The role of lipids in the interaction of Acholeplasma laidlawii cells with lymphocytes.

The role of A. laidlawii membrane lipids in the organism's interaction with mouse spleen lymphocytes is analyzed. A. laidlawii cells were grown in a lipid-poor medium with unsaturated fatty acids that allowed cells with different degrees of membrane lipid microviscosity to be obtained. The intensity of the binding of A. laidlawii cells and derived liposomes with lymphocytes depended directly on the degree of fatty acid unsaturation. Cholesterol incorporation into the A. laidlawii membrane reduced the fluidity of the lipid bilayer and decrease the binding activity. The intensity of cholesterol transfer from lymphocytes to A. laidlawii also depended on the degree of fatty acid unsaturation in A. laidlawii cells. Cells enriched with cholesterol took up considerably less of this sterol from lymphocytes. The loss of cholesterol as well as the enrichment of lymphocytes by A. laidlawii membrane fatty acids resulted in a decrease in the microviscosity of lymphocyte membrane lipids. It was concluded that the ability of A. laidlawii cells or derived liposomes to stimulate the transport of carbohydrates into lymphocytes depended on the degree of unsaturation of fatty acid incorporated into A. laidlawii. Cholesterol also decreased the stimulatory effect, probably by lowering carbohydrate carrier mobility.

Obtaining and diagnostic use of immunospecific serum to protein of human decidual tissue.

A method of diagnosis of early periods of pregnancy has been developed. Immunospecific serum to human decidual tissue protein D1 was used. The technique is relatively simple, easily reproducible and can be used for a differential gynecologic diagnosis as well as for a diagnosis of pregnancy.

Transport activity of mouse spleen lymphocytes after their interaction in vitro with Acholeplasma laidlawii cells.

Transport of two non-metabolized carbohydrates (3-O-methyl-D-glucose, 3-O-MG, and 2-deoxy-D-glucose, 2-DG) into mouse spleen lymphocytes after their interaction with Acholeplasma laidlawii cells has been studied. Incubation of A. laidlawii cells and particularly the liposomes prepared from A. laidlawii membrane lipids enhances the rates of the both carbohydrates transport. This treatment resulted in increasing of the Vmax values of 3-O-MG and 2-DG without changing the Km values. This stimulation can be explained by the increasing of the mobility of membrane carbohydrates carriers as a result of the exchange of lipid components between Acholeplasma and lymphocyte membranes. Actually, it has been shown that liposomes derived from A. laidlawii cells grown on the medium with great amount of unsaturated oleic acid stimulate the transport activity more actively than liposomes prepared from the cells grown on the medium with bovine serum or with oleic acid plus cholesterol. It should be suggested that an activation of carbohydrates transport into lymphocytes caused by alteration of the carriers lipid microenvironment.

Immunological and functional activity of spleen lymphocytes from mice infected by Acholeplasma laidlawii cells and Rauscher virus.

The duration of Acholeplasma laidlawii antigen persistence in mice, resistant to Rausher leukemia virus, after infection with both A. laidlawii cells and Rausher virus has been studied. The antigen persistence was accompanied by marked depression of immune response which was especially severe in case of mixed acholeplasmavirus infection. Such immunosuppression and observed infiltration of the spleen with immature leukemic cells can be regarded as a preleukosis. Immunosuppression was accompanied by an increase of the transport of carbohydrates inthe lymphocytes. This stimulation an be explained by the exchange of lipid components between acholeplasma and lymphocyte membranes resulted in increase of lymphocyte membrane fluidity, or it may be due to the mitogenic effect of A. laidlawii cells and virus, accompanied by the same membrane effect.

Interactions of Acholeplasma laidlawii cells with mouse spleen lymphocytes.

Interaction between Acholeplasma laidlawii cells labelled with oleic acid and mouse spleen lymphocytes depended on the time of incubation, on the temperature and on the quantitative ratio between both cells. Uncouplers and EDTA did not influence the intensity of attachment. The resistance of binding to cytochalasin B amd glutaraldehyde as well as localization of A. laidlawii antigens on the lymphocyte surface and experiments with [14C] uridine-labelled mycoplasmas are an evidence against the participation of pinocytosis in this interaction. Prolonged attachment of intact A. laidlawii to washed lymphocytes can be excluded on the basis of an extremely low amount of CFU recovered from disrupted lymphocytes as well as by experiments with uridine-labelled A laidlawii. Specific receptors didn't take part in the binding, because proteolytic enzymes and neuraminidase treatment proved to be nonefficient. Increased binding of lymphocytes with liposomes prepared from mycoplasma lipids as well as the transfer of cholesterol from lymphocyte membrane to mycoplasma membrane demonstrate the participation of membrane lipids in this binding. It should also be mentioned that after the attachment between both cell types and fusion of A. laidlawii cells with lymphocytes takes place. The transfer of unsaturated fatty acids from mycoplasmas into lymphocyte membrane as well as lymphocyte membrane cholesterol into mycoplasma membranes are the consequence of fusion between both cells. The experiments with uncharged hydrophobic fluorescent probe 4-DMC are the direct proof of fusion and mutual exchange of lipid membrane components.

