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Biomedical subjects

S Rottem

Publications and source records attributed to S Rottem.

At least 109 records · Page 6Linked to original sources
PubMed1984

Cloning of L-2 DNA in Escherichia coli pOL4 plasmid.

A physical map of L-2 DNA was constructed using restriction endonucleases. Based on this map the five HincII-generated L-2 DNA fragments (A-E) were cloned into the SmaI site of Escherichia coli vector plasmid pOL4, that was designed to analyze promoters and transcriptional terminators. The insertion of the HincII-generated L-2 DNA fragments into this plasmid clearly demonstrated that a fragment (fragment E) with a size of 1.1 kbp carried a sequence that initiated transcription in E. coli.

Acholeplasma↗
PubMed1984

Volume regulation in Mycoplasma gallisepticum.

Cells of Mycoplasma gallisepticum incubated in 250 mM NaCl in the absence of glucose for several hours show swelling and eventual lysis. This swelling is believed to be due to colloid osmotic and Donnan forces, since it is prevented by the addition of nondiffusable solutes such as sucrose or MgSO4. The addition of glucose during the swelling stage (but before lysis) caused shrinkage and return to initial volume. Experiments on Na+ and H+ movement are consistent with the operation of an ATP-driven H+ pump and a Na+/H+ exchange reaction.

Adenosine Triphosphate↗
PubMed1984

Bilirubin incorporation into spiroplasma membranes and methylation of spiroplasmal DNA.

Spiroplasma floricola (BNR-1), Spiroplasma sp. MQ-1 and S. apis (B-31) grown in media containing horse serum exhibited intense yellow pigmentation. Yellow pigments were not observed in S. citri (R8A2) and Spiroplasma sp. strains BC-3 and PPS-1 grown in the same medium. The reddish-yellow pigment showed up in lipid extracts of both spiroplasma membranes and horse serum. It exhibited the typical features of bilirubin: specific absorption spectrum from 390 to 500 nm with a peak at 453 nm, and a characteristic sequence of color changes on addition of HNO3 to its solution in chloroform. The pigment comigrated with commercial bilirubin from bull gall and stained greenish blue when subjected to mild oxidation by iodine. S. floricola contained 5.4 micrograms bilirubin/mg cell protein or 9.7 micrograms bilirubin/mg membrane protein. High-performance liquid chromatography (HPLC) showed the presence of significant amounts of 5-methylcytosine and very little 6-methyladenine in the DNA of S. floricola, S. apis and Spiroplasma sp. strains PPS-1 and MQ-1. S. citri and Spiroplasma sp. strain BC-3 contained 6-methyladenine and very little, if any, 5-methylcytosine. The methylated cytosine residues in Spiroplasma sp. MQ-1 were almost exclusively located in the sequence CpG, as in eukaryotes.

5-Methylcytosine↗
PubMed1984

Uptake and transbilayer distribution of phosphatidylcholines in Mycoplasma gallisepticum and their effect on cell morphology.

Mycoplasma gallisepticum cells grown in a serum-free medium incorporated large amounts of egg-phosphatidylcholine (Egg-PC), dioleoylphosphatidylcholine (DOPC) or dipalmitoylphosphatidylcholine (DPPC) added to the growth medium. Egg-PC and DOPC were incorporated at a high rate and to a large extent and were modified by the organisms, whereas DPPC was incorporated at a lower rate and to a lesser extent and was not modified by the cells. The lactoperoxidase-mediated radioiodination applied to study the transbilayer distribution of phosphatidylcholine (PC) in the membranes revealed that the PC in cells grown with DOPC is almost equally distributed in the outer and inner leaflets of M. gallisepticum membranes, while the PC in DPPC-grown cells is preferentially located in the outer leaflet and that in Egg-PC-grown cells is found in the inner leaflet. Thus, in Egg-PC- or DPPC-grown cells the equilibrium in structure and properties between the inner and outer leaflets is disturbed, resulting in dramatic effects on the morphology of M. gallisepticum cells.

Biological Transport↗
PubMed1983

Induction of macrophage-mediated cytolysis of neoplastic cells by mycoplasmas.

