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Biomedical subjects

S Rottem

Publications and source records attributed to S Rottem.

At least 127 records · Page 7Linked to original sources
PubMed1981

Proton motive force across the membrane of Mycoplasma gallisepticum and its possible role in cell volume regulation.

A proton motive force (delta (-) microH+) of 70 to 130 mV was measured across the membrane of Mycoplasma gallisepticum cells. The membrane potential was measured utilizing the lipid-soluble cation tetraphenylphosphonium. The method was validated by showing that in the presence of valinomycin the ratio of the concentrations (in/out) of tetraphenylphosphonium agreed well with those for K+ and Rb+. The pH gradient was calculated from the measured distribution ratio of benzoic acid. The proton motive force was approximately the same in cells harvested at early exponential, midexponential, and stationary phases of growth. The proportion of pH gradient to membrane potential varied with external pH. In the absence of glucose, cells incubated in an isosmotic NaCl solution showed low adenosine triphosphate and delta (-) microH+ levels and a tendency to swell and lyse compared with cells incubated with added glucose. It is concluded that energy is required for normal cell volume regulation.

Adenosine Triphosphate↗
PubMed1981

Eliminating mycoplasmas from contaminated cell cultures.

Mycoplasmas are bothersome contaminants in cell cultures. Cells derived from various sources are subject to contamination and although many procedures have been suggested to eliminate the contaminating mycoplasmas, there is no simple, rapid and effective method for eliminating them. We have devised a method for the selective killing of mycoplasmas based on the differences between the nucleic acid metabolism of mycoplasmas and that of tissue culture cells. Whereas the nutritional requirements of mycoplasmas for nucleic acid precursors can be met by purine and pyrimidine bases, mammalian cells do not incorporate the free bases. The approach was to selectively incorporate the free base analogue 5-bromouracil (5-BrUra) into the mycoplasmas followed by photosensitization of the DNA containing 5-BrUra by low concentrations of the fluorochrome 33258-Hoechst. Such treatment renders the mycoplasma DNA very susceptible to breaks induced by visible light. The unusually high A + T content in mycoplasma DNA makes these organisms suitable candidates for the induction of breakage by the combined action of 5-BrUra, 33258-Hoechst and light because 33258-Hoechst has a high affinity for A + T base pairs and an even higher affinity for A-BrUra base pairs. We report that Mycoplasma hyorhinis and Acholeplasma laidlawii contaminating Chinese hamster cells were selectively killed by our technique, and cured clones of cells were easily obtained from the treated cell cultures. The technique proved efficient in curing BHK-21 and RAG cells from unknown contaminating mycoplasmas suggesting that this technique could be generally applied to eliminate contaminating mycoplasma strains from mammalian cell culture.

Animals↗
PubMed1980

Phospholipid and cholesterol uptake by Mycoplasma cells and membranes.

The ability of growing mycoplasma cells and their isolated membranes to take up exogenous phospholipids was correlated with their ability to take up cholesterol. Horse serum or vesicles made of phosphatidylcholine and cholesterol served as lipid donors. Growing cells of five Mycoplasma species took up significant quantities of phosphatidylcholine and sphingomyelin as well as free and esterified cholesterol. In contrast, growing cells of three Acholeplasma species failed to take up any of the exogenous phospholipids, and only incorporated low amounts of free cholesterol and no esterified cholesterol. Hence, the ability of mycoplasmas to take up large quantities of cholesterol appears to be correlated with an ability to take up exogenous phospholipids. Isolated membranes of Mycoplasma capricolum and Acholeplasma laidlawii took up lower amounts of cholesterol than did membranes of growing cells and did not take up phospholipids. Inhibition of M. capricolum growth decreased the ability of the cells to take up exogenous phospholipids and cholesterol. The possibility that the contact between the lipid donors and the membrane involves specific receptors best exposed in actively growing cells is discussed.

Acholeplasma↗
PubMed1980

Iron storage in Mycoplasma capricolum.

