Biomedical subjects
W S Wu
Publications and source records attributed to W S Wu.
Gas chromatographic determination and negative-ion chemical ionization mass spectrometric confirmation of 4,4'-methylenebis(2-chloroaniline) in urine via thin-layer chromatographic separation.
An analytical method for monitoring 4,4'-methylenebis(2-chloroaniline) (MOCA) in urine was developed. The MOCA extract from a urine sample was derivatized with pentafluoropropionic anhydride and subsequently cleaned-up via TLC before being analysed by a gas chromatograph equipped with an electron capture detector. Heptafluorobutyric anhydride derivatized MOCA was used as the internal standard during quantification. The method showed that the analytical interferences are effectively minimized by using the TLC technique for sample clean-up. Recoveries of spiked MOCA in urine at levels of 7.5, 22 and 62.5 micrograms dm-3 were all over 90% with the limit of detection determined to be 1 microgram dm-3. Confirmation of MOCA in a urine sample was performed by using a gas chromatograph mass spectrometer in negative-ion chemical ionization mode.
Effects of tannic acid on 12-O-tetradecanoylphorbol-13-acetate-induced protein kinase C activation in NIH 3T3 cells.
Tannic acid (TA) is a naturally occurring phenol, which has been found to display an antipromotion effect on mouse skin carcinogenesis. In order to explore the molecular mechanism, we have examined the process of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced protein kinase C (PKC) activation, including phorbol ester binding, enzyme translocation, autophosphorylation and substrate phosphorylation, and finally the TPA-stimulated DNA synthesis. In an initial study, we found that TA slightly inhibited [3H]phorbol dibutyrate (PDBu) binding to intact cells, and only 30% reduction of phorbol ester binding was observed at the highest dose of TA (100 microM). Further analysis by Scatchard plot showed that TA reduced the Bmax of [3H]PDBu from 1.3 pmol/10(6) cells to 1.1 pmol/10(6) cells, but the Kd was increased from 24 to 30 nM. Analysis by western blot indicated that TA did not interfere with the TPA-induced PKC translocation, whereas TA effectively blocked the TPA-evoked phosphorylation of the membrane-bound PKC moiety and its 80 kDa substrate in a dose-dependent manner. We also found that pre- or post-treatment with TA both lead to a similar reduction of 80 kDa protein phosphorylation and that the TPA-stimulated DNA synthesis was also inhibited by TA in a dose-dependent manner, suggesting that the blockage of protein phosphorylation by TA was of biological significance. In conclusion, the work presented here demonstrated that the antitumor promoting effect of TA was not mediated by competing for the binding site with phorbol ester or interrupting the PKC translocation, but rather by effectively blocking phosphorylation by membrane-bound PKC, possibly through altering the biophysical properties of the membrane environment.
Comparative in vitro and in vivo activities of two 9-deazaguanine analog inhibitors of purine nucleoside phosphorylase, CI-972 and PD 141955.
An in-parallel comparison is presented of the in vitro and in vivo properties of two 9-deazaguanine analog inhibitors of purine nucleoside phosphorylase (PNP), CI-972 [8-amino-9-deaza-9-(3-thienylmethyl)guanine] and PD 141955 [9-deaza-9-(3-thienylmethyl)guanine] (published Ki values of 0.83-8.0 and 0.08 microM, respectively). Despite structural similarities, PD 141955 was considerably more potent and active in all systems studied. The respective IC50 values for inhibition of MOLT-4 cell growth in the absence and presence of 10 microM 2'-deoxyguanosine (GdR) were greater than 50 and 5.06 microM for CI-972 and 15.4 and 0.061 microM for PD 141955. PD 141955 induced accumulation of dGTP in GdR-treated MOLT-4 and CEM cells at log-lower concentrations than were required of CI-972, and the magnitude of dGTP accumulation in PD 141955-treated T cell cultures was markedly greater (e.g. 366 vs 100 pmol/10(6) CEM cells at 10 microM). PD 141955 administered orally produced a dose-dependent elevation of plasma inosine and guanosine in rats over a broad concentration range. Mean plasma inosine concentrations following a 150 mg/kg p.o. dose peaked at 6.21 and 13.2 microM in CI-972 and PD 141955-treated rats, respectively. Low levels of inosine were detectable at 50 micrograms/kg following oral administration of PD 141955.
