PubMed Health⌕ Search

Biomedical subjects

S Rottem

Publications and source records attributed to S Rottem.

At least 19 recordsLinked to original sources
PubMed1997

The 57-Kilodalton Phosphoprotein of Spiroplasma melliferum Is Autophosphorylated

The partially purified 57-kDa protein of Spiroplasma melliferum was autophosphorylated when incubated with ATP in the presence of ZnCl2. Autophosphorylation was also apparent by showing the in situ phosphorylation of the 57-kDa protein band separated by polyacrylamide gel electrophoresis under nondenaturing conditions. The autophosphorylation was affected neither by the pH of the reaction mixture nor by the presence of NaF. The steady state level of the phosphorylated 57-kDa protein remained constant for up to 15 min, suggesting the absence of a phosphoprotein phosphatase activity in the preparation. As the initial phosphorylation rate did not decrease upon a 100-fold dilution of the 57-kDa protein under constant substrate concentration, it is suggested that the autophosphorylation is an intramolecular process.

Journal Article↗
PubMed1995

Invasion of HeLa cells by Mycoplasma penetrans and the induction of tyrosine phosphorylation of a 145-kDa host cell protein.

The ability of Mycoplasma penetrans to invade eukaryotic cells was studied using a HeLa cell line. The bactericidal antibiotic, gentamicin, in combination with low concentrations of Triton X-100, was utilized to kill mycoplasmas that had not entered the cells, allowing the quantitation of internalized organisms. The intracellular location of the mycoplasma was also documented by transmission electron microscopy. The actin polymerization inhibitor cytochalasin-D markedly inhibited the internalization process, whereas the tyrosine phosphorylation inhibitors, staurosporin and genistein had only a slight effect. As against the invasion of enteropathogenic Escherichia coli which depends on tyrosine phosphorylation of a 90-kDa (Hp90) HeLa cell protein, internalization of M. penetrans by HeLa cells was independent of the phosphorylation of Hp90. Nonetheless, tyrosine phosphorylation of a 145-kDa HeLa cell protein was found to be associated with the interaction of M. penetrans with HeLa cells.

Bacterial Adhesion↗
PubMed1995

The cell membrane of Mycoplasma penetrans: lipid composition and phospholipase A1 activity.

Analysis of Mycoplasma penetrans membrane lipids revealed that, in addition to large amounts of unesterified cholesterol, M. penetrans incorporated exogenous phospholipids, preferentially sphingomyelin, from the growth medium. The major phospholipids synthesized de novo by M. penetrans were phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG). In vivo labeling of PG and DPG by growing the cells with radioactive palmitate or oleate, followed by snake venom phospholipase A2 treatment, enabled us to assess the positional distribution of fatty acids in these lipids. Saturated fatty acids were found preferentially in position 2 of the glycerol backbone, and not in position 1 as found elsewhere in nature, while unsaturated fatty acids prefer position 1. M. penetrans membranes contain phospholipase activity of the A1 type, removing a fatty acid from the sn-1 ester bond of phospholipids. The activity was neither stimulated by Ca2+ nor inhibited by EGTA and had a broad pH spectrum. The substrate specificity of the enzyme was investigated with various natural lipids and with a fluorescent analog of the phosphatidylcholine. The enzyme was equally active toward phosphatidylcholine and phosphatidylglycerol, but did not hydrolyze diphosphatidylglycerol. The enzyme did not act on triacylglycerol, diacylglycerol or cholesteryl ester, but low activity was detected toward monoacylglycerol. The enzyme was heat-sensitive and detergent-sensitive, and was almost completely inhibited by p-bromophenacylbromide (50 microM), but was not affected by SH reagents. This study is the first one reporting phospholipase A1 activity in Mollicutes. A possible role of this enzyme in forming lipid mediators upon the interaction of M. penetrans cells with eukaryotic cells is suggested.

Acquired Immunodeficiency Syndrome↗
PubMed1995

An unusual polar lipid from the cell membrane of Mycoplasma fermentans.

