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Biomedical subjects

S Chow

Publications and source records attributed to S Chow.

At least 55 records · Page 3Linked to original sources
PubMed1995

Flow cytometric determination of glutathione in clinical samples.

Glutathione-based processes are believed to be important determinants of resistance to cancer chemotherapy, and measurement of glutathione (GSH) in tumor tissue is therefore of clinical relevance. Flow cytometric methods have been developed for measuring cellular GSH content that appear to correlate well with biochemical determinations. These flow cytometric techniques are rapid, allow tumor cells to be distinguished from stromal elements, and show considerable intratumoral heterogeneity in human tumor cell GSH content. Although large prospective studies are required to determine the correlations between GSH content and treatment outcome in cancer patients, this approach is a powerful alternative to standard biochemical assays for GSH.

Aneuploidy↗
PubMed1995

Phylogenetic relationships between tuna species of the genus Thunnus (Scombridae: Teleostei): inconsistent implications from morphology, nuclear and mitochondrial genomes.

In order to infer phylogenetic relationships between tuna species of the genus Thunnus, partial sequences of the mitochondrial cytochrome b and ATPase genes were determined in all eight species. Supplemental restriction analysis on the nuclear rRNA gene was also carried out. Pacific northern bluefin tuna (Thunnus thynnus orientalis) was found to have mtDNA distinct from that of the Atlantic subspecies (T. t. thynnus) but very similar to that from the species albacore (T. alaluga). In contrast, no differentiation in nuclear genome was observed between the Atlantic and Pacific northern bluefin tunas. The Atlantic northern bluefin and southern bluefin tunas possessed mtDNA sequences very similar to species of yellowfin tuna group and not so similar to albacore and bigeye tunas which were morphologically assigned to the bluefin tuna group. The molecular data indicate that (1) mtDNA from albacore has been incorporated into the Pacific population of northern bluefin tuna and has extensively displaced the original mtDNA, and (2) albacore is the earliest offshoot, followed by bigeye tuna in this genus, which is inconsistent with the phylogenetic relationships between these tuna species inferred from morphology.

Animals↗
PubMed1995

Discordant P-glycoprotein antigen expression and transport function in acute myeloid leukemia.

Expression of the multidrug resistance efflux pump P-glycoprotein (Pgp) was measured in a series of AML patients using two flow cytometry methods. Transport function was assessed by measuring the modulating effect of the Pgp inhibitor cyclosporin A (CsA) on the cellular accumulation of daunorubicin, and Pgp antigen expression by surface immunofluorescence using the MRK-16 antibody. Both methods showed a wide range of values for Pgp expression between individual patients, but in contrast to a series of cell lines expressing Pgp there was no correlation between antigen expression and transport function in the clinical samples. As previously reported for chronic lymphocytic leukemia (CLL), pretreatment with neuraminidase markedly improved MRK-16 staining in some cases, indicating that abnormal glycosylation can cause epitope masking in AML blasts. Because experience with cell lines shows that Pgp expression is a continuous variable which correlates with the level of drug resistance, rather than the 'positive' or 'negative' which are frequently reported by clinical flow cytometry laboratories, we used a calibration procedure to estimate the actual number of Pgp molecules expressed in the AML samples. Despite the additional refinements of neuraminidase treatment and antigen quantification, the correlation between Pgp antigen expression and daunorubicin accumulation remained extremely weak (r = 0.11; P = 0.63). It is suggested that the assay for transport function can detect molecules that affect daunorubicin accumulation but are antigenically distinct from classical P-glycoprotein. Heterogeneity of multidrug resistance efflux pumps might in part explain the relatively weak prognostic significance of immunofluorescence detection of Pgp in AML patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Evaluation of methods for measuring cellular glutathione content using flow cytometry.

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with approximately 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labeling.

Animals↗
PubMed1994

Cellular pharmacology of D-3-azido-3-deoxy-myo-inositol, an inhibitor of phosphatidylinositol signaling having antiproliferative activity.

D-3-Azido-3-deoxy-myo-inositol (3AMI) is an inhibitor of the growth of v-sis-transformed NIH 3T3 cells but not of wild-type NIH 3T3 cells, whose effects may be mediated through the phosphatidylinositol-3'-kinase pathway. We studied some properties of the cellular pharmacology of 3AMI using high-specific-activity [3H]-3AMI. The uptake of [3H]-3AMI by wild-type NIH 3T3 and v-sis NIH 3T3 cells was similar. [3H]-3AMI was a substrate for phosphatidylinositol synthetase, with the maximal velocity (Vmax) being 1.0 nmol min-1 mg-1 and the Michaelis constant (Km) being 23 mM. Corresponding values obtained for [3H]-myo-inositol as a substrate were 5.5 nmol min-1 mg-1 and 3.2 mM. [3H]-3AMI was incorporated into the cellular inositol lipids of v-sis NIH 3T3 cells to a similar extent as that observed for [3H]-myo-inositol but was not incorporated into the inositol lipids of wild-type NIH 3T3 cells. The [3H]-3AMI incorporated by the v-sis NIH 3T3 cells was present in the phosphatidylinositol and phosphatidylinositol phosphate fractions but not in bisphosphorylated phosphatidylinositol. myo-Inositol antagonized the growth-inhibitory effects of 3AMI. The v-sis NIH 3T3 cells were found to be more sensitive than the wild-type NIH 3T3 cells to growth inhibition (without 3AMI) caused by the removal of myo-inositol from the medium. The results of the study suggest that 3AMI is an antimetabolite of myo-inositol. The relative sensitivity of v-sis NIH 3T3 and some other cells to 3AMI may be a reflection of increased myo-inositol requirements for the growth of these cells as compared with wild-type NIH 3T3 cells.

