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S Rottem

Publications and source records attributed to S Rottem.

At least 73 records · Page 4Linked to original sources
PubMed1990

Mycoplasma capricolum membranes induce tumor necrosis factor alpha by a mechanism different from that of lipopolysaccharide.

Heat-inactivated (60 degrees C, 45 min) Mycoplasma capricolum strain JR cells activate murine macrophages to secrete high levels of tumor necrosis factor alpha (TNF alpha) and to lyse tumor target cells efficiently. Fractionation of the intact M. capricolum cells, obtained from cells harvested at the exponential phase of growth, shows that their capacity to induce TNF alpha secretion by macrophage resides exclusively in the membrane fraction. The macrophage-mediated cytolysis following activation by M. capricolum membranes was significantly inhibited by specific anti-recombinant murine TNF alpha antibodies. M. capricolum membranes are a potent inducer of TNF alpha as the commonly used bacterial lipopolysaccharide, indicated by their dose-response curve for macrophage activation. Our study further showed that M. capricolum membranes and lipopolysaccharide synergize to augment TNF alpha secretion by C57BL/6-derived macrophages markedly. Moreover, lipopolysaccharide-unresponsive C3H/HeJ-derived macrophages, were pronouncedly activated by M. capricolum membranes, which do not contain lipopolysaccharide. These findings suggest that the mechanism by which M. capricolum membranes activate macrophages differs from that of lipopolysaccharide. Results of preliminary experiments show that human monocytes as well secrete TNF alpha following activation by M. capricolum membranes. Thus, in contrast with the prohibitive toxicity of lipopolysaccharide to animals and humans, M. capricolum membranes, which contain no lipopolysaccharide and are nontoxic in nature, may be of therapeutic value in the treatment of cancer.

Animals↗
PubMed1990

Changes in uterine blood flow during human pregnancy.

A transvaginal duplex Doppler ultrasonography system was used to measure blood flow characteristics in the ascending uterine artery before and during pregnancy. The system uses a 5 MHz Doppler transducer coupled to a 6.5 MHz imaging probe. There was a steady increase in volume flow rate in the left ascending uterine artery from a mean of 94.5 ml/min before pregnancy to a mean of 342 ml/min in late gestation (reflecting a 3.5-fold increase). With the assumption of equal flow on both uterine arteries, the fraction of the cardiac output that is distributed to these vessels was calculated to be 3.5% in early pregnancy and to reach 12% near term. The mean diameter of this vessel in the nonpregnant state was 1.6 mm, increasing to 3.7 mm toward term. The resistance to flow, expressed as the peak systolic to end-diastolic flow velocity ratio, declined from a mean of 5.3 in the nonpregnant state to a mean of 2.3 near term. These changes reflect the perpetual growth and development of the uteroplacental circulation, which provides the metabolic demands of the growing fetus throughout gestation.

Arteries↗
PubMed1990

Partial biochemical characterization of spiroplasma membrane component inducing tumor necrosis factor alpha.

We have recently found that membranes of Spiroplasma spp. strain MQ-1 (hereafter referred to as MQ-1) induce both tumor necrosis factor alpha (TNF alpha) secretion by bone marrow macrophages and blast transformation of lymphocytes via a mechanism different from that operated by bacterial lipopolysaccharide (LPS). This report presents evidence indicating that the MQ-1-derived membrane component(s) which activates bone marrow macrophages to secrete TNF alpha is, at least in part, protein. This conclusion is supported by our findings that TNF alpha secretion was reduced following exposure of MQ-1 membranes to elevated temperatures, extreme acidic pH treatment and incubation with protease K or pronase. Furthermore, following lipid extraction of MQ-1 membranes, most of both induction of TNF alpha secretion and blast transformation activities appeared in the 'protein' fraction. When membranes were chromatographed on a phenyl-Sepharose column, two major peaks were obtained, one containing most of the TNF alpha induction activity and the other the mitogenic activity. Neither peak coeluted with the peak of bulk membrane lipids. The possibility that the spiroplasma membrane component inducing TNF alpha secretion is acylated protein is discussed.

Animals↗
PubMed1990

Transmembrane diffusion channels in Mycoplasma gallisepticum induced by tetanolysin.

