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Thioesterase activity was found in all mycoplasmas tested. Activity was highest in Acholeplasma species, whereas most of the sterol-requiring Mycoplasma species showed little activity. The thioesterase activity of Acholoplasma laidlawii... more
Thioesterase activity was found in all mycoplasmas tested. Activity was highest in Acholeplasma species, whereas most of the sterol-requiring Mycoplasma species showed little activity. The thioesterase activity of Acholoplasma laidlawii is confined to the cell membrane. The enzyme could not be released from the membrane by either low- or high-ionic-strength solutions, with or without ethylenediaminetetraacetic acid, nor solubilized by detergents. The enzyme has a general specificity for long-chain saturated and unsaturated fatty acid thioesters. The preferred substrates among the saturated fatty acyl derivatives are the myristyl and palmityl derivatives. Arrhenius plots of thioesterase activities in A. laidlawii membranes enriched with elaidic or palmitic acids showed discontinuities at 12 and 18 degrees C, respectively. The possible regulatory significance of the thioesterase activity for the fatty acid synthetase and the possibllity that the activity of the enzyme is controlled by...
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The cell membrane of Mycoplasma hominis was isolated by lysing the cells with digitonin. Electron microscopy and chemical, density gradient, and electrophoretic analyses of the membrane proteins showed the membranes so obtained, like... more
The cell membrane of Mycoplasma hominis was isolated by lysing the cells with digitonin. Electron microscopy and chemical, density gradient, and electrophoretic analyses of the membrane proteins showed the membranes so obtained, like those isolated by osmotic lysis, to be relatively free of cytoplasmic contaminants. Sensitivity to digitonin lysis depended on temperature but was not affected by Mg 2+ ions and was only slightly affected by the age of the culture. Accordingly, it seems that digitonin may be used for the isolation of cell membranes from sterol-requiring mycoplasmas that tend to be fairly resistant to osmotic lysis.
Research Interests: Bacteriology, Biology, Transmission Electron Microscopy, Saponins, Medicine, and 15 moreBiological Sciences, Magnesium, Cholesterol, Microbial genetic and drug resistance, Cell Fractionation, Temperature, Membrane, Tritium, Mycoplasma, Densitometry, Phosphotungstic Acid, Sterols, Culture Media, Cell Membrane, and Medical and Health Sciences
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... more
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 t...
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Research Interests: Bacteriology, Biology, Phospholipids, Medicine, Biological Sciences, and 14 moreLipids, Carbon Isotopes, Cholesterol, Triglycerides, Glycerol, GAS LIQUID CHROMATOGRAPHY, Mycoplasma, Species Specificity, Quntitative Thin Layer Chromatography, Culture Media, Silicon Dioxide, Esters, Cell Membrane, and Medical and Health Sciences
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Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin . Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92: 714–722. 1966.—Adenosine triphosphatase activity of Mycoplasma laidlawii, M.... more
Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin . Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92: 714–722. 1966.—Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum , and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2′-(3′)-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. ...
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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... more
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.
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ABSTRACT
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This chapter focuses on membrane fusion. Several studies presented an indication of a fusion process. Apostolov and Windsor demonstrated by ultrathin sectioning and freeze-etching the intimate contacts between erythrocytes and Mycoplasma... more
This chapter focuses on membrane fusion. Several studies presented an indication of a fusion process. Apostolov and Windsor demonstrated by ultrathin sectioning and freeze-etching the intimate contacts between erythrocytes and Mycoplasma gallisepticum and suggested that these areas represent a fusion process. The mycoplasma-liposome fusion process has been intensively characterized and was utilized for the transfer of plasmids encapsulated in lipid vesicles into Spiroplasma floricola. For measuring fusion, a number of sensitive assays based on fluorescence dequenching have been developed. They utilize the mixing of aqueous contents entrapped within the lipid vesicles and fusion is determined by monitoring the generation of a fluorescent product as the vesicles fuse. Another approach is based on membrane mixing. Hoekstra et al. have developed an assay relying on the relief of self-quenching of fluorescence of a fluorescent fatty acid, octadecylrhodamine B (R18) that is spontaneously inserted into native membranes at high concentrations. The extent of R18 dequenching directly correlates with the mycoplasma ratios in the medium. In many cases, lipid vesicle–mycoplasma fusion or mycoplasma–eukaryotic cell fusion was found to depend on polyethylene glycol.