[Lipid regulation of carbohydrate transport system in Mycoplasma]. / Reguliatsiia lipidami aktivnosti uglevodtransportnoi sistemy u mikoplazm.

Cultivation of Acholeplasma laidlawii cells in media containing unsaturated fatty acids results in changes of the physiological state of the membrane lipid bilayer due to preferable incorporation of an unsaturated fatty acid into lipids. The lipids are capable to regulate the transport activity since the transport rates for glucose, 3-O-methyl-C-glucose, glucerol and erythritol change considerably when the cells are cultivated in media containing different unsaturated fatty acids. The transport activity is also affected by the length of the carbon chain, the degree of the fatty acid saturation and the presence of cholesterol. At the same time the activation energy of the transport activity also changes, which suggests that the regulation by lipids (presumably local changes of the physical properties of lipid domen) is involved in the process of the carrier association with the substrate and/or in translocation of this complex through the membrane.

[Glucose induced transport of H+, K+ and lactate in the cells of Acholeplasma laidlawii]. / Indutsirovannyi gliukozoi transport H+, K+ i laktata v kletkakh mikoplazmy Acholeplasma laidlawii.

Currents of H+ and K+ ions initiated under the effect of facultative anaerobe of A. laidlawii, valinomycin, nigericin and carbonyl cyanide chloride phenylhydrasone on the membrane are recorded by means of the system of cation sensitive electrodes. It is stated that during glucose transport a redistribution of H+ and K+ and lactate between the cellular content and the medium takes place. Preincubation of cells with 3-o-methyl-D-glucose essentially decreases the effect of induced ion transport. It is concluded that ion transport observed in the experiments with glucose is related not to glucose transport through the cell membrane but to the release of its metabolic products from the cells.

[Energetics of active carbohydrate transport into mycoplasma cells]. / Energetika aktivnogo transporta uglevodov v kletki mikoplazm.

Possible energy supply pathways of active transport of non-metabolized 3-O-methylglucose (3-O-mG) into Acholeplasma laidlawii cells are studied. 3-O-MG transport is sensitive to arsenate, inhibitors of glucolysis and membrane Mg-dependent ATPase (dicyclohexylcarbodiimide) and to uncoupling agents. It is suggested that glycolysis is the main energy source, which main enzymes are found in cell-free extracts. A. laidlawii cells are capable to glycolytic ATP synthesis using glucose as a substrate but not fructose, arginine and ethanol. ATP synthesis is also sensitive to arsenate and glycolytic inhibitors and is resistant to uncoupling agents, dicyclohexylcarbodiimide, valinomycin and nigericin. Probably, hydrolysis of glycolytic ATP by membrane-bound ATPase results in the formation on a membrane of both components of proton-motive force, which supply energy for the active 3-O-MG transport. It is supported with the data on the inhibitor analysis of transport using ionic-selecive antibiotics and lipid-soluble ions.

[Basic evidence for the active transport of carbohydrates in the membrane vesicles of Acholeplasma laidlawii cells]. / Osnovnye dokazatel'stva aktivnogo transporta uglevodov v membrannye vezikuly iz kletok Acholeplasma laidlawii

A. laidlawii membrane vesicles are able to accumulate C14-glucose as well as maltose and fructose against the concentration gradient in the absence of exogeneous entergetic sources. Sugar transport is inhibited by anaerobiosis and by the electron transfer inhibitors such as rotenone and amytal, and by proton conductors such as carbonylcyanide-m-chlorophenylhydrazone. Arsenate and dicyclohexylcarbodimide (inhibitor of membrane-bound ATPase) do not inhibit the sugar transport. It is concluded that sugar transport in the membrane vesicles can be driven by the high-energy state of the membrane or the membrane potential.

[Some biochemical properties of Acholeplasma laidlawii mutants defective in glucose carrier]. / Biokhimicheskie svoistva mutantov Acholeplasma laidlaw II

Some membrane properties of glucose transport system of Acholeplasma laidlawii mutants uncapable of uptaking glucose as well as those of its non-metabolizing analogue, 3-0-methylglucose are studied. In contrast to the wild strain the mutant PTL-S cells fail to uptake carbohydrates with the saturation. There is no competition between carbohydrates during transport in this mutant no sugar exit is observed, and the sensitivity of transport to thiol reagents and pronase B is considerably affected. All other investigated membrane functions were intact. The data given indicate the presence of a specific defect in glucose transport system.

On the possible role of respiratory activity of Acholeplasma laidlawii cells in sugar transport.