The permeability properties of Mycoplasma gallisepticum cells treated with a purified preparation of tetanolysin were investigated by determining the initial swelling rates of cells suspended in an isoosmotic solution of electrolytes or nonelectrolytes. The swelling, initiated by the tetanolysin, depended on the tetanolysin concentration and was markedly affected by the molecular size of the various osmotic stabilizers utilized. Thus, the initial swelling rates in an isoosmotic solution of monosaccharides were much higher than those in isoosmotic solutions of di-, tri-, or tetrasaccharides. Cell swelling induced by tetanolysin was much lower with energy-depleted M. gallisepticum cells, with arsenate-treated cells, or when the membrane potential (delta psi) was collapsed by valinomycin (10 microM) plus KCl (100 mM). Swelling was not affected by the proton-conducting ionophore carbonyl cyanide-m-chlorophenylhydrazone (1 to 10 microM) or by nigericin (5 microM). These results support the concept that the damage induced by tetanolysin is due to the formation of water-filled pores within the membranes of energized M. gallisepticum cells. Such pores allow the diffusion of hydrophilic molecules into the cells and may vary in size, depending on the tetanolysin concentration utilized.

Cell Membrane↗
PubMed1990

A 57-kilodalton protein associated with Spiroplasma melliferum fibrils undergoes reversible phosphorylation.

Phosphorylation of a major 57-kilodalton protein substrate was observed in cell lysates of Spiroplasma melliferum BC3 incubated with [gamma-32P]ATP. Only serine phosphates have been isolated from the acid hydrolysate of the phosphorylated protein. The 57-kilodalton protein substrate was found, to a large extent, in the cytosolic fraction and, to a lesser extent, associated with cell membranes and was detected in the Triton X-100-insoluble fraction that contained fibrils.

Bacterial Proteins↗
PubMed1990

Ultrasound diagnosis of fetal anomalies.

Obstetric ultrasound permits the antenatal diagnosis of structural fetal anomalies. Every pregnant woman should have the option of a second trimester ultrasound examination in centers where examinations are available.

Brain↗
PubMed1989

First-trimester and early second-trimester diagnosis of nuchal cystic hygroma by transvaginal sonography: diverse prognosis of the septated from the nonseptated lesion.

Fetal cystic hygroma is a congenital malformation of the lymphatic system appearing as a single or multiloculated fluid-filled cavity, most often in the nuchal region. The malformation is believed to arise from failure of the lymphatic system to communicate with the venous nuchal system. Sometimes the lesion progresses to fetal hydrops, causing fetal death. To further delineate the cause and natural history of this disorder, we have prospectively studied eight cases of cystic hygroma of the neck, detected at gestational ages of 9 to 15 weeks by transvaginal sonography. Three of the eight fetuses survived (37.5%) and were normal at birth. Either hydrops fetalis or intrauterine fetal death occurred in the other five fetuses. In one of these five, therapeutic abortion was induced because of trisomy 21. In another fetus of these five, trisomy 18 was diagnosed by amniocentesis. This pregnancy ended in intrauterine fetal death. The ultrasonic evaluation of the cystic hygromas revealed that those that were reabsorbed in the three ultimately normal viable fetuses were nonseptated cysts, whereas all the four cystic hygromas ending in fetal death or associated with aneuploidy were septated, multilocular hygromas. In another fetus with nonseptated hygroma, nonimmune hydrops developed, which resulted in premature delivery and early neonatal death.

Down Syndrome↗
PubMed1989

A cation/proton antiport activity in Acholeplasma laidlawii.

Sealed membrane vesicles of Acholeplasma laidlawii were obtained by controlled lysis of carotenoid-rich intact cells. An imposed delta pH was created by loading membrane vesicles or intact Acholeplasma laidlawii cells with 0.25 M NH4Cl and diluting them into 0.25 M choline chloride. The passive efflux of NH3 from the membrane vesicles or cells resulted in the creation of a delta pH (inside acid) that could be visualized by the quenching of the fluorescence of the weak base acridine orange. Whereas with isolated membrane vesicles, the fluorescence was dequenched by the addition of Na+, with intact cells, K+ in addition to Na+ was required. These results strongly suggest a Na+/H+ exchange activity that in intact Acholeplasma laidlawii cells is K+-dependent. The possible role of the Na+/H+ exchange activity in pH homeostasis at the more alkaline pH range, as well as in the extrusion of excess Na+ from the cells is discussed.

Acholeplasma laidlawii↗
PubMed1989

Biological activities of monoclonal antibodies to Mycoplasma pneumoniae membrane glycolipids.

A purified preparation of membranes was obtained by using a unique method of treating Mycoplasma pneumoniae with the ATPase inhibitor, diethylstilbestrol. This method was shown to yield highly purified membranes with little or no cytoplasmic contamination. These membranes were used to immunize mice for subsequent productions of monoclonal antibodies (MAbs). Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay with whole-cell M. pneumoniae and lipid extract antigens. Four stable MAbs were obtained and characterized. MAb CP3-46F5 reacted with a protein of a molecular weight of approximately 52,000 as determined by Western blot (immunoblot). MAbs CP3-50C2, CP3-53C5, and CP3-53C8 did not react with any antigens on Western blots but did bind to at least 10 distinct glycolipid bands as determined by orcinol staining on thin-layer chromatograms of M. pneumoniae lipid extracts. The MAbs did not react with similarly prepared lipid extracts from Mycoplasma genitalium, Mycoplasma neurolyticum, and Mycoplasma gallisepticum. These MAbs did not inhibit M. pneumoniae metabolism or attachment to WiDr cell cultures. The anti-glycolipid MAbs recognize determinants specific to M. pneumoniae, unlike polyclonal hyperimmune sera against M. pneumoniae, which cross-react with lipid extracts of M. genitalium.

