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S Wu

Publications and source records attributed to S Wu.

At least 289 records · Page 16Linked to original sources
PubMed1997

A stereoselective cobalt-containing nitrile hydratase.

Nitrile hydratase from Pseudomonas putida NRRL-18668 has been purified and characterized. The purified enzyme catalyzes the hydration of 2(S)-(4'-chlorophenyl)-3-methylbutyronitrile at least fifty times faster than that of 2(R)-(4'-chlorophenyl)-3-methylbutyronitrile. This enzyme is a member of the class of nitrile hydratase that contains cobalt. Visible absorption and CD spectra suggest the cobalt exists as a non-corrin low-spin Co3+ ion in a tetragonally-distorted octahedral ligand field. Chemical reduction of the native enzyme results in a species with the EPR signature of a low-spin Co2+ complex. Like the other cobalt-containing nitrile hydratases, this enzyme is relatively stable, maintaining its activity below 35 degrees C, and it shows a broad activity optimum between pH 7.2 and 7.8. The structural genes for this enzyme have been cloned and sequenced. The deduced amino acid sequences for the alpha and beta subunits show 48-63% and 35-41% homology, respectively, to other sequenced nitrile hydratases. In particular, the cysteine residues in the alpha subunit that have been suggested to coordinate the metal ion in the iron-containing nitrile hydratases [Brennan, B. A., Cummings, J. G., Chase, D. B., Turner, I. M., Jr., & Nelson, M. J. (1996) Biochemistry 35, 10068-10077] are conserved in this enzyme, suggesting that this nitrile hydratase, like the enzyme from Rhodococcus rhodochrous J1, is a member of a newly described class of metalloenzymes with Co3+-thiolate ligation [Brennan, B. A., Alms, G., Nelson, M. J., Durney, L. T., & Scarrow, R. C. (1996) J. Am. Chem. Soc. 118, 9194-9195].

Amino Acid Sequence↗
PubMed1997

All-trans-retinoic acid blocks cell cycle progression of human ovarian adenocarcinoma cells at late G1.

We prepared single cell clones from two ovarian carcinoma cell lines, CA-OV3 and SK-OV3, and analyzed the effect of all-trans-RA treatment on cell division, DNA synthesis, and cell cycle stage distribution of these single cell clones. Our results show that despite the well-known heterogeneous nature of these cell lines, all single cell clones of SK-OV3 cells are resistant to the growth inhibitory effects of all-trans-RA. In contrast, all single cell clones of CA-OV3 cells were growth inhibited by all-trans-RA. However, the extent of growth inhibition did vary somewhat from clone to clone. Additional studies employing flow cytometry showed that all-trans-RA blocked CA-OV3 cell cycle progression in the G1 stage. Finally, all-trans-RA was able to inhibit G1 progression in growth-arrested CA-OV3 cells following stimulation with fetal bovine serum, insulin, IGF-1, or estrogen. Since each of these growth factors is known to act via distinct signal transduction pathways, our results suggest that all-trans-RA blocks G1 progression by targeting a downstream process or event which occurs at a point after the insulin/IGF-1, estrogen, and serum signal transduction pathways converge.

Adenocarcinoma↗
PubMed1997

Regulation of renal vitamin D-24-hydroxylase by phosphate: effects of hypophysectomy, growth hormone and insulin-like growth factor I.

Dietary phosphate (Pi) regulates the production of 1,25-dihydroxyvitamin D3. This control is blunted in hypophysectomized (HPX) rats but can be restored by growth hormone (GH) or IGF-I. The regulation of the vitamin D catabolism by Pi, GH and IGF-I is less clear. In the present study, we found that the activity and transcript levels of the catabolic enzyme, vitamin D-24-hydroxylase (24-OHase) were decreased 3- and 5-fold, respectively, during Pi restriction in normal rats, but this effect is greatly reduced in HPX rats. Examination of the serum chemistries revealed that HPX rats on the high Pi diet had lower serum Pi levels than normal rats on this diet, presumably due to the known defective reabsorption of Pi by HPX rats. Treatment of HPX rats, adapted to a 0.6% P diet, with GH (150 micrograms) or IGF-I (80 micrograms) suppressed 24-OHase mRNA levels by 88% and 64%, respectively, by 20 hours and these effects were preceded by decreases in serum Pi. Our findings show that the 24-OHase is regulated by dietary Pi and this control is modulated by hypophysectomy, GH and IGF-I.

