Search PubMed⌕ Search

Biomedical subjects

J Wu

Publications and source records attributed to J Wu.

At least 1,045 records · Page 58Linked to original sources
PubMed1996

Antioxidation of human low density lipoprotein by unconjugated and conjugated bilirubins.

We demonstrate here that both unconjugated bilirubin (Bu) and conjugated bilirubin (Bc) can protect human low density lipoprotein(LDL) against oxidation by oxyradicals generated by 2,2'-azo-bis (2 amidinopropane) dihydrochloride at 37 degrees. The oxidation was assessed by agarose gel electrophoresis and was further corroborated by assaying the malondialdehydes and lipid peroxides formed throughout oxidation. On a per mole basis, Bu and less so Bc was more effective than ascorbate in preventing LDL oxidation. Since oxidative modification of human LDL was implicated in plaque formation in blood vessels leading to atherogenesis, the data suggested that either bile pigment may help reduce the risk of atherogenesis.

Amidines↗
PubMed1996

Inhibition of the association of RNA polymerase II with the preinitiation complex by a viral transcriptional repressor.

Transcriptional repression is an important component of regulatory networks that govern gene expression. In this report, we have characterized the mechanisms by which the immediate early protein 2 (IE2 or IE86), a master transcriptional regulator of human cytomegalovirus, down-regulates its own expression. In vitro transcription and DNA binding experiments demonstrate that IE2 blocks specifically the association of RNA polymerase II with the preinitiation complex. Although, to our knowledge, this is the first report to describe a eukaryotic transcriptional repressor that selectively impedes RNA polymerase II recruitment, we present data that suggest that this type of repression might be widely used in the control of transcription by RNA polymerase II.

Base Sequence↗
PubMed1996

Expression of human mitochondrial NADP-dependent isocitrate dehydrogenase during lymphocyte activation.

In the process of identifying genes involved in optimization of lymphocyte activation, we have cloned the human mitochondrial NADP-dependent isocitrate dehydrogenase (mNADP-IDH) cDNA. The cDNA and its deduced amino acid (AA) sequence had a high degree of homology with those of the porcine and bovine. The heart and muscle had the highest constitutive expression of the gene. The expression of steady-state mRNA in the resting T and B lymphocytes was low but was induced after mitogen stimulation. The mRNA levels peaked around 48 h and remained elevated at 72 h. At the protein level, the mitochondrial but not cytosolic NADP-IDH activity was augmented after the mitogen stimulation. There was no cell cycle-dependent fluctuation of mNADP-IDH expression in synchronized Jurkat cells. In T and B cells, rapamycin (RAPA) could repress the mitogen-stimulated mNADP-IDH expression, although most of the early or late phase activation-related genes including a G-protein beta subunit-related gene H12.3 were not affected by the drug. The restricted expression of the gene in certain tissues and the activation-related expression in lymphocytes suggest that this gene might be necessary for optimal functions in heart, muscle, and the activated lymphocytes.

Amino Acid Sequence↗
PubMed1996

A strategy to locate cysteine residues in proteins by specific chemical cleavage followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A simple methodology has been developed to characterize the number and location of free cysteine and cystine groups in peptides and proteins, using chemical modification and matrix-assisted laser desorption/ ionization time-of flight mass spectrometry (MALDITOF MS). This new approach employs a specific reaction between free sulfhydryls and 2-nitro-5-thiocyanobenzoic acid (NTCB) to selectively cyanylate cysteine thiols. The N-terminal peptide bond of the modified cysteinyl residue can then be cleaved under alkaline conditions to form an amino-terminal peptide and a series of 2-iminothiazolidine-4-carboxylyl peptides which can be mapped to the sequence by MALDI-MS. The number and location of cysteines can be deduced from mass analysis of the peptide mixture resulting from NTCB chemical processing of the original protein of known sequence under nonreducing conditions. Similar experiments are then performed following disulfide bond reduction to further characterize both cysteine and cystine groups. Experimental conditions are described for protein disulfide bond reduction, sulfhydryl cyanylation, and cleavage reactions performed both in solution and on Zetabind membranes.

Cysteine↗
PubMed1996

P-glycoprotein regulates a volume-activated chloride current in bovine non-pigmented ciliary epithelial cells.

