(1/194) Detection of serum proteins in the electrophoretic patterns of total proteins of mycoplasma cells.

The contamination of mycoplasma cell preparations by serum proteins originating from culture medium was studied. A. laidlawii and M. arthritidis cells were grown in the presence of [14C]-aminoacids, and the cells were washed with 0-9% NaC1 by threefold centrifugation. Total proteins of the washed cells were analysed by SDS gel electrophoresis. Coomassie-stained electrophoretic patterns were compared with autoradiographs of the same gels. The stained electrophoretic pattern of washed A. laidlawii grown without serum was identical with autoradiographs of the same cells grown without or with serum. That of washed A. laidlawii grown with serum differed from the corresponding autoradiography by the presence of extra protein bands I, II, III, and IV with molecular weights of over 160,000, 80,000-87,000, 55,000 and 25,000, respectively. The same extra bands were found in stained electrophoretic patterns of washed: (a) A. laidlawii cells grown without serum and mixed with serum in the stationary phase, (b) M. arthritidis cells, as compared with their autoradiographs, (c) serum precipitate. The bands III and IV may be due to the heavy and light chains of gamma-globulin, the band II might belong to transferrin or to some component of complement. Acidification of serum to pH 5 brought about 100-fold rise of amount of serum precipitate, the number of bands in the electrophoretic pattern of the precipitate being also increased. Stained electrophoretic patterns of cells purified by twofold centrifugation in step sucrose density gradient (1-20-1-27 g./cm.3 for A. laidlawii, and 1-15-1-25 for M. arthritidis) contained no extra bands and matched completely with their autoradiographs. It was concluded that contamination of washed mycoplasma cells by serum proteins is mainly due to co-precipitation of aggregated serum proteins together with cells during centrifugation rather than to adsorption of serum proteins on the cell surface.  (+info)

(2/194) Bactericidal/permeability-increasing protein inhibits growth of a strain of Acholeplasma laidlawii and L forms of the gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes.

Bactericidal/permeability-increasing protein (BPI) inhibited growth of cell wall-deficient Acholeplasma laidlawii and L forms of certain strains of Staphylococcus aureus and Streptococcus pyogenes. However, the same strains of S. aureus and S. pyogenes with intact cell walls were not susceptible to the growth-inhibitory effects of BPI.  (+info)

(3/194) Replication of mycoplasmavirus MVL51: attachment of MVL51 parental DNA to host cell membrane.

The replication of the single-stranded circular DNA of MVL51 mycoplasmavirus has been studied with respect to the roles of free and membrane-associated viral DNA intermediates. Replication involves the formation of parental replicative intermediate (RF) molecules on, at most, two to three membrane sites per cell, symmetric RF replication at the membrane and apparent asymmetric RF replication in the cytoplasm leading to single-stranded progeny chromosomes.  (+info)

(4/194) The nonbilayer/bilayer lipid balance in membranes. Regulatory enzyme in Acholeplasma laidlawii is stimulated by metabolic phosphates, activator phospholipids, and double-stranded DNA.

In membranes of Acholeplasma laidlawii a single glucosyltransferase step between the major, nonbilayer-prone monoglucosyl-diacylglycerol (MGlcDAG) and the bilayer-forming diglucosyl-diacylglycerol (DGlcDAG) is important for maintenance of lipid phase equilibria and curvature packing stress. This DGlcDAG synthase is activated in a cooperative fashion by phosphatidylglycerol (PG), but in vivo PG amounts are not enough for efficient DGlcDAG synthesis. In vitro, phospholipids with an sn-glycero-3-phosphate backbone, and no positive head group charge, functioned as activators. Different metabolic, soluble phosphates could supplement PG for activation, depending on type, amount, and valency. Especially efficient were the glycolytic intermediates fructose 1,6-bisphosphate and ATP, active at cellular concentrations on the DGlcDAG but not on the preceding MGlcDAG synthase. Potencies of different phosphatidylinositol (foreign lipid) derivatives differed with numbers and positions of their phosphate moieties. A selective stimulation of the DGlcDAG, but not the MGlcDAG synthase, by minor amounts of double-stranded DNA was additive to the best phospholipid activators. These results support two types of activator sites on the enzyme: (i) lipid-phosphate ones close to the membrane interphase, and (ii) soluble (or particulate)-phosphate ones further out from the surface. Thereby, the nonbilayer (MGlcDAG) to bilayer (DGlcDAG) lipid balance may be integrated with the metabolic status of the cell and potentially also to membrane and cell division.  (+info)

(5/194) Binding of glycoglycerolipid derived from membranes of Acholeplasma laidlawii PG8 and synthetic analogues to lymphoid cells.

