Search PubMed⌕ Search

Biomedical subjects

S R Williams

Publications and source records attributed to S R Williams.

At least 181 records · Page 10Linked to original sources
PubMed1986

31P NMR analysis of intracellular pH of Swiss Mouse 3T3 cells: effects of extracellular Na+ and K+ and mitogenic stimulation.

Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional 31P and 19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4 mM 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pHc on external Na+ concentration (CoNa). PHc also depended on intracellular Na+ concentration (CcNa). Increasing ccNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducing CoNa produced a larger acid shift in pHc than with external K+ present. Comparison of separate preparations indicated that pHc was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pHc of Swiss mouse 3T3 cells using 31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.

Animals↗
PubMed1986

Identification of processing events in the synthesis of platelet-derived growth factor-like proteins by human osteosarcoma cells.

The human osteosarcoma-derived cell line U-2 OS expresses c-sis mRNA and synthesizes platelet-derived growth factor (PDGF)-like proteins. Pulse-chase experiments indicate that proteins of 23 kDa and 180 kDa are synthesized first. The 23-kDa protein undergoes dimerization and proteolysis, giving rise to the 30-kDa dimeric protein secreted by the cells. The 180-kDa protein is proteolytically cleaved in a complex series of steps that give rise to several intracellular species. It is also the likely precursor of high molecular mass PDGF-like or PDGF-associated proteins secreted by these cells. The processing and secretion of the 180-kDa protein is slower than that of the 23-kDa protein. Subcellular fractionation and studies with the antibiotic monensin indicate that the processing events occur in the Golgi-endoplasmic reticulum compartment of U-2 OS cells.

Cell Line↗
PubMed1986

Tissue metabolism studied in vivo by nuclear magnetic resonance.

N.m.r. is finding increasing application as a non-invasive method of studying metabolism in vivo. 31P n.m.r. is particularly useful for studies of energy metabolism, while other nuclei, in particular 1H, 13C and 19F, can provide additional information about certain aspects of cellular metabolism that are not amenable to investigation by 31P n.m.r. The major problem with n.m.r. is lack of sensitivity. The main implications of this are first that signals can only be detected from metabolites that are present at fairly high concentrations, and secondly that the spatial resolution for localized studies, particularly of internal regions of the body, is limited. N.m.r. could considerably enhance our understanding of metabolic abnormalities in disease, but the precise role of such metabolic studies in a clinical setting remains to be evaluated.

Animals↗
PubMed1986

Intracellular pH in Dictyostelium: a 31P nuclear magnetic resonance study of its regulation and possible role in controlling cell differentiation.

Intracellular pH (pHi) has been measured in Dictyostelium discoideum cells by 31P nuclear magnetic resonance. Ax2 cells, newly harvested from growth medium, maintained a pHi of 7.33 +/- 0.04 (17) at an extracellular pH ranging from 3.5 to 6.5. Below pH 3.5 the cells tend to lyse, whereas at pH values above 6.5 their pHi rises though they remain viable. pHi regulation in acid medium is not dependent on external Na+ or any other inorganic ion and so most probably involves the electrogenic plasma membrane proton pump. No significant change in pHi was detected during development through to the slug stage. Mature stalk cells gave a very acidic phosphate signal (pH less than or equal to 5.5) which was probably vacuolar in origin. Indirect experiments had suggested that pHi might regulate the development of Dictyostelium cells, with low pHi favouring stalk cell and high pHi favouring spore cell differentiation. In particular, two inhibitors of the plasma membrane proton pump, diethylstilbestrol and zearalenone, had been shown to be stalk cell inducers. In the present studies measurements of pHi of cells exposed to these inducers failed to detect the expected drop in pHi. In addition, DIF-1 (a low Mr factor), the natural inducer of stalk cell formation, caused, if anything, a slight alkalinization of the cells. Thus the original theory linking pHi and cell differentiation is not supported by these results and therefore appears to require some modification. Finally, extract experiments revealed the existence of two unidentified abundant phospho-compounds with resonant frequencies close to inorganic phosphate. The existence of these compounds can complicate the interpretation of spectra gained from living Dictyostelium cells.