The major unidentified polar lipid (compound X), recently demonstrated in the cell membrane of Mycoplasma fermentans, was purified by preparative silicic acid column chromatography. Chemical analyses of acid-hydrolyzed compound X revealed that, in addition to fatty acids, it contains glycerol, choline and phosphate in a molar ratio of approximately 1:1:2, and an amino acid that has a retention time similar to that of homoserine. The methylated fatty acid fraction of compound X was subjected to gas-liquid chromatography and revealed methyl palmitate and methyl stearate in a 4.6:1 molar ratio. The structure of compound X was further analyzed by combining mass spectrometry, 31P-NMR and 1H-NMR. The positive and negative fast atom bombardment spectra showed a major component of M(r) 1048 and a minor component of M(r) 1076. Two different phosphate groups were identified in each of the components by 31P-NMR. Fast atom bombardment, tandem mass spectrometry, negative and positive chemical ionization mass spectrometry together with mass spectra analyses of the water-soluble and ether-soluble products obtained by methanolysis has shown that, in addition to palmitic and stearic acid residues, the presence of glycerol, ribitol, cholinephosphate and homoserinephosphate residues. It is suggested that the apparent structure of compound X is either a phosphatidylcholine attached via a phosphotriester bond to a ribitolphosphohomoserine moiety or a phosphatidylhomoserine attached via a phosphotriester bond to a ribitolphosphocholine moiety. The major molecular species is the dipalmitoylderivative (M(r) 1048), whereas the minor molecular species is a stearoyl palmitoyl derivative (M(r) 1076).

Cell Membrane↗
PubMed1995

Early detection of structural anomalies and markers of chromosomal aberrations by transvaginal ultrasonography.

Transvaginal ultrasonography is emerging as the newest method for the early detection of structural malformations and markers of chromosomal aberrations. According to a multicenter registry on the onset of fetal anomalies, many defects are potentially detectable at an early transvaginal scan. Moreover, cystic hygroma and other nuchal signs thought to indicate a very significant risk for a genetic problem detected during the first trimester may resolve and not be detected at the later abdominal scan. Current research presented in this review indicates a possible transition from an abdominal scan at 18-22 weeks to two scans: an early transvaginal scan at 12-14 weeks for the detection of gross congenital anomalies and markers of chromosomal aberrations, and then an abdominal scan at 22 weeks for cardiac and other anomalies.

Chromosome Aberrations↗
PubMed1994

Membrane lipids of Mycoplasma fermentans.

Membranes of Mycoplasma fermentans, incognitus strain, were isolated by a combination of osmotic lysis and sonication. Analysis of membrane lipids revealed, in addition to free and esterified cholesterol, six major polar lipids dominated by a de novo synthesized compound (compound X), which accounts for 64% of the total lipid phosphorus. Compound X was labeled by palmitate, but not by oleate. Mass spectrometry and gas liquid chromatography analyses of compound X revealed two molecular species with molecular masses of 1048 and 1076 representing, a dipalmitoyl- and a stearoyl-palmitoyl-glycerodiphosphatidylcholine. Compound X has the ability to stimulate human monocytes to secret TNF alpha and to enhance the fusion of small unilamellar vesicles with MOLT-3 lymphocytes.

Humans↗
PubMed1994

Use of Merocyanine 540 and Hoechst 33258 for the selective killing of contaminating mycoplasmas in cell cultures.

Mycoplasma infection can substantially affect the biological properties of cells in vitro. We have devised a method for the selective killing of mycoplasmas, e.g., A. laidlawii, M. fermentans, M. hyorhinis and M. arginini, from experimentally infected cell cultures. This approach is based on the differential binding of the lipophilic fluorescent probe Merocyanine 540 followed by illumination with visible light. The efficiency of the procedure depends on the Merocyanine 540 concentration, the intensity of illumination, and the presence of oxygen in the medium. When A. laidlawii contaminated corneal endothelial cell cultures were treated simultaneously with Merocyanine 540 and DNA-binding fluorochrome Hoechst 33258 and then illuminated, a significant degree of eradication was observed, even after one cycle of treatment. This combined treatment is therefore recommended as an effective method of purging mycoplasmas from contaminated cultures.

