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Biomedical subjects

Y Wu

Publications and source records attributed to Y Wu.

At least 325 records · Page 18Linked to original sources
PubMed2000

Effect of limited proteolysis on phospholipase C-gamma1 kinetics.

Phospholipase C-gamma1 is a tightly regulated, multidomain protein that generates the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Kinetic analysis reveals that phospholipase C-gamma1 displays apparent allosteric behavior. A previous study determined that proteolytic cleavage of the SH domain region of phospholipase C-gamma1 yields an activated form of the enzyme (A. W. Fernald, G. A. Jones, and G. Carpenter Biochem. J. 302, 508, 1994). In this study, we show that activation of phospholipase C-gamma1 by proteolysis decreases both the cooperativity and the half-maximal value of the enzyme for substrate. Kinetic analysis revealed that the mole fraction of phosphatidylinositol 4,5-bisphosphate (PIP(2)) that resulted in half-maximal PIP(2) hydrolysis (S(0.5)) was lower for proteolyzed than uncleaved phospholipase C-gamma1 (0.08 mole fraction vs 0.18 mole fraction of PIP(2)). The cooperativity index was lower for proteolyzed than full-length phospholipase C-gamma1 (n = 2.5 vs n = 4). Kinetic analysis also revealed that the estimated dissociation constant was lower for phospholipase C-gamma1 that had been subjected to proteolysis (0.1 mM vs 1.0 mM PIP(2) for cleaved vs uncleaved phospholipase C-gamma1, respectively). It was previously hypothesized that activation of phospholipase C-gamma1 requires a conformational change that results in increased accessibility of substrate to the active site and that the SH domain of the enzyme is involved in the activation event. These experiments support the hypothesis that a portion of the protein covers the active site, allosterically inhibiting the enzyme, and that the removal of this "lid" domain activates the enzyme.

Allosteric Regulation↗
PubMed2000

Biosynthesis of ganglioside mimics in Campylobacter jejuni OH4384. Identification of the glycosyltransferase genes, enzymatic synthesis of model compounds, and characterization of nanomole amounts by 600-mhz (1)h and (13)c NMR analysis.

We have applied two strategies for the cloning of four genes responsible for the biosynthesis of the GT1a ganglioside mimic in the lipooligosaccharide (LOS) of a bacterial pathogen, Campylobacter jejuni OH4384, which has been associated with Guillain-Barré syndrome. We first cloned a gene encoding an alpha-2, 3-sialyltransferase (cst-I) using an activity screening strategy. We then used nucleotide sequence information from the recently completed sequence from C. jejuni NCTC 11168 to amplify a region involved in LOS biosynthesis from C. jejuni OH4384. The LOS biosynthesis locus from C. jejuni OH4384 is 11.47 kilobase pairs and encodes 13 partial or complete open reading frames, while the corresponding locus in C. jejuni NCTC 11168 spans 13.49 kilobase pairs and contains 15 open reading frames, indicating a different organization between these two strains. Potential glycosyltransferase genes were cloned individually, expressed in Escherichia coli, and assayed using synthetic fluorescent oligosaccharides as acceptors. We identified genes encoding a beta-1, 4-N-acetylgalactosaminyl-transferase (cgtA), a beta-1, 3-galactosyltransferase (cgtB), and a bifunctional sialyltransferase (cst-II), which transfers sialic acid to O-3 of galactose and to O-8 of a sialic acid that is linked alpha-2,3- to a galactose. The linkage specificity of each identified glycosyltransferase was confirmed by NMR analysis at 600 MHz on nanomole amounts of model compounds synthesized in vitro. Using a gradient inverse broadband nano-NMR probe, sequence information could be obtained by detection of (3)J(C,H) correlations across the glycosidic bond. The role of cgtA and cst-II in the synthesis of the GT1a mimic in C. jejuni OH4384 were confirmed by comparing their sequence and activity with corresponding homologues in two related C. jejuni strains that express shorter ganglioside mimics in their LOS.

Amino Acid Sequence↗
PubMed2000

Chromatographic separation of 3,4-difluorophenylacetic acid and its positional isomers using five different techniques.

