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Biomedical subjects

M Shinitzky

Publications and source records attributed to M Shinitzky.

At least 37 records · Page 2Linked to original sources
PubMed1995

Characterization of the lipid surrounding the delta 6-desaturase of rat liver microsomes.

The delta 6-desaturase system was isolated from rat liver microsomes by high hydrostatic pressure (1500 bars) and the enzyme components were then separated by size chromatography. The lipids extracted by organic solvents from the pressure shed fractions were phosphatidylcholine (PC) and cholesterol at a mole ratio of 4:1. The acyl chains of the shed PC were 56% saturated and 21% polyunsaturated resulting predominantly from 13% higher and 15% lower contents of palmitic and arachidonic acid, respectively, as compared to those of microsomal PC. The weight ratio of phospholipids to protein in the shed desaturase fraction was 0.2 which corresponds to an average of 31 phospholipid molecules around each desaturase molecule. Differential scanning calorimetry of the lipids associated with the desaturase system showed a phase transition at 41 degrees C. Fluorescence anisotropy studies of the desaturase surrounding lipids indicated the same transition point. We concluded that the delta 6-desaturase has an associated lipid surrounding of PC and cholesterol at an approx. 4:1 mole ratio that constitutes a gel phase at physiological temperature. We suggest that this state is essential for optimal desaturase activity and that the specific acyl chains of the lipid annulus provide a regulatory sensor of the delta 6-desaturase activity.

Animals↗
PubMed1994

Shedding and isolation of the delta 6-desaturase system from rat liver microsomes by application of high hydrostatic pressure.

A procedure using high hydrostatic pressure without detergent has been applied in this study to subfractionate the components of the delta 6-desaturase system from the rat liver endoplasmic reticulum. Microsomes were suspended in a buffer containing liposomes made of phosphatidylcholine. The mixture was placed in a sealed pressure bomb and subjected to hydrostatic pressure of up to 1500 bars. Under these conditions the total desaturase activity was found in the liposomal fraction thus indicating that the three components of the desaturase system, namely NADH-cytochrome b5 reductase, cytochrome b5 and the delta 6-desaturase co-extracted. Size chromatography and FPLC of the released proteins followed by SDS-PAGE confirmed the independent release of the three components corresponding to the delta 6-desaturase, system. delta 6-Desaturase activity could be fully regenerated by mixing the aqueous dispersions of the three components without further purification. Our results indicate that these components are physically facing a similar lipid environment in the microsomal membrane.

Animals↗
PubMed1994

Elevated protein tyrosine phosphatase activity and increased membrane viscosity are associated with impaired activation of the insulin receptor kinase in old rats.

Insulin resistance is very common in the elderly, and may be associated with glucose intolerance or frank diabetes. In previous studies we demonstrated that insulin resistance in old Wistar rats is associated with decreased autophosphorylation and activation of the hepatic insulin receptor kinase (IRK) in vivo. We now show that this defect can be reproduced in vitro, where the extent of insulin-induced activation of IRK in liver membranes of old rats was decreased by approximately 50% compared with young controls. The defect could be largely abolished after solubilization of the membranes with Triton X-100. We also show that: (a) the viscosity of membranes from the old rats was significantly (P < 0.001, n = 4) higher (by 15%) compared with young controls; (b) incubation of plasma membranes from old animals with lecithin liposomes, which lowered their cholesterol levels, partially abolished the defect in IRK activation; and (c) Triton extracts of liver membranes prepared from old rats did not interfere with the activation of IRK derived from young controls. Additionally, non-membrane components did contribute to the development of this defect. We observed a significant (approximately 30%) (P < 0.001, n = 18) elevation of cytosolic protein tyrosine phosphatase (PTP) activity directed against the beta subunit of the insulin receptor in livers of old rats. No such elevation of PTP activity could be demonstrated with synthetic substrates. Our findings are consistent with a model in which increased membrane viscosity as well as enhancement of a cytosolic PTP activity both markedly inhibit the activation in vivo of the hepatic IRK in old animals.

Aging↗
PubMed1993

Formation of 1,3-cyclic glycerophosphate by the action of phospholipase C on phosphatidylglycerol.

