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Shlomo Rottem - One of the best experts on this subject based on the ideXlab platform.
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genomic features and insights into the biology of Mycoplasma fermentans
Microbiology, 2011Co-Authors: Hagai Rechnitzer, Elzbieta Brzuszkiewicz, Axel Strittmatter, Heiko Liesegang, Inna Lysnyansky, Rolf Daniel, Gerhard Gottschalk, Shlomo RottemAbstract:We present the complete genomic sequence of Mycoplasma 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 encoding DnaK-DnaJ-GrpE and Tig, forming the putative complex of chaperones, are intact, providing the only known control over protein folding. Eighteen nucleases and 17 proteases and peptidases were detected as well as three genes for the thioredoxin-thioreductase system. Overall, this study presents insights into the physiology of M. fermentans, and provides several examples of the genetic basis of systems that might function as virulence factors in this organism.
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α enolase resides on the cell surface of Mycoplasma fermentans and binds plasminogen
Infection and Immunity, 2007Co-Authors: Amichai Yavlovich, Hagai Rechnitzer, Shlomo RottemAbstract:Plasminogen (Plg) binding to the cell surface of Mycoplasma fermentans results in a marked increase in the maximal adherence of the organism to HeLa cells, enhanced Plg activation by the urokinase-type Plg activator, and the induction of the internalization of M. fermentans by eukaryotic host cells (A. Yavlovich, A. Katzenell, M. Tarshis, A. A. Higazi, and S. Rottem, Infect. Immun. 72:5004-5011, 2004). In this study, the M. fermentans Plg binding protein was isolated by affinity chromatography of Triton X-100-solubilized M. fermentans membranes by utilizing a column of a Plg-biotin complex attached to avidin that was eluted with epsilon-aminocaproic acid. The eluted approximately 50-kDa protein was identified by mass spectrometric techniques as alpha-enolase. The possibility that alpha-enolase, a key cytoplasmatic glycolytic enzyme, resides also on the cell surface of M. fermentans was supported by an immunoblot analysis using polyclonal anti-alpha-enolase antiserum, which showed that alpha-enolase was present in a purified M. fermentans membrane preparation, as well as by immunochemical criteria and by immunoelectron microscopy analysis. Our observation that Plg blocked the binding of anti-alpha-enolase antibodies to a 50-kDa polypeptide band resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of M. fermentans membrane or soluble preparations further supports our notion that Mycoplasmal surface alpha-enolase is a major Plg binding protein of M. fermentans.
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binding of host extracellular matrix proteins to Mycoplasma fermentans and its effect on adherence to and invasion of hela cells
Fems Microbiology Letters, 2007Co-Authors: Amichai Yavlovich, Shlomo RottemAbstract:In the present study, we show that intact Mycoplasma fermentans cells have a wealth of adhesive interactions with components of the extracellular matrix. Mycoplasma fermentans intensively bind plasminogen, and to a lesser extent, fibronectin, heparin, and laminin. The binding of collagen type III, IV, or V was low. The binding of plasminogen, collagen type III, or collagen type V markedly enhanced the adherence of M. fermentans to HeLa cells, whereas the binding of fibronectin, heparin, laminin, or collagen IV induced only a small effect on Mycoplasma adherence. Utilizing plasminogen-treated M. fermentans preparations, we detected microorganisms within host HeLa cells by the gentamicin protection assay or by confocal laser scanning microscopy of immunofluoresent preparations. However, no intracellular M. fermentans was detected when M. fermentans preparations treated with fibronectin, heparin, laminin, or collagen type III, IV, or V were utilized.
