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Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 1,153 records · Page 64Linked to original sources
PubMed1994

Alterations in nitric oxide synthase activity, superoxide anion generation, and platelet aggregation in systemic hypertension, and effects of celiprolol.

A decrease in endothelium-derived relaxing factor or nitric oxide has been proposed as a potential mechanism of increased vascular resistance in hypertension. An increase in the generation of superoxide anions, which degrade nitric oxide and induce platelet aggregation, may also compromise regional blood flow in hypertension. Recent studies show that human neutrophils generate nitric oxide, which has a similar biologic profile to the endothelium-derived relaxing factor. This study measured nitric oxide synthase activity and superoxide generation in human neutrophils and platelet aggregation in patients with essential hypertension. Nitric oxide synthase activity, measured as conversion of 3H-L-arginine to 3H-L-citrulline, in peripheral blood neutrophils was decreased in hypertensive subjects (percent conversion of 3H-L-arginine: 4.2 +/- 0.5 vs 9.0 +/- 3.0 in control subjects; p < 0.01). Neutrophil superoxide anion generation, measured as conversion of ferricytochrome C to ferrocytochrome C, in response to phorbol-12-myristate 13-acetate (100 ng/ml) was higher in hypertensive subjects (17.5 +/- 8.1 vs 13.2 +/- 3.0 nmoles/10(6) cells/10 minutes in control subjects; p < 0.05). Patients were treated with a selective beta blocker, celiprolol, for 8 weeks. Supine blood pressure decreased from 177/103 mm Hg (mean +/- SD 18/7) to 160/92 mm Hg (mean +/- 10/5; p < 0.02), while heart rate was unchanged (73 +/- 11 vs 69 +/- 10 beats/min). Epinphrine and adenosine diphosphate-induced platelet aggregation was also increased in hypertensive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1994

Ionic strength dependence of the reaction between methanol dehydrogenase and cytochrome c-551i: evidence of conformationally coupled electron transfer.

The quinoprotein methanol dehydrogenase and cytochrome c-551i are two soluble acidic proteins that form a physiological complex in which electrons are transferred from pyrroloquinoline quinone to heme. The oxidation of methanol dehydrogenase by the cytochrome was studied as a function of ionic strength using stopped-flow spectroscopy. The dissociation constant (Kd) for complex formation decreased 2-fold with increasing ionic strength from 0.21 to 1.3 M and increased at higher ionic strengths. The rate constant for the electron transfer reaction (kET) increased 2-fold with increasing ionic strength from 0.21 to 1.3 M and decreased at higher ionic strengths. The variation of Kd and kET over this range of ionic strengths was described by Van Leeuwen theory, which takes into account monopole-dipole and dipole-dipole forces, in addition to the monopole-monopole force, to predict the interactions between large molecules. Analysis of the kinetic results in terms of these electrostatic interactions indicated the probable orientations for protein-protein binding and electron transfer. To explain the ionic strength dependence of the observed kET, a model is presented in which the true kET is reduced by a factor Kc, an equilibrium constant that describes some rearrangement of the proteins after a nonoptimal collision to produce the most efficient orientation for electron transfer. This model is consistent with the notion that the large reorganizational energy obtained from temperature-dependence studies of this electron transfer reaction [Harris, T. K., & Davidson, V. L. (1993) Biochemistry 32, 14145-14150] is due to such an intracomplex rearrangement.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗
PubMed1994

B7-1/CD80-transduced tumor cells elicit better systemic immunity than wild-type tumor cells admixed with Corynebacterium parvum.