Unexpected cytolysis was encountered when nonactivated murine peritoneal macrophages were cultured with [3H]TdR-prelabeled syngeneic or allogeneic tumor cells at a 10:1 ratio. The level of specific cytolysis reached 70% within 48 hr of cocultivation. Similar killing was observed whether the macrophages were derived from untreated, thioglycollate-treated, or germ-free mice. Cytolytic activity was also demonstrated when bone marrow-derived or peritoneal macrophages from 9- and 5-day in vitro cultures, respectively, were employed rather than freshly harvested peritoneal macrophages. Thus, the macrophage-mediated killing was neither the result of in vivo preactivation nor a consequence of the presence of lymphocytes in the assay. Moreover, macrophages derived from different strains caused similar effects. Our study revealed that the neoplastic target cell cultures susceptible to cytolysis by nonactivated macrophages were contaminated with mycoplasma. A mycoplasma was isolated from the supernatant of a culture of the A9HT fibrosarcoma line, identified as Mycoplasma orale, and cultivated. Addition of viable mycoplasma from that isolate to mixed cultures of thioglycollate-elicited macrophages and [3H]TdR-prelabeled mycoplasma-free target cells resulted in specific cytolysis of transformed A9 cells, but not of normal mouse fibroblasts. The level of macrophage-dependent cytolysis correlated with the number of viable mycoplasma cells added and was higher than that attained by activation with LPS at optimal concentration. Similar specific cytolysis was observed with heat-killed mycoplasmas. Our results demonstrate that mycoplasmas may cause selective macrophage-mediated cytolysis of neoplastic but not of normal target cells, perhaps via activation of the macrophages. It is suggested that undetected infection of experimental systems by mycoplasmas may account for some reports on lysis of neoplastic cells by nonactivated macrophages.

Animals↗
PubMed1982

Phospholipid interconversions in Mycoplasma capricolum.

Mycoplasma capricolum cells increase their phospholipid content by incorporating exogenous phospholipids from the growth medium. Growing the cells in media with increasing serum concentrations resulted in a massive incorporation of phosphatidylcholine and sphingomyelin (up to about 50% of total phospholipids) into the cell membrane. The incorporation of the exogenous phospholipids had essentially no effect on the rate of cell growth and did not decrease the overall phospholipid biosynthesis of the cells. Thus, the ratio of phospholipid to protein in membranes from cells grown with 5% horse serum was 0.5 (mumol/mg) compared to 0.3 (mumol/mg) in cells grown without serum, and the relative content of charged polar lipids was apparently decreased. The consequence of the incorporation of exogenous phosphatidylcholine was an alteration in the relative amount of the major end-products of the de novo phospholipid biosynthesis; a marked increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol was observed. The possibility that the increase in the ratio of diphosphatidylglycerol to phosphatidylglycerol is part of a control mechanism to maintain a mixture of bilayer and non-bilayer lipids is discussed.

Animals↗
PubMed1982

Possible association of segregated lipid domains of Mycoplasma gallisepticum membranes with cell resistance to osmotic lysis.

Freeze-fracturing of cholesterol-rich Mycoplasma gallisepticum membranes from cells grown in a medium containing horse serum revealed particle-free patches. The patches appeared in cells quenched from either 4 or 37 degrees C. Particle-free patches also occurred in membranes of cells grown in a serum-free medium supplemented with egg-phosphatidylcholine but not in membranes of cells grown with dioleoylphosphatidylcholine. The appearance of particle-free patches was attributed to the presence of disaturated phosphatidylcholine (PC) molecules in M. gallisepticum membranes, which were synthesized by the insertion of a saturated fatty acid at position 2 of lysophosphatidylcholine derived from exogenous PC present in the growth medium. Consequences of the synthesis of the disaturated PC also included a decrease in osmotic fragility and the ability of the cells to be permeated by K+. Electron paramagnetic resonance and fluorescence polarization measurements revealed that the fluidity of the lipid domain in the protein-rich M. gallisepticum membranes was almost identical to that of an aqueous dispersion of M. gallisepticum membrane lipids. Furthermore, the electron paramagnetic resonance spectra of the membranes were single-component spectra showing no indication of immobilized regions. The possibility that the osmotic resistance of M. gallisepticum cells is associated with the particle-free patches rather than with a restricted membrane fluidity caused by membrane proteins is discussed.

Bacterial Proteins↗
PubMed1982

Isolation of mycoplasma membranes by dicyclohexylcarbodiimide-induced lysis.

A simple procedure was devised to prepared membranes from Mycoplasma gallisepticum cells. The cells were lysed in an isosmotic NaCl solution by dicyclohexylcarbodiimide, which blocks ATPase activity and interferes with the regulation of cell volume. The procedure can be used to isolate membranes of other osmotically resistant mycoplasmas.

Bacteriolysis↗
PubMed1982

Lysophospholipase-catalyzed hydrolysis of lysophospholipids in Mycoplasma gallisepticum membranes.