3T3 Cells↗
PubMed1994

Alcohol consumption by C57BL/6, BALB/c, and DBA/2 mice in a limited access paradigm.

Alcohol consumption by three inbred mice strains in a limited access condition was examined. Access to "Richter" tubes containing alcohol solution was restricted to 60 min per day in a drinking cage. Alcohol solution was given in escalating concentrations starting at 3% and ending at 12% w/v over several days. During the 12% phase, C57 mice consumed an average of 1.68 g/kg, while BALB and DBA mice consumed an average of 0.66 and 0.25 g/kg, respectively. The C57BL/6 mice achieved an average blood alcohol level (BAL) of 60 mg%, whereas the other two strains displayed negligible levels. The relationship between alcohol intake in a continuous and limited access as well as the utility of the limited access paradigm are discussed.

Alcohol Drinking↗
PubMed1993

The effects of selective blockade of delta and mu opiate receptors on ethanol consumption by C57BL/6 mice in a restricted access paradigm.

The effects of naltrexone, naltrindole (a selective delta opiate receptor antagonist) and beta-funaltrexamine (beta-FNA; a selective mu opiate receptor antagonist) on alcohol intake by C57BL/6 mice in a restricted access paradigm were examined. During the pretreatment baseline phase, mice consumed an average of 1.3 g/kg during 1 h access sessions to a 12% alcohol solution. Treatment with naltrexone reduced alcohol consumption to about 50% of that of the saline controls. Treatment with beta-FNA had no effect on alcohol consumption whereas naltrindole reduced consumption to the same extent as that observed with naltrexone. The pattern of findings indicate that naltrexone's ability to reduce alcohol consumption can be attributed to blockade of delta opiate receptors. Implications for treatment in human clinical trials are indicated.

Alcohol Drinking↗
PubMed1993

Studies of a sperm/placenta cross-reacting antigen, STX-10.

A monoclonal antibody, HSA-10 initially produced against acrosome-reacted human sperm was also shown to cross-react with human placenta/trophoblast. Transmission electron microscopy, as well as indirect immunofluorescent assay, demonstrated that HSA-10 was found to react with antigen on the inner acrosome of human sperm. The cognate antigen, designated as STX-10, was found to exist as an aggregate in the native form when analyzed by Sephacryl S-300 gel filtration chromatography. When purified by HSA-10-immunoaffinity chromatography from human placenta extract, STX-10 was found to be predominantly a group of glycoproteins with a subunit molecular mass in the range of 75 +/- 5 kDa, whereas an additional group of three proteins with subunit molecular mass less than 20 kDa were copurified from human sperm extract. A sandwich enzyme immunoassay was designed to quantitatively determine the immunoactivity of STX-10 in solution, using HSA-10 monoclonal antibody for coating and for signal detection via enzyme conjugation. Based on this assay, it was found that STX-10 could be detected only in human sperm and placenta extract, but not in any other human somatic tissues, such as serum, brain, heart, muscle, kidney and liver. The immunoactivity of STX-10 was found to be sensitive to proteolytic digestion, low pH, in the presence of reducing agent, but resistant to treatment with sodium periodate. This observation suggests that HSA-10 specific epitope is a peptide in nature and not a carbohydrate moiety. Results of antifertility studies revealed that HSA-10 significantly inhibited human sperm penetration to zona-free hamster ova. Thus, the results of this study are consistent with those of WHO Workshop evaluations that seem to suggest that STX-10 is a highly gamete-specific antigen localized on the inner acrosome of human sperm and in human trophoblast/placenta. Therefore, it may play an important role during human fertilization and embryo development.

Animals↗
PubMed1992

Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid.