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Reconstituted Proteolipid Vesicles Prepared from Mycoplasma fermentans Membranes Are Able to Bind and Fuse with Molt-3 Cells
Current Microbiology, 2006Co-Authors: Hagai Rechnitzer, Shlomo RottemAbstract:We describe and characterize reconstituted proteolipid vesicles (rPLV) prepared from solubilized Mycoplasma fermentans membranes and studied their binding to and fusion with host Molt-3 cells. The rPLV were prepared following membrane solubilization by Triton X-100 and detergent removal by SM-2 resin beads. The vesicles thus obtained had a rather uniform diameter of about 1 μm and were sealed as monitored by measuring in an assay that measures the quenching by sodium dithionite of a hydrophobic fluorescent probe incorporated into the rPLV membrane. The rPLV adhered to Molt-3 cells and, based on measurements of lipid mixing, fused with the host cells at a similar rate and to about the same extent as intact M. fermentans . Preliminary experiments showed that a chimeric protein, GnRH-PE66, could be encapsulated within these rPLV, opening the way to develop a system for the transfer of high-molecular weight soluble molecules, encapsulated in the rPLV, to target eukaryotic cells.
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the reducing antioxidant capacity of Mycoplasma fermentans
Fems Microbiology Letters, 2006Co-Authors: Amichai Yavlovich, Ron Kohen, Isaac Ginsburg, Shlomo RottemAbstract:Mycoplasma fermentans is an extracellular microorganism capable of adhering to the surface of host cells. It has been recently shown that plasminogen binding to M. fermentans in the presence of the urokinase-type plasminogen activator promotes the invasion of host cells by this organism. In this report, we show that viable Mycoplasmas persist within the infected HeLa cells for prolonged periods of time despite the expectation that within host cells the organism may be exposed to oxidative stress. Using cyclic voltammetry and luminol-enhanced chemiluminescence assays, we detected a potent reducing antioxidant activity in M. fermentans. The reducing antioxidant activity was heat stable, not affected by proteolysis and was almost totally lost upon dialysis suggesting that the activity is due to a nonproteinaceus low molecular weight antioxidant. This antioxidant was partially purified by Bio-Gel column chromatography followed by high-pressure liquid chromatographic analysis. We suggest that the high reducing antioxidant capacity in M. fermentans is a principal defense mechanism playing a major role in the battle of the organism against oxidative stress within the host cells.
Shyhching Lo - One of the best experts on this subject based on the ideXlab platform.
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Mycoplasma fermentans infection promotes immortalization of human peripheral blood mononuclear cells in culture
Blood, 2004Co-Authors: Shimin Zhang, James Waikuo Shih, Shien Tsai, Timothy T Wu, Bingjie Li, Shyhching LoAbstract:Chronic infection or colonization by Mycoplasma(s) could gradually and significantly alter many biologic properties of mammalian host cells in culture, including induction of malignant transformation. We examined effects of Mycoplasma fermentans infection on the continuing survival and immortality of human peripheral blood mononuclear cells (PBMCs) from healthy blood donors. Without specific supplemental growth factors, human PBMCs normally die rapidly, with few cells other than macrophages/monocytes surviving after 2 weeks in cultures. Only occasional Epstein-Barr virus (EBV)-positive B lymphocytes would continue to proliferate and undergo spontaneous immortalization. Our present study revealed that infection of human PBMCs in culture with the incognitus and PG18 strains of M fermentans, but surprisingly not with some other strains tested in parallel, markedly enhanced the rate of EBV-positive B lymphocytes to undergo immortalization (74% vs 17%). Compared with spontaneously immortalized PBMCs, the PBMCs immortalized in cultures infected with the Mycoplasmas often had prominent karyotype changes with chromosomal loss, gain, or translocations. Furthermore, many of these immortalized B lymphocytes were found to be monoclonal in nature. The in vitro findings would be of relevance to lymphoproliferative disorders that occurred in patients with immune suppression. The Mycoplasma-mediated promotional effect in cell immortalization and its potential clinical implications warrant further study.