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The mycoplasmas form a large group of prokaryotic microorganisms with over 190 species distinguished from ordinary bacteria by their small size, minute genome, and total lack of a cell wall. Owing to their limited biosynthetic... more
The mycoplasmas form a large group of prokaryotic microorganisms with over 190 species distinguished from ordinary bacteria by their small size, minute genome, and total lack of a cell wall. Owing to their limited biosynthetic capabilities, most mycoplasmas are parasites exhibiting strict host and tissue specificities. The aim of this review is to collate present knowledge on the strategies employed by mycoplasmas while interacting with their host eukaryotic cells. Prominant among these strategies is the adherence of mycoplasma to host cells, identifying the mycoplasmal adhesins as well as the mammalian membrane receptors; the invasion of mycoplasmas into host cells including studies on the role of mycoplasmal surface molecules and signaling mechanisms in the invasion; the fusion of mycoplasmas with host cells, a novel process that raises intriguing questions of how microinjection of mycoplasma components into eukaryotic cells subvert and damage the host cells. The observations of d...
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We have previously demonstrated that the AIDS-associated Mycoplasma fermentans as well as Mycoplasma capricolum membranes activated bone marrow macrophages to secrete tumor necrosis factor alpha (TNF alpha) and induce blast transformation... more
We have previously demonstrated that the AIDS-associated Mycoplasma fermentans as well as Mycoplasma capricolum membranes activated bone marrow macrophages to secrete tumor necrosis factor alpha (TNF alpha) and induce blast transformation of splenic lymphocytes. Herein, we show that the membrane component of Mycoplasma capricolum capable of inducing TNF alpha secretion is a hydrophobic protein. This is supported by our findings that the TNF alpha inducing activity was eluted by a phenyl-Sepharose column in a peak distinct from bulk membrane lipids. The hydrophobic nature of the protein is indicated by the activity of the "hydrophobic protein" fraction of the membranes, and the pattern of elution obtained by the phenyl-Sepharose column. Fractionation of the M. capricolum membranes, solubilized by CHAPS (3-[(3-cholamidopropyl)-dimethylammoniol]1-propane sulfate) on a gel filtration column revealed a major peak of TNF alpha inducing activity of about 75,000 daltons, and a min...
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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... more
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, resultin...
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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... more
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.
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Sealed vesicles were obtained by fusing Mycoplasma gallisepticum membranes with asolectin-cholesterol vesicles. The fusion was induced by freezing and thawing followed by a brief sonication treatment and was detected using a fluorescence... more
Sealed vesicles were obtained by fusing Mycoplasma gallisepticum membranes with asolectin-cholesterol vesicles. The fusion was induced by freezing and thawing followed by a brief sonication treatment and was detected using a fluorescence sedimentation behavior in a sucrose density gradient and were shown to be impermeable to small solutes. The advantages of such fused preparations for transport studies in mycoplasmas are discussed.
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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... more
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 membrane...
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Research Interests: Fluorescence Microscopy, Biology, Confocal Microscopy, Medicine, Lipids, and 15 moreHumans, Escherichia coli, Cholesterol, Liposomes, Biotin, Histones, Lipid bilayers, Bovine Serum Albumin, Erythrocyte Membrane, Erythrocytes, Lipid Bilayer, Enzyme Linked Immunosorbent Assay, Biochemistry and cell biology, Biotinylation, and Cell Membrane
Mollicutes are the smallest and simplest self-replicating prokaryotes. These microorganisms lack a rigid cell wall and are bound by a single membrane, the plasma membrane (Razin et al., 1998). Wall-less prokaryotes were first described... more
Mollicutes are the smallest and simplest self-replicating prokaryotes. These microorganisms lack a rigid cell wall and are bound by a single membrane, the plasma membrane (Razin et al., 1998). Wall-less prokaryotes were first described 100 years ago and now over 180 species, widely distributed among humans, animals, insects and plants are known (Razin et al., 1998). The lack of a cell wall is used to distinguish these microorganisms from ordinary bacteria and to include them in a separate class named Mollicutes. Most human and animal mollicutes are Mycoplasma and Ureaplasma species of the family Mycoplasmataceae. The trivial name mycoplasmas will be used by us to denote any organisms within this family. Mycoplasmas have an extremely small genome size of 0.58–1.35 mb (compared with the 4.64 mb of E. coli). Over the last three years the genomes of Mycoplasma genitalium (0.58mb) and Mycoplasma pneumoniae (0.816mb) have been sequenced (Himmelreich et al., 1996; Fraser et al., 1995), showing only 470 and 500 protein coding regions respectively. Their small genomes impose on these organisms limited metabolic options for replication and survival (Himmelreich et a1.,1997; Pollack et al., 1997).
