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C Wei

Publications and source records attributed to C Wei.

At least 127 records · Page 7Linked to original sources
PubMed1994

Reaction and free radical kinetics of prostaglandin H synthase with manganese protoporphyrin IX as the prosthetic group.

Prostaglandin H synthase (PGHS) is a hemoprotein with both cyclooxygenase and peroxidase activities. Several aspects of the peroxidase and cyclooxygenase activities and the formation of substrate-induced free radical species were characterized with ovine seminal vesicle PGHS reconstituted with manganese protoporphyrin IX (Mn-PGHS) for comparison with the enzyme-containing heme (Fe-PGHS). Compared to Fe-PGHS, the Km of Mn-PGHS peroxidase for ethyl hydroperoxide was much higher, but that for 15-hydroperoxyeicosatetraenoic acid (15-HPETE) was little changed. The Mn-PGHS peroxidase Vmax value with 15-HPETE was about 4% that with Fe-PGHS. Mn-PGHS oxidized 0.95 +/- 0.05 and Fe-PGHS oxidized 2.06 +/- 0.09 mol of TMPD/mol of 15-HPETE. Reaction of 15-HPETE with Mn-PGHS resulted in approximately equal proportions of two lipid products: 15-hydroxyeicosatetraenoic acid (15-HETE) and a compound identified as 15-ketoeicosatetraenoic acid. Fe-PGHS produced only 15-HETE. Thus, 15-HETE can serve as an efficient two-electron reductant of oxidized Mn-PGHS intermediates. The rate of accumulation of oxidized Mn-PGHS intermediate was dependent on the substrate, decreasing in the following order: 15-HPETE > 11,14-eicosadienoate > arachidonate > EtOOH. The cyclooxygenase specific activity increased in a saturable fashion as the concentration of Mn-PGHS was increased, reaching a value higher than that for Fe-PGHS. Computer simulations of the reaction kinetics indicated that this dependence on the Mn-PGHS level was a consequence of the low rate of formation of oxidized peroxidase intermediate. Incubation of Mn-PGHS with either 15-HPETE or arachidonate resulted in the rapid production of a free radical species. The initial accumulation of radical coincided with the synthesis of PGG2 and PGH2, indicating that the radical was kinetically competent to participate in cyclooxygenase catalysis. Reaction with tetranitromethane, a reagent that selectively nitrates tyrosyl residues, destroyed the cyclooxygenase activity of Mn-PGHS, resulting in a much narrower free radical EPR signal. These effects indicate that tyrosine residues are essential for cyclooxygenase activity and that they influence the radical structure in Mn-PGHS.

Animals↗
PubMed1994

Interaction between nitric oxide and prostaglandin H synthase.

Prostaglandin H synthase (PGHS) is a hemeprotein, and thus its catalytic activity potentially could be modulated by direct interaction with nitric oxide (NO). We have monitored spectroscopic and activity changes in pure ovine PGHS isoform-1 to investigate its interaction with NO in more detail. The binding kinetics for NO and the ferric heme in resting PGHS were analyzed by stopped-flow spectrophotometry at 21 degrees C. The rate constants for association and dissociation were estimated to be 6.5 x 10(4) M-1 s-1 and 60 s-1, respectively, leading to an equilibrium dissociation constant (Kd) of 0.92 mM. NO thus has a relatively weak affinity for heme in ferric PGHS, the resting oxidation state of this hemeprotein. NO did react strongly and completely with ferrous PGHS under anaerobic conditions, displacing the proximal histidine ligand to the prosthetic group. Dissolved NO at up to 2 mM produced only slight decreases in the cyclooxygenase activity of microsomal, detergent-extracted, or homogeneous preparations of ovine PGHS. The NO donors sodium nitroprusside and glyceryl trinitrate at levels of up to 1 mM also had little effect on the activity of the PGHS preparations. Thus, there was no evidence for significant direct interaction of PGHS with NO at concentrations likely to be encountered in vivo.

Animals↗
PubMed1994

Prediction of outcome in omphalocele and gastroschisis by intraoperative measurement of intravesical pressure.

A simple and accurate measurement of intraabdominal pressure is essential to predict a successful closure of defects in omphalocele and gastroschisis. Intravesical pressure (IVP) is a close estimation of intraabdominal pressure and can be measured safely by placing a catheter in the urinary bladder during surgery. Three neonates with gastroschisis and four with omphalocele were studied. Pressure-related complications such as ascites leakage, ventral hernia, impaired venous return of the lower extremities, and oliguria developed only in the patients with IVP > 20 mmHg after fascial closure. Prolonged hospitalization, ventilation support and intensive care were required for these patients.

