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Biomedical subjects

N Williams

Publications and source records attributed to N Williams.

At least 343 records · Page 19Linked to original sources
PubMed1984

Proton ATPase of rat liver mitochondria: a rapid procedure for purification of a stable, reconstitutively active F1 preparation using a modified chloroform method.

A method is described for the purification of rat liver F1-ATPase by a modification of the chloroform extraction procedure originally described by Beechey et al. (Biochem. J. (1975) 148, 533). Purified liver membrane vesicles are extracted with chloroform in the presence of ATP and EDTA. The procedure yields pure F1 in only 2-3 h without the necessity of ion-exchange chromatography. The enzyme exhibits the alpha, beta, gamma, delta, and epsilon bands characteristic of F1-ATPase. It has a high ATPase specific activity, and is reconstitutively active, catalyzing high rates of ATP synthesis. Significantly, it can be readily crystallized. If desired, the enzyme can be passed over a gel filtration column to place it in a stabilizing phosphate-EDTA buffer, lyophilized and stored indefinitely at -20 degrees C.

Animals↗
PubMed1984

The proton adenosinetriphosphatase complex of rat liver mitochondria. Temperature-dependent dissociation-reassociation of the F1-ATPase subunits.

The soluble F1 moiety of the rat liver mitochondrial proton ATPase dissociates into two easily separable fractions when cold treated and then warmed. One fraction is soluble in potassium phosphate buffer, pH 7.4, whereas the other is insoluble. Neither of these two fractions alone can catalyze ATP hydrolysis under assay conditions optimal for the native F1-ATPase. The insoluble fraction when resolved via sodium dodecyl sulfate--polyacrylamide gel electrophoresis is shown to be composed of only alpha and gamma subunits. When this fraction is chromatographed on Sephadex G-75, it is resolved into an alpha gamma complex and into the alpha subunit alone. The soluble fraction when resolved in the same electrophoretic system is shown to contain the remaining subunits, beta, delta, epsilon, and some gamma. This fraction is resolved into two major components by chromatography on Sepharose CL-6B, a beta gamma complex and beta subunit alone. The cold-dissociated enzyme can be readily associated when the temperature is raised to 20 degrees C. In the presence of either ATP or MgATP the enzyme completely regains its original ATPase specific activity. In contrast, Mg2+ is only about 15% effective in restoring ATPase activity. The results presented here define conditions for the dissociation and reassociation of the major subunits comprising the F1-ATPase of rat liver and thus provide a unique system among mammalian enzymes for testing the function of individual subunits. In addition, they strongly indicate that neither the alpha nor beta subunits, nor complexes of these subunits with the gamma subunit, are capable of catalyzing ATP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1984

The role of erythropoietin, thrombopoietic stimulating factor, and myeloid colony-stimulating factors on murine megakaryocyte colony formation.

Various growth factors including purified erythropoietin, colony-stimulating factor (CSF-1), and granulocyte-macrophage CSF (GM-CSF) were tested for their ability to stimulate megakaryocytopoiesis. Four separate preparations of erythropoietin were tested in highly defined cell culture medium. One unit of purified material stimulated small but significant numbers of megakaryocyte colonies, both in serum-containing and in serum-free cultures. All other erythropoietin preparations failed to induce megakaryocyte colony formation. Purified erythropoietin showed no synergistic activity with either WEHI-3 cell conditioned medium (WEHI-3CM, a source of both megakaryocyte CSF and megakaryocyte-potentiating activity) or P388D1 cell conditioned medium (P388D1CM, a preparation containing megakaryocyte potentiator). Partially purified thrombopoietic stimulatory factor did not stimulate directly megakaryocyte colony formation, but acted together with WEHI-3CM, augmenting the number of clonable progenitors detected. Optimal activity was observed at 12-25 micrograms protein per plate. Myeloid growth factors (CSF-1 and GM-CSF) were inactive in the murine megakaryocyte assay. The data show lineage specificity for the myeloid stimulators, but a purified erythropoietin preparation was found to stimulate a small level of megakaryocytopoiesis.

Animals↗
PubMed1983

Colony-stimulating factors and regulation of macrophage tumoricidal and microbicidal activities.

Conditioned medium from antigen- or mitogen-stimulated spleen cells, lymphokines, contained factors that induced formation of granulocyte and macrophage colonies in cultures of bone marrow cells (CSF). Lymphokines also contained factors that induced macrophage non-specific tumoricidal activity against fibrosarcoma 1023, antibody-dependent tumoricidal activity against lymphoma 18-8, and antimicrobial activities against amastigotes of the protozoan parasite, Leishmania tropica. The factors that regulated macrophage effector functions, however, were different from those that induced colony formation, and could be distinguished from CSF by Sephadex gel chromatography or heat sensitivity. To further analyze a role for CSF in induction of macrophage effector activities, conditioned medium from several nonlymphoid cell sources (L-929, WEHI-3, and endotoxin-treated lung cells) were assayed for CSF activities and capacity to induce tumoricidal and microbicidal activities. Conditioned medium that contained either macrophages CSF (CSF-1) or the factor that induced formation of both macrophage and granulocyte colonies failed to activate macrophages for effector activities against fibrosarcoma 1023, lymphoma 18-8, and L. tropica amastigotes (either resistance to infection or intracellular destruction). These data suggest that CSF has no direct role in activation of macrophages for tumoricidal and microbicidal activities against these targets.