Animals↗
PubMed1997

Structure/function relationship study of Tyr14 and Arg22 in trichosanthin, a ribosome-inactivating protein.

Amino acids Tyr14 and Arg22 in trichosanthin are residues on helix A1 close to the active-site cleft. They are invariant in various type-I and type-II ribosome-inactivating proteins. In this study, Tyr14 was changed to Phe and Arg22 to Lys and Leu. Modified proteins were purified, and activities compared by assaying their median inhibitory concentration (ID50) on a rabbit-reticulocyte-lysate protein-synthesis system. While the ID50 of wild-type trichosanthin was 0.02 nM, those for [Phe14], [Lys22], [Leu22] and [Phe14, Leu22]trichosanthin were 0.10, 0.03, 0.25 and 0.15 nM, respectively. Therefore, compared with Tyr14, Arg22 appears to play a more important role in trichosanthin. Structural studies on [Leu22]trichosanthin showed that two water molecules occupy the space left by the side chain of Arg22, and hydrogen bonds exist between these water molecules and nearby residues to retain the conformation. The use of intermolecular rather than intramolecular hydrogen bonds may have an adverse effect on stability or folding of the protein and results in a mild decrease in activity.

Abortifacient Agents, Nonsteroidal↗
PubMed1997

An integrin polymorphism that defines reactivity with alloantibodies generates an anchor for MHC class II peptide binding: a model for unidirectional alloimmune responses.

Polymorphic proteins are often the targets of T and B lymphocytes in alloimmune responses. The polymorphic residue 33 of integrin beta3 is responsible for both a B cell response and a T cell response. Leu at position 33 controls the epitopes recognized by alloantibodies generated in individuals homozygous for Pro at 33. The alloantibody response shows a tight class II MHC restriction, and T cells have been identified that respond to the alloantigen. As part of the T cell response, the alloantigen could be recognized as foreign, and the autoantigen would be ignored due to self-tolerance. Alternatively, the polymorphism could change the ability of the peptide to bind. We tested these two possibilities using synthetic peptides corresponding to the polymorphic region and insect cell-derived class II MHC. The Leu33-containing peptide bound to the restricting MHC allele, whereas the Pro33 peptide did not. Thus, the presence of Leu at position 33 generates an anchor specific for the restricting MHC allele and helps define the peptide binding motif of this allele, HLA-DRB3*0101 (DR52a). These data indicate that certain alloresponses can be viewed entirely as foreign because the polymorphism generates a functional anchor residue in a peptide that otherwise would not bind. On the basis of these results, we would predict a unidirectional alloantibody response as the lack of binding of the Pro33 peptide would preclude T cell help. This could explain the observation that alloantibodies to the Pro33 allele are very rare.

Antigens, CD↗
PubMed1997

Genetic immunotherapy of established tumors with adenovirus-murine granulocyte-macrophage colony-stimulating factor.

Increased local production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by genetically modified tumor cells can induce specific antitumor cellular immunity. We constructed a recombinant adenovirus expressing murine GM-CSF and tested it for therapeutic efficacy in a syngeneic murine lung cancer model system. In vitro transduction of Lewis lung carcinoma cells with adenovirus-mGM-CSF suppressed tumor formation in syngenic mice (C57BL/6), and transduced and irradiated Lewis lung carcinoma cells induced regression of pre-established wild-type tumors without in vitro selection for transductants. Low, but significant, levels of specific antitumor cytotoxic T lymphocytes (CTL) were observed in mice inoculated with GM-CSF but not with reporter virus-transduced tumor cells. GM-CSF-transduced cells induced the accumulation of dendritic cells at the site of tumor, consistent with a mechanism involving improved tumor antigen presentation. These data suggest that transduction of tumor cells with recombinant GM-CSF adenovirus may be an effective and practical cancer gene therapeutic strategy.