The separation of five positional isomers from 3,4-difluorophenylacetic acid was investigated using normal- and reversed-phase high-performance liquid chromatography, capillary zone electrophoresis, gas chromatography and supercritical fluid chromatography. Operating parameters of each technique, such as temperature, type of stationary phase, mobile phase pH, ionic strength, organic modifiers and additives were varied in order to elucidate the separation mechanisms. Based on the advantages and disadvantages of each methodology, a simple and practical RPLC method was selected. The method was validated in terms of linearity, limit of detection, accuracy, recovery, ruggedness and precision.

Acetates↗
PubMed2000

Requirement of calmodulin binding by HIV-1 gp160 for enhanced FAS-mediated apoptosis.

Accelerated apoptosis is one mechanism proposed for the loss of CD4+ T-lymphocytes in human immunodeficiency virus type 1 (HIV-1) infection. The HIV-1 envelope glycoprotein, gp160, contains two C-terminal calmodulin-binding domains. Expression of gp160 in Jurkat T-cells results in increased sensitivity to FAS- and ceramide-mediated apoptosis. The pro-apoptotic effect of gp160 expression is blocked by two calmodulin antagonists, tamoxifen and trifluoperazine. This enhanced apoptosis in response to FAS antibody or C(2)-ceramide is associated with activation of caspase 3, a critical mediator of apoptosis. A point mutation in the C-terminal calmodulin-binding domain of gp160 (alanine 835 to tryptophan, A835W) eliminates gp160-dependent enhanced FAS-mediated apoptosis in transiently transfected cells, as well as in vitro calmodulin binding to a peptide corresponding to the C-terminal calmodulin-binding domain of gp160. Stable Tet-off Jurkat cell lines were developed that inducibly express wild type gp160 or gp160A835W. Increasing expression of wild type gp160, but not gp160A835W, correlates with increased calmodulin levels, increased apoptosis, and caspase 3 activation in response to anti-FAS treatment. The data indicate that gp160-enhanced apoptosis is dependent upon calmodulin up-regulation, involves the activation of caspase 3, and requires calmodulin binding to the C-terminal binding domain of gp160.

Alanine↗
PubMed2000

Hydrogen peroxide dose dependent induction of cell death or hypertrophy in cardiomyocytes.

Cardiomyocyte hypertrophy and cell death are often observed in the end stages of heart failure. The triggers of these two cellular processes are not known under most pathological conditions. Oxidants are by-products of aerobic metabolism. The level of oxidants increases as a result of ischemic reperfusion. Using H9C2 and primary cultured neonatal rat cardiomyocytes, we found that a 2-h pulse treatment with H(2)O(2) at 250 microM or lower caused activation of DEVD sequence specific caspases. The activity of DEVD-ase peaked with 200 microM H(2)O(2) at 24 h. While a fraction of the cells detached and showed nuclear condensation, the majority of the cells (>55%) survived the treatment and appeared to enlarge when cultured for 5 days. These cells showed increases in cell surface area, cell volume, and protein content. With 200 microM H(2)O(2), treated cells appeared to be six times bigger in volume and contained three times more protein per cell than untreated cells. The enlarged cells showed enhanced actin stress fibers and disrupted myofibrils. Our data indicate that while H(2)O(2) can cause cell death, the surviving cardiomyocytes undergo hypertrophy.

Actins↗
PubMed2000

Deletion of the GPG motif in the HIV type 1 V3 loop does not abrogate infection in all cells.

The three amino acids glycine, proline, and glycine (GPG) constitute a conserved motif at the center of the V3 loop of HIV-1 surface glycoprotein 120. It has been indicated that deletion of this GPG motif is lethal for viral infectivity and abrogates the ability of the virus to form syncytia. In the present work, we studied the effects of GPG deletion on viral infectivity, cell tropism, syncytium formation, and initiation of apoptosis by constructing a mutant provirus based on the infectious clone pBRu-2. Successful infection and replication of GPG-deleted virus were detected in MT-2 cells, although the mutant virus showed lower infectivity. Infection could also be observed in the C8166, C91-PL, Molt-3, and THP-1 cell lines, and in PBMC-derived dendritic cells (DCs), but not in CEM-SS, HUT78, H9, Jurkat, and U937 cell lines or in PBMCs. Mutant virus also induced syncytia and apoptosis in the MT-2 cells. An intact GPG motif is probably necessary for unimpaired induction of fusion in some HIV-1-permissive cells. However, once the virus enters the cells, the GPG sequence does not seem to be indispensable for syncytium formation or apoptosis induction in MT-2 cells. Our data also imply that cell surface molecules other than CD4 and CXCR4 may be involved in entry of the GPG-deleted virus.