Animals↗
PubMed1983

The nature of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated hemopoiesis, colony stimulating factor (CSF) requirement for colony formation, and the effect of TPA on [125I]CSF-1 binding to macrophages.

The tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to act both independently of and synergistically with the mononuclear phagocyte specific colony stimulating factor (CSF-1) to stimulate the formation of macrophage colonies in cultures of mouse bone marrow cells. In contrast, TPA did not synergize with other CSF subclasses that stimulate the formation of eosinophil, eosinophil-neutrophil, neutrophil, neutrophil-macrophage, and macrophage colonies, nor with either of the two factors required for megakaryocyte colony formation, megakaryocyte CSF, and megakaryocyte colony potentiator. In serum-free mouse bone marrow cell cultures TPA retained the ability to independently stimulate macrophage colony formation. However, TPA-stimulated colony formation was suboptimal and delayed in serum-free cultures that could support optimal colony formation in the presence of CSF-1. In addition, TPA did not directly compete with [125I]CSF-1 at 4 degrees C for its specific, high-affinity receptor on mouse peritoneal exudate macrophages. However, a 2-hour preincubation of the cells with TPA at 37 degrees caused almost complete loss of the receptor. Thus, TPA is able to mimic CSF-1 in its effects on CSF-1 responsive cells in some aspects (the spectrum of target cells, the morphology of resulting colonies, and the ability to down-regulate the CSF-1 receptor) but it is not able to mimic CSF-1 in other ways (TPA alone cannot stimulate the full CSF-1 response, TPA does not stimulate the most primitive CSF-1 responsive cells, and TPA does not bind to the CSF-1 receptor).

Animals↗
PubMed1983

Is a calmodulin-opiopeptide interaction related to the mechanism of opioid action?

The effect of several opioids: methadone, etorphine, beta-endorphin and D-ala2met enkephalin on Ca++/calmodulin stimulation of enzyme activities either in pure solution (cyclic nucleotide phosphodiesterase) or in striatal membranes (protein kinases in synaptic membranes) were compared to see if a direct opioid/calmodulin interaction could eliminate the stimulation of enzyme activity as part of the mechanism by which opioids alter ion flow and neurotransmitter release. In other experiments, in which endogenous phosphorylation of proteins in striatal synaptic membranes was altered by opioid treatments, the possibility of restoring protein kinase activity to normal levels in the membrane preparation by supplementation with calmodulin at optimal Ca++ concentration was examined. Some opioids (methadone and D-ala2met enkephalin) did not inhibit calmodulin-stimulated phosphodiesterase, which suggests that they were not able to bind to calmodulin. In addition, it was not possible to restore decreases in protein kinase activity to normal levels by adding calmodulin to the assay in the presence of optimal Ca++. We conclude that a direct binding of opioids to calmodulin is not a general mechanism of opioid action, although the binding may participate in the action of some neuropeptides, including beta-endorphin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Evaluation of a new range of air drawover vaporizers. The 'PAC' series--laboratory and 'field' studies.

The 'Ether Pac' and 'Fluo Pac' temperature compensated vaporizers have been evaluated in the laboratory and the 'field'. Rigorous testing has demonstrated that these vaporizers are robust and reliable. Shaking, tilting and overturning do not significantly affect their performance. Both vaporizers deliver lower concentrations of the vapour than the setting on the vaporizers at low tidal volumes (100 ml). The 'Ether Pac' vaporizer output declines progressively with ambient temperatures below 23 degrees C and a similar result occurs with the 'Fluo Pac' at temperatures below 20 degrees C. Clinical trials in Nepal, Kenya, Burma and the UK have demonstrated that, when halothane is used, oxygen enrichment is necessary during spontaneous and controlled ventilation. When ether is used with controlled ventilation oxygen enrichment is probably not necessary even with ambient pressures as low as 619 mmHg.

Adolescent↗
PubMed1982

Two-factor requirement for murine megakaryocyte colony formation.

WEHI-3 cell-conditioned medium with the capacity to stimulate megakaryocyte colony formation was separated by Sephadex G-150 column chromatography. The development of colonies containing megakaryocytes was observed only when mixing experiments were performed. Individual fractions did not support megakaryocyte colony growth. The two factors in WEHI-3 CM required for megakaryocyte colony growth had apparent average molecular weights of 35,000 daltons (megakaryocyte CSF) and 100,000 daltons (megakaryocyte potentiator). The results were confirmed in serum-free conditions in which colonies were directly identified in the cultures by acetylcholinesterase staining. Two growth factors may be necessary for the genesis of megakaryocytic colonies.