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high frequency dna rearrangements in the chromosomes of clinically isolated Mycoplasma fermentans
Current Microbiology, 1998Co-Authors: Wensi S Hu, M M Hayes, James Waikuo Shih, Richard Wang, Shyhching LoAbstract:Mycoplasma fermentans is currently being examined as an agent potentially associated with human disease. Several strains of M. fermentans were isolated from patients with respiratory tract disease and AIDS. Two of these clinical strains, M64 and SK6, were triple-filter-cloned and designated as the parental clones in this study. Genomic DNA of randomly picked subclones in four and five subsequent generations passed from the parental M64 and SK6 clones were analyzed by using a radiolabeled M. fermentans-specific insertion sequence (IS)-like element as the probe. The hybridization patterns of DNA restriction fragments revealed high frequencies of chromosomal changes accompanied with excision or new insertion of the IS-like element in M. fermentans chromosome. The findings indicate M. fermentans has an effective mechanism(s) to produce a rapid gene rearrangement that may be mediated by one or more copies of the IS-like element.
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in vitro antibiotic susceptibility testing of different strains of Mycoplasma fermentans isolated from a variety of sources
Antimicrobial Agents and Chemotherapy, 1993Co-Authors: M M Hayes, H Kotani, D J Wear, Shyhching LoAbstract:The in vitro susceptibilities to antibiotics of 24 strains of Mycoplasma fermentans (isolated from human immunodeficiency virus type 1-infected AIDS patients, non-AIDS patients with acute respiratory disease, and tissue culture) were determined. MICs for 90% of the strains tested (micrograms per milliliter) were obtained for chloramphenicol (1.25), ciprofloxacin (0.078), clindamycin (0.078), doxycycline (0.625), erythromycin (> 10), gentamicin (> 10), levofloxacin (0.078), lincomycin (0.156), streptomycin (> 10), and tetracycline (0.625).
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fatal systemic infections of nonhuman primates by Mycoplasma fermentans incognitus strain
Clinical Infectious Diseases, 1993Co-Authors: Shyhching Lo, Douglas J Wear, James Waikuo Shih, Richard Wang, Perry B Newton, Jose F RodriguezAbstract:Four silvered leaf monkeys inoculated with Mycoplasma fermentans (incognitus strain) showed wasting syndromes and died in 7-9 months. Infected animals had a late and transient antibody response to Mycoplasmal infection. Three monkeys revealed periodic Mycoplasmal antigenemia. The one that had the most persistent antigenemia failed to mount a detectable antibody response and was the first to die of the infection. The control monkey was killed 8 months later, after the last of the infected animals had died, and revealed no evidence of seroconversion or antigenemia
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a family of strain variant surface lipoproteins of Mycoplasma fermentans
Infection and Immunity, 1993Co-Authors: K S Wise, P Theiss, Shyhching LoAbstract:The wall-less procaryote Mycoplasma fermentans is currently being examined as an agent potentially associated with human disease, including infectious processes affecting immunocompromised individuals. To delineate and understand the interactions of M. fermentans with its host, specific membrane surface components were characterized as markers for detecting the organism and for assessing heterogeneity in antigenic surface architecture within this Mycoplasma species. Detergent phase fractionation of metabolically labeled organisms of type strain PG18 identified a family of prominent integral membrane proteins; several of these labeled with 35S-cysteine and 3H-palmitate, which are characteristics of procaryotic lipoproteins. Specific monoclonal and polyclonal antibodies raised to strain PG18 components further distinguished seven of these membrane proteins, which were localized on the organism9s surface by monitoring their selective susceptibility during trypsin treatment of intact cells. With these antibodies, Western immunoblot profiles of surface membrane antigens expressed on strain PG18 were compared with those expressed on the recently identified Incognitus strain of M. fermentans, as well as with several other human and animal Mycoplasma species. While the antibodies were specific for M. fermentans, marked differences were observed between the strains in the size of one surface lipoprotein and in the apparent levels of several antigens expressed in the cultured populations analyzed. Some monoclonal antibodies to strain PG18 and a previously described monoclonal antibody to strain Incognitus showed apparent selectivity for the strain used for immunization. Monoclonal antibodies developed here recognize stable epitopes defining a family of surface lipoproteins and provide critical tools to determine the basis of surface variation in this Mycoplasma species and to assess the location and antigenic phenotypes of organisms in the human host.
Talma Brenner - One of the best experts on this subject based on the ideXlab platform.