1. Out of 20 exogeneous substrates only ethanol and, to a much lesser extent, lactate and pyruvate were shown to be capable of stimulating the respiration of Acholeplasma laidlawii cells. However, none of these substrates changed the initial rate of active transport of 3-O-methyl-D-glucose (3-O-MG). 2. From inhibitory analyses and spectroscopic data, it is apparent that the respiratory chain of A. laidlawii has no cytochromes and is probably not responsible for oxidative phosphorylation. 3. Valinomycin and nigericin stimulated cell respiration only in the presence of K+-ions, while monensin stimulated it in the presence of Na+-ions. 4. 3-O-MG transport was shown to be sensitive to uncouplers, ATPase inhibitors and arsenate are resistant to a majority of respiratory inhibitors tested. This suggested that there was no relationship between respiration and carbohydrate transport in the A. laidlawii cells. Further evidence was provided by the absence of respiratory stimulation during the transport of non-metabolizing carbohydrates.

[Lipid composition of Acholeplasma laidlawii cells]. / Lipidnyi sostav kletok Acholeplasma laidlawii

Lipid composition of the cells of Acholeplasma laidlawii was studied by column and thin layer chromatography. Lipids of the cells contain phospholipids (41.3%), glycolipids (28%) and neutral lipids. Phospholipids are represented by monoacylglycerophosphoryldiglucosyldiglyceride, glycerophosphoryldiglucosyldiglyceride, phosphatidylglycerine and phosphatidic acid. Glycolipids contained diglucosyldiglyceride and monoglucosyldiglyceride. The content of cholesterine was low (1.3%) while the content of carotenoids was A442X1000/mg lipids -- 150. Lipid components of Acholeplasma laidlawii and related strains of A. laidlawii A and B are compared.

[3-O-methl-D-glucose transport system in the cells of Acholeplasma laidlawii]. / Sistema transporta 3-O-metil-D-gliukozy v kletkakh Acholeplasma laidlawii

In cells of Acholeplasma laidlawii a constitutive system of transport of glucose derivate 3-O-methyl-D-glucose was found. This derivate penetrated into cells, without modification and accumulated in free state. The transport system was of enzymatic nature and exhibited sharp substrate specificity. The transport of 3-O-methyl-D-glucose was an active process and occurred against the concentrated gradient.

Properties of the 3-o-methyl-D-glucose transport system in Acholeplasma laidlawii.

Transport of 3-O-methyl-D-glucose (3-O-MG) by Acholeplasma laidlawii cells was studied. The 3-O-MG transport system appeared to be constitutive in cells grown on 3-O-MG and glucose; the transport process depended on the concentration of substrate used and exhibited typical saturation kinetics, with an apparent Km of 4.6 muM. 3-O-MG was transported as a free carbohydrate and was not metabolized further in the cell. Dependence on pH and temperature and the results of efflux and "counterflow" experiments demonstrated the carrier nature of the transport system. 6-Deoxyglucose and glucose competitively inhibited 3-O-MG transport, whereas maltose inhibited in non-competitively. p-Chloromercuribenzoate, p-chloromercuribenzene sulfonate, N-ethylmaleimide, and iodoacetate inhibited transport of 3-O-MG. Cells were able to accumulate 3-O-MG against a concentration gradient. Some electron transfer inhibitors (rotenone and amytal), arsenate, dicyclohexylcarbodiimide, and proton conductors such as 2,4-dinitrophenol, carbonylcyanide, m-chlorophenylhydrazone, pentachlorophenol, and tetrachlorotrifluoromethylbenzimidazole inhibited this process.

[Respiratory activity of Acholeplasma laidlawii cells and its role in the active transport of carbohydrates]. / Dykhatel'naia aktivnost' kletok Acholeplasma laidlawii i ee rol' v aktivnom transporte uglevodov

The respiratory activity of the Acholeplasma laidlawii cells was studied in order to elucidate a possible mechanism of coupling of transport with energy. The respiration of the cells is stimulated by ethanol, glucose, NADH, lactate, and pyruvate. The substrates of the Krebs cycle have no effect on the respiration. The respiratory activity, stimulated by ethanol and glucose, is inhibited by the inhibitors of the respiratory chain, SH reagents, and the inhibitors of glycolysis. The results of experiments with inhibitors suggest that the respiratory chain in the A. laidlawii cells is reduced and terminated by flavoprotein. This is confirmed by the results of spectroscopic analysis of cytochromes. Respiration coupled with phosphorylation did not play any important role in the active transport of carbohydrates. Probably, the energy, necessary for the transport of carbohydrates, is supplied by the substrate phosphorylation. This explains the activation of respiration by glucose, which is so sensitive to arsenate. The respiration of the A. laidlawii cells is not stimulated by some carbohydrates (fructose, 3-O-methyl-D-glucose).