Amino Acid Motifs↗
PubMed2000

The phytophthora genome initiative database: informatics and analysis for distributed pathogenomic research.

The Phytophthora Genome Initiative (PGI) is a distributed collaboration to study the genome and evolution of a particularly destructive group of plant pathogenic oomycete, with the goal of understanding the mechanisms of infection and resistance. NCGR provides informatics support for the collaboration as well as a centralized data repository. In the pilot phase of the project, several investigators prepared Phytophthora infestans and Phytophthora sojae EST and Phytophthora sojae BAC libraries and sent them to another laboratory for sequencing. Data from sequencing reactions were transferred to NCGR for analysis and curation. An analysis pipeline transforms raw data by performing simple analyses (i.e., vector removal and similarity searching) that are stored and can be retrieved by investigators using a web browser. Here we describe the database and access tools, provide an overview of the data therein and outline future plans. This resource has provided a unique opportunity for the distributed, collaborative study of a genus from which relatively little sequence data are available. Results may lead to insight into how better to control these pathogens. The homepage of PGI can be accessed at http:www.ncgr.org/pgi, with database access through the database access hyperlink.

Databases, Factual↗
PubMed2000

Ca2+-activated non-selective cation current in rabbit ventricular myocytes.

Oscillatory currents (OCs) were studied in isolated rabbit ventricular myocytes with whole cell mode voltage clamp using Na+-free intracellular and extracellular solutions under conditions where K+ currents were anticipated to be eliminated or minimized. All OCs were dependent on release of Ca2+ from the sarcoplasmic reticulum (SR) because they were associated with intracellular Ca2+ ([Ca2+]i) transients, and were suppressed by high concentrations of BAPTA (20 mmol l-1) or pretreatment with the SR antagonist agents ryanodine (10 micromol l-1) or thapsigargin (1 micromol l-1). The reversal potential (Vrev) for OCs shifted with changes in the calculated Vrev for Cl- (ECl) but was between ECl and the calculated Vrev for elemental monovalent cations (ECat), indicating that more than one Ca2+-activated current contributed to OCs. Addition of the Ca2+-activated Cl- current (ICl(Ca)) antagonist, niflumic acid, shifted the OC Vrev to ECat, suggesting that ICl(Ca) and a Ca2+-activated non-selective cation current (ICAN) contributed to the observed OCs. A reduced niflumic acid-insensitive Ca2+-activated OC persisted following marked symmetrical reduction of Cl- in the intracellular and extracellular solutions. Subsequent removal of all extracellular monovalent cations, by N-methyl-D-glucamine (NMDG) substitution, eliminated OCs and the inward holding current suggesting that ICAN and ICl(Ca) accounted for all or most of the Ca2+-activated OC in the absence of Na+. The OC Vrev was equal to ECl in the absence of monovalent elemental cations. Under these conditions niflumic acid eliminated all OCs. Macroscopic OC is partially due to ICAN in rabbit ventricular myocytes.

Animals↗
PubMed2000

Increased expression of intestinal P-selectin and pulmonary E-selectin during intravenous total parenteral nutrition.

HYPOTHESIS: Intravenous total parenteral nutrition (TPN) induces intestinal polymorphonuclear neutrophil recruitment with increased intestinal intercellular adhesion molecule-1 expression. While intercellular adhesion molecule-1 causes firm adhesion of leukocytes to the endothelial cells, P- and E-selectin mediate leukocyte recruitment via rolling. Therefore, manipulation of nutrition may also affect P- and E-selectin expression in organs. DESIGN: Prospective randomized experimental trials. SETTING: Laboratory. MATERIALS: Male mice. INTERVENTIONS: Fifty-three mice were randomized to chow, intravenous TPN, or intragastric TPN. MAIN OUTCOME MEASURES: After 5 days of diet, mice were administered iodine 125-labeled anti-P-selectin antibody (or iodine 125-labeled anti-E-selectin antibody) and iodine 131-labeled nonbinding antibody to quantify P-selectin (or E-selectin) expression in organs (lung, liver, kidney, small intestine, colon, stomach, pancreas, mesentery, heart, and skeletal muscle). RESULTS: P-selectin in small intestine, colon, stomach, and pancreas in the intravenous TPN group increased significantly as compared with the chow and the intragastric TPN groups. E-selectin expression was up-regulated after intravenous TPN in the lung but not in other sites. CONCLUSIONS: In a time frame (5 days) when intercellular adhesion molecule-1 expression and neutrophil recruitment are increased, intestinal expression of P-selectin remains up-regulated. Early lung inflammatory changes are reflected by increases in E-selectin. This change may reflect early pulmonary dysfunction with intravenous TPN, but its significance requires further study.