1. The whole-cell patch clamp technique was used to investigate the swelling-activated currents in bovine non-pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The I-V relationship was shifted in the direction expected for a Cl- current when the external Cl- was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), also inhibited this current. 5. The volume-activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time-dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl- current. 8. An antibody (C219) to the MDR1 gene product, P-glycoprotein, caused a functional block of the swelling-activated Cl- current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB-1 demonstrated the presence of P-glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non-pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)-independent, ATP-dependent Cl- current and that the activity of this current is associated with P-glycoprotein. 11. It is suggested that this Cl- current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Molecular cloning of the cDNA of mouse mitochondrial NADP-dependent isocitrate dehydrogenase and the expression of the gene during lymphocyte activation.

The current report documents the molecular cloning of the mouse mitochondrial NADP-dependent isocitrate dehydrogenase (mNADP-IDH) cDNA. The cDNA was 1,863 bp in length and contained one open reading frame encoding a 523-residue polypeptide with a predicted molecular weight of 58 kDa. The cDNA and the deduced amino acid (AA) sequence of the mouse mNADP-IDH had a high degree of homology with those of porcine, bovine, alfalfa, and yeast. The recombinant mNADP-IDH expressed in Escherichia coli had active enzymatic function, as well as an expected molecular weight. The heart had the highest constitutive expression of the steady-state mNADP-IDH mRNA, followed by the kidney, while the expression of the gene in other tissues was low. The enzymatic activity of different tissues was in agreement with their mNADP-IDH mRNA levels. The resting lymphocytes had low constitutive expression of the gene, but the steady-state mRNA could be induced 48 h after mitogen stimulation. At the protein level, the resting lymphocytes had low enzymatic activity of mNADP-IDH, but the activity was augmented fivefold after mitogen stimulation. The cytosolic NADP-IDH, on the contrary, remained low or undetectable before and after the mitogen stimulation. Based on our current findings as well as the known roles of the mNADP-IDH in anabolism and in the isocitrate shuttle, it is conceivable that the mNADP-IDH is necessary for optimizing proliferation in lymphocytes.

Amino Acid Sequence↗
PubMed1996

The immunosuppressive effect of rapamycin on mouse small bowel transplantation.

The efficacy of rapamycin (RAPA) was tested on small bowel transplantation in the mouse and compared with cyclosporine (CsA). Four groups were involved, each one included three combinations (n > or = 6) for evaluation of host-versus-graft (HVG, C57BL/6 X BALB/c F1 (CB6F1)-to-BALB/c), graft-versus-host (GVH, BALB/c-to-CB6F1), and combined HVG and GVH responses (C57BL/6-to-BALB/c). Grafts were transplanted to recipients heterotopically. Groups were as follows: group 1: naive controls; groups 2 and 3: recipient mice treated with RAPA 2 mg/kg/day and 4 mg/kg/day orally for 14 days, respectively; group 4: recipient mouse treated with CsA 4 mg/kg/day orally for 14 days. In the HVG model, the mean survival time (MST) of recipients was significantly longer in group 2 (32.9 +/- 17.7 days, P=0.006), group 3 (32.7 +/- 10.4 days, P=0.0001), and group 4 (37.9 +/- 11.8 days, P=0.0001), compared with naive controls in group 1 (8.5 +/- 1.6 days). In the GHV model, the MST of recipients in group 2 (41.8 +/- 19.9 days, P=0.002), group 3 (48.2 +/- 21.4 days, P=0.001) and group 4 (56.5 +/- 30.6 days, P=0.003) were significantly prolonged compared with control group 1 (8.5 +/- 1.6 days). In combined HVG and GVH responses, MST of recipient in group 2 (20.9 +/- 4.9 days, P=0.0001), group 3 (27.0 +/- 4.3 days, P=0.008), and group 4 (35.2 +/- 23.9 days, P=0.0001) were also significantly longer than that in controls (6.9 +/- 1.4 days), but in all three combinations, there were no statistically significant differences between groups 2 and 3, groups 2 and 4, or groups 3 and 4 (P>0.05). RAPA is a potent immunosuppressant able to significantly prolong small bowel allograft survival in mice using a short-term treatment. There is no statistically significant difference in recipient survival between low and high doses of RAPA treatment and the CsA standard dose used in this study.

Animals↗
PubMed1996

Expression of prokaryotic HhaI DNA methyltransferase is transforming and lethal to NIH 3T3 cells.