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choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes
Fems Microbiology Letters, 2001Co-Authors: Gil Benmenachem, Awni Mousa, Talma Brenner, Florence Pinto, Ulrich Zahringer, Shlomo RottemAbstract:A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a Km of 2.2×10−5 M and a Vmax of 0.15 nmol 10 min−1 mg−1 cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated Mycoplasmas and could be reversed by the addition of free choline to the growth medium.
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Mycoplasma fermentans—Induced Inflammatory Response of Astrocytes: Selective Modulation by Aminoguanidine, Thalidomide, Pentoxifylline and IL-10
Inflammation, 1999Co-Authors: Ruth Gallily, Marianne Kipper-galperin, Talma BrennerAbstract:Exposure of primary rat glial cells, mostly astrocytes, to heat-inactivated Mycoplasma fermentans triggers the production of tumor necrosis factor α (TNFα) nitric oxide (NO) and prostaglandin E_2 (PGE_2). To attenuate the production of these proinflammatory mediators, four agents: aminoguanidine, pentoxifylline, thalidomide and IL-10 were added to astrocyte cultures. Aminoguanidine (1 and 3 mM), an inhibitor of inducible nitric oxide synthase (iNOS), suppressed the production of the three mediators. TNFα was the most sensitive to thalidomide, showing dose-response inhibition at concentrations of 20 μg/ml, 50 μg/ml and 250 μg/ml. PGE_2 was affected only by concentrations of 50 μg/ml and 250 μg/ml, whereas NO responded solely to the highest amount of this inhibitor. The cytokine IL-10, at 10 U and 50 U, inhibited only TNFα production. Our results imply that selective suppression of proinflammatory mediators by various agents may prove feasible for amelioration of central nervous system inflammatory diseases.
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Mycoplasma fermentans induced inflammatory response of astrocytes selective modulation by aminoguanidine thalidomide pentoxifylline and il 10
Inflammation, 1999Co-Authors: Ruth Gallily, Marianne Kippergalperin, Talma BrennerAbstract:Exposure of primary rat glial cells, mostly astrocytes, to heat-inactivated Mycoplasma fermentans triggers the production of tumor necrosis factor α (TNFα) nitric oxide (NO) and prostaglandin E2 (PGE2). To attenuate the production of these proinflammatory mediators, four agents: aminoguanidine, pentoxifylline, thalidomide and IL-10 were added to astrocyte cultures. Aminoguanidine (1 and 3 mM), an inhibitor of inducible nitric oxide synthase (iNOS), suppressed the production of the three mediators. TNFα was the most sensitive to thalidomide, showing dose-response inhibition at concentrations of 20 μg/ml, 50 μg/ml and 250 μg/ml. PGE2 was affected only by concentrations of 50 μg/ml and 250 μg/ml, whereas NO responded solely to the highest amount of this inhibitor. The cytokine IL-10, at 10 U and 50 U, inhibited only TNFα production. Our results imply that selective suppression of proinflammatory mediators by various agents may prove feasible for amelioration of central nervous system inflammatory diseases.
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Mycoplasma fermentans glycolipid triggers inflammatory response in rat astrocytes
Brain Research, 1998Co-Authors: Gil Benmenachem, Shlomo Rottem, Mark Tarshis, Varda Barash, Talma BrennerAbstract:Abstract Mycoplasma fermentans glycolipid (MfGL-II) is a major lipid in the membranes of this AIDS-associated Mycoplasma and constituting up to 20% of the total phospholipids of this organism. It was recently shown that MfGL-II, mainly through its phosphocholine moiety, is responsible for the attachment of M. fermentans to host cells. We now show that MfGL-II is also associated with the secretion of inflammatory mediators by cells of the central nervous system. Stimulation of primary rat astrocytes by MfGL-II caused activation of protein kinase C, secretion of nitric oxide (NO) and prostaglandin E 2 , and augmented glucose utilization and lactate formation in a dose-dependent manner. In an attempt to define the minimal structural requirements for MfGL-II activity, the two O -acylated fatty acids in the molecule were removed. Deacylation pronouncedly reduced the stimulatory activity of the glycolipid, suggesting that the fatty acyl residues are essential. Incubation of MfGL-II with polyclonal anti-MfGL-II antiserum or with monoclonal anti-phosphocholine antibody diminished NO release, whereas incubation of MfGL-II with normal rabbit serum had no effect. It is, therefore, likely that the terminal phosphocholine moiety plays an important role in MfGL-IIs stimulation of glial cells.
