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Biomedical subjects

M Shinitzky

Publications and source records attributed to M Shinitzky.

At least 19 recordsLinked to original sources
PubMed1992

Improved chemotactic ability of neonatal polymorphonuclear cells induced by mild membrane rigidification.

Membrane lipid fluidity of peripheral blood polymorphonuclear cells (PMNs) of 24 newborn infants, 2-4 days after birth, was determined by steady-state fluorescence polarization with 1,6-diphenyl 1,3,5-hexatriene (DPH) as a probe and compared with that of PMNs from 23 adults. Measurements with intact cells, which correspond to all cellular lipid domains, did not display any statistically significant difference between PMNs of the two groups. However, application of bixinoyl glucosamine, a membrane-impermeable fluorescence quencher, revealed that the PMN plasma membrane of the newborn is about 23% more fluid than that of the adult. Total cholesterol-to-phospholipid ratio of newborn PMNs was found to be lower by about 10% than that of the adult, which could account for the difference in their plasma membrane fluidity. The possible implication of this finding for the deficit in chemotactic ability of leukocytes from newborns was tested with neonatal PMNs that have incorporated cholesteryl hemisuccinate (CHS), an efficient plasma membrane rigidifier. In all neonatal PMNs tested a mild incorporation of CHS (0.5-1 min incubation in 50 micrograms/ml dispersion) caused a significant improvement in their net chemotaxis, from an average value of 28 +/- 7 to 43 +/- 11. Longer incubations with CHS caused a gradual decrease in chemotactic ability that approached the basal level after about 5 min incubation. The net chemotaxis in adult PMNs was significantly higher than that of neonatal PMNs (72 +/- 13) and was gradually inhibited by incorporation of CHS without any initial augmentation. Based on these results it was estimated that about 27% of the chemotactic deficit of neonatal PMNs is mediated by their immature fluid membrane.

Adult↗
PubMed1991

Potentiation of delayed-type hypersensitivity response to syngeneic tumors in mice prevaccinated with cells modified by hydrostatic pressure and crosslinking.

Delayed-type hypersensitivity (DTH) to the chemically induced EL4 and virally induced ARadLV 136 leukemia cells was determined by the radioactive ear test. Prior to the DTH test, mice were prevaccinated with cells treated either with hydrostatic pressure or with the membrane-impermeant crosslinker adenosine dialdehyde or with a combination of these. For both tumor cells, DTH against unmodified cells was markedly potentiated by prevaccination with cells treated with hydrostatic pressure combined with adenosine dialdehyde. In vitro cytotoxicity data indicated that maximal lysis of target cells is induced by vaccination with adenosine-dialdehyde-treated cells, as well as by pressure in combination with adenosine dialdehyde treatment. In addition, increased [3H]thymidine uptake was observed in effector T cells induced by prevaccination with tumor cells modified by adenosine dialdehyde or pressure or both. This novel and innocuous potentiation of an antitumor immune response may have a practical utility in the treatment of human cancer.

Adenosine↗
PubMed1991

Resistance to NK cell-mediated cytotoxicity (in K-562 cells) does not correlate with class I MHC antigen levels.

Natural Killer (NK) cells probably function as an early line of defense against virus-infected cells and tumor cells. In all cases, the killing by NK cell-mediated cytotoxicity (NK-CMC) is not MHC-restricted and the factors which determine the sensitivity to NK-CMC have not yet been identified. A positive correlation between resistance to NK-CMC and the level of class I MHC antigen (MHC I) expression on target cells has been reported in many studies, and in some cases a functional linkage between the two has been claimed. Several other studies have shown that there is no such correlation. By employing several experimental systems, we demonstrate here a lack of correlation between the level of MHC I and the sensitivity of K-562 cells to NK-CMC. Transfer of MHC I to MHC I-negative cells via vesicles had no effect on their resistance to NK-CMC. In addition, a decrease in resistance to NK-CMC and increase of MHC I levels was observed following target-cell membrane modulation by both application of cholesterol and hydrostatic pressure. Finally, no correlation between sensitivity to NK-CMC and MHC I expression was found in three sublines of K-562 cells. Since NK-CMC is a multistage process, it is concluded that components other than class I MHC antigens have a more prominent role in modulating the sensitivity of target cells to NK-CMC.

Cell Death↗
PubMed1991

Platelet autoantibodies in dementia and schizophrenia. Possible implication for mental disorders.