Animals↗
PubMed1989

Role of Na+ cycle in cell volume regulation of Mycoplasma gallisepticum.

The mechanism for the extrusion of Na+ from Mycoplasma gallisepticum cells was examined. Na+ efflux from cells was studied by diluting 22Na+-loaded cells into an isoosmotic NaCl solution and measuring the residual 22Na+ in the cells. Uphill 22Na+ efflux was found to be glucose dependent and linear with time over a 60-s period and showed almost the same rate in the pH range of 6.5 to 8.0. 22Na+ efflux was markedly inhibited by dicyclohexylcarbodiimide (DCCD, 10 microM), but not by the proton-conducting ionophores SF6847 (0.5 microM) or carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 microM) over the entire pH range tested. An ammonium diffusion potential and a pH gradient were created by diluting intact cells or sealed membrane vesicles of M. gallisepticum loaded with NH4Cl into a choline chloride solution. The imposed H+ gradient (inside acid) was not affected by the addition of either NaCl or KCl to the medium. Dissipation of the proton motive force by CCCP had no effect on the growth of M. gallisepticum in the pH range of 7.2 to 7.8 in an Na+-rich medium. Additionally, energized M. gallisepticum cells were stable in an isoosmotic NaCl solution, even in the presence of proton conductors, whereas nonenergized cells tended to swell and lyse. These results show that in M. gallisepticum Na+ movement was neither driven nor inhibited by the collapse of the electrochemical gradient of H+, suggesting that in this organism Na+ is extruded by an electrogenic primary Na+ pump rather than by an Na+-H+ exchange system energized by the proton motive force.

Cell Membrane↗
PubMed1989

Volume regulation in Mycoplasma gallisepticum: evidence that Na+ is extruded via a primary Na+ pump.

The primary extrusion of Na+ from Mycoplasma gallisepticum cells was demonstrated by showing that when Na+-loaded cells were incubated with both glucose (10 mM) and the uncoupler SF6847 (0.4 microM), rapid acidification of the cell interior occurred, resulting in the quenching of acridine orange fluorescence. No acidification was obtained with Na+-depleted cells or with cells loaded with either KCl, RbCl, LiCl, or CsCl. Acidification was inhibited by dicyclohexylcarbodiimide (50 microM) and diethylstilbesterol (50 microM), but not by vanadate (100 microM). By collapsing delta chi with tetraphenylphosphonium (200 microM) or KCl (25 mM), the fluorescence was dequenched. The results are consistent with a delta chi-driven uncoupler-dependent proton gradient generated by an electrogenic ion pump specific for Na+. The ATPase activity of M. gallisepticum membranes was found to be Mg2+ dependent over the entire pH range tested (5.5 to 9.5). Na+ (greater than 10 mM) caused a threefold increase in the ATPase activity at pH 8.5, but had only a small effect at pH 5.5. In an Na+-free medium, the enzyme exhibited a pH optimum of 7.0 to 7.5, with a specific activity of 30 +/- 5 mumol of phosphate released per h per mg of membrane protein. In the presence of Na+, the optimum pH was between 8.5 and 9.0, with a specific activity of 52 +/- 6 mumol. The Na+-stimulated ATPase activity at pH 8.5 was much more stable to prolonged storage than the Na+-independent activity. Further evidence that two distinct ATPases exist was obtained by showing that M. gallisepticum membranes possess a 52-kilodalton (kDa) protein that reacts with antibodies raised against the beta-subunit of Escherichia coli ATPase as well as a 68-kDa protein that reacts with the anti-yeast plasma membrane ATPases antibodies. It is postulated that the Na+ -stimulated ATPases functions as the electrogenic Na+ pump.

Acridine Orange↗
PubMed1988

Phosvitin kinase activity in Acholeplasma axanthum.

Incubating the soluble fraction derived from A. axanthum with phosvitin and [gamma-32P]ATP results in the phosphorylation of phosvitin. Casein, histone and kemptide were practically ineffective substrates, whereas a 55 kDa protein of M. gallisepticum was efficiently phosphorylated. The enzymatic activity has an optimal pH in the pH range 6.0-6.2 and requires divalent cations. The activity was inhibited by ammonium sulfate, heparin and sulphydryl blocking reagents, but was not affected by calmodulin with or without Ca2+ or by cyclic AMP.