Analysis of Variance↗
PubMed2000

Detection of oxidative species for 4-phenoxyphenol derivatives during the electrospray ionization process.

Analyses by flow injection as well as liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were performed with four 4-phenoxyphenol derivatives. When ambient temperature nitrogen gas was used to facilitate solvent evaporation, [M + H]+, [M + NH4]+, and [2M + NH4]+ ions were observed as the major ions. As the nitrogen gas temperature increased from ambient to 250 and 450 degrees C, [M]+*, [M - 1]+ and [M + 15]+ ions were the predominant ions. Heat-induced oxidation was found to be the primary source for the formation of oxidative species. Aqueous solvents were found to be essential for the formation of the [M + 15]+ ions. The [M]+* and [M + 15]+ ions were further characterized by tandem mass spectrometry. Based on the MS/MS data, it was proposed that the [M + 15]+ ions were the in-source generated 1,2-quinone ions.

Mass Spectrometry↗
PubMed2000

In vitro characterization of mesenchymal stem cell-seeded collagen scaffolds for tendon repair: effects of initial seeding density on contraction kinetics.

Mesenchymal stem cells (MSCs) were isolated from bone marrow, culture-expanded, and then seeded at 1, 4, and 8 million cells/mL onto collagen gel constructs designed to augment tendon repair in vivo. To investigate the effects of seeding density on the contraction kinetics and cellular morphology, the contraction of the cell/collagen constructs was monitored over time up to 72 h in culture conditions. Constructs seeded at 4 and 8 million cells/mL showed no significant differences in their gross appearance and dimensions throughout the contraction process. By contrast, constructs seeded at 1 million cells/mL initially contracted more slowly and their diameters at 72 h were 62 to 73% larger than those seeded at higher densities. During contraction, MSCs reoriented and elongated significantly with time. Implants prepared at higher seeding densities showed more well aligned and elongated cell nuclei after 72 h of contraction. Changes in nuclear morphology of the MSCs in response to physical constraints provided by the contracted collagen fibrils may trigger differentiation pathways toward the fibroblastic lineage and influence the cell synthetic activity. Controlling the contraction and organization of the cells and matrix will be critical for successfully creating tissue engineered grafts.

Animals↗
PubMed2000

RET and GDNF gene scanning in Hirschsprung patients using two dual denaturing gel systems.

Hirschsprung disease (HSCR) is a congenital disorder characterised by intestinal obstruction due to an absence of intramural ganglia along variable lengths of the intestine. RET is the major gene involved in HSCR. Mutations in the GDNF gene, and encoding one of the RET ligands, either alone or in combination with RET mutations, can also cause HSCR, as can mutations in four other genes (EDN3, EDNRB, ECE1, and SOX10). The rare mutations in the latter four genes, however, are more or less restricted to HSCR associated with specific phenotypes. We have developed a novel comprehensive mutation detection system to analyse all but three amplicons of the RET and GDNF genes, based on denaturing gradient gel electrophoresis. We make use of two urea-formamide gradients on top of each other, allowing mutation detection over a broad range of melting temperatures. For the three remaining (GC-rich) PCR fragments we use a combination of DGGE and constant denaturing gel electrophoresis (CDGE). These two dual gel systems substantially facilitate mutation scanning of RET and GDNF, and may also serve as a model to develop mutation detection systems for other disease genes. In a screening of 95 HSCR patients, RET mutations were found in nine out of 17 familial cases (53%), all containing long segment HSCR. In 11 of 78 sporadic cases (14%), none had long segment HSCR. Only one GDNF mutation was found, in a sporadic case.