Mycoplasma gallisepticum strains have a membrane-bound lysophospholipase which hydrolyzes lysophospholipid generated in these membranes by treatment with an external phospholipase. This paper studies the hydrolysis of the membranous lysophospholipids by an enzyme residing in the same membrane (intramembrane utilization) or in adjacent membranes (intermembrane utilization). To study intermembrane hydrolysis, the phospholipids of M. gallisepticum were labeled with [3H]oleic acid. Membranes were prepared, heated at 65 degrees C, and subsequently treated with pancreatic phospholipase A2. This resulted in membranes whose enzyme was heat inactivated, but which contained lysophospholipid. When these membranes were mixed with M. gallisepticum cells or membranes, the lysophospholipid was hydrolyzed by the membranous lysophospholipase. To study intramembrane hydrolysis, [3H]oleyl-labeled membranes of M. gallisepticum were treated with pancreatic phospholipase A2 at pH 5.0. At this pH, lysophospholipid was generated but not hydrolyzed. Adjustment of the pH to 7.4 resulted in hydrolysis of the lysophospholipid by the membranous lysophospholipase. These procedures permitted measuring the initial rates of intramembrane and intermembrane hydrolysis of the lysophospholipid, showing that the time course and dependence on endogenous substrate concentration were different in the intramembrane and intermembrane modes of utilization. They also permitted calculation of the molar concentration of the lysophospholipid in the membrane and its rate of hydrolysis, expressed as moles per minute per cell or per square centimeter of cell surface.

Cell Fractionation↗
PubMed1982

Structural characteristics of tetanolysin and its binding to lipid vesicles.

Tetanolysin binding to lipid vesicles was found to depend on the molar ratio of cholesterol to phospholipid, being low in vesicles containing up to 20 mol% cholesterol and high in vesicles containing more than 33 mol%. High concentrations of purified tetanolysin preparations formed arc- and ring-shaped structures. The structures were not readily detectable in diluted preparations unless incubated with lipid vesicles containing high molar ratios of cholesterol to phospholipid. It is suggested that the toxin is concentrated on the vesicles to local concentrations high enough to form the arcs and rings.

Bacterial Toxins↗
PubMed1981

Symmetrical distribution and rapid transbilayer movement of cholesterol in Mycoplasma gallisepticum membranes.

The exchange of cholesterol between [14C]cholesterol-labeled Mycoplasma gallisepticum cells and an excess of sonicated egg phosphatidylcholine/cholesterol vesicles (molar ratio of 0.9) was measured. More than 90% of the radioactive cholesterol underwent transfer from intact cells to the vesicles. The kinetics of the transfer was biphasic. About 50% of the radioactive cholesterol was exchanged with a half-time of about 4 h. The residual was exchanged at a slower rate with a half-time of about 9 h at 37 degrees C. Bovine serum albumin had a pronounced effect in enhancing both the fast and slow rates of cholesterol exchange, but did not affect the pool sizes significantly. The half-time for equilibration of the two pools in the presence of 2% albumin, calculated using a reversible two-pool method of analysis, was 6.2 h. The effect of albumin was also obtained with isolated membrane preparations and with cells treated with growth inhibitors, suggesting that this effect is independent of albumin preservation of cell viability. The rate enhancement of albumin was concentration dependent with maximal effects observed with greater than or equal to 2%, where the rates of exchange of both the rapidly and slowly exchanging pools were twice as fast. The mechanism by which albumin may affect the exchange rates is discussed.

Cell Membrane↗
PubMed1981

Determination of cholesterol asymmetry by rapid kinetics of filipin-cholesterol association: effect of modification in lipids and proteins.

The rapid kinetic behavior of filipin association with cholesterol was unaffected by binding of water-soluble proteins to vesicle and mycoplasma membranes and by proteolytic digestion of mycoplasma membrane proteins. The kinetic properties were, however, dependent on the membrane phospholipids, in that the initial rate of filipin association with cholesterol was enhanced by phospholipase A2 treatment by the incorporation of lysophosphatidylcholine, and by increasing the degree of unsaturation in phospholipid vesicles and mycoplasma membranes. The second-order rate constant was also dependent on th mol % of cholesterol in small unilamellar vesicles but not in large unilamellar vesicles. The ratio of rate constants in intact mycoplasma cells relative to isolated membranes provides an estimate of cholesterol distribution in membranes [Bittman, R., & Rottem, S. (1076) Biochem. Biophys. Res. Commun. 71, 318; Clejan, S., Bittman, R., & Rottem, S. (1978) Biochemistry 17, 4579]. This ratio was unaffected by proteolytic digestion of intact cells and by the incorporation of exogenous phospholipids into the Mycoplasma capricolum cell membrane. However, on cross-linking of surface proteins of M. capricolum by dimethylsuberimidate, cholesterol was localized predominantly in the outer half of the bilayer. On aging of mycoplasma cultures, the cholesterol distribution remained constant in membranes of M. capricolum cells but was enriched in the outer leaflet of the Mycoplasma gallisepticum cell membrane. The results of these experiments are discussed in relation to the use of the rapid kinetics of filipin binding as a probe of cholesterol distribution.