Abdominal Muscles↗
PubMed1994

[Targeting treatment of human HCC xenografts implanted in nude mice using bifunctional antibody HC-1 combined with mitomycin C].

Bifunctional antibody (BFA) HC-1 possessing one binding site for mitomycin C (MMC) and a companion site directed against human hepatocellular carcinoma (HCC) cell membrane was constructed by chemical conjugation of two Fab' fragments of McAb MMC-1 and McAb HCMP-1. BFA could be specifically attached to tumor xenograft of nude mice bearing human HCC and thus simultaneously capture mitomycin C. The attachment of these complexes was detected by radioimmunoimaging in nude mice bearing human HCC using 131-I labelled BFA HC-1. Clear imaging of the tumor was obtained in 6 days after i.p. injection. On the 8th day after the injection, tumor/liver (T/L) ratio was 8.04 +/- 0.45. When the BFA HC-1 was used to be combined with MMC for the targeting treatment of human HCC implanted in nude mice, the highly significant suppression of tumor growth was achieved. After two months of treatment, xenografts of 40% (4/10) mice disappeared and 60% (6/10) of the mice survived. Those mice treated only with MMC became more and more sick even if the grafted tumors shrank, and all of them died within 2 months after therapy. The controls were treated with nonspecific IgG. Tumors grew very fast, and most of the controls died in 1 month after the first injection. The results suggest that BFA HC-1 could concentrate MMC on the human HCC cells, and it is a kind of suitable carrier for the targeting treatment of human HCC.

Animals↗
PubMed1993

Overexpression of asparagine synthetase in albizziin-resistant murine diploid embryonic stem cells.

Gene amplification is commonly observed in primary tumors and established drug-resistant cell lines, both of which are generally aneuploid. However, this process is undetectable (frequency < 10(-9) in normal diploid mammalian cell lines. To investigate whether gene amplification can occur in pluripotent diploid cells, we have selected drug-resistant mutants of mouse embryonic stem (ES) cells. We had previously found that Chinese hamster ovary (CHO) and human cell lines selected in albizziin (Alb), an amino acid analog of L-glutamine, overexpress asparagine synthetase (AS) due to gene amplification. The same drug selection system was applied to ES cells to isolate single-step and multistep drug-resistant mutants. Albizziin-resistant ES cells exhibited elevated levels of AS; however, drug resistance in ES cells was associated with mRNA overexpression without gene amplification. AS gene amplification was observed in only one drug-resistant cell line and was preceded by AS mRNA overexpression. Gene amplification in the latter coincided with the loss of the pluripotent nature of the ES cells.

Amino Acids, Diamino↗
PubMed1992

p23rab2, a ras-like GTPase with a -GGGCC C-terminus, is isoprenylated but not detectably carboxymethylated in NIH3T3 cells.

With the development of a specific anti-rab2 antiserum, p23rab2, a ras-like GTPase with a -GGGCC C-terminus, has been localized mainly to the particulate (P100) fraction in NIH3T3 cells, although a small amount of this protein also appears in the soluble fraction. The endogenous p23rab2 is isoprenylated in intact cells, and recombinant murine p23rab2 is also isoprenylated in an in vitro system using reticulocyte lysate. Both the cytosolic and membrane-bound forms of p23rab2 are isoprenylated in intact cells. Recombinant p23rab2 is specifically geranylgeranylated at one or both of the C-terminal cysteine residues in the in vitro system. Blocking isoprenoid synthesis with lovastatin results in an accumulation of a totally cytosolic unisoprenylated form, indicating that isoprenylation is a prerequisite for membrane association of p23rab2. Surprisingly, unlike p21ras and p25rab3A, no carboxymethylation of p23rab2 is detectable in either the soluble or particulate fractions.

3T3 Cells↗
PubMed1990

Analysis of fibrinogen A alpha-fusion proteins. Mutants which inhibit thrombin equivalently are not equally good substrates.