Ulrich Zahringer - One of the best experts on this subject based on the ideXlab platform.
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physicochemical characterization and biological activity of a glycoglycerolipid from Mycoplasma fermentans
FEBS Journal, 2003Co-Authors: Klaus Brandenburg, Ulrich Zahringer, Frauke Wagner, Mareike Muller, Holger Heine, Jorg Andra, Michel H J Koch, Ulrich SeydelAbstract:We report a comprehensive physicochemical characterization of a glycoglycerolipid from Mycoplasma fermentans, MfGl-II, in relation to its bioactivity and compared this with the respective behaviors of phosphatidylcholine (PC) and a bacterial glycolipid, lipopolysaccharide (LPS) from deep rough mutant Salmonella minnesota strain R595. The β⇆α gel-to-liquid crystalline phase transition behavior of the hydrocarbon chains with Tc = 30 °C for MfGl-II as well as for LPS exhibits high similarity between the two glycolipids. A lipopolysaccharide-binding protein (LBP)-mediated incorporation into negatively charged liposomes is observed for both glycolipids. The determination of the supramolecular aggregate structure confirms the existence of a mixed unilamellar/cubic structure for MfGl-II, similar to that observed for the lipid A moiety of LPS. The biological data clearly show that MfGl-II is able to induce cytokines such as tumor necrosis factor-α (TNF-α) in human mononuclear cells, although to a significantly lower degree than LPS. In contrast, in the Limulus amebocyte lysate test, MfGl-II is completely inactive, and in the CHO reporter cell line it does not indicate any reactivity with the Toll-like receptors TLR-2 and -4, in contrast to control lipopeptides and LPS. These data confirm the applicability of our conformational concept of endotoxicity to nonlipid A structures: an amphiphilic molecule with a nonlamellar cubic aggregate structure corresponding to a conical conformation of the single molecules and a sufficiently high negative charge density in the backbone.
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choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes
Fems Microbiology Letters, 2001Co-Authors: Gil Benmenachem, Awni Mousa, Talma Brenner, Florence Pinto, Ulrich Zahringer, Shlomo RottemAbstract:A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a Km of 2.2×10−5 M and a Vmax of 0.15 nmol 10 min−1 mg−1 cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated Mycoplasmas and could be reversed by the addition of free choline to the growth medium.
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the phosphocholine motif in membranes of Mycoplasma fermentans strains
Fems Microbiology Letters, 2001Co-Authors: Gil Benmenachem, Ulrich Zahringer, Shlomo RottemAbstract:Mycoplasma fermentans strains differ in the profile of choline-containing phosphoglycolipids (PGL) present in their cell membrane. MfGL-II [Zahringer et al. (1997) J. Biol. Chem. 272, 26262–26270] was found to be the major PGL in most strains tested. However, in the pulmonary isolates, M52 and M39 the major choline-containing PGLs were MfGL-I [Matsuda et al. (1994) J. Biol. Chem. 269, 33123–33129] and MfEL, a unique choline-containing ether lipid recently identified by us [Wagner et al. (2000) Eur. J. Biochem. 267, 6276–6286]. Both MfGL-I and MfEL were metabolically labeled by growing the cells with radioactive choline but only MfGL-I reacted with anti-phosphocholine antibodies. All tested strains fused with Molt-3 cells at almost the same rate and to about the same extent and in all the strains membrane proteins that reacted with anti-phosphocholine antibodies were detected, indicating that some membrane proteins are decorated with phosphocholine moieties.