Adenoviridae↗
PubMed1997

A model for the double-stranded RNA (dsRNA)-dependent dimerization and activation of the dsRNA-activated protein kinase PKR.

Binding of double-stranded RNA (dsRNA) to PKR induces autophosphorylation and activation. However, the requirement for dsRNA in promoting dimerization and the requirement for dimerization in PKR activation are controversial. We have studied the dsRNA binding and dimerization requirements for the activation of PKR in vivo. Co-expression and immunoprecipitation experiments detected an interaction between the K296P mutant and a bacteriophage T7-epitope-tagged K64E mutant of dsRNA binding domain. In contrast, the K64E/K296P double mutant did not form a detectable dimer with the wild-type dsRNA binding domain. These results support that dimerization of intact PKR with the isolated dsRNA binding domain requires dsRNA binding activity. Expression of the isolated PKR kinase domain (residues 228-551) reduced translation of the reporter mRNA even in the presence of PKR inhibitors. Furthermore, the isolated kinase domain (residues 228-551) undergoes autophosphorylation and sequentially transphosphorylates both mutant K296P PKR and wild-type eIF-2alpha in vitro. In contrast, the isolated kinase domain (residues 264-551) lacking the third basic region was not active. These observations lead us to propose that the dsRNA binding domains on intact PKR inhibit kinase activity and that dsRNA binding to intact PKR induces a conformational change to expose dimerization sites within the dsRNA binding domain thereby promoting dimerization and facilitating trans-phosphorylation and activation.

Animals↗
PubMed1997

Bacteroides fragilis toxin rearranges the actin cytoskeleton of HT29/C1 cells without direct proteolysis of actin or decrease in F-actin content.

Enterotoxigenic strains of B. fragilis associated with childhood diarrhea produce a 20 kD zinc metalloprotease toxin (BFT). BFT is reported to cleave G-actin in vitro and also causes dramatic rounding and rearrangement of the F-actin cytoskeleton in human intestinal epithelial cell lines (HT29) and HT29/C1). To test the hypothesis that the proteolysis of cellular actin by BFT in vivo may contribute to these alterations in morphology and cytoskeletal architecture, we assessed the F-actin content and the arrangement of the F- and G-actin cytoskeleton in BFT-treated HT29/C1 cells by spectrofluorimetry, confocal microscopy, and immunoblotting. BFT-treated cells were compared to cells treated with C. difficile toxin A (CDA) or cytochalasin D. Using spectrofluorimetric quantification, the F-actin content of BFT- and cytochalasin D-treated cells was unchanged in contrast to a significant decrease in CDA-treated cells. By confocal microscopy, the arrangement of F- and G-actin in all treated cells was markedly different than control cells. There was no change in the immunoblotting pattern of actin in the Triton-soluble or -insoluble cellular fractions of BFT-treated HT29/C1 cells. We conclude that BFT alters the F- and G-actin cytoskeletal architecture of HT29/C1 cells without direct proteolysis of actin or decrease in F-actin content.

Actins↗
PubMed1997

The mechanism of free radical generation in brain capillary endothelial cells after anoxia and reoxygenation.