Platelets isolated from blood of demented and schizophrenic patients were found to bear surface antibodies at a considerably higher titer than those found on platelets from normal age-matched groups or patients with affective disorders. The platelet count in demented and schizophrenic patients correlated inversely with the level of the platelet associated antibodies (PAA) which suggested an autoimmune route of opsonization. In most individual cases of dementia or schizophrenia PAA and platelet count were found to oscillate with time between high PAA-low platelet number and low PAA-high platelet number in approximately inverse correlation. PAA isolated from demented patients were found to cross-react with platelets from normals and with brain tissue from rats. Furthermore, molecular weights of specific brain antigens were identified by binding to PAA. These observations support the possibility that PAA might be implicated in the etiology of some mental dysfunctions associated with dementia and schizophrenia.

Adult↗
PubMed1990

A method for preparing biologically active aqueous cyclosporin A solutions avoiding the use of detergents or organic solvents.

The immunosuppressive agent cyclosporin A (CsA) is highly hydrophobic and is, therefore, being taken for in vitro use directly from ethanolic stock solutions. In the present study it is demonstrated that ethanol itself affects several immunological activities in vitro, thus interfering with the immunosuppressive effects of CsA. As an alternative, it is demonstrated that CsA can be solubilized and retain its activity in aqueous solutions of polyvinyl pyrrolidone (PVP). Unlike ethanol, PVP has no effect on the various immunological activities in vitro and therefore the immunosuppressive effects of CsA can be evaluated without interference.

2',5'-Oligoadenylate Synthetase↗
PubMed1990

Application of high-pressure to subfractionate membrane protein-lipid complexes: a case study of protein kinase C.

Current procedures for solubilization of membrane proteins involve the use of detergents. A procedure using high hydrostatic pressures without detergent has been applied in this study to subfractionate membrane proteins and their endogenously associated lipids. Rat brain membrane preparations were suspended in hypotonic buffer containing the membrane fluidizer benzyl alcohol in a sealed pressure cell and subjected to hydrostatic pressures of up to 1500 atmospheres (approx 22,000 psi) in a French press. Under these conditions, specific membrane proteins including protein kinase C, phospholipase A2, calmodulin-binding proteins, G-proteins, and microtubule-associated proteins all coextracted and were associated to lipid particles, suggesting inherent physical contact. Two populations of membrane-associated protein kinase C were identified according to molecular weight estimations. The first coeluted with the lipid particles composed predominantly of phospholipids, while the second contained much less lipid and was similar to the soluble monomer, i.e., cytosolic protein kinase C. This procedure provides an important technique for selective subfractionation of membrane proteins in their native lipid environment which could be used for structure-function studies.

Animals↗
PubMed1990

Repellents for Escherichia coli operate neither by changing membrane fluidity nor by being sensed by periplasmic receptors during chemotaxis.

A long-standing question in bacterial chemotaxis is whether repellents are sensed by receptors or whether they change a general membrane property such as the membrane fluidity and this change, in turn, is sensed by the chemotaxis system. This study addressed this question. The effects of common repellents on the membrane fluidity of Escherichia coli were measured by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene in liposomes made of lipids extracted from the bacteria and in membrane vesicles. Glycerol, indole, and L-leucine had no significant effect on the membrane fluidity. NiSO4 decreased the membrane fluidity but only at concentrations much higher than those which elicit a repellent response in intact bacteria. This indicated that these repellents are not sensed by modulating the membrane fluidity. Aliphatic alcohols, on the other hand, fluidized the membrane, but the concentrations that elicited a repellent response were not equally effective in fluidizing the membrane. The response of intact bacteria to alcohols was monitored in various chemotaxis mutants and found to be missing in mutants lacking all the four methyl-accepting chemotaxis proteins (MCPs) or the cytoplasmic che gene products. The presence of any single MCP was sufficient for the expression of a repellent response. It is concluded (i) that the repellent response to aliphatic alcohols can be mediated by any MCP and (ii) that although an increase in membrane fluidity may take part in a repellent response, it is not the only mechanism by which aliphatic alcohols, or at least some of them, are effective as repellents. To determine whether any of the E. coli repellents are sensed by periplasmic receptors, the effects of repellents from various classes on periplasm-void cells were examined. The responses to all the repellents tested (sodium benzoate, indole, L-leucine, and NiSO4) were retained in these cells. In a control experiment, the response of the attractant maltose, whose receptor is periplasmic, was lost. This indicates that these repellents are not sensed by periplasmic receptors. In view of this finding and the involvement of the MCPs in repellent sensing, it is proposed that the MCPs themselves are low-affinity receptors for the repellents.