Tumor cells genetically modified by transduction of B7 (B7-1/CD80), a natural ligand for the T-cell costimulatory molecules CD28 and CTLA-4, can elicit potent tumor immunity, and they can be effective for treatment of established cancers in animal models. In this study, three tumor lines, the EL4 lymphoma, the P815 mastocytoma, and the MCA102 sarcoma were transduced with recombinant retrovirus containing the murine B7 gene, and their potency to induce systemic immunity protective against challenge with wild-type tumor was compared to that of the same tumor cells admixed with the commonly used adjuvant Corynebacterium parvum. While admixture of tumor cells with C. parvum resulted in complete regression of tumors in syngeneic mice, it did not induce protective immunity against a subsequent challenge of wild-type cells from any of the 3 tumors tested. In contrast, B7-transduced EL4 and P815 tumors regressed locally and induced a potent systemic immunity to wild-type tumors and a higher level of cytotoxic T-cell activity than did tumor cells admixed with C. parvum. No systemic immunity was induced by B7-transduced nonimmunogenic MCA102 sarcoma cells. Our results demonstrate that immunogenic tumor cells transduced with the B7 gene are superior to tumor cells mixed with C. parvum for the induction of systemic tumor immunity.

Animals↗
PubMed1994

Purification, characterization and properties of an NADH oxidase from Desulfovibrio vulgaris (Hildenborough) and its coupling to adenylyl phosphosulfate reductase.

An NADH oxidase was purified from Desulfovibrio vulgaris. This FMN-containing enzyme reacts with oxygen forming hydrogen peroxide with a specific activity of 0.21 mumoles.min-1.mg-1. The molecular weight of the protein was determined to be 65 kDa on 12.5% SDS/PAGE. It shows very low NADH: rubredoxin oxidoreductase activity specifically towards the rubredoxin from the same organism. However, adenylyl phosphosulfate reductase can be fully reduced by NADH with the purified enzyme, suggesting that NADH could be an electron donor for respiratory sulfate reduction.

Amino Acid Sequence↗
PubMed1994

Structure and chromosomal localization of the gene (BDKRB2) encoding human bradykinin B2 receptor.

The bradykinin B2 receptor (BDKRB2) has high affinity for the intact kinins, which mediate a wide spectrum of biological effects, including pain, inflammation, vasodilation, and smooth muscle contraction and relaxation. In the present study, we have cloned and sequenced the gene encoding human bradykinin B2 receptor from a human genomic library. The B2 receptor gene contains three exons separated by two introns. The first and second exons are noncoding, while the third exon contains the full-length coding region, which encodes a protein of 364 amino acids forming 7 transmembrane domains. The human B2 gene shares high sequence identity with rat and mouse B2 receptor genes and significant similarity with the gene encoding the angiotensin II type I receptor in the nucleotide sequence and exon-intron arrangement. In the 5' flanking region, a consensus TATA box and several putative transcription factor-binding sites have been identified. Genomic Southern blot analysis showed that the B2 receptor is encoded by a single-copy gene that was localized to chromosome 14q32 by in situ hybridization. In a Southern blot analysis following reverse transcription and polymerase chain reaction, the human B2 receptor was found to be expressed in most human tissues.

Amino Acid Sequence↗
PubMed1994

A mutation in the effector region of Ras2 can be partially suppressed by alteration of a 'nonessential' region of Ras.

Phenotypically normal revertants of budding yeast cells that contain the hyperactive RAS2Val19 allele often result from second-site mutations within the RAS2 locus itself. Several such intragenic revertants harboring a suppressed RAS2Val19 allele as their only RAS gene were analyzed. All such suppressors resulted from single amino acid substitutions that affected either: (i) the effector region of Ras2, (ii) the C-terminal CAAX box of Ras2, or (iii) residues known to be critical for GTP binding in Ras proteins. While these suppressor mutations completely suppressed the hyperactive phenotype induced by the Val19 substitution, they did not block the ability of Ras2 to promote growth at normal temperatures. These results suggest that in yeast, attenuation of Ras proteins can effectively block hyperactive phenotypes without completely blocking the growth-promoting function. A spontaneous intragenic mutation that restored function to an effector mutant was mapped to a 'nonessential' region of Ras proteins. Based on this genetic interaction with the effector region and the report that deletions of this region affect Ras/GAP interaction, we suggest that this region may have a functional role in Ras activation of target effectors.