Considerable quantities or iron were incorporated into the Mycoplasma capricolum cell membrane. Mossbauer studies showed that the iron is in a form which becomes magnetically ordered at low temperatures. The iron-enriched cells contained membrane-bound electron-dense particles of about 6.0 nm in diameter.

Cell Membrane↗
PubMed1979

Mössbauer spectroscopic studies of iron in Proteus mirabilis.

Mössbauer measurements of Proteus mirabilis cells grown in 57Fe-enriched media show that considerable quantities of iron are incorporated, particularly in the stationary phase of growth. A major part of the iron is in a form which shows magnetically split spectra at low temperatures. Fractionation of cells reveals that iron is present in both the soluble and particulate fractions, with the larger proportion being found in the soluble fraction. The iron in the particulate fraction is predominantly found in the outer membrane.

Iron↗
PubMed1979

Lipid distribution in Acholeplasma laidlawii membrane. A study using the lactoperoxidase-mediated iodination.

The lactoperoxidase-mediated radioiodination has been applied to study the transbilayer distribution of phospho- and glycolipids in Acholeplasma laidlawii membranes. After radioiodination, about 5% of the 125I-iodine was found in membrane lipids. A comparison of the labeling intensities of the various lipid species between iodinated intact cells and isolated membranes revealed that the glycolipids monoglucosyldiglyceride and diglucosyldiglyceride are located almost exclusively in the outer half of the bilayer, whereas the phospholipids phosphatidylglycerol and diphosphatidylglycerol as well as the phosphoglycolipids glycerophosphoryl-diglucosyldiglyceride and glycerophosphoryl-monoglucosyldiglyceride are almost equally distributed in the outer and inner halves of A. laidlawii membranes.

Acholeplasma laidlawii↗
PubMed1979

Is the vertical disposition of Mycoplasma membrane proteins affected by membrane fluidity?

The influence of the physical state of the membrane lipid matrix on the vertical disposition of membrane proteins was studied with Acholeplasma laidlawii. Changes in membrane fluidity were brought about by altering the fatty acid composition of membrane lipids, by changing the growth temperature, by aging of cultures and by inducing changes in the membrane lipid-to-protein ratio through treatment with chloramphenicol. The lactoperoxidase-mediated iodination technique was used to label membrane proteins exposed to the aqueous surroundings. The degree of exposure of the iodine-binding sites of membrane proteins on the external surface of intact cells was found to undergo significant changes on varying growth conditions, but the changes could not be consistently correlated with changes in membrane fluidity, nor were they discernible on iodination of isolated membranes.

Acetates↗
PubMed1979

Outer membrane proteins of smooth and rough strains of Proteus mirabilis.

The outer membranes of the smooth Proteus mirabilis S1959 strain and its rough R13, R110, R51 and R45 mutants were isolated by sonication of the cells and sucrose density gradient centrifugation. The outer membrane of the rough strains had a lower density than that of their parent smooth strain, but the protein-to-phospholipid ratios were the same. The electrophoretic patterns of outer membrane polypeptides of the S and R strains in sodium dodecylsulfate/polyacrylamide gels were identical, with two major polypeptide bands, C1 and C2 (Mr 39,000 and 38,000) predominating. The C1 polypeptide band was a heat-modifiable polypeptide, which migrated as a band at Mr 33,000 when membranes were solubilized at 37 degrees C or 50 degrees C, and at Mr 39,000 when solubilization was at 100 degrees C. Susceptibility of outer membrane polypeptides to proteolytic digestion was found to be higher in isolated outer membrane preparations of the rough strains than in the smooth strain, suggesting that the availability of the polypeptide chains to proteolytic activity depends on the length of the polysaccharide chains of the outer membrane lipopolysaccharide.

Cell Membrane↗
PubMed1979

Determination of lipopolysaccharide by a bioluminescence technique.