Alcohols↗
PubMed1990

Reduction of circulating HIV antigens in seropositive patients after treatment with AL-721.

Sixteen patients seropositive for human immunodeficiency virus (HIV) at different stages participated in an open trial using the lipid mixture AL-721. Seven patients were antigen negative and asymptomatic and the other nine antigen positive, three of whom were virtually asymptomatic. AL-721 was given per os once a day at a dose of 10 g in a fat-free breakfast for up to 16 months. The presence of HIV antigens in the serum was monitored by enzyme immunoassay (EIA) using two commercial kits. In five of the nine antigen-positive patients the concentration of HIV antigens was reduced to basal level after about 3 months of AL-721 treatment; three of these responders were asymptomatic, one had persistent generalized lymphadenopathy, and the other had Kaposi's sarcoma. In one of the antigen-positive patients who did not respond to AL-721, the addition of AZT to the treatment resulted in a marked decrease in antigen level. Another AIDS patient receiving this combination therapy from onset of the trial showed a similar decrease. The other two nonresponding patients were at a stage of full-blown AIDS. These results suggest that AL-721, an innocuous compound, may be effective in reducing the serum HIV level in patients at the preclinical stage.

Adult↗
PubMed1989

Monoclonal antibodies against the S2-serotonin receptor from rat brain that cross-react with dopamine and opiate receptors.

Balb/c mice were immunized with rat striatal integral membrane proteins. After hybridization of splenocytes with myeloma cells, hybridoma lines secreting antibodies against serotonin, dopamine and opiate receptors were detected by inhibition of ligand binding to brain membrane preparations. Antibodies from two positive lines, Mab/a9 and Mab/a18, were able to inhibit ligand binding to the S2-serotonin (Kd range: 10-100 nM), the mu-opiate (Kd range: 0.4-3 microM) and the delta-opiate receptors (Kd range: 0.7-1.1 microM), while Mab/a9 was also found to inhibit ligand binding to the D2/D4-dopamine receptor (Kd approximately 50 nM). An apparent molecular mass of 60 kDa could be ascribed to the delta-opiate receptor and apparent molecular masses of 29 and 36 kDa to the mu-opiate receptor by ligand elution from immuno-precipitates.

Animals↗
PubMed1987

Therapeutic vaccination against adjuvant arthritis using autoimmune T cells treated with hydrostatic pressure.

An ideal treatment for autoimmune diseases would be a nontoxic means of specifically neutralizing the autoreactive lymphocytes responsible for the disease. This goal has been realized in experimental autoimmunity models by immunizing rats or mice against their own autoimmune cells such that the animals generate an immune response specifically repressive to the disease-producing lymphocytes. This maneuver, termed lymphocyte vaccination, was demonstrated to be effective using some, but not all, autoimmune helper T-lymphocyte lines. We now report that T lymphocytes, otherwise incapable of triggering an immune response, can be transformed into effective immunogens by treating the cells in vitro with hydrostatic pressure. Clone A2b, as effector clone that recognized cartilage proteoglycan and caused adjuvant arthritis in Lewis rats, is such a cell. Untreated A2b could not trigger an immune response, but inoculating rats with pressure-treated A2b induced early remission of established adjuvant arthritis as well as resistance to subsequent disease. Specific resistance to arthritis was associated with anti-idiotypic T-cell reactivity to clone A2b and could be transferred from vaccinated rats to naive recipients using donor lymphoid cells. Aggregation of T-lymphocyte membrane components appeared to be important for an immune response because the effects of hydrostatic pressure could be reproduced by treatment of A2b with chemical cross-linkers or with agents disrupting the cytoskeleton. Populations of lymph node cells from antigen-primed rats, when treated with hydrostatic pressure, could also induce suppression of disease. Thus, effective vaccines can be developed without having to isolate the autoimmune T lymphocytes as lines or clones. These results demonstrate that effector T lymphocytes suitably treated may serve as agents for specifically controlling the immune system.