Amino Acid Substitution↗
PubMed2000

Base-catalyzed epimerization of the butenolide in annonaceous acetogenins.

The elusive epimerization process in the chiral butenolide moiety of Annonaceous acetogenins was examined under several sets of conditions commonly used for elimination leading to the alpha, beta-unsaturated lactone and the results provide practical guidance in choosing elimination conditions.

4-Butyrolactone↗
PubMed2000

BLNK is associated with the CD72/SHP-1/Grb2 complex in the WEHI231 cell line after membrane IgM cross-linking.

Tyrosine phosphorylation of CD72 strongly correlates with B cell antigen receptor signals leading to apoptosis. We have previously shown that CD72 carrying two immunoreceptor tyrosine-based inhibition motifs (ITIM) is an in vivo substrate of SHP-1. CD72 forms a complex with SHP-1 and Grb2 via its tyrosine-phosphorylated ITIM when the WEHI231 cell line, which is representative of immature B cells, undergoes apoptosis. The CD72 complex formation was also demonstrated in normal primary B cells, suggesting that the complex formation in apoptotic B cells is a universal mechanism. In this study, we further investigated the molecular components of the CD72 complex in WEHI231 cells in order to understand the molecular mechanism involved in the signaling pathway mediated through the complex. Our experiments demonstrate that BLNK, a recently identified adaptor molecule predominantly expressed in B cells, is associated with the CD72 complex via the Src homology 3 domain(s) of Grb2 in the cell line after membrane IgM (mIgM) engagement. The results suggest that the mIgM-mediated signal strongly correlates with the formation of the CD72 / SHP-1 / Grb2 / BLNK complex.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The use of liquid chromatography-mass spectrometry for the identification of drug degradation products in pharmaceutical formulations.

Recent applications of LC-MS in the analysis of drug degradation products in pharmaceutical formulations are reviewed. Drug degradation products are categorized according to their formation mechanism: oxidation, hydrolysis, dimerization and adduct formation with excipients and packaging materials. The oxidative ring opening and dimerization of an indole derivative are discussed in detail. The examples used in this review clearly demonstrate that LC-MS is a very powerful technique for the analysis of low-level degradates in formulations without the time-consuming isolation process. At the same time, limitations and precaution of using LC-MS techniques for unknown identification are also addressed. In some cases, the LC-MS data could become misleading if the ionization process and gas-phase behavior of the analytes are not well understood.

Chemistry, Pharmaceutical↗
PubMed2000

Gradient-induced acoustic and magnetic field fluctuations in a 4T whole-body MR imager.

Both the acoustic and magnetic fluctuation frequency response functions for a Siemens AS25 body gradient coil inside a 4 Tesla whole-body MR system were measured and analyzed in this study. In an attempt to correlate the acoustic noise inside the gradient coil with magnetic field oscillations, triangular and trapezoidal gradient impulses of varying amplitudes and widths were used to excite the gradient coil. The acoustic and magnetic responses to these inputs were measured. The results show the existence of discrete resonances in both acoustic and uniform magnetic field fluctuation spectra, while gradient magnetic field fluctuation spectra show no such resonances. In addition, the dominant amplitude peaks in spectra fluctuate similarly with respect to trapezoidal gradient impulse flat-top widths. This implies that these phenomena are correlated, and that the trapezoidal impulse flat-top width may be used as a way to suppress both acoustic noise and uniform magnetic field oscillations.

Electromagnetic Fields↗
PubMed2000

Analysis of acyclovir by high performance capillary electrophoresis with on-column amperometric detection.

The separation of acyclovir (ACV) by high performance capillary electrophoresis (HPCE) with on-column amperometric detection using alpha-amino-5-mercapto-3,4-dithiazole (AMD) as internal standard is described. The calibration line was linear in the range of 0.5-20 mg/L of ACV. The detection limit was 0.15 mg/L of ACV. Its recovery ranged from 98 to 101% with relative standard deviations (RSDs) from 1.9 to 3.2% (n = 5). This method was successfully used for determining ACV in some pharmaceuticals and human urine. Comparable results with HPCE with ultraviolet (UV) detection and amperometric detection were obtained.

Acyclovir↗