We studied the mechanism of reoxygenation injury of cerebral microvessels in cultured rat brain capillary endothelial cells (BCECs). BCECs were isolated from rat cerebral cortices by a two step enzymatic treatment. The monolayers of BCECs were subjected to anoxia for 20 minutes followed by reoxygenation for 3 hours. Cell damage was assessed by measuring the leakage of intracellular lactic dehydrogenase (LDH). The control group was anoxia/reoxygenated BCECs without any protective reagents. To study the protective effect of free radical scavengers and antioxidants, superoxide dismutase, catalase, deferoxamine, oxypurinol, indomethacin, or NG-nitro-L-arginine methyl ester (L-NAME) was applied during anoxia/reoxygenation. Thus 7 experimental conditions were established. Lactic dehydrogenase (LDH) leaked from reoxygenated BCECs due to cell membrane damage. This leakage was almost totally suppressed by superoxide dismutase, indicating that reoxygenation injury of BCECs is mediated by superoxide generation. The other scavengers and antioxidants partially suppressed LDH leakage. Reduction of Ca2+ in the culture medium from 1.6 mM to 0.016 mM also suppressed LDH leakage. These results indicate that BCECs subjected to anoxia/reoxygenation become potent generators of superoxide anion, which is thought to be responsible for reoxygenation injury. The superoxide generation partially depends on the xanthine oxidase and cyclooxygenase pathways. As L-NAME partially suppressed LDH leakage peroxynitrite may contribute to reoxygenation injury of BCECs. The extracellular Ca2+ concentration also plays a critical role in the reoxygenation injury of BCECs.

Animals↗
PubMed1997

The role of calcium ion in anoxia/reoxygenation damage of cultured brain capillary endothelial cells.

Capillary endothelial cells are critical targets in both ischemia and reperfusion of the brain. Arachidonic acids and oxygen free radicals have been shown to cause disruption of blood-brain barrier (BBB) by destruction of capillary endothelial cell membrane. However, the exact mechanism of BBB breakdown by cerebral ischemia/reperfusion remains undetermined. The aim of the present study is to clarify the mechanism of intracellular calcium ion ([Ca2+]i) change in brain capillary endothelial cells under anoxia/reoxygenation. Brains capillary endothelial cells were isolated from ten male Sprague-Dawley rats by a two step enzymatic process. [Ca2+]i was measured by means of a confocal laser scanning microscope using Indo 1-A/M as a calcium indicator. The endothelial cells were subjected to anoxia and reoxygenization under different conditions. [Ca2+]i increased gradually during anoxia and slightly decreased after reoxygenation. Indomethacin and SOD suppressed the elevation of [Ca2+]i during anoxia. NG-nitro-L-arginine methyl ester and catalase moderately suppressed the elevation, however nifedipine did not suppress it at all. In this model, rapid [Ca2+]i change was not observed during the reoxygenation phase. The results indicate that the anoxia induced elevation of [Ca2+]i in the brain capillary endothelial cells depends on superoxide and peroxynitrite generation.

Animals↗
PubMed1997

Impaired learning of active avoidance in water-intoxicated rats.

Brain edema is an important clinical condition. Pathophysiological findings on behavioral changes may be helpful for a comprehensive understanding of brain edema. However, only few reports on behavioral studies of brain edema have so far appeared. Experiments using psychological techniques on animals are rather time-consuming and may not be suitable for the study of transient conditions, as brain edema caused by trauma, vascular accidents, or others. We have developed a method for avoidance learning of rats using a running wheel apparatus with computer assistance. This model was employed in studies on brain edema from water introxication in rats. As a result, avoidance learning was significantly impaired by water intoxication. Either direct overhydration of the brain or indirect effects, as a decrease in cerebral blood flow, or both, are suggested as mechanisms underlying the impairment of behavior.

Animals↗
PubMed1997

The mechanism of reversible osmotic opening of the blood-brain barrier: role of intracellular calcium ion in capillary endothelial cells.

Despite clinical and experimental interest in the osmotic opening of the blood-brain barrier (BBB), the mechanism underlying the phenomenon remain undetermined. The aim of this study is to investigate the mechanism of intracellular Ca2+ change in brain microvascular endothelial cells subjected to hyperosmotic stress. Cultured rat brain capillary endothelial cells were obtained by two-step enzymatic purification. Intracellular Ca2+ was measured by a confocal laser scanning microscope. After exposing the endothelial cells to 1.4 M mannitol for 30 seconds, the change of intracellular Ca2+ concentration was monitored. Intracellular Ca2+ concentration increased rapidly and reached its peak value within 10 seconds after the application of mannitol. The Ca2+ concentration returned to the basal level within 200 seconds. A calcium channel blocker nifedipine (100 microM, 10 microM) did not block the increase. A specific blocker (KB-R7943) of Na+/Ca2+ exchange did not affect the rapid elevation of intracellular Ca2+. However, it blocked the return phase almost completely. The results indicated that the Na+/Ca2+ exchanger pumped out the increased intracellular Ca2+ during the return phase. Reversible osmotic disruption and reconstruction of the BBB is not due to simple mechanical shrinkage of the endothelial cells but is due to the intracellular Ca(2+)-activated complex mechanism. The manipulation of the reconstruction phase, which depends on Na+/Ca2+ exchanger, may have clinical implications.