Animals↗
PubMed1986

Inhibition of IL-2 secretion and IL-2 receptor appearance of activated lymphocytes pretreated with hydroxylated sterols.

When murine BALB/c splenocytes are pretreated for 2 h with different concentrations of 25-hydroxycholesterol (25-HC) or 7 beta-hydroxycholesterol (7-HC), washed and stimulated either with irradiated C57BL/6 splenocytes or with Con A, IL-2 secretion is inhibited in a dose-dependent way as well as the subsequent cell proliferation. Using the same treatment and stimulation conditions, IL-2 receptor appearance on human T lymphocytes, as characterized by anti-Tac antibody binding, is also inhibited in a dose-dependent way. In contrast, the hydrophilic derivative of 7 beta-hydroxycholesterol, the 3.7 bishemisuccinate sodium salt (7-HC BHS), did not influence any of the 3 tested parameters.

Animals↗
PubMed1986

Promotion of tumor antigenicity in EL-4 leukemia cells by hydrostatic pressure.

Subjection of EL-4-leukemia cells to hydrostatic pressure of 1200-1500 atm for 15 min increased their weak basal immunogenicity to a potent practical level. Injection of such pressure-treated and irradiated EL-4 cells into syngeneic naive C57Bl/6 mice significantly delayed tumor development and increased survival after subsequent challenge with untreated EL-4 cells. Application of pressure of 1500 atm for a longer period of time (e.g., 120 min) resulted in cell death and a smaller increase in tumor immunogenicity which could be partially accounted for by passive shedding of membrane material. Unlike previously studied tumor cells, incorporation of cholesteryl hemisuccinate (CHS) into the plasma membrane of EL-4 cells increased their apparent tumor immunogenicity only slightly. In addition, isolated EL-4 plasma membranes, untreated, CHS-treated or pressure-treated, as well as the material shed thereof by hydrostatic pressure, were all of weak immunogenicity. Modulation in the projection of surface antigens upon pressure treatment could account for the observed increase in tumor immunogenicity and was monitored via the Thy 1.2 antigen. Fluorescence cell sorting analysis indicated that upon application of 1500 atm for different periods of time the projection of Thy 1.2 progressively and irreversibly increased to a maximal level of about 140% at 15 min. At longer pressurization the availability of Thy 1.2 to antibody binding decreased sharply to levels below that of the untreated cells. It is suggested that pressure promotion of tumor immunogenicity is induced by changes in projection and surface distribution of the relevant antigens.

Animals↗
PubMed1979

Gross structural changes in isolated liver cell plasma membranes upon binding of insulin.

The addition of 10(-9) M insulin to a suspension of rat liver plasma membranes increases the overall lipid microviscosity, eta, by about 10--20%. The effect is confined to physiological concentrations of the hormone and is highly specific. The specificity was demonstrated in experiments where insulin analogues were added to liver plasma membranes and where insulin was added to human erythrocyte membranes. In both of these experiments practically no change in eta was detected. Upon in vitro enrichment of the membrane cholesterol, eta exceeded the level mediated by insulin binding, and the addition of 10(-9) M insulin to the cholesterol-enriched membranes did not further increase eta. Concomitant to the increase in eta upon insulin binding, the overall degree of exposure of the membrane protein, presumably to both sides of the membrane, is substantially increased. This effect is in line with the notion of vertical displacement of membrane proteins induced by changes in eta. The observed structural modulation can account for the effect of insulin on unrelated membrane responses, as well as for the negative cooperativity of insulin binding.

Animals↗
PubMed1979

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.

Cholesterol↗
PubMed1979

Possible role for glycosphingolipids in the control of immune responses.

Preparations of gangliosides from bovine brain contain material which acts as a strong mitogen on murine spleen cells. This material is highly lipophilic and co-purifies with the ganglioside fraction. It contains saccharides of a similar composition to those found in monosialogangliosides, as well as a spinogsine base and an appreciable amount of peptide. The common brain gangliosides GM1, GD1a and GD1b, on the other hand, are not mitogenic and act as suppressors of the mitogenic activity of bacterial lipopolysaccharide on murine spleen cells. Both the mitogenically active and suppressive fractions of bovine brain glycosphingolipid were found to act exclusively on B lymphocytes. Since gangliosides and related compounds are components of plasma membranes and of amphipathic nature, they may passively migrate between the lymphocyte subpopulations and thus act as physiological modulators of immune responses.

Animals↗