Cell Membrane↗
PubMed1981

Effects of sterol structure and exogenous lipids on the transbilayer distribution of sterols in the membrane of Mycoplasma capricolum.

Stopped-flow kinetic measurements of the association of filipin with sterols in intact cells and isolated membranes of Mycoplasma capricolum were used to study the effects of varying the phospholipid in the membrane. The phospholipid composition and content of the membrane were varied by growing cells in an albumin-containing medium with cholesterol, palmitic and oleic acids, and various concentrations of exogenous phospholipids. The exogenous phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidic acid) were incorporated up to levels of approximately 50% of the total membrane phospholipids but had no effect on the distribution of cholesterol between the two halves of the membrane bilayer. The sterol structure was varied by growing the cells with 10/micrograms/mL of either cholesterol, beta-cholestanol, 4,6-cholestadien-3 beta-ol, ergosterol, beta-sitosterol, or stigmasterol. With cholesterol, beta-cholestanol, and 4,6-cholestadien-3 beta-ol, approximately 65% of the sterol was found to be present in the outer half of the lipid bilayer. With ergosterol, beta-sitosterol, and stigmasterol, about 89% of the sterol is localized in the outer half of the membrane bilayer. Thus, the behavior of the alkyl-substituted sterols differs from that of cholesterol. The extent to which a sterol is distributed asymmetrically between the two halves of the bilayer is not related to the extent to which maximum growth is produced. These results suggest that growth-supporting sterols need not be translocated extensively.

Cell Membrane↗
PubMed1981

Lipid and protein membrane components associated with cholesterol uptake by Mycoplasmas.

Membranes of Mycoplasma species take up 2--4 times more exogenous cholesterol than membranes of Acholeplasma species. To test whether the lower cholesterol uptake capacity of Acholeplasma is due to the high glycolipid content of their membranes, the phospholipids of Acholeplasma laidlawii and Mycoplasma capricolum membranes were hydrolyzed by phospholipase A2. Digestion removed about 30% of the polar lipids of A. laidlawii, leaving the glycolipids and phospholglycolipids intact, and about 70% of the polar lipids of M. capricolum, the residue consisting mostly of sphingomyelin. Cholesterol uptake by the treated membranes from phosphatidylcholine/cholesterol vesicles decreased in rough proportion to the amount of polar lipid removed, indicating that the glycolipids in A. laidlawii membranes can participate in cholesterol uptake. Trypsin digestion of growing cells and isolated membranes of M. capricolum decreased cholesterol uptake by about one-half. Similar treatment of A. laidlawii cells and membranes had no effect on cholesterol uptake. These findings suggest the existence of protease-sensitive receptors on the cell surface of M. capricolum responsible for tighter contact with the cholesterol/phosphatidylcholine vesicles. It is proposed that the ability of Mycoplasma species to take up large quantities of exogenous cholesterol and phospholipids depends on the presence of protein receptors for cholesterol donors, receptors which are absent in Acholeplasma species.

Acholeplasma laidlawii↗
PubMed1981

The organization of cholesterol esters in membranes of Mycoplasma capricolum.

The organization of cholesterol esters in Mycoplasma capricolum membranes was studied by differential scanning calorimetry. Cells grown in the presence of horse serum incorporated large amounts of cholesterol esters into their membranes. The cholesterolester-containing membranes after incubation at low temperature showed an endotherm characteristic of a cholesterol ester crystalline leads to isotropic liquid transition that was identical in membranes both before and after thermal protein denaturation. This transition was not observed in membranes of cells grown in medium in which the horse serum was replaced by bovine albumin, fatty acids and unesterified cholesterol unless cholesterol esters were added to the growth medium. In membrane preparations obtained both from cells grown in horse serum and from cells grown with bovine albumin plus cholesterol and fatty acids, the free cholesterol content was sufficient to eliminate the bilayer order/disorder transition observed in isolated membrane phospholipids. Our studies indicate that the majority of cholesterol esters in M. capricolum membranes is not present in attached serum lipoprotein particles, nor is intimately associated with membrane protein, but exists as relatively large cholesterol ester droplets or pockets tightly associated with the membrane. The cholesterol esters in these pockets appear relatively pure, although the presence of small amounts of other membrane components is likely.

Calorimetry, Differential Scanning↗