Application of tryptamine as a derivatizing agent for the determination of airborne isocyanates. Part 5. Investigation of tryptamine-coated XAD-2 personal sampler for airborne isocyanates in workplaces.
The development of an efficient solid sorbent personal sampler with increased convenience for sample collection in workplaces is described. Several solid sorbents were coated with tryptamine, and sampling tubes were prepared with the coated sorbents. These tubes were evaluated for the collection of phenyl isocyanate vapour generated in a commercial test atmosphere generation system that permits the simultaneous collection of up to 12 uniformly loaded samples. Tryptamine-coated XAD-2 resin was shown to be the most efficient solid sorbent for the collection of airborne phenyl isocyanate. The optimum amount of tryptamine needed for coating XAD-2 resin was investigated.
Differential induction of c-fos and c-jun proto-oncogenes and AP-1 activity by tumor promoter 12-O-tetradecanoyl phorbol 13-acetate in cells at different stages of tumor promotion in vitro.
A two-stage (initiation and promotion) model of chemical transformation of mouse embryonic fibroblasts was used to elucidate the molecular mechanisms of tumor promotion in vitro. C3H10T1/2 cells which had been initiated with a subcarcinogenic dose (0.5 micrograms ml-1) of benzo[a]pyrene (B[a]P) were isolated after 12-O-tetradecanoyl phorbol 13-acetate (TPA) treatment lasting 12, 24 or 36 days. These series of partially promoted cells were designated T-12, T-24 and T-36 cells. T-12 and T-24 cells exhibited higher anchorage-independent growth on soft agar in the presence of TPA than did the initiated or T-36 cells. Cytosolic protein kinase C (PKC) in the resting state was slightly depleted in T-24 and T-36 cells. Proteolytic down-regulation of membrane-bound PKC was enhanced in T-12 cells compared with the initiated cells after 3 h of TPA treatment. Induction of c-fos and c-jun proto-oncogene expression increased two- to threefold in T-12 cells. Moreover, the basal level of c-fos mRNA progressively increased in T-12, T-24 and T-36 cells. As compared with other cell types, T-12 cells had the highest AP-1 DNA-binding activity at both the basal level and at 30 min after TPA treatment. These results indicate that deregulation of the TPA-induced cellular responses occurs in the cells at various stages of tumor promotion, and might be associated with transforming processes.
Purification of cytochrome cd1 nitrite reductase from Pseudomonas stutzeri JM300 and reconstitution with native and synthetic heme d1.
Cytochrome cd1 nitrite reductase has been purified from Pseudomonas stutzeri strain JM 300. This enzyme appears to be a dimer with a subunit molecular mass of 54 kDa and its isoelectric point is determined to be 5.4. The N terminus of amino acid sequence has strong homology with that of nitrite reductase from P. aeruginosa. The apoprotein of this enzyme has been reconstituted with native and synthetic heme d1. The nitrite reductase activity measured by NO and N2O gas evolution can be restored to 82% of the activity of the original enzyme when the protein was reconstituted with the native heme d1 and to 77% of the activity when reconstituted with the synthetic heme d1. The absorption spectra of both reconstituted enzymes are essentially identical to that of the original nitrite reductase. These results further substantiate the novel dione structure of heme d1 as proposed. The loss of NO2- reducing activity in the absence of heme d1 and its restoration by addition of heme d1 provides further evidence that heme d1 plays a key role in the conversion of NO2- to NO and N2O.
Modelling heme d1. The spectral properties of copper(II) porphyrindiones.