Animals↗
PubMed1993

Arginine utilization by Mycoplasma fermentans is not regulated by glucose metabolism: a 13C-NMR study.

13C-NMR studies on the effect of glucose metabolism on arginine hydrolysis in Mycoplasma fermentans cells have been performed using a continuous perfusion technique. With this procedure we were able to show, in the presence of glucose, the rapid accumulation of lactic acid and, in the presence of arginine, the formation of citrulline that is apparently further metabolized. As the accumulation of lactate and the breakdown of arginine were observed in the simultaneous presence of both substrates, it is suggested that the glucose utilization has little or no effect on the deimination of arginine to citrulline.

Arginine↗
PubMed1993

Beware of mycoplasmas.

Mycoplasma infection of cell cultures is widespread and has major detrimental effects on cellular physiology and metabolism. Since cell culture is used extensively, both in research and in industrial production processes, questions of primary concern arise, such as: how can mycoplasma contamination be detected; what are the effects of such contamination on cellular functions; what methods are available for eliminating contamination?

Animals↗
PubMed1993

Cholesterol is required for the fusion of single unilamellar vesicles with Mycoplasma capricolum.

Small unilamellar vesicles (SUV) were prepared from the total lipid extract of Mycoplasma capricolum. The SUV were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C, and in the presence of 5% polyethylene glycol, an increase in the R18 fluorescence with time was observed when the R18-labeled SUV were introduced to a native M. capricolum cell suspension. The fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes of M. capricolum, was interpreted as a result of lipid mixing during fusion between the SUV and the mycoplasma cells. The presence of cholesterol in the SUV was found to be obligatory to allow SUV-mycoplasma fusion to occur. Adaptation of M. capricolum cells to grow in a medium containing low cholesterol concentration provided cells in which the unesterified cholesterol content was as low as 17 micrograms/mg cell protein. The fusion activity of the adapted cells was very low or nonexistent. Nonetheless, when an early exponential phase culture of the adapted cells was transferred to a cholesterol-rich medium, the cells accumulated cholesterol and regained their fusogenic activity. The cholesterol requirement for fusion in the target mycoplasma membrane was met by a variety of planar sterols having a free beta-hydroxyl group, but differing in the aliphatic side chain, e.g., beta-sitosterol or ergosterol, even though these sterols, having a bulky side chain, are preferentially localized in the outer leaflet of the lipid bilayer. It is suggested that the role of cholesterol in mycoplasma-SUV fusion is not at the level of bulk bilayer viscosity but rather, affecting local lipid-lipid or lipid-protein interactions that are relevant to the fusion event.

Adaptation, Physiological↗
PubMed1993

Mycoplasma fermentans (incognitus strain) cells are able to fuse with T lymphocytes.

Characteristics of the fusion of Mycoplasma fermentans (incognitus strain) with cultured lymphocytes were investigated. The rate and extent of fusion were monitored continuously in an assay that measured lipid mixing on the basis of dequenching of a fluorescent probe, octadecylrhodamine (R18), incorporated into mycoplasmas. Fusion of M. fermentans was detected with CD4+ (Molt-3) cells, CD4- (12E1) cells, and primary peripheral-blood lymphocytes. The level of fusion was relatively low (8%-12%). Detection of a similarly low level of fusion by fluorescence microscopy suggested the involvement of a specific lymphocyte subpopulation. After a short lag period, fusion at 37 degrees C proceeded exponentially for approximately 30 minutes and was virtually complete at 60 minutes. Throughout the process, lymphocytes remained intact. Fusion was stimulated by CaCl2 but not by MgCl2; its inhibition by antisera to M. fermentans and by pretreatment of M. fermentans with proteolytic enzymes implied that the mycoplasmas possessed a proteinase-sensitive receptor involved in fusion. Mycoplasmas were rendered nonfusogenic by treatment with the uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone; 5 microM) but were unaffected by treatment with chlorpromazine (10 microM) or DCCD (dicyclohexylcarbodiimide; 50 microM); these findings suggested that a proton gradient across the cell membrane is required for fusion.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Presence of protein constituents of the gram-positive bacterial phosphotransferase regulatory system in Acholeplasma laidlawii.

