Membrane lipids of Mycoplasma fermentans

Abstract 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 TNFα and to enhance the fusion of small unilamellar vesicles with MOLT-3 lymphocytes.     

Control of membrane lipids in Mycoplasma gallisepticum: effect on lipid order.

Adaptation of Mycoplasma gallisepticum, a sterol-requiring Mycoplasma sp., to growth in a serum-free medium supplemented with cholesterol in decreasing concentrations and with various saturated or unsaturated fatty acids enabled us to control both the cholesterol levels and the membrane fatty acid composition. An estimate of the membrane physical state from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicated that the membrane lipids of native M. gallisepticum were highly ordered. Elongation of the saturated fatty acid chains from 14 to 18 carbon atoms caused only a small increase in the membrane lipid ordering, whereas the introduction of a cis double bond reduced it significantly. Lipid-phase transitions were observed in low-cholesterol-adapted organisms, whose membrane lipids were still highly ordered at the growth temperature.     

Detection of Mycoplasma hominis and Mycoplasma orale in cell cultures by immunofluorescence.

Mycoplasma contaminants of animal and human cell cultures were rapidly detected and identified by an indirect immunofluorescent technique. Cells suspected of being contaminated by mycoplasmas were grown as monolayers on chamber slides in a culture medium selected to promote mycoplasmal growth. Before fixation by acetone, the monolayers were subjected to a hypotonic treatment to cause swelling of the mycoplasmas. Detection and identification were then performed by indirect immunofluorescence using rabbit antisera to various mycoplasma species. The correlation between results obtained by the standard isolation procedure and those obtained by this method was very close.     

Attachment of Mycoplasma salivarium and Mycoplasma orale to glass surfaces

Mycoplasma salivarium ATCC 23064 and 24 other oral strains, and Mycoplasma orale ATCC 15539 and 22 other oral strains were examined quantitatively for attachment to glass surfaces by using ELISA method. Although all of the tested strains attached to glass surfaces, M. salivarium attached far more readily than M. orale. The results suggested that electrostatic bonds were involved in the attachment and that bivalent metal ions also played a role.     

Membrane lipid composition and cellular function

Membrane fatty acid composition, phospholipid composition, and cholesterol content can be modified in many different kinds of intact mammalian cells. The modifications are extensive enough to alter membrane fluidity and affect a number of cellular functions, including carrier-mediated transport, the properties of certain membrane-bound enzymes, binding to the insulin and opiate receptors, phagocytosis, endocytosis, depolarization-dependent exocytosis, immunologic and chemotherapeutic cytotoxicity, prostaglandin production, and cell growth. The effects of lipid modification on cellular function are very complex. They often vary from one type of cell to another, and they do not exert a uniform effect on all processes in a single cell line. Therefore, it is not yet possible to make any generalizations or to predict how a given system will respond to a particular type of lipid modification. Many of the functional responses probably are caused directly by the membrane lipid structural changes, which affect either bulk lipid fluidity or specific lipid domains. The conformation or quaternary structures of certain transporters, receptors, and enzymes probably are sensitive to changes in the structure of their lipid microenvironment, leading to changes in activity. Prostaglandin production is modulated by the availability of substrate fatty acids stored in the membrane phospholipids, but the underlying chemical mechanism still involves a change in membrane lipid structure. While this is the most likely mechanism, the possibility that the membrane lipid compositional change is an independent event that occurs concurrently but is not causally related to the functional perturbations also must be considered.     

The lipid composition of Mycoplasma laidlawii strain B

1. Total lipid was extracted from Mycoplasma laidlawii strain B with chloroform-methanol mixtures and fractionated into neutral lipid, glycolipid and phospholipid components by chromatography on silicic acid. 2. Saponification of the glycolipid fraction, which represented nearly half of the total lipid, yielded two glycosides for which the structures O-alpha-d-glucopyranosyl-(1-->1)-d-glycerol and O-alpha-d-glucopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl-(1-->1)-d-glycerol were established. 3. The ratio of monoglucosyl diglyceride to diglucosyl diglyceride increased with the age of the culture, though the total glycolipid concentration remained virtually constant. The glycolipid concentration was unaffected by the addition of cholesterol to the culture medium. 4. The phospholipid fraction consisted of two components, phosphatidylglucose and phosphatidylglycerol. Organisms harvested at acidic pH also contained O-amino acyl esters of phosphatidylglycerol. No lipids containing inositol could be detected.     

Membrane lipid composition defines membrane protein spectrum

In addition to glycerophospholipids, bacterial membranes often include amino acid-containing acyloxyacyl lipids. The functional implications of these aminolipids are largely unknown. However, a recent study by Stirrup et al. expands our understanding and shows that they are major determinants for membrane properties and the relative abundance of distinct membrane proteins in bacterial membranes.     

Attachment of Mycoplasma hominis and M. orale to Human Diploid Lung Fibroblasts

The process of attachment of Mycoplasma hominis and M. orale to HAIN-55 cells, derived from normal embryonic human lung, was investigated quantitatively. The attachment reached its maximum within about 2–4 hr at 37 C and increased linearly as a function of the number of organisms present in the system. The relative attachment efficiency of M. hominis was approximately 1% under our experimental conditions. Trypsin and EDTA were effective in detaching particles of M. hominis and M. orale from the surfaces of HAIN-55 cells. Therefore it was suggested that some proteinaceous substance and salt bridges might be involved in the attachment of these mycoplasmas to HAIN-55 cells.     