Cell Differentiation↗
PubMed1985

Cloned mouse ribonucleotide reductase subunit M1 cDNA reveals amino acid sequence homology with Escherichia coli and herpesvirus ribonucleotide reductases.

We have isolated and sequenced overlapping cDNA clones containing the entire coding region of mouse ribonucleotide reductase subunit M1. The coding region comprises 2.4 kilobases and predicts a polypeptide of 792 amino acids (Mr 90,234) which shows striking homology with ribonucleotide reductases from Escherichia coli and the herpesviruses, Epstein-Barr virus and herpes simplex virus. The homologies reveal three domains: an N-terminal domain common to the mammalian and bacterial enzymes, a C-terminal domain common to the mammalian and viral ribonucleotide reductases, and a central domain common to all three. We speculate on the functional basis of this conservation.

Amino Acid Sequence↗
PubMed1984

Human purine nucleoside phosphorylase cDNA sequence and genomic clone characterization.

The isolation of a cDNA clone containing the complete coding region for human purine nucleoside phosphorylase (PNP) has been described previously. In this report we present the nucleotide sequence of this cDNA clone and compare the derived amino acid sequence, encoding a protein of 32 kilodaltons, with the published amino acid composition. Using a fragment of the cDNA clone as a probe, human PNP genomic clones from a bacteriophage lambda library have been isolated and the structural organization of the wild type PNP gene determined.

Base Sequence↗
PubMed1984

Cloning of human adenosine deaminase cDNA and expression in mouse cells.

A previously isolated partial cDNA sequence encoding human adenosine deaminase (ADA) was used to probe a cDNA library prepared from human cultured cell mRNA. Clones containing a combined overlapping length of 1462 bp were isolated and sequenced. One of these was found to include the entire ADA coding region. An open reading frame consisting of 363 codons was identified, predicting a polypeptide of Mr 40762. A mammalian expression plasmid was constructed, positioning the ADA coding sequence to be under transcriptional control of the mouse metallothionein promoter. Transfection of cultured mouse L-cells with this plasmid resulted in the acute expression of human ADA enzymatic activity, as assayed by isoelectric focusing.

Adenosine Deaminase↗
PubMed1984

Evidence for functional platelet-derived growth factor receptors on MG-63 human osteosarcoma cells.

The specific interaction of platelet-derived growth factor (PDGF) with the human osteosarcoma cell line MG-63 was studied. Scatchard analysis of 125I-PDGF binding to MG-63 cells indicated there were 32,000 specific PDGF-binding sites per cell with a Kd of 2.4 X 10(-11) M. Unlabeled PDGF blocked the specific binding of labeled PDGF to MG-63 cells at concentrations greater than 1 ng/ml. When assayed for phosphorylation of MG-63 membrane vesicles, PDGF was shown to stimulate a dose-dependent phosphorylation of a protein (phosphoprotein with a molecular weight of 185,000) which was stable in 1 M NaOH. In the absence of PDGF, a prominent alkali-stable phosphoprotein with a molecular weight of 116,000 was noted. PDGF also stimulated a dose-dependent increase in [3H]aminoisobutyric acid uptake, [3H]thymidine incorporation, and cell proliferation. When tested for secretion of PDGF-like factors, the mitogenic activity of MG-63-conditioned serum-free medium was not blocked by anti-PDGF antiserum. Concentrated MG-63-conditioned medium did not compete with 125I-PDGF for specific receptor sites on diploid fibroblasts. Therefore, MG-63 osteosarcoma cells have functional PDGF receptors and do not secrete PDGF-like mitogens.

Amino Acids↗
PubMed1983

Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli.