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ether lipids in the cell membrane of Mycoplasma fermentans
FEBS Journal, 2000Co-Authors: Frauke Wagner, Shlomo Rottem, Heinzdieter Held, Stefan Uhlig, Ulrich ZahringerAbstract:Two new ether lipids, 1-O-alkyl/alkenyl-2-O-acyl-glycero-3-phosphocholine and its lyso form, 1-O-alkyl/alkenyl-glycero-3-phosphocholine, were identified in the cell membrane of Mycoplasma fermentans using chemical analyses, GLC-MS, MALDI-TOF MS, and 1D and 2D NMR spectroscopy. The lipids are heterogeneous with respect to both acyl and alkyl/alkenyl residues. The acyl residues at position 2 of glycerol are hexadecanoyl and octadecanoyl in a molar ratio of 3.6 : 1 with a trace amount of octadecenoyl. The alkyl/alkenyl residues at position 1 of glycerol are hexadecyl (78%), octadecyl (7%), octadecenyl (14%), and hexadecenyl (traces). In the octadecenyl residue, the double bond has a cis configuration and is located at either position 1′ (plasmalogen-type lipid) or 9′ in a ratio ≈ 1 : 1. This is the first report of the presence of alkyl and vinyl (alk-1′-enyl) ether lipids in the cell membrane of aerobically grown Mycoplasmas. Lipids of this type have been found in some Gram-positive bacteria, thus supporting the hypothesized close taxonomical relationship of these bacteria to Mycoplasmas. The ether lipids of M. fermentans are structurally similar to platelet activating factor; it was demonstrated that the 2-O-acetylated lyso form lipid can mimic platelet-activating factor activity in isolated perfused and ventilated rat lungs.
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primary structure of a new phosphocholine containing glycoglycerolipid of Mycoplasma fermentans
Journal of Biological Chemistry, 1997Co-Authors: Ulrich Zahringer, Gil Benmenachem, Frauke Wagner, Ernst Th Rietschel, Joseph Deutsch, Shlomo RottemAbstract:Abstract The chemical structure of a novel phosphocholine-containing glycoglycerolipid, the major polar lipid in the cell membrane of Mycoplasma fermentans PG18, was investigated by chemical analyses, gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as one- and two-dimensional homo- and heteronuclear NMR spectroscopy and identified as 6′-O-(3"-phosphocholine-2"-amino-1"-phospho-1",3"-propanediol)-α-d-glucopyranosyl-(1′→3)-1,2-diacyl-glycerol (MfGL-II). Palmitate (16:0) and stearate (18:0), in a 3.6:1 molar ratio, constitute the major fatty acids present. MALDI-TOF mass spectrometry revealed two major pseudomolecular ions atm/z 1049.5 [MI + H]+and 1077.3 [MII + H]+ representing a dipalmitoyl as the major component and a palmitoyl-stearoyl structure as a minor component. This is the first report of 2-amino-1,3-propanediol-1,3-bisphosphate present in a natural product. This glycoglycerolipid is the second phosphocholine-containing glycoglycerolipid found in M. fermentans.
Gil Benmenachem - One of the best experts on this subject based on the ideXlab platform.
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the physico chemical characteristics of the phosphocholine containing glycoglycerolipid mfgl ii govern the permeability properties of Mycoplasma fermentans
FEBS Journal, 2001Co-Authors: Gil Benmenachem, Shlomo Rottem, Tomas Bystrom, Hagai Rechnitzer, Leif Rilfors, Goran LindblomAbstract:Mycoplasma fermentans seems to be involved in several pathogenic condtions in humans, and is among other things capable of fusing with T-cells and lymphocytes. The choline-containing phosphoglycoli ...
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choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes
Fems Microbiology Letters, 2001Co-Authors: Gil Benmenachem, Awni Mousa, Talma Brenner, Florence Pinto, Ulrich Zahringer, Shlomo RottemAbstract:A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a Km of 2.2×10−5 M and a Vmax of 0.15 nmol 10 min−1 mg−1 cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated Mycoplasmas and could be reversed by the addition of free choline to the growth medium.
