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Biomedical subjects

K Resch

Publications and source records attributed to K Resch.

At least 127 records · Page 7Linked to original sources
PubMed1985

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose.

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.

Animals↗
PubMed1985

Are cyclic nucleotides involved in the initiation of mitogenic activation of human lymphocytes?

In order to obtain more insight into the possible role of cyclic AMP or cyclic GMP in modulating the initial cellular processes following activation of lymphocytes, we measured the effects of the T-cell mitogen concanavalin A and other substances including hormones on the cyclic nucleotide levels in human peripheral blood lymphocytes. The enzyme activities of the corresponding nucleotide cyclases, adenylate cyclase and guanylate cyclase were measured in both isolated plasma membranes or the cytosol of resting or concanavalin A stimulated rabbit thymocytes. Concanavalin A in a mitogenic concentration of about 5-10 micrograms/ml caused small, but consistent increases in cAMP but no changes in cGMP levels during the first hour of activation. Concomitantly, the specific activity of plasma membrane-bound adenylate cyclase was always increased at least 1.5-fold 30 min after stimulation of rabbit thymocytes with concanavalin A, but no effect could be detected on the specific activities of plasma membrane-bound or soluble guanylate cyclase. At high, supraoptimal concentrations of concanavalin A (more than 20 micrograms/ml) cAMP levels dramatically increased in human lymphocytes within minutes, but cGMP levels again were unaffected. Forskolin and beta-adrenergic hormones elevated cAMP in human lymphocytes, whereas cGMP levels were increased by the addition of sodium nitroprusside or alpha-adrenergic hormones. Sodium nitroprusside, in concentrations which elevated cGMP in human lymphocytes, had no influence on the incorporation of [3H]uridine into RNA of resting or concanavalin A stimulated human lymphocytes. Addition of forskolin resulted in an increase of cAMP levels and a dose-dependent decrease of [3H]uridine incorporation into RNA of concanavalin A-stimulated lymphocytes with no effect on resting lymphocytes. The data suggest that cGMP does not play a role in the initial phase of mitogenic activation of lymphocytes, whereas cAMP may be involved in the blast transformation process as an inhibitory signal.

Adenylyl Cyclases↗
PubMed1985

Characterization of cellular and extracellular plasma membrane vesicles from a non-metastasizing lymphoma (Eb) and its metastasizing variant (ESb).

Plasma membrane fragments from two variants of a murine lymphoma, Eb and ESb, with different metastatic capacity were investigated. Plasma membranes were isolated from tumor cells recovered from the peritoneal cavity. They differed in their lipid composition, indicating a more fluid state of the plasma membranes derived from the highly metastatic tumor line ESb. Extracellular membrane vesicles could be isolated from the ascites of the tumor-bearing mice. The shedding capacity of ESb cells was much higher than that of Eb cells. The extracellular membranes by chemical analysis and the measurement of marker enzymes proved to be derived from the plasma membranes. However, they differed from the plasma membranes from which they were derived in several aspects: the lipid to protein ratio was diminished; the activities of some plasma membrane-associated enzymes were lower while others were identical in plasma membranes and extracellular membranes; the content of saturated fatty acids in phospholipids was enhanced in extracellular membranes. These effects were more pronounced in the highly metastasizing tumor line ESb. It is thus concluded that shedding of extracellular membranes is not a random process. The biochemical differences found in the plasma membranes and the extracellular membranes of the two tumor lines are discussed with respect to the different metastatic capacity of the tumors.

Animals↗
PubMed1985

Functional role of lipid metabolism in activated T-lymphocytes.

Exogenous long-chain fatty acids are readily taken up by unstimulated lymphocytes derived from the thymus of calves or rabbits and esterified to complex lipids, primarily phospholipids and triacylglycerols. Compared to saturated fatty acids, unsaturated fatty acids are incorporated preferentially. Furthermore, unsaturated fatty acids are transferred from triacylglycerols to phospholipids. The transfer cannot be observed with palmitic acid. With regard to individual phospholipid species, oleic acid and linoleic acid are found primarily in phosphatidylcholine. Arachidonic acid, however, is transferred to phosphatidylethanolamine and phosphatidylinositol as well. This suggests an arachidonic-specific transfer between individual phospholipids. Stimulation of the cells with the mitogen concanavalin A results in an enhanced incorporation of the fatty acids and an enhanced transfer from triacylglycerols to phospholipids. Triacylglycerols may thus be regarded as a labile intracellular storage pool that may be activated upon mitogenic stimulation.

Animals↗
PubMed1985

Inhibition of lymphocyte activation by cyclosporin A: interference with the early activation of the membrane phospholipid metabolism in rabbit lymphocytes stimulated with concanavalin A, anti-rabbit immunoglobulin or the Ca2+ ionophore A 23187.