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We present the complete genomic sequence ofMycoplasma fermentans, an organism suggested to be associated with the pathogenesis of rheumatoid arthritis in humans. The genome is composed of 977 524 bp and has a mean G+C content of... more
We present the complete genomic sequence ofMycoplasma fermentans, an organism suggested to be associated with the pathogenesis of rheumatoid arthritis in humans. The genome is composed of 977 524 bp and has a mean G+C content of 26.95 mol%. There are 835 predicted protein-coding sequences and a mean coding density of 87.6 %. Functions have been assigned to 58.8 % of the predicted protein-coding sequences, while 18.4 % of the proteins are conserved hypothetical proteins and 22.8 % are hypothetical proteins. In addition, there are two complete rRNA operons and 36 tRNA coding sequences. The largest gene families are the ABC transporter family (42 members), and the functionally heterogeneous group of lipoproteins (28 members), which encode the characteristic prokaryotic cysteine ‘lipobox’. Protein secretion occurs through a pathway consisting of SecA, SecD, SecE, SecG, SecY and YidC. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The genes encod...
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Summary: The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. The ability of M. penetrans to both adhere to and invade Molt-3 lymphocytes was markedly increased in the presence of... more
Summary: The AIDS-associated Mycoplasma penetrans is capable of inducing its own uptake by non-phagocytic cells. The ability of M. penetrans to both adhere to and invade Molt-3 lymphocytes was markedly increased in the presence of polyethylene glycol 8000 (PEG). The effect of PEG was more pronounced in the more alkaline pH range, where the binding kinetics were much faster and almost unaffected by temperature (4-37 C). Incubation of [14C]oleic-acid-labelled Molt-3 cells with viable M. penetrans resulted in a substantial release of radioactive fatty acids, whereas treating the host cells with heat-inactivated mycoplasmas, isolated M. penetrans membrane preparations, or M. penetrans growth medium, had no effect. Total lipid analysis of Molt-3 lymphocytes infected by M. penetrans revealed an augmented level of the neutral lipid fraction that was associated with a decrease in the relative amounts of polar lipids, mainly a decrease in the amount of phosphatidylserine and diphosphatidylgl...
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Choline-containing lipids were identified and characterized in the cell membrane of Mycoplasma fermentans and were shown to participate in the adhesion to the surface of eukaryotic cells, to stimulate mycoplasma fusion with eukaryotic... more
Choline-containing lipids were identified and characterized in the cell membrane of Mycoplasma fermentans and were shown to participate in the adhesion to the surface of eukaryotic cells, to stimulate mycoplasma fusion with eukaryotic cells, and to induce cytokine secretion by cells of the immune system. These findings suggest that choline-containing lipids are important mediators of tissue pathology in the infectious process caused by M. fermentans.
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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... more
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.
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Research Interests: International organizations, Transmission Electron Microscopy, Biological Sciences, Cell line, Humans, and 12 moreEscherichia coli, Bacteria, Mechanism, Phosphorylation, Proteins, HeLa cells, International Organizations, Bacterial Adhesion, Tyrosine, Tyrosine Phosphorylation, Gentamicins, and Medical and Health Sciences
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... Williamsport, Pa.) prewashed with chloroform. Neutral and polar lipids were eluted in succession with 20 ml of chloroform and 20 ml of methanol, respectively (Ansell and Hawthorne, 1966). Polar lipid fractions, which account ...
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ABSTRACT
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... 9. Razin, S., and Rottem, S. (1976) In Biochemical Analysis of Membranes (Maddy, AH, ed.), pp 326, Chapman and Hall, London. 1O.Rottem, S., Markowitz, 0., and Razin, S. (1978) Eur. J. Biochem. 85, 451456. 11.Lowry, OH, Rosebrough, NJ,... more
... 9. Razin, S., and Rottem, S. (1976) In Biochemical Analysis of Membranes (Maddy, AH, ed.), pp 326, Chapman and Hall, London. 1O.Rottem, S., Markowitz, 0., and Razin, S. (1978) Eur. J. Biochem. 85, 451456. 11.Lowry, OH, Rosebrough, NJ, Farr, AL, and Randall, RJ (1951) J ...
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ABSTRACT
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Research Interests: Chemical Engineering, Chemistry, Medicine, Biological Sciences, Cell fusion, and 14 moreBacteria, Physical sciences, Membrane Fusion, Temperature, Fusion, Membrane Lipids, Liposome, Mycoplasma, Elsevier, Hydrogen-Ion Concentration, Polyethylene Glycols, Edetic Acid, Biochemistry and cell biology, and magnesium chloride
Research Interests: Chemical Engineering, Kinetics, Biology, Phospholipids, Medicine, and 15 moreBiological Sciences, Physical sciences, Temperature, Solubility, Mycoplasma, Surface Active Agents, Bacillus Cereus, Detergents, Freezing, Biochemistry and cell biology, Phospholipase C, Cell Membrane, Sodium Dodecyl Sulfate, Papain, and Pronase
1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The... more
1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis 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 is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.