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the phosphocholine motif in membranes of Mycoplasma fermentans strains
Fems Microbiology Letters, 2001Co-Authors: Gil Benmenachem, Ulrich Zahringer, Shlomo RottemAbstract:Mycoplasma fermentans strains differ in the profile of choline-containing phosphoglycolipids (PGL) present in their cell membrane. MfGL-II [Zahringer et al. (1997) J. Biol. Chem. 272, 26262–26270] was found to be the major PGL in most strains tested. However, in the pulmonary isolates, M52 and M39 the major choline-containing PGLs were MfGL-I [Matsuda et al. (1994) J. Biol. Chem. 269, 33123–33129] and MfEL, a unique choline-containing ether lipid recently identified by us [Wagner et al. (2000) Eur. J. Biochem. 267, 6276–6286]. Both MfGL-I and MfEL were metabolically labeled by growing the cells with radioactive choline but only MfGL-I reacted with anti-phosphocholine antibodies. All tested strains fused with Molt-3 cells at almost the same rate and to about the same extent and in all the strains membrane proteins that reacted with anti-phosphocholine antibodies were detected, indicating that some membrane proteins are decorated with phosphocholine moieties.
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Mycoplasma fermentans glycolipid triggers inflammatory response in rat astrocytes
Brain Research, 1998Co-Authors: Gil Benmenachem, Shlomo Rottem, Mark Tarshis, Varda Barash, Talma BrennerAbstract:Abstract Mycoplasma fermentans glycolipid (MfGL-II) is a major lipid in the membranes of this AIDS-associated Mycoplasma and constituting up to 20% of the total phospholipids of this organism. It was recently shown that MfGL-II, mainly through its phosphocholine moiety, is responsible for the attachment of M. fermentans to host cells. We now show that MfGL-II is also associated with the secretion of inflammatory mediators by cells of the central nervous system. Stimulation of primary rat astrocytes by MfGL-II caused activation of protein kinase C, secretion of nitric oxide (NO) and prostaglandin E 2 , and augmented glucose utilization and lactate formation in a dose-dependent manner. In an attempt to define the minimal structural requirements for MfGL-II activity, the two O -acylated fatty acids in the molecule were removed. Deacylation pronouncedly reduced the stimulatory activity of the glycolipid, suggesting that the fatty acyl residues are essential. Incubation of MfGL-II with polyclonal anti-MfGL-II antiserum or with monoclonal anti-phosphocholine antibody diminished NO release, whereas incubation of MfGL-II with normal rabbit serum had no effect. It is, therefore, likely that the terminal phosphocholine moiety plays an important role in MfGL-IIs stimulation of glial cells.
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primary structure of a new phosphocholine containing glycoglycerolipid of Mycoplasma fermentans
Journal of Biological Chemistry, 1997Co-Authors: Ulrich Zahringer, Gil Benmenachem, Frauke Wagner, Ernst Th Rietschel, Joseph Deutsch, Shlomo RottemAbstract:Abstract The chemical structure of a novel phosphocholine-containing glycoglycerolipid, the major polar lipid in the cell membrane of Mycoplasma fermentans PG18, was investigated by chemical analyses, gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as one- and two-dimensional homo- and heteronuclear NMR spectroscopy and identified as 6′-O-(3"-phosphocholine-2"-amino-1"-phospho-1",3"-propanediol)-α-d-glucopyranosyl-(1′→3)-1,2-diacyl-glycerol (MfGL-II). Palmitate (16:0) and stearate (18:0), in a 3.6:1 molar ratio, constitute the major fatty acids present. MALDI-TOF mass spectrometry revealed two major pseudomolecular ions atm/z 1049.5 [MI + H]+and 1077.3 [MII + H]+ representing a dipalmitoyl as the major component and a palmitoyl-stearoyl structure as a minor component. This is the first report of 2-amino-1,3-propanediol-1,3-bisphosphate present in a natural product. This glycoglycerolipid is the second phosphocholine-containing glycoglycerolipid found in M. fermentans.