The heme d1 macrocycle of Ps. aeruginosa dissimilatory nitrite reductase is an iron porphyrin-3,8-dione with a 17-acrylate substituent. We have compared the RR properties of Cu-d1, the copper(II) TME of extracted heme d1, with those of models that differ only with respect to the acrylate: Cu-17-acrylate mesoporphyrin-3,8-dione (2) and Cu-mesoporphyrin-3,8-dione (3). The RR spectrum of Cu-d1 is very similar to that of 2, including v(C = O) at approximately 1720 cm-1. Replacement of the acrylate with propionate changes the spectrum markedly. For example, the v(C = O) mode of 3 shifts to 1712 cm-1, and peaks of Cu-d1 and 2 at approximately 1400 and approximately 1535 cm-1 are shifted or absent from the spectrum of 3. FTIR spectra of 2 and 3 also differ in their voxo(C = O) frequencies. The acrylate thus has a surprisingly strong influence on the electronic structural and spectral properties of heme d1. These data provide a foundation for studies of the novel biological porphyrindione macrocycles.
Application of tryptamine as a derivatising agent for airborne isocyanate determination. Part 3. Evaluation of total isocyanates analysis by high-performance liquid chromatography with fluorescence and amperometric detection.
Determination of total airborne isocyanates using tryptamine as the derivatising agent was investigated. Tryptamine derivatised isocyanates were analysed by reversed-phase high-performance liquid chromatography (HPLC). The column was equipped with dual detectors of fluorescence emission and amperometric oxidation. The characteristics of fluorescence emission and amperometric oxidation of tryptamine were retained even after its reaction with isocyanates. With this unique behaviour, all tryptamine derivatised isocyanates can be quantified using HPLC by employing a single, pure derivative, such as tryptamine derivatised hexamethylene diisocyanate as the calibration standard. This is especially important for analysing polymeric isocyanates when identical calibration standards are not always available. The applicability of this method for air sampling was evaluated by comparison with the established method of Bagon et al. involving 1-(2-methoxyphenyl)piperazine. Simulation of air sampling was performed in a Test Atmosphere Generation System by the vaporisation of toluene diisocyanate. Satisfactory results were obtained, indicating the applicability of this technique for the determination of total airborne isocyanates.
The effect of high cholesterol, high fat diet on rabbit plasma beta-VLDL and its interaction with macrophage.
Rabbits were fed on 1% cholesterol and 10% rape seed oil (group A) or lard (group B). Plasma cholesterol and triglyceride level of group A was strikingly lower than that of group B. No difference was found in the lipid composition of beta-VLDLs between both groups. However, apo E content of beta-VLDL from group A was higher and apo B (higher molecular weight) was lower than that of group B respectively. beta-VLDL of group A caused greater accumulation of cholesterol in mouse peritoneal macrophage than that of group B. Therefore, we assume that the good effect of poly-unsaturated fatty acid on atherosclerosis results not from decreasing accumulation of cholesterol in macrophages but from enhancing elimination of beta-VLDL by the liver.
The role of lipoprotein lipase in metabolism of normolipidemic very low density lipoprotein by macrophages.
It was demonstrated by using lipoprotein lipase (LPL) inhibitor benzene boronic acid (BBA) that LPL played a major role in the accumulation of triglycerides (TG) in mouse peritoneal macrophages exposed to rabbit normal very low density lipoproteins (N-VLDL). There wwere less free fatty acids (FFA) and much more N-VLDL-TG left in the media containing BBA than in the controls. TG accumulation in the cells was greatly diminished or even prohibited by BBA. Thus, we obtained direct evidence that LPL played a more important role than the receptor did in the uptake of N-VLDL-TG by mouse macrophages. The mechanisms of LPL action were discussed.
The effect of macrophage-conditioned medium on metabolism of normal very low density lipoprotein by fibroblasts.