Base Sequence↗
PubMed1994

A detailed structural characterization of ribonuclease B oligosaccharides by 1H NMR spectroscopy and mass spectrometry.

The structures of ribonuclease B oligosaccharides have previously been shown to be high mannose type by methylation analyses and sequential exoglycosidase digestion. Due to the unique nature of these oligosaccharides, in that all mannosyl residues are attached by alpha-(1-->2)-linkages beyond the branch points, methylation analysis fails to solve the exact structures beyond Man5. Therefore, we have undertaken this study using 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. In this study, bovine pancreatic ribonuclease B was first reduced and carboxymethylated, and was then deglycosylated by peptide/N-glycosidase F (PNGase F). The released oligosaccharides were fractionated by Bio-Gel P-4 chromatography to give five pools, Man5 through Man9. The structures of the oligosaccharide pools were then studied by laser desorption time of flight mass spectrometry and 1H NMR spectroscopy at 300 MHz. For Man5, Man-A and Man-B are attached in alpha-(1-->3)- and alpha-(1-->6)-linkages to the alpha-(1-->6)-linked Man-4' of the pentasaccharide core structure. For Man6, Man-C is linked alpha-(1-->2) to the alpha-(1-->3)-linked Man-4. Man7 exists as three structural isomers, and has the additional mannosyl residue (Man-D) linked alpha-(1-->2) to Man-A, Man-B, and Man-C is linked alpha-(1-->2) to the alpha-(1-->3)-linked Man-4. Man-7 exists as three structural isomers, with the additional two mannosyl residues linked alpha-(1-->2) to Man-A, Man-B, and Man-C. For each position, Man-A, Man-B, and Man-C, the extent of occupancy by one of the additional alpha-(-->)-linked mannosyl residues was 15, 94, and 91%, respectively. Man9 is a single component, with the three additional mannosyl residues linked alpha-(1-->2) to Man-A, Man-B, and Man-C, respectively. The relative molar proportions of Man5 to Man9 are 57, 31, 4, 7, and 1%, respectively. This report presents for the first time the complete structural characterization of the oligosaccharides from ribonuclease B.

Amidohydrolases↗
PubMed1994

Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90.

Nuclear Factor of Activated T-cells (NF-AT) is a crucial transcription factor required for T-cell expression of interleukin 2. Purified NF-AT contains 45-kDa and 90-kDa subunits (Corthésy, B., and Kao, P. N. (1994) J. Biol. Chem. 269, 20682-20690). Partial internal amino acid sequences derived from each subunit indicate that these proteins are novel. The amino acid sequences were used to clone the cDNAs encoding each subunit. The cDNAs predict proteins of novel structures: NF45 has limited similarity to prokaryotic transcription factor sigma-54 and to human DNA topoisomerase II; NF90 has limited similarity to Drosophila Staufen in a domain predicted to bind double-stranded RNA. RNA encoding NF45 and NF90 exists in nonstimulated Jurkat T-cells and in all other cell types examined (HeLa, HepG2, K562). Immunofluorescence microscopy was used to demonstrate that both proteins are located in the nucleus of Jurkat T-cells. Clones NF45 and NF90 with a polyhistidine fusion tag were transiently expressed and processed in the native environment of Jurkat T-cells. Histidine-tagged NF45 and NF90 proteins, affinity-purified on nickel chelate columns, encode a NF-AT DNA-binding activity that is enhanced following T-cell stimulation, and this enhancement is blocked when T-cells are stimulated in the presence of cyclosporin A or FK506.

Amino Acid Sequence↗
PubMed1994

The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth.

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revealed the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) and are not found in homologous genes in other organisms. These insertion sequences do not resemble RNA introns, and are not spliced out at the mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid residues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugametos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein coding sequences. The C. reinhardtii clpP gene was further shown to be essential for cell growth, as demonstrated through targeted gene disruption by particle gun-mediated chloroplast transformation. Only heteroplasmic transformants could be obtained, even under mixotrophic growth conditions. The heteroplasmic transformants were stable only under selection pressure for the disrupted clpP, rapidly segregated into wild-type cells when the selection pressure was removed, and grew significantly more slowly than wild-type cells under phototrophic conditions.