Animals↗
PubMed1997

Feasibility of simultaneous dual-isotope myocardial perfusion acquisition using a lower dose of sestamibi.

The feasibility of simultaneous dual-isotope myocardial perfusion imaging was assessed using a ca. 12 mCi dose of technetium-99m sestamibi (MIBI) and ca. 3 mCi thallium-201. Planar and single-photon emission tomographic (SPET) data from 40 patients (41 studies) imaged with both a single- and a dual-isotope protocol were analyzed. Rest injected 201T1 (pure-T1) images were acquired using 20% windows at 70 and 166 keV about 15 min after the 201T1 injection. Patients were then stressed, and at peak stress 99mTc-sestamibi was injected. About 30 min later 99mTc data were recorded with a 20% window center at 140 keV, and simultaneous 201T1 (dual-T1) data were recorded with a single 20% window centered at 80 keV. Total myocardial counts based on SPET data in the dual-T1 images were increased by 18.61% +/- 2.91% (SEM) (range: -12.8% to 84.1%) compared to pure-T1 images. Region of interest analysis revealed the greater increase in counts in the apical region and the least in the lateral wall. Pure T1 and dual-T1 images were visually evaluated for image quality (IQ) on a five-point scale (0 = unacceptable to 4 = excellent). Dual-T1 IQ was lower than that of pure-T1 in 61% of cases, and similar in 37% (12% of the pure-T1 and 41% of the dual-T1 images fell into the 0 and 1 categories). Thallium perfusion abnormalities were of similar extent in 70% of segments, less severe in 18%, and more severe in 12%. There was an inverse correlation with patient weight, such that patients weighing more than 180 lbs had substantially worse images than those below this cutoff value. While dual-tracer images are of lower quality, they are interpretable if the patient is not severely overweight.

Body Weight↗
PubMed1997

Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli: activity requires a novel downstream protein.

The stereoselective nitrile hydratase (NHase) from Pseudomonas putida 5B has been over-produced in Escherichia coli. Maximal enzyme activity requires the co-expression of a novel downstream gene encoding a protein (P14K) of 127 amino acids, which shows no significant homology to any sequences in the protein database. Nitrile hydratase produced in transformed E. coli showed activity as high as 472 units/mg dry cell (sixfold higher than 5B), and retained the stereoselectivity observed in the native organism. Separated from the end of the beta subunit by only 51 bp, P14K appears to be part of an operon that includes the alpha and beta structural genes of nitrile hydratase, and other potential coding sequences.

Amino Acid Sequence↗
PubMed1997

Phase I clinical trial of all-trans-retinoic acid with correlation of its pharmacokinetics and pharmacodynamics.