Animals↗
PubMed1982

Immature megakaryocytes in the mouse: in vitro relationship to megakaryocyte progenitor cells and mature megakaryocytes.

An assay describing conditions for the maturation of single immature megakaryocytes in vitro is reported. Enriched populations of small, relatively immature megakaryocytes have been found to develop into single, mature megakaryocytes by 60 hours in semisolid agar cultures. Continued incubation of these cells did not lead to the formation of colonies within 5-7 days. Maturation was indicated by increasing cell size and cytoplasmic and acetylcholinesterase content. Factors stimulating the development of immature megakaryocytes were found in preparations of human embryonic kidney cell-conditioned media (a source of in vivo Thrombopoietic Stimulatory Factor), peritoneal exudate cell-conditioned medium, lung-conditioned medium, or bone marrow cellular sources of activity (adherent cells or cells that sediment at 5-6 mm hr-1). Immature megakaryocytes cultured serum free responded to sources of an auxiliary megakaryocyte potentiating activity by developing into single, large megakaryocytes but did not respond to a megakaryocyte colony-stimulating factor devoid of detectable potentiator activity present in WEH1-3-conditioned medium. In contrast, serum-free proliferation of the megakaryocyte progenitor cell required both megakaryocyte colony-stimulating factor and the auxiliary potentiator activity. In the presence of megakaryocyte colony-stimulating factor alone, progenitor cells did not form colonies of easily detectable megakaryocytes. However, groups of cells comprised entirely of small acetylcholinesterase containing immature megakaryocytes were observed, thus establishing that megakaryocyte colony development passes through a stage of immature cells prior to detectable megakaryocyte development and that some acetylcholinesterase-containing cells can undergo cellular division.

Animals↗
PubMed1982

Molecules stimulating early red cell, granulocyte, macrophage, and megakaryocyte precursors in culture: similarity in size, hydrophobicity, and charge.

Molecules in conditioned medium from stimulated lymphocyte populations or from certain cell lines are known to stimulate cells committed to various hemopoietic lineages as well as pluripotential cells to form colonies in culture. In this study, the relationship between molecules active on pluripotential cells and early cells committed to granulocyte, macrophage, megakaryocyte, or red cell production was explored using techniques of chemical separation. After separation on the basis of charge, or after sequential purification using methods of high resolving power based on hydrophobicity and size, these activities remained associated with one another. The observations provide support for a model which proposes that pluripotential hemopoietic precursor cells as well as their early committed progeny may all be responsive to a single lineage-indifferent factor. Responsiveness to "lineage-specific" factors such as erythropoietin is proposed to be a feature only of later cells after they have made the appropriate receptors as part of their differentiation program.

Animals↗
PubMed1982

The role of calmodulin in opioid-induced changes in the phosphorylation of rat striatal synaptic membrane proteins.

Chronic morphine treatment of rats decreased the level of phosphorylation of synaptic membrane proteins of the striatum assayed in vitro. Although the patterns of phosphorylated proteins separated on SDS-gel electrophoresis from morphine-tolerant rats resembled patterns produced by lowering Ca2+ levels in the assay, supplementation of the protein kinase assay with Ca2+ and its binding protein, calmodulin, did not restore full kinase activity. The addition of methadone or etorphine to the protein kinase in vitro however, was able to block the Ca2+-calmodulin stimulation of phosphorylation in both synaptic membranes and intact synaptosomes. These data suggest that opioids produce an irreversible (or slowly reversible) defect in the Ca2+-dependent protein kinase system of striatal membranes.

Animals↗
PubMed1982

Differences in the regulation of megakaryocytopoiesis in the murine bone marrow and spleen.

Murine splenic megakaryocytopoiesis has been analysed and compared to that of the bone marrow. Quantification of megakaryocytes by acetylcholinesterase staining indicated a reduction in the total numbers of megakaryocytes in the spleen, with the largest decrease being in the total numbers of immature megakaryocytes. On a per organ basis, the spleen also contained a lower number of the megakaryocyte progenitor cells (CFU-Mk) than the bone marrow. The splenic and bone marrow progenitor cells had similar in vitro responses to megakaryocyte colony-stimulating activities. However, the splenic progenitor cells developed a lower number of megakaryocytes per colony, compared to bone marrow. This lower number of cell divisions was not compensated by increased endomitotic activity, since the splenic colony megakaryocytes had a similar distribution of DNA to those derived from marrow megakaryocytes. Cell cycle analysis indicated that, in contrast to marrow cells, splenic megakaryocyte progenitor cells are a rapidly-cycling population. This change in cell cycle status, together with altered proportions of progenitor cells, immature and mature megakaryocytes, suggests that the regulation and kinetics of megakaryocyte development are different in spleen and bone marrow.

Animals↗