent interaction to form a protein splicing domain that is functionally sufficient and structurally independent from the centrally located endonuclease domain of the intein.

Amino Acid Sequence↗
PubMed1998

A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type.

Slow- and fast-twitch myofibers of adult skeletal muscles express unique sets of muscle-specific genes, and these distinctive programs of gene expression are controlled by variations in motor neuron activity. It is well established that, as a consequence of more frequent neural stimulation, slow fibers maintain higher levels of intracellular free calcium than fast fibers, but the mechanisms by which calcium may function as a messenger linking nerve activity to changes in gene expression in skeletal muscle have been unknown. Here, fiber-type-specific gene expression in skeletal muscles is shown to be controlled by a signaling pathway that involves calcineurin, a cyclosporin-sensitive, calcium-regulated serine/threonine phosphatase. Activation of calcineurin in skeletal myocytes selectively up-regulates slow-fiber-specific gene promoters. Conversely, inhibition of calcineurin activity by administration of cyclosporin A to intact animals promotes slow-to-fast fiber transformation. Transcriptional activation of slow-fiber-specific transcription appears to be mediated by a combinatorial mechanism involving proteins of the NFAT and MEF2 families. These results identify a molecular mechanism by which different patterns of motor nerve activity promote selective changes in gene expression to establish the specialized characteristics of slow and fast myofibers.

3T3 Cells↗
PubMed1998

Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803.

A split intein capable of protein trans-splicing is identified in a DnaE protein of the cyanobacterium Synechocystis sp. strain PCC6803. The N- and C-terminal halves of DnaE (catalytic subunit alpha of DNA polymerase III) are encoded by two separate genes, dnaE-n and dnaE-c, respectively. These two genes are located 745,226 bp apart in the genome and on opposite DNA strands. The dnaE-n product consists of a N-extein sequence followed by a 123-aa intein sequence, whereas the dnaE-c product consists of a 36-aa intein sequence followed by a C-extein sequence. The N- and C-extein sequences together reconstitute a complete DnaE sequence that is interrupted by the intein sequences inside the beta- and tau-binding domains. The two intein sequences together reconstitute a split mini-intein that not only has intein-like sequence features but also exhibited protein trans-splicing activity when tested in Escherichia coli cells.

Amino Acid Sequence↗
PubMed1998

Structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors.

A series of influenza neuraminidase inhibitors with the cyclohexene scaffold containing lipophilic side chains have been synthesized and evaluated for influenza A and B neuraminidase inhibitory activity. The size and geometry of side chains have been modified systematically in order to investigate structure-activity relationships of this class of compounds. The X-ray crystal structures of several analogues complexed with neuraminidase revealed that the lipophilic side chains bound to the hydrophobic pocket consisted of Glu276, Ala246, Arg224, and Ile222 of the enzyme active site. The structure-activity relationship studies of this series have also demonstrated remarkably different inhibitory potency between influenza A and B neuraminidase. This indicated that the lipophilic side chains had quite different hydrophobic interactions with influenza A and B neuraminidase despite their complete homology in the active site. Influenza B neuraminidase appeared to be much more sensitive toward the increased steric bulkiness of inhibitors compared to influenza A neuraminidase. From the extensive structure-activity relationship investigation reported in this article, GS 4071 emerged as one of the most potent influenza neuraminidase inhibitors against both influenza A and B strains.

Acetamides↗
PubMed1998

Induction of antioxidants by adriamycin in mouse heart.

Cardiac oxidative injury is a major limiting factor for clinical application of Adriamycin (ADR) in cancer chemotherapy. ADR depresses some antioxidant systems, thereby further enhancing the cardiotoxicity. Previous studies have shown that ADR inhibits the overall synthesis of DNA, RNA, and protein. It was presumed that the depressed antioxidant activity resulted from the inhibited gene expression. However, there were no experimental data to demonstrate the relationship between the change in antioxidant activities and that in their gene expression. Therefore, the present study was undertaken to examine the effects of ADR on the activities and mRNA abundances of antioxidants in mouse heart. FVB mice (7 weeks old) were treated with ADR (15 mg/kg) by a single i.p. injection. Four days after the treatment, cardiac antioxidant activities and mRNA abundances were measured. The results showed that ADR increased the levels of mRNAs for Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase, glutathione peroxidase (GSHpx), and gamma-glutamylcysteine synthetase (gamma-GCS). On the other hand, ADR increased the activities of catalase and gamma-GCS, and slightly decreased total glutathione concentrations in the heart. Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, and glutathione peroxidase activities were not changed significantly. In addition, ADR increased both mRNA and protein levels of metallothionein in the heart. The data demonstrate that up-regulation of antioxidant gene expression occurred in response to ADR in the mouse heart, although the antioxidant activities were not all increased.

Animals↗
PubMed1998

Stepwise in vitro affinity maturation of Vitaxin, an alphav beta3-specific humanized mAb.

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the alphav beta3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-alphav beta3 interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to alphav beta3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.

Antibodies, Monoclonal↗
PubMed1998

Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis.