Rabbit lymphocytes from the mesenteric lymph nodes were stimulated with concanavalin A, goat anti-rabbit immunoglobulin, or the Ca2+ ionophore A 23187. The stimulated incorporation of labeled uridine into RNA as well as of labeled thymidine into DNA was suppressed within a dose range of 40-1000 ng/ml cyclosporin A in both Con A-stimulated T lymphocytes and in anti-immunoglobulin-stimulated B lymphocytes, without affecting the resting cells. A 23187-stimulated rabbit lymphocytes proved to be more sensitive to cyclosporin A. At 40 ng/ml the immunosuppressive drug was effective in inhibiting elevated incorporation of labeled nucleosides into macromolecules in ionophore-stimulated cells. Cyclosporin A, at the same concentrations that were effective in inhibiting stimulated RNA and DNA synthesis, suppressed one of the earliest events occurring in stimulated lymphocytes, i.e., enhanced incorporation of unsaturated fatty acids into membrane phospholipids. Whereas cyclosporin A significantly inhibited the incorporation of arachidonic acid into phosphatidylcholine and phosphatidylethanolamine in concanavalin A-, anti-immunoglobulin-, and A 23187-stimulated cells, it proved to be ineffective in inhibiting the incorporation of arachidonate into phosphatidylinositol. The data indicate that cyclosporin A inhibits both T- and B-cell stimulation by interfering with a common target, e.g., the early activation of membrane phospholipid metabolism of rabbit lymphocytes.

Animals↗
PubMed1985

Studies on the mechanism whereby cyclosporin A inhibits T-lymphocyte activation.

The immunosuppressive drug Cyclosporin A (CyA) inhibited the ConA-induced DNA synthesis in C57B1/6 spleen cells at a concentration of 40 ng/ml totally; this inhibition could not be overcome by the addition of highly purified interleukin-1. ConA-induced RNA synthesis was also inhibited by concentrations of 40 or 200 ng/ml CyA, although total inhibition could not be achieved. In contrast, lipopolysaccharide-induced proliferation could not be inhibited. CyA at a concentration of 40 ng/ml also inhibited the ConA-induced production of interleukin-2 by mouse spleen cells, this inhibition was not due to a toxic mechanism. On the contrary, the proliferative response of T cell blasts from a long-term T cell line (M2) to interleukin-2 containing supernatants was not inhibited by concentrations of 40 or 200 ng/ml CyA; only at 20-100-fold higher concentrations partial inhibition could be observed. One of the earliest events in the course of lymphocyte activation, the enhanced incorporation of unsaturated fatty acids into the lymphocyte plasma membranes; was also inhibited by concentrations of CyA, which abrogated the ConA-induced DNA synthesis. The inhibition of the enhanced incorporation of 14C-oleic acid and 14C-linoleic acid, which are incorporated by the membrane-bound lysolecithin-acyltransferase, thus suggests a molecular site of action for CyA.

Animals↗
PubMed1985

Induction of prostanoid synthesis in human platelets by the late complement components C5b-9 and channel forming antibiotic nystatin: inhibition of the reacylation of liberated arachidonic acid.

Treatment of human platelets by the purified late complement components C5b-9 results in a dose- and time-dependent release of prostaglandin E (PGE) and thromboxane B2 (TXB2). To study the mechanism underlying the complement-induced prostanoid synthesis, we examined whether C5b-9 affected the enzyme acyl-coA:lysolecithin acyltransferase (E.C.2.3.1.2.3) that catalyzes the reinsertion of liberated arachidonic acid, the precursor molecule of the prostanoids. With C5b-9 doses sufficient to induce prostanoid synthesis, the activity of lysolecithin acyltransferase, measured as conversion of lysophosphatidyl choline to phosphatidyl choline, was inhibited. For comparison, another channel-forming substance, nystatin, was studied. Nystatin had an effect similar to C5b-9: PGE and TXB2 release was stimulated, whereas acyltransferase activity was inhibited. These finding support the concept that inhibition of lysolecithin acyltransferase might be the prerequisite for prostanoid production.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1984

Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes.

The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.

Adenylyl Cyclases↗
PubMed1984

Densitometric quantitation of individual phospholipids from natural sources separated by one-dimensional thin-layer chromatography.

A method for the quantitation of small amounts of phospholipids derived from biological sources is described. Total phospholipid is determined by mineralization followed by the estimation of liberated phosphate by means of malachite green. The main phospholipid species are separated by one-dimensional thin-layer chromatography. The individual phospholipids are detected by charring with CuSO4/H3PO4. They may be directly quantitated by scanning the thin-layer chromatography plates with a laser densitometer.