ATP-Dependent Proteases↗
PubMed1994

Crystal structure of recombinant human platelet factor 4.

The crystal structure of human platelet factor 4 (PF4) has been solved to a resolution of 2.4 A by molecular replacement and refined to an R-factor of 24.1%. The structure consists of four polypeptide chains which form a tetrameric unit. N-terminal residues, previously defined as a random coil or extended loop region, form antiparallel beta-sheet-like structures that form noncovalent associations between dimers. These antiparallel beta-sheet-like structures are positioned lateral to the beta-bilayer motif and stabilize the tetrameric unit. A positively charged ring of lysine and arginine side chains encircles the PF4 tetramer sphere, presenting multiple potential sites and orientations for heparin binding. The electrostatic interactions of multiply charged amino acid side chains and hydrogen bonding interactions at the AB/CD dimer interface serve to stabilize the tetrameric structure further.

Amino Acid Sequence↗
PubMed1994

Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes.

BCL-2 is a 26-kDa integral membrane protein that represses apoptosis by an unknown mechanism. Recent findings indicate that Ca2+ release from the endoplasmic reticulum (ER) mediates apoptosis in mouse lymphoma cells. In view of growing evidence that BCL-2 localizes to the ER, as well as mitochondria and the perinuclear membrane, we investigated the possibility that BCL-2 represses apoptosis by regulating Ca2+ fluxes through the ER membrane. A cDNA encoding BCL-2 was introduced into WEHI7.2 cells and two subclones, W.Hb12 and W.Hb13, which express high and low levels of BCL-2 mRNA and protein, respectively, were isolated. WEHI7.2 cells underwent apoptosis in response to treatment with the glucocorticoid hormone dexamethasone, whereas W.Hb12 and W.Hb13 cells were protected from apoptosis, revealing a direct relationship between the level of BCL-2 expression and the degree of protection. Significantly, BCL-2 also blocked induction of apoptosis by thapsigargin (TG), a highly specific inhibitor of the ER-associated Ca2+ pump. TG completely inhibited ER Ca2+ pumping in both WEHI7.2 and W.Hb12 cells, but the release of Ca2+ into the cytosol after inhibition of ER Ca2+ pumping was significantly less in W.Hb12 cells than in WEHI7.2 cells, indicating that BCL-2 reduces Ca2+ efflux through the ER membrane. By reducing ER Ca2+ efflux, BCL-2 interfered with a signal for "capacitative" entry of extracellular Ca2+, preventing a sustained increase of cytosolic Ca2+ in TG-treated cells. These findings suggest that BCL-2 either directly or indirectly regulates the flux of Ca2+ across the ER membrane, thereby abrogating Ca2+ signaling of apoptosis.

Apoptosis↗
PubMed1994

Costimulation of tumor-reactive CD4+ and CD8+ T lymphocytes by B7, a natural ligand for CD28, can be used to treat established mouse melanoma.

Interactions between the costimulatory molecule B7 on APC and its counter-receptor CD28 on T lymphocytes play a key role in the induction of cell-mediated immune responses. We studied the role of costimulation of tumor-reactive T cells by B7 in the immune destruction of the K1735-M2 mouse melanoma into which the gene encoding the human melanoma-associated Ag, p97, had been transfected. Previous work has demonstrated that the p97 transfectant cl62 is immunogenic but still grows progressively in immunocompetent C3H/HeN mice and that adoptive transfer of p97-specific CD4+ T cells can induce the regression of small established cl62 tumors metastatic to the lungs. We have now shown that expression of B7 in cl62 after retroviral-mediated gene transfer eliminated its ability to grow in immunocompetent mice but not in T cell-deficient nude mice. Mice immunized with B7-transduced p97+ cells had an increased activity of both CD4+ T cells, which could proliferate in response to the p97 Ag, and CD8+ CTL, which could lyse a broad spectrum of cultured syngeneic p97+ and p97- tumor lines but not allogeneic tumor lines or syngeneic lymphoblasts. Both CD4+ and CD8+ T cell subsets were required for tumor rejection, and the depletion of CD4+ T cells in vivo decreased the tumoricidal activity of CD8+ CTL. Treatment of mice bearing an 8-day established s.c. cl62 melanoma by i.p. injection of B7+ cells from 2A, a highly immunogenic p97 transfectant, resulted in complete tumor regression and cure, injection of B7- 2A cells did not. The therapeutic effect was specific for the cl62 tumor. Our results demonstrate that costimulation by B7 can amplify both CD4+ and CD8+ T cell responses against small tumors toward therapeutic benefit.