We have examined the interaction of thrombin with fibrinogen A alpha chain residues 7-16. Using genetically engineered constructions, we have synthesized in Escherichia coli a fibrinogen A alpha 1-50 fusion protein and seven mutant proteins with single amino acid substitutions. These are: Asp7----Ala, Phe8----Tyr, Glu11----Ala, Gly12----Val, Gly13----Val, Gly14----Val, and Arg16----Leu. Competitive immunoassay of cell lysates showed that all the mutations but one, Arg16----Leu, altered the structure of the protein such that cross-reactivity with the A alpha-specific monoclonal antibody, Y18, was significantly reduced. The fusion proteins were purified and analyzed as thrombin inhibitors and substrates. All the fusion proteins are competitive inhibitors of the amidolytic hydrolysis of Spectrozyme TH, a thrombin-specific chromogenic substrate, with inhibition constants corresponding to that for fibrinogen. We conclude that these 7 amino acid substitutions do not alter thrombin binding to the fusion proteins. The fusion proteins were tested as substrates by monitoring thrombin-dependent peptide release. The natural sequence and three mutants, Asp7----Ala, Glu11----Ala, and Gly14----Val, are good substrates. The other mutants are either poor substrates or are not cleaved by thrombin within A alpha 1-50. These results indicate that residues between Asp7 and Arg16 are critical to efficient peptide hydrolysis, whereas residues outside this region are critical to thrombin binding.

Amino Acid Sequence↗
PubMed1990

Conformation and activity of recombinant human fibroblast interferon-beta.

Conformation of highly purified recombinant human fibroblast interferon-beta (rHuIFN-beta) was correlated with its biological activity. The extent of ordered secondary structure was determined by circular dichroic (CD) spectroscopy in various buffer conditions to establish conditions of protein stability and its potential for helix formation. The highest "helicity" (about 50 +/- 5% of alpha-helices) and the highest antiviral activities (4-10 x 10(7) units/mg) were found in 50% ethylene glycol, 1 M NaCl and 0.05 M Na3PO4, pH 7.2 (Buffer I); 80 mM citric acid, 20 mM Na2HPO4, pH 2.9 (Buffer II); and 25 mM NH4OAc, 125 mM NaCl, pH 5.1 (Buffer III). Both helicity and antiviral activity of the IFN-beta decrease in parallel with denaturation by urea, heat, and/or by repeated cycles of freezing and thawing. Low pH (pH 2.9 Buffer II) exhibits a distinct stabilizing effect on the structure and antiviral activity of IFN-beta against heat denaturation.

Amino Acid Sequence↗
PubMed1989

Modulation of ras expression by anti-sense, nonionic deoxyoligonucleotide analogs.

Anti-ras oligodeoxyribonucleoside methylphosphonates (ONMP's) complementary to the initiation codon region have been synthesized to explore their efficacy and specificity on ras-p21 translation. ONMP (IC-0) precisely complementing the first initial 11 nucleotides of the Balb-ras initiation codon region acts in a dose-dependent manner to inhibit p21 translation by a rabbit reticulocyte lysate. At 100 microM, IC-0 inhibits the cell-free translation of p21 close to completion. The two control oligomers containing one or two nucleotide mismatches were significantly less effective than IC-0 at the equivalent concentration. In living cells, a perfectly matched ONMP directed against the initiation codon region inhibited Ha-ras p21 expression by 90% at a concentration of 50 microM.

Animals↗
PubMed1985

Nitroreductase-dependent mutagenicity of p-nitrophenylhydroxylamine and its N-acetyl and N-formyl hydroxamic acids.

p-Nitrophenylhydroxylamine (NPH) and two hydroxamic acids derived from it were synthesized and subjected to mutagenicity testing in Salmonella typhimurium strains TA98, TA98NR, TA1538 and TA1538NR. In addition, p-dinitrobenzene (DNB), p-nitroaniline (NA) and p-nitroacetanilide (AcNA) were simultaneously examined for mutagenic action against these four tester strains. NPH, its N-acetyl (AcNPH) and N-formyl (FoNPH) derivatives, and also DNB displayed strong mutagenic action to the nitroreductase-containing strains, TA98 and TA1538. NPH was the most potent chemical in this series against both of these strains, while the two hydroxamic acids AcNPH and FoNPH, and also DNB displayed approximately the same degree of mutagenicity. In the nitroreductase-deficient strains, TA98NR and TA1538NR, the mutagenicity of these four compounds was markedly reduced. The necessity for nitroreduction in order to activate these promutagens is fairly certain; however, the lack of mutagenicity of NA and AcNA towards all four tester strains made the interpretation of these data somewhat more complicated. Several possible bioactivation pathways were presented, with one mechanism in particular being proposed. This mechanism requires only that the strong electron-withdrawing nitro group be converted to an electron-donating group by bacterial nitroreductase. Such a mechanism is unique for the bioactivation of nitro aromatics by nitroreductase, since the enzymatic reduction need not produce the intermediary hydroxylamine metabolite.

Acetylation↗