We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled clones was used to transform PNP-deficient Escherichia coli cells, and those transformants that phenotypically expressed PNP were isolated on selective media. The presence of human PNP in the selected bacterial cells was detected by immunoprecipitation with human PNP antibody.

Base Sequence↗
PubMed1982

A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain, and the ionophore R02-2985 (X537A) in the isolated, perfused rat heart.

1. Some metabolic effects of increased mechanical activity by the Langendorff-perfused rat heart have been characterized using 31P-NMR. Mechanical activity was increased by infusion of ouabain (0.9-7.0.10(-5) M), the ionophore R02-2985 (1.10(-5) M) or epinephrine (5.10(-8) M). 2. Similar metabolic changes accompanied infusion of each of the positive inotropic agents into hearts perfused with buffer containing 11 mM glucose as the substrate. In each case phosphocreatine concentrations decreased. During the period of epinephrine infusion the phosphocreatine began to recover its original concentration, although there were no significant changes in mechanical activity. 3. Comparisons of the metabolic changes accompanying the positive inotropic and chronotropic effects of epinephrine were made between hearts perfused with either glucose (11 mM), acetate (5 mM) or lactate (5 mM). A time-dependent decrease in phosphocreatine concentrations also accompanied infusion of epinephrine into hearts perfused with lactate as the sole exogenous substrate, but no statistically significant metabolite changes were observed after identical epinephrine infusions with acetate as the substrate. 4. Calculations of the concentration of free ADP assuming equilibrium in the creatine phosphokinase reaction allows estimation of the cytosolic phosphate potential ([ATP]/[ADP][Pi]), which appears to be dependent on the number of factors, including the nature of the exogenous substrate and the level of mechanical activity. 5. Thus, we conclude that there is no general correlation between the phosphate potential and the mitochondrial respiratory rate in the perfused rat heart.

Animals↗
PubMed1982

The effects of insulin on myocardial metabolism and acidosis in normoxia and ischaemia. A 31P-NMR study.

1. The effect of insulin on the perfused rat heart during normoxia and total ischaemia was studied by 31P-NMR. 2. During normoxic perfusion, insulin increased the phosphocreatine to ATP ratio at the expense of Pi, when glucose was the substrate. No change was observed when acetate was used as the sole substrate. the intracellular pH (as measured from the position of the 2-deoxyglucose 6-phosphate resonance peak) was unaffected by insulin treatment. 3. Infusion of insulin prior to ischaemia caused an increase in the rate and extent of acidosis during the period of no flow while the rate of ATP depletion was decreased. 4. Freeze-clamped studies showed an increase in glycogen levels upon insulin treatment of the glucose perfused rat heart. During ischaemia, a decrease in glycogen content concomitant with an increase in lactate was observed. The accessibility of glycogen to phosphorylase during ischaemia is increased as a result of insulin treatment. The control of glycolysis during ischaemia is discussed with respect to the content and structure of glycogen in heart tissue.

Acidosis↗
PubMed1982

Employer-based health insurance.

Employer-based health insurance (insurance that is purchased by employers for their employees and financed through employer or joint employer-employee contributions) is currently subsidized in part by the federal government through tax exclusions for employer contributions to employee health insurance plans. This subsidization costs the federal government close to 10 billion dollars a year in lost revenues. Many proposed national health insurance plans assign a key role to employer-based health insurance as a vehicle for financing health care. Federal subsidization of employer-based health insurance and plans that assign employers a key role in the administration of a national health insurance plan both assume that private industry acts to realize federal health policy goals-- particularly cost containment--in administering health insurance plans. Little is known, however, about how employers go about selecting the plans they offer their employees or about the incentives and disincentives regarding cost of care than are created by employer-based health insurance. Existing evidence suggests that rather than helping to contain health care costs, employer-based health insurance may be partly responsible for their present escalation. In addition, employer-based health insurance may not be the most equitable way to implement a national health insurance plan.

Blue Cross Blue Shield Insurance Plans↗