Janus kinases (Jaks) play an important role in signal transduction via cytokine and growth factor receptors. A targeted inactivation of Jak2 was performed. Jak2-/- embryos are anemic and die around day 12.5 postcoitum. Primitive erythrocytes are found, but definitive erythropoiesis is absent. Compared to erythropoietin receptor-deficient mice, the phenotype of Jak2 deficiency is more severe. Fetal liver BFU-E and CFU-E colonies are completely absent. However, multilineage hematopoietic stem cells (CD34low, c-kit(pos)) can be found, and B lymphopoiesis appears intact. In contrast to IFNalpha stimulation, Jak2-/- cells do not respond to IFNgamma. Jak2-/- embryonic stem cells are competent for LIF signaling. The data provided demonstrate that Jak2 has pivotal functions for signal transduction of a set of cytokine receptors required in definitive erythropoiesis.

Anemia↗
PubMed1998

[Detection of SMN gene deletions in spinal muscular atrophy].

OBJECTIVE: Survival motor neuron gene(SMN) and neuronal apoptosis inhibitory protein gene (NAIP) have been identified as the candidates of progressive spinal muscular atrophy (SMA)-determining genes. The aims of this study were to investigate the absence of SMN gene exon 7 in Chinese SMA patients, to confirm the relationship between the deletion of the SMN and SMA further, and to establish methods for gene diagnosis and prenatal diagnosis of SMA. METHODS: PCR-SSCP with silver staining method was used to detect the genomic DNA of 37 SMA patients and 30 normal individuals for deletions of SMN exon 7. RESULTS: Homozygous deletion of the SMN exon 7 was identified in 86.7%(13/15) of type I SMA patients and 86.4%(19/22) of type II patients. In the 88 controls (including parents of patients and normal individuals), homozygous absence of SMA exon 7 was only found in a mother of a patient. CONCLUSION: The data support that homozygous absence of SMN exon 7 is strongly associated with SMA. The percentage of homozygous deletions in this study is almost as high as that reported by other researchers. This method is useful, reliable and effective for gene diagnosis and prenatal diagnosis of SMA.

Cyclic AMP Response Element-Binding Protein↗
PubMed1998

Molecular cloning and tissue expression of porcine beta-defensin-1.

Beta-defensins constitute an emerging family of cysteine-rich antimicrobial peptides, which are particularly prominent at mucosal epithelial sites in mammals. Here we report the identification of a novel beta-defensin from porcine tissues, porcine beta-defensin-1 (pBD-1). The cDNA sequence of pBD-1 encoded a 64 amino acid prepro-peptide, which contained the beta-defensin consensus sequence of six invariantly spaced cysteine residues. Northern blot analysis showed that pBD-1 was expressed abundantly in tongue epithelia and that the expression was regulated developmentally. Using RT-PCR, pBD-1 mRNA was detected throughout the respiratory and digestive tracts and also in thymus, spleen, lymph node, brain, liver, kidney, urinary bladder, testis, skin, heart, muscle, bone marrow, peripheral blood neutrophils, alveolar macrophages, and umbilical cord. The wide expression of pBD-1 suggests that this endogenous peptide antibiotic may contribute to both mucosal and systemic host defenses in pigs, which may have implications for the use of porcine tissues and organs in xenotransplantation.

Amino Acid Sequence↗
PubMed1998

Discovery of human antibodies to cell surface antigens by capture lift screening of phage-expressed antibody libraries.

An assay for the rapid identification and cloning of antibody fragments (Fabs) reactive with cell surface antigens was established and used to identify Fabs selectively reactive with tumor cell surface antigens. The Fabs were produced by a phage expression system and screened by a modified plaque lift approach in which nitrocellulose filters were coated with an anti-immunoglobulin reagent and blocked with bovine serum albumin prior to application to the phage-infected bacterial lawn. Subsequently, capture lifts were incubated with biotinylated antigen and reactive Fabs were identified with streptavidin conjugates. This screening method, termed capture lift, results in the immobilization of greater quantities of Fab and decreases the binding of unrelated host proteins, resulting in a more sensitive plaque lift assay. The capture lift permits the simultaneous analysis of thousands of antibody clones and, more importantly, can be used with crude detergent-solubilized cell extracts, permitting the discovery of Fabs which bind integral membrane proteins present in heterogeneous mixtures of antigens. Optimal conditions were identified utilizing phage-expressed BR96 Fab and a horseradish peroxidase conjugate of Lewis Y, a soluble cross-reactive antigen. Subsequently, it was demonstrated that the assay was functional with postnuclear detergent extracts isolated from surface-biotinylated tumor cells expressing the BR96 tumor antigen. Purification of the target antigen was not required. To demonstrate the application of the capture lift assay for the discovery of Fabs reactive with novel cell surface antigens a phage-expressed human antibody library constructed from tumor-infiltrating B lymphocytes was screened. Multiple antibody clones which reacted with detergent-solubilized biotinylated surface antigens were identified. Upon further characterization a portion of these displayed selectivity for tumor cells, as demonstrated by the binding of Fab to fixed and live tumor cells but not normal fibroblasts.

Animals↗