Biomedical subjects

W Marsh

Publications and source records attributed to W Marsh.

17 recordsLinked to original sources

PubMed1990

A quantitative assay for neomycin phosphotransferase activity in plants.

A sensitive, simple, and quantitative assay for determining neomycin phosphotransferase (NPT) activity in plant cell extracts is described. The procedure retains the simplicity of previously published methods, yet offers up to a 140-fold increase in sensitivity. This increase is due to (1) the addition of bovine serum albumin (BSA) to the assay mixture, (2) desalting of crude maize extracts to remove a low-molecular-weight inhibitor of the enzyme, and (3) use of a different extraction buffer and an improved extraction procedure to liberate more enzyme from the cells. This method has been used successfully to detect and quantitate both stable and transient expression of NPT in transgenic tobacco and maize tissue.

Animals↗

PubMed1987

Adriamycin resistance in HL60 cells and accompanying modification of a surface membrane protein contained in drug-sensitive cells.

HL60 cells resistant to Adriamycin contain a 32P-labeled, Mr 150,000 surface membrane protein (p150) which is not detected in cells sensitive to drug. The levels of phosphorylation of this protein increase with increasing levels of resistance. Analysis of plasma membranes prepared from cells labeled with [14C]glucosamine shows, however, that both sensitive cells and those exhibiting an 80-fold increase in drug resistance contain essentially identical levels of a highly glycosylated Mr 150,000 protein. Identical results are obtained when cells are labeled with [14C]galactose or [14C]mannose. Limited proteolytic digestion of [14C]glucosamine-labeled p150 from sensitive and resistant cells shows that the glycopeptides formed are identical. Additional studies involving binding of proteins to insolubilized lectin indicate that 32P-labeled p150 is glycosylated. Polyacrylamide gel electrophoresis of p150 followed by silver staining shows no difference in the levels of this protein in sensitive and 80-fold drug-resistant cells. Further studies show that two-dimensional tryptic peptide maps of 125I-labeled p150 of sensitive and resistant cells are essentially the same. It has also been found that treatment of cells with 12-O-tetradecanoylphorbol-13-acetate followed by [14C]glucosamine labeling results in a selective decrease in the glycosylation of p150 of sensitive and resistant cells. TPA has an identical effect on the phosphorylation of p150 in cells resistant to drug. HL60 cells have also been examined for the presence of the Mr 170,000 to 180,000 P-glycoprotein. Using immunoblot analysis with a monoclonal antibody directed against the P-glycoprotein we did not detect the presence of this protein in membranes of drug-sensitive or -resistant HL60 cells. The results of this study suggest that Adriamycin resistance in HL60 cells may be related to a modified form of a protein contained in cells sensitive to drug. Proteins active in drug resistance in this system may be distinct from those described for other cell lines.

Cell Line↗

PubMed1987

Liver transplantation in older patients.

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Adult↗

PubMed1987

Orthotopic liver transplantation for alpha-1-antitrypsin deficiency: an experience in 29 children and ten adults.

Thirty-nine patients (29 children and ten adults) underwent OLT for liver disease associated with A1AD from March 1980 to March 1986. Thirty of thirty-six patients (83%) with available data were homozygous phenotype PiZZ. The other six were Pi heterozygotes, being either PiMZ or PiSZ. The mean A1A activity in homozygous and heterozygous patients was 38.8 mg/dL and 114.3 mg/dL respectively. Eight patients died during the first 3 months after OLT (20%). The 5-year actuarial survival is 83% and 60% in pediatric and adult recipients respectively. Today 30 (76%) of the recipients are alive, with follow-ups of 8 to 64 months (average 27 months). The quality of life in the surviving patients is excellent.

Adolescent↗

PubMed1986

Dimethylsulfoxide, retinoic acid and 12-O-tetradecanoylphorbol-13-acetate induce a selective decrease in the phosphorylation of P150, a surface membrane phosphoprotein of HL60 cells resistant to adriamycin.