In neoplastic cells, levels of DNA methyltransferase activity are often increased, and evidence is accruing to suggest an important role for this event in tumorigenesis. To evaluate this possibility further, and to investigate the contribution of increasing de novo, as opposed to maintenance, DNA methylation in mammalian cells, we expressed the bacterial HhaI methyltransferase in cultured murine fibroblasts. This enzyme is a pure de novo DNA methyltransferase that methylates the internal C in the sequence GCGC. We find that both constitutive and induced expression of the wild-type HhaI results, primarily, in lethality to the cells. However, surviving cell clones that express low levels of M. HhaI demonstrate increased tumorigenicity as assessed by soft agar cloning efficiency (8.6% for sense HhaI-transduced PA 317 cells versus 0.4% for antisense controls; 1.7% for sense HhaI-transfected NIH 3T3 cells versus 0% for a mutant HhaI control) and tumorigenicity in nude mouse heterotransplants (75% for sense HhaI-transduced PA 317 cells versus 18.5% for antisense controls). DNA isolated from the clonogenic sense HhaI clones, versus clones expressing the mutant HhaI gene, has no increase in overall CpG methylation but an average of 27% (range, 16.7-38.9) increase in methylcytosine content at GCGC sites. These findings suggest that eukaryotic cells tolerate a narrow window of increase de novo DNA methylating capacity, above which cell death occurs and within cell transformation results. Our results further emphasize the potential role of increased DNA methyltransferase activity in the evolution of cancer.

3T3 Cells↗
PubMed1996

Identification of the epitope for monoclonal antibody 4B1 which uncouples lactose and proton translocation in the lactose permease of Escherichia coli.

Monoclonal antibody 4B1 binds to a conformational epitope on the periplasmic surface of the lactose permease of Escherichia coli, uncoupling lactose and H+ translocation in a manner indicating that it blocks deprotonation [Carrasco, N., Viitanen, P., Herzlinger, D., & Kaback, H. R. (1984) Biochemistry 23, 3681; Herzlinger, D., Viitanen, P., Carrasco, N., & Kaback, H. R. (1984) Biochemistry 23, 3688]. In this paper, 4B1 binding to purified lactose permease is shown to exhibit a KD of about 5 x 10(-10) M by surface plasmon resonance. Furthermore, the combined use of mutants containing 6 contiguous His residues in each periplasmic loop in the permease and Cys-scanning mutagenesis in conjunction with chemical labeling demonstrates that 4B1 binds specifically to the periplasmic loop between helices VII and VIII and that Phe247 and Gly254 are the primary determinants. Remarkably, although 4B1 binding uncouples lactose and H+ translocation, none of the amino acid residues in periplasmic loops, particularly Phe247 or Gly254, play an important role in the transport mechanism. Moreover, binding of avidin to biotinylated Glu255-->Cys in the loop containing the epitope has no effect on transport activity. Therefore, the uncoupling effect of 4B1 involves highly specific interactions which in all likelihood exert a torsional effect on the loop, resulting in a conformational change in helix VII and/or VIII that alters the pKas of residues involved in lactose-coupled H+ translocation.

Amino Acid Sequence↗
PubMed1996

Spatially heterogeneous discrete waves in predator-prey communities over a patchy environment.

A model motivated by the earlier work of J. D. Murray and D. Stirzaker is proposed to describe the dynamics of predator-prey communities over a patchy environment. The model is a system of delay differential equations. It is shown that if only one time lag is incorporated into the model, then a branch of spatially homogeneous periodic solutions occur as a primary Hopf bifurcation. However, if two time lags are used to measure different delayed factors in the process of growth, decay, and predator consumption of the prey population, then stable spatially heterogeneous periodic solutions (discrete waves or phase-locked oscillations) may exist. The utilized method is based on center manifold, normal form, and equivariant Hopf bifurcation theory.

Animals↗
PubMed1996

The gene encoding p120cas, a novel catenin, localizes on human chromosome 11q11 (CTNND) and mouse chromosome 2 (Catns).

The p120cas gene encodes a protein tyrosine kinase substrate that associates with the cell-cell adhesion protein complex containing E-cadherin and its cytoplasmic cofactors alpha-catenin, beta-catenin, and plakoglobin. Like other components of the cadherin/catenin complex, defects in p120cas may contribute to cell malignancy. We have determined the chromosomal location of the p120cas gene in human and mouse using fluorescence in situ hybridization and interspecific backcross analysis, respectively. The human p120cas gene (CTNND) is localized immediately adjacent to the centromere on the long arm of chromosome 11 in band 11q11. The murine p120cas gene (Catns) was assigned to the middle of chromosome 2. Neither locus is currently known to be associated with disease or malignancy.