Animals↗
PubMed1994

Isolation and characterization of a high molecular weight cytochrome from the sulfate reducing bacterium Desulfovibrio gigas.

A high molecular weight c-type cytochrome (Hmc) was purified and characterized from Desulfovibrio gigas. The molecular weight was estimated to be 67 kDa by SDS-PAGE and its N-terminus is homologous to those of the 16 hemes containing high molecular weight cytochrome c from Desulfovibrio vulgaris strains Hildenborough and Miyazaki. The purified hemoprotein shows c-type cytochrome absorption spectrum with e533 (red) = 368 mM-1.cm-1. A band at 640 nm, characteristic of high-spin hemes, was detected. The EPR spectra show the presence of two high-spin heme species, plus several non-equivalent low-spin hemes. The heme reduction potentials, at pH 7.6, range from -50 mV to -315 mV. In contrast to what has been described for D. vulgaris Hmc, the protein isolated from D. gigas directly accepts electrons from hydrogenase and further reduces other redox proteins.

Amino Acid Sequence↗
PubMed1994

Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and HNF-1 beta regulate transcription via two elements in an intestine-specific promoter.

Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and HNF-1 beta are transcription factors that contain a divergent homeodomain, bind to DNA as homo- or heterodimers, and act to regulate transcription of many genes that are expressed primarily in liver. We show that HNF-1 alpha and HNF-1 beta act as regulatory factors for transcription of sucrase-isomaltase (SI), the gene for which is expressed exclusively in absorptive enterocytes of the small intestine. HNF-1 alpha and HNF-1 beta bind to two evolutionarily conserved sites in the SI promoter, previously named SIF2 and SIF3, with different relative affinities. HNF-1 alpha binds to the SIF3 element with greater affinity than to the SIF2 element, whereas HNF-1 beta binds to both elements equally. Co-transfection experiments demonstrated that HNF-1 alpha is able to increase transcriptional initiation from a minimal SI intestine-specific promoter. In contrast, HNF-1 beta has no functional effect on transcription of the SI promoter. The functional and DNA binding differences of HNF-1 alpha and HNF-1 beta on elements in the SI promoter suggest that these transcription factors may play a role in the complex spatial patterns of SI gene expression in the intestine.

Base Sequence↗
PubMed1994

NF-AT components define a family of transcription factors targeted in T-cell activation.

The NF-AT transcription complex is required for the expression of a group of proteins that collectively coordinate the immune response. Here we purify two proteins encoded by separate genes that represent the pre-existing (p) and cytosolic (c) components of NF-AT. Expression of the full-length complementary DNA encoding NF-ATc activates the interleukin (IL-2) promoter in non-T lymphocytes, whereas a dominant negative of NF-ATc specifically blocks activation of the IL-2 promoter in T lymphocytes, indicating that NF-ATc is required for IL-2 gene expression. NF-ATc RNA expression is largely restricted to lymphoid tissues and is induced upon T-cell activation. The other protein, NF-ATp, is highly homologous to NF-ATc over a limited domain which shows similarity to the Dorsal/Rel family, but has a wider tissue distribution. Agents that increase intracellular Ca2+ or activate protein kinase C independently modify NF-ATc, indicating that distinct signalling pathways converge on NF-ATc to regulate its function.

Amino Acid Sequence↗