Studies have been carried out to analyze protein phosphorylation in membranes isolated from adriamycin resistant HL60 cells which have been grown for various time periods in the presence of dimethylsulfoxide (DMSO), retinoic acid (RA) or 12-O-tetradecanoylphorbol-13-acetate (TPA). The results show that membranes isolated from cells treated with these agents are defective in the phosphorylation of P150, a membrane phosphoprotein associated with drug resistance in HL60 cells. This response is highly selective since only a few membrane proteins show decreased phosphorylation levels under these conditions. Magnesium dependent protein kinase activity in membranes from cells treated with DMSO, RA or TPA is not altered relative to untreated membranes under conditions where there is a major decrease in P150 phosphorylation. Additional studies also show that treatment of resistant cells with TPA results in a major decrease in the in vivo phosphorylation of P150. These results thus demonstrate that agents capable of inducing differentiation in HL60 cells can selectively modulate the phosphorylation of P150. This system should be of value in clarifying mechanisms involved in the phosphorylation of this protein.

Dimethyl Sulfoxide↗

PubMed1986

Isolation and characterization of adriamycin-resistant HL-60 cells which are not defective in the initial intracellular accumulation of drug.

Two human leukemia cell lines (Molt-4 and HL-60) have been used for establishing cells which exhibit a low level resistance to Adriamycin. Analysis of drug uptake patterns shows that the Molt-4 resistant cells are defective in the initial intracellular accumulation of drug. In contrast to Molt-4 the levels of drug which accumulate in the sensitive and resistant HL-60 cells during a 60-min incubation period are essentially the same. However, when incubations are continued there is a major reduction in intracellular drug levels in the resistant cell. Further studies show that resistant cells incubated in the presence of drug for extended time periods efflux drug at a rate considerably greater than that exhibited by the sensitive parent line. Similar efflux patterns are obtained with nuclei isolated from drug-sensitive and -resistant cells. Additional studies using an in vitro phosphorylation system demonstrate distinct protein changes in membranes of Molt-4 and HL-60 resistant cells. Thus, we have found that a membrane fraction from the Molt-4 resistant line contains a Mr 170,000 protein which is not detected in a similar fraction from cells sensitive to drug. HL-60 resistant membranes contain two proteins with molecular weights of 150,000 and 120,000 which are also not found in membranes from drug-sensitive cells. The results of this study suggest that drug resistance in HL-60 cells is related to an efflux mechanism which is triggered only after cells are exposed to drug for prolonged periods.

Cell Line↗

PubMed1985

In vitro phosphorylation and the identification of multiple protein changes in membranes of Chinese hamster lung cells resistant to adriamycin.

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Amino Acids↗

PubMed1985

Evidence for the involvement of two distinct membrane proteins in adriamycin resistance in Chinese hamster lung cells.

Chinese hamster lung cells resistant to Adriamycin were labeled with inorganic [32P]orthophosphate and thereafter incubated with low levels of N-ethylmaleimide. Plasma membranes and endoplasmic reticulum were isolated and the phosphorylated proteins were analyzed after polyacrylamide gel electrophoresis. The results demonstrate that both plasma membranes and endoplasmic reticulum from resistant cells contain two highly phosphorylated proteins [Mr 180,000 (p180) and Mr 220,000 (p220)] which are present in very low levels in these membrane fractions prepared from drug sensitive cells. p220 is present in much higher levels in the endoplasmic reticulum as compared to the plasma membranes whereas p180 is equally distributed in these two membrane fractions. When resistant cells revert to drug sensitivity there is a parallel loss in the phosphorylation levels of p180 and p220. Labeling of membrane proteins with 125I in the presence of chloramine-T also reveals that p180 and p220 are present in significantly greater levels in resistant membranes as compared to similar fractions prepared from drug sensitive cells. Partial digests of phosphorylated p180 and p220 produced with chymotrypsin or V8 protease reveal that each protein has a distinct phosphopeptide pattern. Both p180 and p220 are phosphorylated exclusively at serine residues. The results of this study therefore suggest that resistance to Adriamycin in Chinese hamster lung cells requires the involvement of two distinct proteins which are both bound to cell membranes.

Animals↗

PubMed1974