Animals↗
PubMed1996

Mass spectrometric analyses of beta-ketolactone oligomers, macrocyclic or catenane structures?

Mass spectrometry is used to develop an analytical method for a new class of organic oligomeric material, beta-ketolactones. Three different series with varying oligomeric sizes are examined. The oligomers may form at least two different structures, a macrocyclic and a catenane ring. Fast atom bombardment coupled with Fourier transform mass spectrometry provides a rapid and convenient method that provides both molecular weight information and abundant fragment ions that are structurally relevant. All the compounds are found to work well with the mass spectrometric analysis. Electrospray ionization coupled with triple-quadrupole mass spectrometry was also evaluated and found to yield results that are similar to FAB. On the basis of the FAB and the low-energy collisionally activated dissociation spectra, we conclude that these compounds are macrocyclic rings and not catenanes.

Chromatography, Liquid↗
PubMed1996

Immunolocalization of osteonectin in avian tibial dyschondroplastic cartilage.

Osteonectin is an acidic calcium-binding protein found in cartilage, bone matrix, vascular endothelium, and areas of tissue repair. Using immunocytochemistry, osteonectin has been localized in all zones of the normal avian epiphyseal growth plate with notably high amounts in the hypertrophic zone. In the proximal portion of this zone the staining was intracellular, while in the distal calcifying portion of the hypertrophic zone staining was both intracellular and extracellular. Osteonectin was also detected in the growth plate associated with lesions of chickens with tibial dyschondroplasia (TD). Intense intracellular staining was observed in hypertrophic chondrocytes proximal to the lesion; staining was markedly diminished in the TD lesion; extracellular matrix was devoid of staining. Staining intensity was high along the peripheral edges of the lesion that were undergoing vascularization and resorption. This was the only area in the dysplastic cartilage where staining was observed in the extracellular matrix as well as intracellularly. Similar patterns were viewed in all TD lesions examined, whether they were spontaneous or induced by dietary treatments or genetic selection.

Animals↗
PubMed1996

Genetic variation in the morphology of the septo-hippocampal cholinergic and GABAergic system in mice. I. Cholinergic and GABAergic markers.

In the present study, variations of cholinergic and GABAergic markers in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB) and the hippocampus of eight different inbred mouse strains were investigated. By means of immunocytochemistry against the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT), the cholinergic neurons were visualized and the number of ChAT-positive neuronal profiles in the MS/vDB was counted. Cholinergic and GABAergic septo-hippocampal projection neurons were detected with a combined retrograde tracing and immunocytochemical approach. In order to quantify the cholinergic innervation of various hippocampal sub-regions, we estimated the density of acetylcholinesterase (AChE)-containing fibers as visualized by AChE histochemistry. Additionally, the densities of muscarinic receptors (mainly the subtypes M1 and M2) in different hippocampal areas of seven inbred strains were measured by means of quantitative receptor autoradiography. We found significant strain differences for the number of ChAT-positive neurons in the MS/vDB; in the numbers of cholinergic septo-hippocampal projection neurons; in the density of cholinergic fibers in hippocampal subfields CA3c, CA1, and in the dentate gyrus; and in the density of muscarinic receptors in the hippocampus. In contrast the GABAergic component of the septo-hippocampal projection did not differ between the strains investigated. The number of ChAT-reactive neurons in the MS/vDB was not correlated with either hippocampal cholinergic markers. This might be attributed to different collateralization of cholinergic neurons or to different projections of these neurons to other brain regions. These results show a strong hereditary variability within the septo-hippocampal cholinergic system in mice. In view of the role of the cholinergic system in learning and memory processes, strain differences in cholinergic markers might be helpful in explaining behavioral variation.

Animals↗
PubMed1996

Genetic variation in the morphology of the septo-hippocampal cholinergic and GABAergic systems in mice: II. Morpho-behavioral correlations.

