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Biomedical subjects

K Resch

Publications and source records attributed to K Resch.

At least 109 records · Page 6Linked to original sources
PubMed1986

Lipid composition of functional domains of the lymphocyte plasma membrane.

Plasma membrane vesicles from calf T-lymphocytes were fractionated by affinity chromatography on Con A-Sepharose. One subfraction eluted freely from the affinity column (fraction 1), while a second one adhered specifically to the column (fraction 2). While both fractions were derived exclusively from the plasma membrane, fraction 2 carried the high-affinity receptor for the mitogen concanavalin A and was distinct from fraction 1 with respect to its polypeptide pattern and the content of some plasma membrane-associated enzymes, suggesting the existence of functional plasma membrane domains. These functionally distinct fractions showed different lipid composition. The adherent fraction was enriched in phosphatidylcholine, while the relative amount of phosphatidylethanolamine and phosphatidylserine was reduced. Furthermore, the relative amount of saturated fatty acids was enhanced in the phospholipids of the adherent plasma membrane fraction. This could be shown in total phospholipids, as well as in separated individual phospholipids. We could therefore demonstrate that lipid heterogeneity may exist in plasma membranes of cells without structural polarity. Similar results were obtained when T-lymphocytes were stimulated with the mitogen concanavalin A. The functional domain, consisting of the high-affinity concanavalin A receptor, several enzymes and distinct lipid compositional pattern, thus seems to constitute a relatively stable structural entity of the lymphocyte plasma membrane.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1986

Control of prostanoid synthesis: role of reincorporation of released precursor fatty acids.

Prostanoid synthesis is limited by the availability of free arachidonic acid. This polyunsaturated fatty acid is liberated by phospholipases and usually is an intermediate of the deacylation-reacylation cycle of membrane phospholipids. In rat peritoneal macrophages, ethylmercurisalicylate (merthiolate) or N-ethylmaleimide (NEM) dose dependently inhibited the incorporation of arachidonic acid into cellular phospholipids, at lower concentrations specifically into phosphatidylcholine. Furthermore, merthiolate could be shown to be a rather selective inhibitor of lysophosphatidylcholine acyltransferase. In contrast, phospholipase A2 activity was not affected over a wide dose range. Consequently, macrophages showed a large increase in prostanoid synthesis (prostaglandin E, prostacyclin and thromboxane) in the presence of both lysophosphatide acyltransferase inhibiting agents. Similar results were obtained with human platelets, in which merthiolate increased the release of thromboxane. Addition of free arachidonic acid also enhanced prostanoid synthesis in macrophages. At optimal concentrations, merthiolate had no further augmenting effect. It is concluded that the rate of prostanoid synthesis is not only controlled by phospholipase A2 activity, but rather by the activity of the reacylating enzymes, mainly lysophosphatide acyltransferase.

Animals↗
PubMed1986

Interleukin 1 induces specific phosphorylation of a 41 kDa plasma membrane protein from the human tumor cell line K 562.

An in vitro system was established to investigate the early effects of interleukin 1 (IL 1) on a clearly defined subcellular system, the plasma membrane. Murine IL 1 was obtained from the macrophage tumor line P 388 D1 and purified to apparent homogeneity. Plasma membranes from the human leukaemic tumor cell line K 562 were highly purified by differential ultracentrifugation. The monokine induced the specific phosphorylation of a 41 kDa plasma membrane protein. This could be demonstrated by incubating the plasma membranes in the presence of IL 1 and (gamma-32P)-ATP, separating the proteins by SDS-PAGE and subsequent autoradiography. A first biochemical reaction caused by IL 1 in the target organelle of the cell, the plasma membrane, was discovered, yielding a clue of how signal transmission may take place finally resulting in the stimulation of the responding cell.

Cell Line↗
PubMed1986

Effects of cyclosporin A on functions of specific murine T cell clones: inhibition of proliferation, lymphokine secretion and cytotoxicity.

Allospecific T lymphocyte clones with different functions were generated from spleen cells of C 57/Bl6 mice following sensitization in vitro by a one-way mixed lymphocyte culture (MLC) with irradiated DBA/2 spleen cells. The clones were propagated in vitro in the presence of interleukin 2 (IL 2) and restimulation with stimulator cells. In these clones Cyclosporin A (CSA) was tested for its suppressive effect on different T lymphocyte functions. The antigen-dependent proliferation of a helper clone (HTL) was totally inhibited by 50 ng/ml CSA. Proliferation induced by simultaneous administration of antigen and IL 2 was partially suppressed in all helper and cytotoxic clones (CTL). The IL 2-driven proliferation in the absence of antigen was also suppressed between 25-70% by the immunosuppressive drug. Secretion of macrophage activating factor (MAF) and interferon (IFN) by HTL and CTL in response to antigen or mitogen was reduced dose dependently by CSA. Concentrations of 50 ng/ml CSA diminished lymphokine secretion to approximately 10% of controls, also when excess IL 2 was present. Cytotoxicity, previously described to be insensitive to the drug, could be suppressed by 50 ng/ml CSA to a various extent, from 40-70%, in different cytotoxic clones when the effector cells were preincubated with CSA for 1 h or more. Conclusively, the data suggest that CSA interferes generally with the activation of T lymphocyte clones.