The determination of the lipid A content of bacterial lipopolysaccharide by using a dim mutant of the luminous bacterium Beneckea harveyi is described. The luminous bacteria emitted light upon the addition of an acid hydrolysate of lipopolysaccharide which contained myristic acid, thus making it possible to detect as little as 1 ng of lipopolysaccharide. By converting the 3-OH-myristic acid to myristic acid, it was possible to further increase the detection sensitivity and to establish a basis for a specific and highly sensitive bioassay for the detection of lipopolysaccharide.

Biological Assay↗
PubMed1979

Carotenoids acts as reinforcers of the Acholeplasma laidlawii lipid bilayer.

Acholeplasma laidlawii cells grown with oleic acid produced much more colored carotenoids than did cells grown with elaidic acid. The amount of carotenoids was decreased 80 to 90% by growing the cells with 0.05 M propionate, resulting in a marked increase in the mobility of both 5-doxylstearate and 12-doxylstearate incorporated into the membranes. The fatty acid composition of the propionate-grown cells differed from that of cells grown without propionate by containing odd-numbered rather than even-numbered saturated fatty acids, but the ratios of saturated to unsaturated fatty acids were the same. To determine whether the carotenoids are the cause for the restricted mobility in the membranes, the carotenoids were selectively removed from A. laidlawii membranes by incubating the membranes with phosphatidylcholine vesicles. The carotenoid-depleted membranes showed an increase in the mobility of the hydrocarbon chains of the spin-labeled fatty acids. Furthermore, the incorporation of carotenoids into artificial membrane vesicles restricted the mobility of the hydrocarbon chain. Our results support the notion that the carotenoids in A. laidlawii act as a rigid insert reinforcing the membrane bilayer.

Acholeplasma laidlawii↗
PubMed1979

Composition and molecular organization of lipids and proteins in the envelope of mycoplasmavirus MVL2.

MVL2 virus was purified from culture supernatants of infected Acholeplasma laidlawii cells by differential centrifugation, followed by velocity centrifugation in sucrose gradients. The purified virus contained 0.08 to 0.1 mumol of lipid phosphorous per ml of viral protein. Thin-layer chromatography of viral lipids revealed the presence of phospho-, glyco-, and phosphoglycolipids identical with those found in the host cell membrane, but the relative amount of phosphatidylglycerol was much lower than that in the virus. The fatty acid composition of lipids incorporated into the virus included lipids synthesized before and after infection. The freedom of motion of spin-labeled fatty acids in MVL2 depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe has the properties of a lipid bilayer. Nevertheless, the lipid hydrocarbon chains in MVL2 appear to be less mobile than those in membranes of the host cells. Polyacrylamide gel electrophoresis of purified MVL2 revealed four major and about five minor polypeptide bands. None of the polypeptide bands gave a positive periodic acid-Schiff reaction. Lactoperoxidase-mediated iodination, followed by proteolytic digestion of intact MVL2 particles, revealed that at least two major polypeptides are localized on the external surface of the viral envelope.

Acholeplasma laidlawii↗
PubMed1979

Binding of lectins to membranes of mycoplasmas from aging cultures.

The binding of iodinated concanavalin A (Con A) and Ricinus communis agglutinin (RCA) to intact cells and isolated membranes of Acholeplasma laidlawii, Mycoplasma hominis and Mycoplasma capricolum decreased with the progression of the culture from the mid- to the late-logarithmic phase of growth. The binding of the lectins to Acholeplasma laidlawii membranes had no significant effect on membrane fluidity, as assessed by electron-paramagnetic resonance spectroscopy of spin-labelled fatty acids, and had no effect on several membrane-associated enzymic activities. Temperature affected the binding of Con A and RCA in an opposite manner: the binding of Con A increased, whereas that of RCA decreased, on raising the temperature from 4 degrees C to 37 degrees C. No significant difference in lectin binding was found between oleate- and elaidate-enriched membranes at low temperatures where the former was in the liquid-crystalline state and the latter in the gel state, suggesting that membranes fluidity does not influence the binding of Con A and RCA to Acholeplasma laidlawii membranes.

Acholeplasma laidlawii↗