Triglyceride was accumulated saturably in cultivated human fibroblasts exposed to increasing concentration of normal very low density lipoprotein (N-VLDL). Characterization of binding and degradation of 125I-N-VLDL by the cells indicated a direct uptake of intact N-VLDL particles via the cell surface receptor. Competition of unlabelled N-VLDL and LDL with 125I-N-VLDL in fibroblasts suggested that LDL receptor may be involved in this process. An unsaturable triglyceride accumulation in fibroblast induced by macrophage-conditioned medium containing N-VLDL was also observed. Intracellular triglyceride content, in this case, is linearly correlated with the concentration of N-VLDL in the medium and results mainly from re-esterification of fatty acid produced by hydrolysis of VLDL-triglyceride by lipoprotein lipase of macrophage.
Application of tryptamine as a derivatizing agent for airborne isocyanate determination. Part 2. Dual function of tryptamine for calibration and derivatisation of poly[methylene(polyphenyl isocyanate)] for quantification by high-performance liquid chromatography.
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Application of tryptamine as a derivatizing agent for airborne isocyanates determination. Part 1. Model of derivatisation of methyl isocyanate characterised by fluorescence and amperometric detection in high-performance liquid chromatography.
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Acute massive unilateral hydrothorax: a rare complication of chronic peritoneal dialysis.
We have described a case of massive unilateral hydrothorax during chronic peritoneal dialysis. The communication between the peritoneal and right pleural cavities was confirmed by injection into the peritoneal cavity of methylene blue, which promptly appeared in the pleural cavities. Peritoneal dialysis was continued, using decreased dialysate and a semisitting position. The possibility of continuing peritoneal dialysis in the patient with acute hydrothorax and in whom hemodialysis may be contraindicated is demonstrated.
Nitration of benzene as method for determining nitrites and nitrates in meat and meat products.
Nitrites and nitrates in meat and meat products are determined by nitration of benzene in the presence of sulfuric acid. Nitrites are converted to nitrates by permanganate oxidation prior to nitration. The heat of dilution originating from the sulfuric acid is sufficient to complete the reaction. The final product, nitrobenzene, is determined by gas-liquid chromatography and electron capture detection. Peak height quantitation is both accurate and reproducible. Interferences from impurities are rarely encountered, and may be differentiated easily. The method also has potential application to samples other than meat.
Metabolism of polyadenylic acid sequences during germination of blastocladiella emersoni: Zoospores.
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QT interval effects of cisapride in the clinical setting.
PURPOSE: The clinical effect of cisapride on QT intervals was prospectively studied. SUBJECTS: Consecutive adult patients were recruited in whom cisapride was indicated for gastroesophogeal reflux, gastric ulcer, duodenal ulcer, diabetic gastroparesis or chronic constipation refractory to laxatives. Exclusion criteria included disorders and medications affecting cardiac conduction, electrolyte homeostasis, drug clearance and membrane stability. METHODS: Seventy-five patients were included and followed at 1 to 2 week intervals. Patients took cisapride 5 mg thrice daily for 1 to 4 weeks (lower dose stage), followed by 10 mg thrice daily for another 1 to 4 weeks (higher dose stage). Twelve-lead ECGs were performed before commencing cisapride (group B), at completion of the lower dose stage (group L) and at completion of the higher dose stage (group H). RESULTS: No patients experienced presyncope or syncope. Seventeen patients failing to comply, and 7 complaining of abdominal discomfort or diarrhea were excluded, leaving 51 participants. Group H's corrected QT interval (QTc) was longer than group B's by 13+/-15 ms (P<0.001), and longer than group L's by 7+/-11 ms (P<0.001). Group L's QTc was longer than group B's by 7+/-21 ms (P<0.05). QT dispersion did not differ significantly among groups. Neither torsade de pointe nor ventricular tachycardia were noted in Holter monitoring of 33 patients during the higher dose stage. CONCLUSION: cisapride dose-dependently prolongs the QT interval. Further study is needed to examine the arrhythmogenicity of cisapride in higher doses and for longer durations.