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Biomedical subjects

M Marsh

Publications and source records attributed to M Marsh.

At least 127 records · Page 7Linked to original sources
PubMed1983

Acidification of macrophage and fibroblast endocytic vesicles in vitro.

We have used the pH-dependent fluorochrome fluorescein-dextran (FD) to study the acidification of prelysosomal vacuoles (endosomes) and lysosomes isolated from cultured macrophages and fibroblasts. FD was internalized by pinocytosis under conditions that allowed its selective localization in endosomes (1- to 5-min pulse) or in lysosomes (5-min pulse, 30-min chase). Fibroblasts were also exposed to FD at 20 degrees C, at which temperature endosome-lysosome fusion is inhibited. Cells were homogenized and labeled organelles were separated by centrifugation in Percoll density gradients. The addition of ATP rapidly decreased the internal pH of both endosomes and lysosomes, as indicated by a decrease in fluorescence intensity. The pH gradient was dissipated by H+ ionophores and ammonium chloride. Acidification was not affected by inhibitors of the mitochondrial F1, F0-ATPase or the Na+, K, K+-ATPase and did not require permeant anions, Na+, or K+. Of the inhibitors tested, only N-ethylmaleimide prevented the ATP-dependent acidification of both compartments. These findings provide direct support for the existence of an acidic prelysosomal compartment that may be acidified via the same type of H+ pump believed to operate in lysosomes and secretory granules.

Adenosine Triphosphatases↗
PubMed1983

Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells.

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH-dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.

Animals↗
PubMed1982

Endocytosis of enveloped animal viruses.

After attaching to the cell surface, virus particles are rapidly internalized by endocytosis and channelled into the lysosomal compartment. The endocytosis occurs by a pinocytic process involving coated pits and coated vesicles. Intermediate pre-lysosomal vacuoles, termed endosomes, are recognized as a part of the intracellular pathway. Our studies have shown that for several of the enveloped viruses (toga viruses, orthomyxoviruses and rhabdoviruses) the endocytic pathway is essential for productive infection. In these cases the viral genome penetrates from the lysosomes where the virus membrane fuses with the lysosomal membrane. The low pH in the lysosomes triggers membrane fusion by causing a conformational change in the virus spike glycoproteins, which results in the expression of potent fusion activity. As a result of the fusion reaction the nucleocapsids are transferred into the cytoplasm. In this paper we review some work in which Semliki Forest virus (SFV) has been used to probe the adsorptive endocytosis pathway in baby hamster kidney (BHK-21) cells. In addition, we present new data on the kinetics by which the contents of the endocytic vacuoles become acidified. In these studies the pH-dependent penetration by SFV has been used as an indicator of pH.

Ammonium Chloride↗
PubMed1982

Monensin inhibits Semliki Forest virus penetration into culture cells.

The carboxylic ionophores monensin and nigericin, at concentrations higher than 10 and 6 muM, respectively, prevent the penetration of the Semliki Forest virus (SFV) genome into the cytosol of baby hamster kidney (BHK-21) cells and thereby inhibit viral replication. In the absence of inhibitors, the entry of SFV is known to proceed by adsorptive endocytosis in coated vesicles, followed by acid-triggered membrane fusion in intracellular vacuoles or lysosomes. The results show that binding of the virus to the cell surface, adsorptive endocytosis, and intracellular transport of viruses to the lysosomes are only marginally affected by the ionophores. No direct virucidal effect is observed, nor is the membrane fusion activity of the virus at low pH directly affected. Sequential addition of monensin and ammonium chloride (a non-related lysosomotropic inhibitor of SFV entry) indicates that both inhibitors affect the same step in the entry pathway. On the basis of these data and the known effects of carboxylic ionophores and lysosomotropic weak bases on cellular pH gradients, we conclude that monensin inhibits penetration by increasing the pH in endocytic vacuoles and lysosomes above pH 6, which is the pH threshold for the viral membrane fusion activity.

Animals↗
PubMed1982

Inhibition of Semliki forest virus penetration by lysosomotropic weak bases.

The effect of five lysosomotropic weak bases (chloroquine, amantadine, tributylamine, methylamine and NH4C1) on Semliki Forest virus (SFV) infection has been studied in BHK-21 cells. When present at concentrations equal to or greater than 0.1, 0.5, 2, 15 and 15 mM respectively, the agents inhibited SFV infection by more than 90%. The effect was reversible and involved a process occurring within the first 60 min of virus-cell contact. The agents did not have a direct virucidal effect nor did they affect virus binding to the cells, receptor-mediated endocytosis of prebound virus, intracellular distribution of virus after endocytosis, or the low pH-induced membrane fusion activity of the virus spike glycoproteins. The step blocked by chloroquine and NH4C1 occurred intracellularly and was identified as the release of the virus nucleocapsid into the cytoplasm or the uncoating process. On the basis of these results, our previous studies on SFV entry, and the known effects of lipophilic amines on lysosomes, we conclude that the agents affect entry by a common mechanism: they prevent the transfer of the virus nucleocapsid into the cytoplasm by increasing the lysosomal pH above the critical value needed to trigger a low pH-dependent fusion reaction between the membranes of the lysosome and the virus.