A phase I trial of all-trans-retinoic acid (ATRA) was conducted to establish the maximum tolerable dose (MTD) of ATRA given once daily to patients with solid tumors. Cancer patients for whom no standard therapy was available were treated with ATRA once daily. Doses were escalated in cohorts of at least three patients. The pharmacokinetics of ATRA were assessed on day 1 for all patients and weekly for 31 patients who received doses of > or = 110 mg/m2 per day. Patients were followed for toxicity and response. Correlations of toxicity frequency and grade with pharmacokinetic parameters were sought. In addition, correlation of changes in ATRA pharmacokinetics with the concentration of ATRA metabolites in plasma were sought. A total of 49 patients received ATRA at doses ranging from 45 to 309 mg/m2 per day. Hypertriglyceridemia was dose-limiting at 269 mg/m2 per day. Other frequent toxicities included mucocutaneous dryness and headache. With chronic dosing, plasma ATRA concentrations fell in 59% of patients. Stable, low, or variable [ATRA] were seen in 16%, 6%, and 16% of patients respectively. Age, gender, smoking, or concurrent medication did not correlate with the pharmacokinetic pattern. Severe toxicities tended to occur with initial peak [ATRA] of > or = 0.5 microgram/ml (1.7 microM), and the toxicity frequency did not change if [ATRA] decreased with continued dosing. No consistent change in 4-oxo-ATRA or retinoid glucuronide concentrations was observed with decreases in plasma [ATRA]. The recommended once-daily ATRA dose is 215 mg/m2, although significant interpatient variability is observed in toxicity and plasma retinoid concentrations. Although not statistically significant, more frequent and severe toxicity tended to occur in patients with higher plasma peak ATRA concentrations. Other factors, such as responses at target tissues, may be at least as important as the plasma ATRA concentration in predicting toxicity and/or response.

Adult↗
PubMed1997

Coordinate expression of inducible nitric oxide synthase and cyclooxygenase-2 genes in uterine tissues of endotoxin-treated pregnant mice.

OBJECTIVES: Our purpose was to investigate the relationship between expression of cyclooxygenase-2 and inducible nitric oxide synthase genes after labor induction with bacterial lipopolysaccharide in a murine model of preterm parturition. STUDY DESIGN: Pregnant C57B1/6 mice were given Escherichia coli lipopolysaccharide (20 micrograms per mouse) by intraperitoneal injection on day 16 of gestation, and the animals were followed up for signs of labor. Control mice received an equivalent volume of 0.9% saline solution. The latency from lipopolysaccharide injections until appearance of the first pup was recorded. Two separate groups of mice were given either aminoguanidine or indomethacin (5 mg/kg intragastric) 24 hours before induction of preterm labor. In a separate set of experiments mice were treated with lipopolysaccharide as described and were killed at intervals from 0.5 to 72 hours and intrauterine tissues (uterus, placenta, and fetal membranes) were removed and snap frozen in liquid nitrogen. Total protein and ribonucleic acid were extracted for Western and Northern blot analysis of cyclooxygenase-2 and inducible nitric oxide synthase protein and messenger ribonucleic acid, respectively. RESULTS: Northern blots from uterine, placental, and fetal membrane tissues of lipopolysaccharide- and saline solution-treated mice revealed that cyclooxygenase-2 and inducible nitric oxide synthase messenger ribonucleic acid transcripts were rapidly (within 0.5 to 2 hours) up-regulated after lipopolysaccharide administration but were unchanged in mice injected with saline solution. Immunoblot analysis with isoform-specific antibodies revealed that both enzymes were expressed in uterus, placenta, and fetal membranes in a coordinated fashion with peak expression seen at 6 to 8 hours. Although the steady-state accumulation of messenger ribonucleic acid transcripts encoding cyclooxygenase-2 and inducible nitric oxide synthase peaked at 6 hours and declined to baseline by 16 hours after injection with lipopolysaccharide, expression of cyclooxygenase-2 and inducible nitric oxide synthase was sustained through the period when premature delivery was observed. Nitric oxide-dependent cyclooxygenase-2 and inducible nitric oxide synthase expression was demonstrated by the elimination of accumulation of both messenger ribonucleic acid transcripts in mice pretreated with aminoguanidine before injection with lipopolysaccharide. CONCLUSIONS: These data indicate that nitric oxide synthesis may be a prerequisite for subsequent stimulation of cyclooxygenase-2 and inducible nitric oxide synthase gene expression. Taken together, the data suggest that cyclooxygenase-2 and inducible nitric oxide synthase are expressed in a coordinated manner in the uterus of endotoxin-challenged pregnant mice and that their enzymatic products may contribute to the signaling of uterine activity or cervical changes culminating in expulsion of the fetus.

Animals↗