Animals↗
PubMed1984

Stimulation of macrophage activity by 12-O-tetradecanoylphorbol-13-acetate.

Treatment of resident murine peritoneal macrophages with 12-O-tetradecanoyl phorbol-13-acetate (TPA) rapidly converted the cells to tumour cytostatic and cytolytic effector cells, as determined by growth inhibition or lysis of T-lymphoma cells (Eb, EL4) in vitro. The effective TPA concentrations were 10(-8) to 10(-7)M. Macrophages became cytotoxic as early as one to two hours after exposure to TPA, and tumour cytotoxicity persisted up to 48 hours. TPA did not interfere with the action of the lymphokine macrophage activating factor (MAF) but acted in synergy with it. Generation of antitumour-active macrophages by TPA was accompanied by other metabolic and functional changes, such as an enhancement of the hexose monophosphate shunt, glucosamine incorporation, RNA and protein synthesis, release of prostaglandin E2, thromboxane and prostacyclin, as well as pinocytosis. These data show that TPA may be a valuable model substance to complement studies of MAF; furthermore, use of TPA may help to clarify the role of activated macrophages during tumour promotion and tumour defense.

Animals↗
PubMed1983

Characterization of functional domains of the lymphocyte plasma membrane.

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.

Acyltransferases↗
PubMed1983

Inhibition of mitogen-induced lymphocyte proliferation by ouabain: interference with interleukin 2 production and interleukin 2 action.

Since the discovery of IL 2 it has been possible to dissect the mitogenic activation of T lymphocytes into two steps: first, the production of IL 2; and second, the response of IL 2-dependent T cell blasts to IL 2. We investigated the influence of ouabain, a known inhibitor of T cell activation, on the production of and the response to IL 2 in mouse T lymphocytes. Ouabain dose-dependently inhibited DNA synthesis in Con A activated C57BL/6 spleen cells; inhibition below background levels occurred at a concentration of 5 X 10(-4) mol/liter. The action of IL 2 was assayed on a mouse T cell line, named M 2, derived from C57BL/6 spleen cells and maintained in continuous IL 2-dependent culture for more than 21 mo. Ouabain dose-dependently inhibited the response of these cells to mouse IL 2 with optimal inhibition at 10(-3) mol/liter ouabain. The production of IL 2 by Con A-stimulated C57BL/6 spleen cells was also totally inhibited by 10(-3) mol/liter ouabain; 10(-4) mol/liter ouabain inhibited IL 2 production partially, and 10(-5) mol/liter and 10(-6) mol/liter ouabain had no inhibitory effect. The ouabain-mediated inhibition of IL 2 production and of IL 2 action was not due to a nonspecific toxicity, because the preincubation of spleen cells with 10(-3) mol/liter ouabain for 24 hr did not impair their ability to produce IL 2, nor was the response of M 2 cells to exogenously added IL 2 abrogated by the preincubation of the cells with 10(-3) mol/liter ouabain. The data suggest that ouabain acts on two steps during the induction of T cell activation.

Animals↗
PubMed1982

Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A.

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.

Acyl Coenzyme A↗
PubMed1981

Inhibition of lymphocyte activation by ouabain. Interference with the early activation of membrane phospholipid metabolism.

Activation of lymphocytes by antigens and mitogens can effectively be prevented by ouabain, a known inhibitor of (Na+ + K+)-ATPase. Recently it was shown that lowering of intracellular levels of monovalent cations is not involved in the inhibitory effect of ouabain. (Na+ + K+)-ATPase was found to be closely associated with acyl-CoA : lysophosphatidylcholine acyltransferase in the plasma membrane of lymphocytes. Both enzymes are activated as an immediate consequence of mitogen binding. Human peripheral lymphocytes were stimulated with concanavalin A. Ouabain suppressed the induction of RNA and DNA synthesis in a concentration-dependent way. Increase of RNA synthesis was suppressed only if the glycoside were added within the first hours of activation. If ouabain was added later, incorporation of uridine remained at the rate that was reached at the time of glycoside administration, pointing to an early event where ouabain may be operative. Ouabain, in a dose-dependent manner similar to that affecting RNA and DNA synthesis, inhibited the increase in the incorporation of oleate into phospholipids in stimulated lymphocytes, whereas the turnover of phospholipid fatty acids in resting lymphocytes was unaffected. Increasing extracellular K+ concentrations reversed the binding of ouabain to lymphocytes. Simultaneously, the inhibition of stimulated RNA synthesis was decreased and the inhibition of oleate incorporation was reversed. These results suggest that the suppression of lymphocyte activation by ouabain is due to the inhibition of membrane phospholipid metabolism mediated by (Na+ + K+)-ATPase.

1-Acylglycerophosphocholine O-Acyltransferase↗