Amantadine↗
PubMed1978

The crystal sheaths from bivalve hinge ligaments.

The aragonite crystals in the molluscan bivalve hinge ligament are surrounded by an organic sheath which is distinct from the remainder of the ligament matrix. Methods have been developed to isolate these sheathed crystals from the ligaments of Spisula solidissima and Mercenaria mercenaria employing a papain digestion of the matrix protein. The sheathed crystals from Spisula have a CaCO3/protein ratio of 11.1 and those from Mercenaria a ratio of 29.6. The sheathed crystals and the empty crystal sheaths have been examined by electron microscopy for structural integrity. The sheath proteins exhibit much smaller proportions of the amino acids glycine and methionine than the hinge ligaments. These are characteristic amino acids of high concentrations in the hinge ligaments of both species. The concentrations of acidic and basic amino acids are increased about two fold in the sheaths over those of the ligaments. Otherwise there is little similarity in the amino acid composition of the sheaths in the two species. However, SDS electrophoresis shows the sheaths of both to contain a major protein component with a molecular weight of about 25,000. The sheath protein from the Mercenaria ligament contains about 5% carbohydrate and that of Spisula sheaths less than 1% carbohydrate.

Amino Acids↗
PubMed1976

The process of cholesterol cholelithiasis induced by diet in the prairie dog: a physicochemical characterization.

The rapid induction of cholesterol cholelithiasis in a new experimental model, the prairie dog, has recently been reported by two groups. In this model they were able to induce gallstones in as brief a period as 2 weeks, using a 1.2 per cent cholesterol diet. This unprecedented time intensity or telescoping of the induction process provided a unique opportunity for observation of physicochemical changes occurring rapidly over a short period of time and to correlate these with degree of biliary cholesterol saturation. To make such observations, 97 adult male and female prairie dogs were used in the present study. Seventy-two were fed the high cholesterol diet and sacrificed at intervals over a 14-day period; the remaining 25 were used as controls. The primary objective of this work was to determine whether or not there was any relationship between the in vivo events induced in prairie dog bile and our recently reported detailed observations of cholesterol precipitation phenomena in synthetic bile analogs. In these studies, solutions of physiologically relevant composition were constructed, which, when plotted according to convention on a tri-linear graph, fell within the zone of metastable or suspended supersaturation. These solutions revealed a consistent and previously undescribed liquid crystal to solid crystal phase transition during their approach to equilibrium at 37 degrees C. The in vivo studies of prairie dog bile following rapid induction of supersaturation revealed identical changes. At first, the supersaturated biles were isotropic followed by a period of turbidity and invariable formation of mesophase. After a few days, the liquid crystals decreased and solid cholesterol crystallites appeared. The most striking aspect of these observations is that the in vitro work has now predicted not only static, but dynamic processes as well, with respect to cholesterol precipitation beginning with the metastably supersaturated state and eventuating in gallstone formation. Ancillary new findings, resulting from the high cholesterol diet, included the induction of human-like hyper-beta-lipoproteinemia and rapid hepatocyte degranulation shown by electron microscopy.

Animals↗
PubMed1975

Nicotinamide adenine dinucleotide degradation in infarcted cardiac muscle.

Nicotinamide adenine dinucleotides (NAD, NADH2, NADP, and NADPH2) levels decrease in myocardial dog tissue after the ligature of the coronary artery branch. The activity of a glycohydrolytic enzyme acting on NAD and releasing nicotinamide in an equivalent amount was of the same order of magnitude in infarcted tissue, irrespective of the time elapsed after the coronary artery occlusion, as it was in normal tissue. Most of the NAD contained in normal heart muscle was hydrolyzed as soon as the tissue was disrupted in a homogenizer, whereas no hydrolysis occurred when the whole fragment was incubated for 1 hour. The enzymatic activity was found mainly in a membranous fraction seperated at 17,000 x g by differential centrifugation. Acid phosphatase, K+ -activated phosphatase, and NA+-K+-ATPase specific activities were greater in this fraction. It is suggested that the structural disorganization of the heart elicited either in vitro or during the infarction process determines the conditions for a reaction between the enzyme which is localized in the membranes and the NAD which is mainly in the cytosol.

Animals↗