A method for measuring lipid peroxidation using time resolved flow cytometry is described. Because of its chemical nature, the naturally fluorescent fatty acid cis-parinaric acid is readily consumed in lipid peroxidation reactions. It could be loaded into Chinese hamster ovary cells in a time and concentration dependent manner at 37 degrees C, with 5 microM for 60' giving consistent, bright fluorescence without evidence of cytotoxicity. Examination of cells by fluorescence microscopy showed diffuse staining of surface and internal membranes. Cells were maintained at 37 degrees C while being examined in an Epics Elite flow cytometer equipped with a 325 nm HeCd laser, and parinaric acid fluorescence at 405 nm was measured over time. Addition of the oxidant tert-butyl hydroperoxide resulted in a burst of intracellular oxidation, shown by simultaneously loading the cells with dichlorofluorescein, and loss of parinaric fluorescence over time. This was followed by cell death, indicated by loss of forward light scatter and uptake of propidium iodide. Pretreatment of the cells with the antioxidant alpha-tocopherol, 200 microM, reduced the rate of loss of parinaric acid fluorescence and delayed the onset of cell death. Simultaneous biochemical determination of the lipid peroxidation breakdown product malondialdehyde confirmed a close temporal relationship with loss of parinaric acid fluorescence, both with and without alpha-tocopherol pretreatment and suggested that the flow cytometric assay for lipid peroxidation is of comparable sensitivity. The mitochondrial stain dodecyl acridine orange and the cyanine dye DiOC(6)3 were combined with cis-parinaric acid staining and could be excited by the latter using resonance energy transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Increased radiation dose at mammography due to prolonged exposure, delayed processing, and increased film darkening.

Four single-emulsion films introduced over the past 2 years--Du Pont Microvision, Fuji MiMa, Konica CM, and Eastman Kodak OM--were compared with Eastman Kodak OM SO-177 (Min-RE) film to evaluate their varying effects on mean glandular dose of reciprocity law failure due to prolonged exposure, delayed processing, and increased film darkening as a result of increased radiation exposure to improve penetration of glandular tissue. Exposures over 1.3 seconds led to increased radiation doses of 20%-30%. Delays in processing of 6 hours decreased processing speed by 11%-32% for all films except Du Pont Microvision. Optical density increases of 0.40 required 20%-30% more skin exposure for all five films. Optimal viewing densities were also evaluated and found to be different for each of the five films. Mammographers need to be aware of these differences in mammographic films to achieve maximum contrast at mammography.

Breast↗
PubMed1990

Expression of ras oncogene leads to down-regulation of protein kinase C.

The effect of mutated c-Ha-ras expression on Ca2+ and phospholipid-dependent protein kinase C (PKC) activity during the process of transformation was analysed using an inducible metallothionein-ras hybrid oncogene system. A close correlation was found between the timing of ras expression and the loss of PKC enzymatic activity measured in a cell-free system. Examination of the subcellular distribution of the enzyme in inducible and constitutive ras-transformants revealed that expression of ras was associated with an apparent translocation of PKC to the plasma membrane concomitant with down-regulation of PKC enzymatic activity in particulate as well as cytosolic fractions. Quantitation of PKC protein utilizing a PKC-specific antiserum showed that ras expression was associated with a decrease in the total amount of PKC protein present in the cell. We conclude that transformation by c-Ha-ras is accompanied by down-regulation of PKC activity and that the basis of this effect may, to a large extent, lie in the down-regulation of the amount of PKC protein.

Animals↗
PubMed1987

The control of lambda DNA terminase synthesis.

Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E. coli. To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured. In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified. It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon. Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits. It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation. The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production. This result suggests a role for cos in the control of late gene expression. The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques.

Bacteriophage lambda↗
PubMed1987

The overproduction of DNA terminase of coliphage lambda.

An artificial operon containing the genes coding for the two subunits of lambda DNA terminase, Nul and A, has been constructed. Derivatives of plasmid pBR322 served as the cloning vehicles. The transcription is driven by the pL promoter of phage lambda, and translation of the terminase genes was made efficient by the replacement of the wild-type ribosome-binding sites for those of lambda genes cII and/or D. The operon also carries the oL operator, and this enables regulation of its expression by a thermosensitive repressor. The synthesis of genes Nul and A products is extremely efficient upon derepression. Within 40 min after induction of the operon, the two subunits comprise about 20% of the total cellular protein mass. Crude extracts prepared from these overproducing strains are at least 100 times more active than extracts prepared from induced lambda lysogens in both promotion of lambda DNA packaging and cosmid cleaving. The ability to produce highly concentrated terminase would enormously facilitate the study of its structure and mechanism of action. These extracts are also extremely useful in techniques such as lambda DNA packaging, cosmid mapping and cosmid linearization to improve efficiency of integration into mouse eggs.

Bacteriophage lambda↗
PubMed1985

Dermatomyositis and pregnancy.

Dermatomyositis is a rare medical disease complicating pregnancy. Three patients are reported with five successful pregnancies complicated by dermatomyositis. Although pregnancy loss may arise when dermatomyositis complicates pregnancy, successful therapy will permit a satisfactory result. Optimal pregnancy success can be anticipated when pregnancy is undertaken with disease in remission and prospective maternal and fetal evaluation is undertaken.

Adult↗
PubMed1981

Hailey-Hailey disease. Report of a case treated surgically.

A 51-year-old man with Hailey-Hailey disease was treated with a split thickness skin graft of the left axilla. The dramatic improvement in the grafted area is contrasted with the continued disease activity in the untreated axilla and other intertriginous areas. Local excision and grafting, with a resultant decrease in sweating and maceration, is an effective form of therapy for Hailey-Hailey disease.

Axilla↗