We investigated the contribution of the septo-hippocampal cholinergic and GABAergic system to spatial and nonspatial aspects of learning and memory that had previously been found to correlate with the extent of the hippocampal intra- and infrapyramidal mossy fiber projection in different inbred mouse strains. The following cholinergic and GABAergic markers were measured in the septi and hippocampi of male mice: the number of cholinergic and parvalbumin-containing neurons in the medial septum/vertical limb of the diagonal band of Broca (MS/vDB), the number of septo-hippocampal cholinergic and GABAergic projection neurons, the density of cholinergic fibers in different hippocampal subfields, and the density of muscarinic receptors (predominantly M1 and M2) in the hippocampus. In addition, animals were behaviorally tested for spatially dependent and activity-dependent variables in a water maze and spatial and nonspatial working and reference memory in different experimental set-ups in an eight-arm radial maze. Using only those variables for which significant strain differences were obtained, we looked for covariations between behavior and neuroanatomy. The density of cholinergic fibers in the dentate gyrus was significantly correlated with activity-dependent learning in the water maze, whereas the number of septo-hippocampal cholinergic projection neurons correlated with spatial and, to a lesser extent, also with nonspatial aspects of radial maze learning. Only weak correlations were found between receptor densities and behavioral traits. From these data we conclude that variations in the septo-hippocampal cholinergic system, like variations in the mossy fiber projection, entail functional consequences for different types of maze learning in mice.

Acetylcholinesterase↗
PubMed1996

The NMR solution structure of the pheromone Er-11 from the ciliated protozoan Euplotes raikovi.

The NMR solution structure of the pheromone Er-11, a 39-residue protein from the ciliated protozoan Euplotes raikovi, was calculated with the distance geometry program DIANA from 449 NOE upper distance constraints and 97 dihedral angle constraints, and the program OPAL was employed for structure refinement by molecular mechanics energy minimization in a water bath. For a group of 20 conformers used to characterize the solution structure, the average of the pairwise RMS deviations from the mean structure calculated for the backbone heavy atoms N, C alpha, and C' of residues 2-38 was 0.30 A. The molecular architecture is dominated by an up-down-up bundle of three short helices with residues 2-9, 12-19, and 22-32, which is closely similar to the previously determined structures of the homologous pheromones Er-1, Er-2, and Er-10. This finding provides structural evidence for the capability shown by these pheromones to compete with each other in binding reactions to their cell-surface receptors.

Amino Acid Sequence↗
PubMed1996

Immunosuppressive effects of the cyclosporin derivative SDZ IMM 125 on kidney allograft in the dog and small bowel and pancreas allografts in the rat.

The efficacy of SDZ IMM 125 in preventing allograft rejection was evaluated in kidney transplantation in the dog and orthotopic small bowel and heterotopic pancreas transplantation in the rat. Seven groups (n = 6) were involved in dog kidney transplantation. Untreated recipients rejected kidney allografts with a mean survival time (MST) of 8.0 +/- 1.8 days. There was a graded dose response in SDZ IMM 125-treated groups: 10 mg/kg/day, MST 11.3 +/- 3.5 days, P = 0.065; 15 mg/kg/day, MST 44.8 +/- 10.8 days, P = 0.0001; and 20 mg/kg/day, MST 47.3 +/- 5.6 days, P = 0.0001, same as in cyclosporin A (CsA)-treated groups: 10 mg/kg/day, MST 25.8 +/- 13.7 days, P = 0.001; 15 mg/kg/day, MST 38.3 +/- 18.1 days, P = 0.002; and 20 mg/kg/day, MST 38.7 +/- 17.7 days, P = 0.002. Low dose of SDZ IMM 125 (10 mg/kg/day) was less effective in prolonging the graft survival than low dose of CsA. High dose (20 mg/kg/day) treatment of both SDZ IMM 125 and CsA led to abnormal recipient liver enzymes alanine aminotransferase and aspartate aminotransferase postoperatively. A histopathological study demonstrated fatty degeneration of hepatic cells in both 20 mg/kg/day SDZ IMM 125- and CsA-treated groups. We also tested the effect of SDZ IMM 125 in orthotopic small bowel transplantation with three combinations, namely host-versus-graft (HVG), graft-versus-host (GVH), and combined HVG and GVH immune responses and pancreas transplantation in the rat with different doses. The results indicate that SDZ IMM 125 is a potent immunosuppressant to prolong the kidney allograft survival in the dog, to alleviate HVG and GVH responses in small bowel transplantation, and to delay pancreas allograft rejection in the rat. Low dose of SDZ IMM 125 is less effective than a similar dose of CsA to prevent kidney allograft rejection in the dog.

Animals↗