Acholeplasma species have been reported to lack a functional phosphoenolpyruvate:sugar phosphotransferase system (PTS). We show here that Acholeplasma laidlawii possesses activities of enzyme I, HPr, HPr(ser) kinase, and HPr(ser-P) phosphatase but lacks detectable activities of enzymes II of the PTS. HPr from this organism was purified, and the regulatory properties of the kinase and phosphatase were characterized and shown to differ from those of previously studied bacteria. The results suggest the presence of an incomplete PTS in A. laidlawii which has the potential to function in a unique regulatory capacity.

Acholeplasma laidlawii↗
PubMed1993

Fusion of Spiroplasma floricola cells with small unilamellar vesicles is dependent on the age of the culture.

Small unilamellar vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride and mixed with intact Spiroplasma floricola cells. The increase in fluorescence observed was interpreted as a result of the dilution of the probe in the unlabeled S. floricola membranes because of lipid mixing upon fusion. The progression of S. floricola cultures to the stationary phase of growth was accompanied by a sharp decrease in the ability of the cells to fuse with small unilamellar vesicles. Low fusogenic activity was also detected in cells from cultures that were aged in a growth medium maintained at pH 7.5 throughout the growth cycle. Chemical analysis of the cell membrane preparations isolated from cells harvested at the various phases of growth revealed that the phospholipid content and composition and the cholesterol/phospholipid molar ratio were changed very little upon aging of the cultures. Likewise, no changes in the fatty acid composition of membrane lipids were detected, with palmitic and oleic acids predominating throughout the cycle. Nonetheless, upon aging of S. floricola cultures, a pronounced increase in the levels of both cholesteryl esters, incorporated from the growth medium, and organic peroxides was observed. A decrease in both fluorescence anisotropy of diphenylhexatriene and merocyanine 540 binding to membranes of aged cells was also detected. The possible influence of these changes on the fusogenic activity of the cells is discussed.

Anaerobiosis↗
PubMed1993

Uterine artery flow velocity waveforms and color flow imaging in women with perimenopausal and postmenopausal bleeding. Correlation to endometrial histopathology.

We performed Doppler studies of the uterine artery in 85 women with postmenopausal and perimenopausal bleeding. Sixty-six women had non-malignant changes and 19 women had malignant changes on histologic examination of the endometrium. Uterine fibroids were detected in 30 women. When malignant changes were detected in the endometrium, uterine artery resistance index was always below 0.83. The mean uterine artery resistance index was significantly lower in the group of women with pathologic changes of the endometrium (RI = 0.77 +/- 0.03) compared to the group with non pathologic changes (RI = 0.85 +/- 0.08 p < 0.01), excluding the women with uterine fibroids. The lowest mean resistance index in the uterine artery was observed in the group of women with uterine fibroids (RI = 0.60 +/- 0.09 p < 0.001). In five of six patients with endometrial carcinoma blood vessels were detected around the myometrium by using color flow imaging. Their RI was always less than 0.5. In five patients, blood vessels were detected in uterine fibroids but their RI was always more than 0.5. All six women with endometrial carcinoma but only seven of 13 women with endometrial hyperplasia, had endometrial thickness greater than 5 mm. Doppler velocimetry of the uterine artery carries a high sensitivity (100%) in detecting pathological changes of the endometrium in patients presenting with postmenopausal or perimenopausal bleeding. The presence of high resistance in this vessel, may allow a more conservative approach in this group of patients.

Aged↗