Fatty acid composition of the milk lipids of Nepalese women: correlation between fatty acid composition of serum phospholipids and melting point.

Milk was collected from 36 Nepalese women, 15 to 32 years of age, in order to investigate relationships between the proportions of intermediate chain-length (C10-C14) fatty acids and critical n-3 and n-6 polyunsaturated fatty acids in the milk lipids they were producing. Serum was also obtained from these lactating women and the fatty acid composition of their serum phospholipid fraction was determined and compared with that of the corresponding milk lipid fraction. Compared to women in technologically advanced parts of the world, the serum phospholipids of the Nepalese women contained nutritionally adequate proportions of linoleic acid (LA) (16.8%), alpha-linolenic acid (ALA) (0.53%), arachidonic acid (AA) (5.69%), and docosahexaenoic acid (DHA) (1.42%). However, although the milk lipids contained adequate proportions of ALA (1.81%), AA (0.43%), and DHA (0.23%), the lipids contained low to moderate percentages of LA (mean, 9.05%). Positive correlations were observed between the proportions of AA (P=0.001, r=0.50) and ALA (P=0.03, r=0.36) in the serum phospholipids and milk lipids of the women. As the proportion of C10-Cl4 fatty acids in the milk lipids increased from 10% to 40%, there was preferential retention of three critical n-3 and n-6 fatty acids (ALA, AA, and DHA) at the expense of two relatively abundant nonessential fatty acids, namely stearic acid and oleic acid. In addition, using fatty acid melting point data and the mol fraction of the 9 most abundant fatty acids in the milk, we estimated the mean melting point (MMP) of the milk lipids of the Nepalese women. The MMPs ranged from 29.3 to 40.5 degrees C (median, 35.5 degrees C). These results indicate that: 1) the levels of AA and ALA in the blood of lactating mothers influence the levels of these fatty acids in the milk they produce; 2) when the mammary gland produces a milk that is rich in C10-Cl4 fatty acids, it somehow regulates triglyceride synthesis in such a way as to ensure that the milk will provide the exclusively breast-fed infant with the amounts of the critical n-3 and n-6 fatty acids it requires for normal growth and development; and 3) the melting point of the milk lipid fraction is determined mainly by the mol % of the intermediate chain-length (C10-C14) fatty acids, oleic acid, linoleic acid, and alpha-linolenic acid.     

Fatty acid composition of platelet phospholipids and plasma lipids in obese Zucker rats

The purpose of this work was to see whether hyperlipaemia observed in genetically obese Zucker rats (fa/fa) was associated with differences in fatty-acid composition of plasma triacylglycerols, plasma phospholipids and of platelet phospholipids, in comparison with the control lean rats (Fa/-). Results showed that plasma triacylglycerols and phospholipids were increased in obese rats. In triacylglycerols, the amount of saturated and monounsaturated fatty acids was highly increased whereas the amount of the n-6 and n-3 polyunsaturated fatty acids was little modified. In plasma phospholipids, saturated and monounsaturated fatty acids were also increased, as were the n-3 fatty acids (except C 18:3 n-3); the n-6 fatty acids were little increased except C 20:3 n-6 which was markedly increased. These results concerning the amounts of fatty acids have their counterpart in their relative proportions of fatty acids. Data thus obtained suggest that conversion of linoleic acid (C 18:2 n-6) into arachidonic acid (C 20:4 n-6) was decreased in obese rats, particularly the delta 5 desaturation step. On the contrary, conversion of linolenic acid (C 18:3 n-3) into higher polyenes seemed increased. Thrombocytosis was not modified in the obese rat, but the volume of the platelets was increased. Platelet phospholipids exhibited the same modifications as plasma phospholipids but with different magnitude. Saturated and monounsaturated fatty acids were little augmented, n-3 fatty acids were more augmented (except C 18:3 n-3 acid which was unchanged); n-6 fatty acids were not modified except C 20:3 n-6 acid which was highly increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in membrane lipid composition of Mycoplasma capricolum affect the cell volume.

The cellular water volume of Mycoplasma capricolum was markedly increased by a decrease in the cholesterol-to-phospholipid molar ratio in the membrane. An increase in cell volume was also observed with the increase in the phospholipid cell membrane content obtained by the incorporation of exogenous phosphatidylcholine from the growth medium.     

Membrane lipid composition of obligately and facultatively alkalophilic strains of Bacillus spp.