A component that binds to human lymphoid cells was isolated from the membranes of Acholeplasma laidlawii PG8. The component was extracted using the Bligh-Dyer method and purified using a silica-gel column and TLC. The active component was identified as 3-O:-[2'-O-(alpha-D-glucopyranosyl)- 6'-O-acyl-alpha-D-glucopyranosyl]-1,2-di-O- acyl-sn-glycerol (GAGDG) using (1)H- and (13)C-NMR and GC-MS. The compositions of the major saturated fatty acids were nC (14) (17.8%), isoC(14) (10.7%) and nC (16) (34.9%) as determined by GC-MS. The amounts of unsaturated species were less than 10% of those of the corresponding saturated acids. GAGDGs which have three tetradecanoyl groups were synthesized. These synthetic GAGDGs, as well as GAGDGs derived from A. laidlawii membranes, had a high binding affinity for MOLT-4 and HUT-78 (human T cell lines), Raji (a B cell line), HL-60 (a monoblastoid cell line) and primary cultured human T cells. The binding affinities of GAGDGs with an isoC(14) acyl group was higher than those with nC(14) and nC(16) acyl groups. The binding to lymphoid cells reveals a novel biological activity of GAGDGs.  (+info)

(6/194) Survival of frozen mycoplasmas.

Cooling to -70 C killed a higher percentage of Acholeplasma laidlawii and Mycoplasma mycoides var. capri cells than cooling to -20 C. However, to preserve cell viability for prolonged periods storage at -70 C was much more preferable. The percentage of cells surviving freezing could be increased by increasing the initial cell concentration or by the addition of dimethyl sulfoxide or glycerol as cryoprotective agents. In the presence of 1.5 M of any one of these agents survival rates of up to 100% could be obtained. The optimal cooling rates for maximal survival of A. laidlawii under the experimental conditions tested were 11 C/min for cooling to -20 C and about 15 C/min for cooling to -70 C. Increasing the warming rate during thawing from 0.6 to 67 C/min increased survival by 3 log. Oleic acid enrichment of A. laidlawii membrane lipids, or reduction in the cholesterol content of M. mycoides var. capri membranes, increased the percentage of organisms surviving freezing. Hence, the composition of membrane lipids appears to have a marked influence on the susceptibility of mycoplasmas to freezing injury.  (+info)

(7/194) Reduced nicotinamide adenine dinucleotide oxidase activity in membranes and cytoplasm of Acholeplasma laidlawii and Mycoplasma mycoides subsp. capri.

The properties of the membrane-bound reduced nicotinamide adenine dinucleotide (NADH) oxidase of Acholeplasma laidlawii were compared with those of the corresponding cytoplasmic activity of Mycoplasma mycoides subsp. capri. The striking differences in pH optima, susceptibility to inhibitors and detergents, and heat inactivation between the NADH oxidase activity, with oxygen as an electron acceptor, and the NADH oxidoreductase activity, with dichlorophenol indophenol (DCPIP) as an alternate electron acceptor, support the presence of more than one catalytic protein in both the membrane-bound and soluble enzyme systems. The detection of more than one band positive for the NADH-nitroblue tetrazolium oxidoreductase reaction on electrophoresis of either the membranes of A. laidlawii or the cytoplasm of M mycoides subsp. capri also points in the same direction. The membrane-bound enzyme system differed, however, form the soluble one because it had a lower ratio of oxidase activity to oxidoreductase activity, and because it was less susceptible to heat inactivation and more readily incorporated incorporated into reaggregated membranes. In addition, the specific activity of the membrane-bound enzyme system increased as the culture aged, whereas that of the soluble system decreased as the culture aged. It is suggested that the different location in the cell could be responsible for some of the differences between the membrane-bound NADH oxidase activity of A. laidlawii and that found in the cytoplasm of M. mycoides subsp. capri.  (+info)

(8/194) Microcalorimetric detection of growth of Mycoplasmatales.

A static ampoule microcalorimeter was used to study the growth of mycoplasmas, acholeplasmas and ureaplasmas. Growth as indicated by thermograms was compared with the results of conventional methods, namely, terminal dilution counts, plate counts, turbidimetric measurements, glucose consumption and pH changes. Removal of oxygen had little effect on mycoplasma growth. The microcalorimetric method is potentially useful for identifying and enumerating the members of the Mycoplasmatales.  (+info)

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