Animals↗
PubMed1986

The receptor for interleukin 1 in plasma membranes of the human leukemia cell K 562: biological and biochemical characterization.

Murine interleukin 1 (IL 1) inhibited concentration dependently the proliferation of murine T cell lymphomas and the human leukemic cell line K 562. The cytostatic action of IL 1 was not associated with cytotoxicity and appeared to be irreversible. Changes in the expression of surface antigens, like a rapid decrease of transferrin receptors or, more delayed, an increase in HLA-A, B, C antigen density suggested that a differentiation step was induced by IL 1. This effect of IL 1 was a direct one and most likely mediated by a specific receptor molecule. In order to characterize the receptor for IL 1, highly purified plasma membranes from K 562 were incubated with murine IL 1, and the phosphorylation pattern of plasma membrane proteins was investigated by the addition of radiolabeled ATP. At 0 degree C, IL 1 induced the specific phosphorylation of a 41 kDa membrane protein in a time- and concentration-dependent manner. Analysis of the phosphoamino acid composition revealed that IL 1 induced specifically the phosphorylation of tyrosine residues of the 41 kDa protein. Crosslinking experiments proved that the 41 kDa protein had an IL 1 binding site, strongly suggesting that the 41 kDa protein was the receptor for IL 1 itself. Affinity labeling with an ATP-analogue showed that this protein possessed an ATP binding and cleaving site. We conclude from this that the receptor for IL 1 in the plasma membranes of K 562 is a transmembranous protein of 41 kDa, which possesses a tyrosine specific protein kinase activity with an autophosphorylating capacity.

Animals↗
PubMed1986

Inhibition of T lymphocyte activation by cyclosporin A: interference with the early activation of plasma membrane phospholipid metabolism.

Rabbit lymph node and thymus lymphocytes were stimulated with concanavalin A (Con A). Cyclosporin A (CSA) inhibited in a dose-dependent way the induction of RNA and DNA synthesis; nearly complete inhibition was observed at a concentration of 200 ng/ml. Results of kinetic studies suggested that the immunosuppressive drug interfered with an early event occurring in activated lymphocytes. Among the earliest changes detectable in activated lymphocytes, the turnover of plasma membrane phospholipids is increased, predominantly of their fatty acid moieties, catalyzed by the membrane-bound lysophosphatide acyltransferase. CSA, at concentrations identical with those inhibiting macromolecular synthesis, also inhibited the Con A-stimulated specific increase in the incorporation of labeled fatty acids into plasma membrane phospholipids. When lymphocytes were stimulated with Con A for 1 hr, incorporation of labeled oleic acid and arachidonic acid approximately doubled in plasma membrane phospholipids. CSA at a concentration of 200 ng/ml prevented the elevated incorporation of labeled fatty acids into plasma membrane phospholipids of Con A-stimulated thymocytes. Concomitantly, the activation of lysolecithin acyltransferase, the key enzyme for the incorporation of long-chain fatty acids into phospholipids, was strongly inhibited. Up to high concentrations, CSA had no effect on the phospholipid metabolism of unstimulated lymphocytes. The results suggest that CSA inhibits the activation of T lymphocytes by interfering with the early activation of plasma membrane phospholipid metabolism.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1986

Macrophage cytotoxicity: interleukin 1 as a mediator of tumor cytostasis.

Purified macrophage interleukin 1 (IL 1) induced a concentration-dependent inhibition of the proliferation of two commonly used tumor cell target lines, the human myeloid K562 and the murine T lymphoma Eb. In contrast, mastocytoma-derived P815 cells were not inhibited. The cytostatic action of IL 1 was not associated with direct cytotoxicity and was only partially reversible. PGE or interferon did not appear to mediate these effects. IL 1 treatment of the multipotential K562 cells revealed no morphologic evidence for the induction of specific differentiation. FACS analysis of IL 1-treated K562 cells showed a rapid decrease in transferrin receptor density, and a more delayed, but highly significant, increase in HLA-A,B,C antigen density. These findings provide one explanation for the frequently reported macrophage cytostatic actions against tumor cells, and indicate as well that IL 1, like interferon, may enhance the expression of Class I MHC antigens. These observations further extend the range of IL 1 actions and underscore the fundamental and direct role of this monokine in macrophage antitumor activity.

Animals↗
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↗