The membrane lipids from two obligately and two facultatively alkalophilic strains of Bacillus spp. were characterized in a comparative study that included B. subtilis. Preparations of membrane lipids were made from pH 10.5-grown cells of all of the alkalophiles and from pH 7.5- or 7.0-grown cells of the two facultative strains and B. subtilis. The two obligate alkalophiles contained high ratios of membrane lipid to membrane protein, and the lipid fraction contained a high proportion of neutral lipid. These characteristics are probably not prerequisites for growth at very high pH since one or another of the facultative strains failed to show these properties at high pH. All of the alkalophiles contained appreciable amounts of squalene and C40 isoprenoids. Among the polar lipids, the alkalophiles all contained high concentrations of anionic phospholipids, including phosphatidylglycerol and especially large amounts of cardiolipin; phosphatidylethanolamine was the other major phospholipid. Small amounts of bis(monoacylglycero)phosphate were found in most, but not all, of the alkalophile preparations. Glycolipids and phosphoglycolipids were absent. The fatty acid composition of the total phospholipid and individual fractions revealed two features that distinguished between the obligate and facultative strains. Membranes from the obligately alkalophilic species contained a high concentration of branched-chain fatty acids, comparable to that in membranes from B. subtilis, as well as a relatively high content of unsaturated fatty acids. By contrast, the facultatively alkalophilic strains contained almost no unsaturated fatty acids and a lower concentration of branched-chain fatty acids than either the obligate alkalophiles or B. subtilis.     

Fish skin surface lipids: phospholipids

Phospholipid components of skin surface secretions of four Indian freshwater fishes were analysed using different solvent systems in one-dimensional and two-dimensional thin-layer chromatography, and quantified using a spectrophotometer. The phospholipid pattern of the skin secretions is similar to that of membrane lipids. Phosphatidyl choline and phosphatidyl ethanolamine are the major components constituting approximately two-thirds of the total phospholipids. Sphingomyelin and phosphatidyl serine are present in relatively low quantities. Phosphatidic acid, phosphatidyl glycerol, lysophosphatidyl choline and lysophosphatidyl ethanolamine, which may be important metabolic intermediates, are present in very low quantities in the skin surface secretions of all four species. It is suggested that the lipids are derived from membranous profiles in the secretions of skin glands, extrusion of membrane-bound vesicles from surface epithelial cells, and exfoliated cells. The results are discussed in relation to the biochemical and biological significance of these lipids in the skin secretions.     

Lanolin-derived lipid mixtures mimic closely the lipid composition and organization of vernix caseosa lipids

The aim of the present study was to use semi-synthetic lipid mixtures to mimic the complex lipid composition, organization and thermotropic behaviour of vernix caseosa (VC) lipids. As VC shows multiple protecting and barrier supporting properties before and after birth, it is suggested that a VC substitute could be an innovative barrier cream for barrier deficient skin. Lanolin was selected as the source of the branched chain sterol esters and wax esters — the main lipid classes of VC. Different lipid fractions were isolated from lanolin and subsequently mixed with squalene, triglycerides, cholesterol, ceramides and fatty acids to generate semi-synthetic lipid mixtures that mimic the lipid composition of VC, as established by high-performance thin-layer chromatography. Differential scanning calorimetry and Fourier transform infrared spectroscopy investigations revealed that triglycerides play an important role in the (lateral) lipid organization and thermotropic behaviour of the synthetic lipid mixtures. Excellent resemblance of VC lipids was obtained when adding unsaturated triglycerides. Moreover, these lipid mixtures showed similar long range ordering as VC. The optimal lipid mixture was evaluated on tape-stripped hairless mouse skin in vivo. The rate of barrier recovery was increased and comparable to VC lipid treatment.     

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale.

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.     

Membrane composition of jetted lipid vesicles: a Raman spectroscopy study

Microfluidic jetting is a promising method to produce giant unilamellar phospholipid vesicles for mimicking living cells in biomedical studies. We have investigated the chemical composition of membranes of vesicles prepared using this approach by means of Raman scattering spectroscopy. The membranes of all jetted vesicles are found to contain residuals of the organic solvent decane used in the preparation of the initial planar membrane. The decane inclusions are randomly distributed over the vesicle surface area and vary in thickness from a few to several tens of nanometers. Our findings point out that the membrane properties of jetted vesicles may differ considerably from those of vesicles prepared by other methods and from those of living cells. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Membrane lipid composition and susceptibility to bile salt damage

Erythrocyte membranes with low sphingomyelin: choline-containing phospholipid ratios haemolyse at low concentrations of the bile salt, glycocholate. Erythrocytes with higher sphingomyelin: choline-containing phospholipid ratios require progessively greater concentrations of the bile salt for lysis.Sublytic concentrations of glycocholate remove phospholipid and acetylcholinesterase from the membranes. Membranes with low sphingomyelin: choline-containing phospholipid ratios lose both particulate (microvesicles of distinct composition) and ‘solubilized’ material, the particulate form predominating. The proportion of particulate material falls with increase of the membrane sphingomyelin: choline-containing phospholipid ratio and those membranes of highest sphingomyelin: choline-containing phospholipid ratio lose material predominantly in ‘solubilized’ form.Sheep erythrocytes treated to increase their content of phosphatidylcholine (and thereby reduce their membrane sphingomyelin: choline-containing phospholipid ratio) become more susceptible to lysis by glycocholate.These observations indicate a correlation between membrane lipid composition and the perturbation of membranes with bile salt; they also point to possible features of membranes capable of surviving exposure to the high bile salt concentrations of the biliary tract.