Acholeplasma↗
PubMed1988

Protein phosphorylation in Mycoplasma gallisepticum.

Incubation of the soluble fraction derived from Mycoplasma gallisepticum cells with [gamma-32P]ATP results in the phosphorylation of several endogenous proteins. One protein with an apparent molecular mass of 55 kDa was the acceptor of more than 95% of the radioactive phosphate. This protein was also found to be radiolabeled in intact cells grown in the presence of [32P]orthophosphate. Acid hydrolysis of the phosphorylated 55-kDa protein followed by two-dimensional electrophoresis revealed that the 32P-labeled material co-migrated with phosphoserine. The in vitro phosphorylation of the 55-kDa protein has an optimum pH of 5.5-6.0 and is not affected by various metabolites of glycolysis, by cAMP or by calmodulin with or without Ca2+. The phosphorylation is dependent upon divalent cations, a dependency that is best fulfilled by the simultaneous addition of Ca2+ and Zn2+ that act in a specific and cooperative manner. Of a variety of possible exogenous protein acceptors tested, the endogenous protein kinase was capable to phosphorylate only phosvitin. The phosphorylation of the 55-kDa protein is reversible through the activity of a phosphoprotein phosphatase present in the soluble fraction of M. gallisepticum. The phosphoprotein phosphatase has an optimum pH of 7.5-8.0, is inhibited by NaF and stimulated to a large extent by inorganic phosphate and arsenate and to a lesser extent by pyrophosphate ATP and ADP. The possible association of the reversible protein phosphorylation to cell shape and gliding motility of M. gallisepticum are discussed.

Adenosine Triphosphate↗
PubMed1988

Effect of 7 beta-hydroxycholesterol on growth and membrane composition of Mycoplasma capricolum.

7 beta-OH cholesterol in a cholesterol rich growth medium (5-10 micrograms/ml) extended the lag period and slowed down the growth rate of Mycoplasma capricolum cells. In a cholesterol poor medium (0.5 micrograms/ml) inadequate to support growth, 7 beta-OH cholesterol exerts a synergistic effect on growth. The 7 beta-OH cholesterol was incorporated unchanged from the growth medium and could be recovered exclusively in the membrane fraction. The incorporation of the 7 beta-OH cholesterol has no effect on the total phospholipid content but the DPG to PG ratio was markedly decreased. Exchange studies with lipid vesicles revealed that whereas most of the cholesterol underwent exchange, only about 20% of the 7 beta-OH cholesterol was exchanged.

Cardiolipins↗
PubMed1988

Animal viruses are able to fuse with prokaryotic cells. Fusion between Sendai or influenza virions and Mycoplasma.

Sendai and influenza virions are able to fuse with mycoplasmata. Virus-Mycoplasma fusion was demonstrated by the use of fluorescently labeled intact virions and fluorescence dequenching, as well as by electron microscopy. A high degree of fusion was observed upon incubation of both virions with Mycoplasma gallisepticum or Mycoplasma capricolum. Significantly less virus-cell fusion was observed with Acholeplasma laidlawii, whose membrane contains relatively low amounts of cholesterol. The requirement of cholesterol for allowing virus-Mycoplasma fusion was also demonstrated by showing that a low degree of fusion was obtained with M. capricolum, whose cholesterol content was decreased by modifying its growth medium. Fluorescence dequenching was not observed by incubating unfusogenic virions with mycoplasmata. Sendai virions were rendered nonfusogenic by treatment with trypsin, phenylmethylsulfonyl fluoride, or dithiothreitol, whereas influenza virions were made nonfusogenic by treatment with glutaraldehyde, ammonium hydroxide, high temperatures, or incubation at low pH. Practically no fusion was observed using influenza virions bearing uncleaved hemagglutinin. Trypsinization of influenza virions bearing uncleaved hemagglutinin greatly stimulated their ability to fuse with Mycoplasma cells. Similarly to intact virus particles, also reconstituted virus envelopes, bearing the two viral glycoproteins, fused with M. capricolum. However, membrane vesicles, bearing only the viral binding (HN) or fusion (F) glycoproteins, failed to fuse with mycoplasmata. Fusion between animal enveloped virions and prokaryotic cells was thus demonstrated.

Acholeplasma laidlawii↗
PubMed1988

The technique of transvaginal sonography with the use of a 6.5 MHz probe.

Ultrasonography has become an important tool in infertility and obstetric practice. Its use in gynecology, while effective, has been less. This article describes the use of a high-frequency 6.5 MHz probe specially designed for intravaginal use. The technique is presented, along with a description of its uses and findings by organ. Patient acceptance has been universal. Its high resolution makes this technique more useful in general gynecologic practice.

Fallopian Tubes↗
PubMed1988

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile.

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.

Bacterial Proteins↗