The action of phospholipase C (PLC) from Bacillus cereus on phosphatidylglycerol (PG), derived from egg yolk phosphatidylcholine (PC), was examined in an ether-water mixture. The PLC cleavage of PG and PC followed a Michaelis-Menten kinetics with apparent Vmax values per 1 microgram enzyme of 0.26 and 0.91 mumol.min-1 and Km values of 10 and 12 mM, respectively. When the same enzymic reaction was carried out in minimally buffered aqueous solution of 1% Triton X-100, the decrease in pH with respect to phospholipid cleavage was as expected with PC but much less pronounced with PG. This could be accounted for by the formation of a cyclic glycerophosphate, rather than alpha-glycerophosphate, in the PLC hydrolysis of PG. Examination of the chemical nature of the water-soluble product of PG by phosphorus nuclear magnetic resonance (31P NMR) revealed a single band at 2.31 ppm, while the bands of alpha-glycerophosphate and beta-glycerophosphate appeared at 5.12 and 4.57 ppm, respectively. Basic hydrolysis of the phospholipase cleavage product of PG (0.1 M NaOH for 1 min at 80 degrees C) followed by neutralization shifted its 31P NMR band to 5.18 ppm, which practically coincided with that of alpha-glycerophosphate. Analogous experiments were carried out with PG labeled with 3H at the carbon 2 of the glycerol headgroup ([3H]PG). Autoradiography of thin layer chromatography (TLC) of the [3H]PG enzymic hydrolyzate displayed a single 3H-labeled compound, which could be converted to alpha-glycerophosphate by basic hydrolysis. These results strongly suggest that the phosphate headgroup of PG is cleaved off by PLC as 1,3-cyclic glycerophosphate. A series of PLC experiments with phosphatidyl dihydroxyacetone and phosphatidyl 1,3-propanediol as model substrates supported this assignment. Two-dimensional homonuclear 1H NMR correlated spectra as well as infrared spectra carried out on the isolated sodium salt of this product could further confirm such a structure. The unique structure and chemical nature of 1,3-cyclic glycerophosphate may bear a distinct physiological function.

Bacillus cereus↗
PubMed1993

Increased projection of MHC and tumor antigens in murine B16-BL6 melanoma induced by hydrostatic pressure and chemical crosslinking.

The B16-BL6 melanoma, like most spontaneously arising tumors, is poorly immunogenic and expresses low levels of major histocompatibility complex (MHC) antigens. Treatment of cells of this tumor in vitro by hydrostatic pressure in the presence of adenosine 2',3'-dialdehyde (oxAdo), a membrane-impermeant crosslinker, caused elevated projection of MHC and a specific tumor antigen as demonstrated by flow-cytometric analysis. Maximum projection of both the MHC and the tumor antigens could be reached by application of 1200 atm for 15 min in the presence of 20 mM oxAdo. It is not yet clear whether this passive increase in availability of antigens on the cell surface originated from a dormant pool of antigens in the plasma membrane or from pressure-induced fusion of antigen-rich intracellular organelles (e.g. the endoplasmic reticulum). The immunogenic properties of the antigen-enriched B16-BL6 cells are described in the following paper.

Animals↗
PubMed1993

Induction of cell-mediated immunity against B16-BL6 melanoma in mice vaccinated with cells modified by hydrostatic pressure and chemical crosslinking.

In the preceding paper we have demonstrated an increase in presentation of both major histocompatibility complex antigens (MHC) and a tumor-associated antigen of the weakly immunogenic B16 melanoma by a straight-forward technique. The method consists in modulating the tumor cell membrane by hydrostatic pressure and simultaneous chemical crosslinking of the cell-surface proteins. In B16-BL6 melanoma, the induced antigenic modulation was found to persist for over 48 h, which permitted the evaluation of the ability of modified B16-BL6 cells to induce immunity against unmodified B16-BL6 cells. In the present study, we have shown that a significant systemic immunity was induced only in mice that were immunized with modified B16-BL6 melanoma cells, whereas immunization with unmodified B16-BL6 cells had only a marginal effect when compared to the results in control sham-immunized mice. The induced immunity was specific since a single immunization affected the growth of B16-BL6 tumors but had no effect on MCA 106, an antigenically unrelated tumor. The addition of interleukin-2 to the immunization regimen had no effect on the antitumor responses induced by the modified B16-BL6 cells. The cell-mediated immunity conferred by immunization with treated B16-BL6 cells was confirmed in experiments in vitro where splenocytes from immunized mice could be sensitized to proliferate by the presence of B16-BL6 cells. In addition, the altered antigenicity of these melanoma cells appeared to correlate with their increased susceptibility to specific effectors. Thus, 51Cr-labeled B16-BL6 target cells, modified by pressure and crosslinking, in comparison to control labeled target cells, were lysed in much greater numbers by effectors such as lymphokine-activated killer cells and allogeneic cytotoxic lymphocytes (anti-H-2b), while such cells remained resistant to lysis by natural killer cells. Our findings indicate that the physical and chemical modifications of the tumor cells that are described here may be considered as a simple yet effective method for the preparation of tumor vaccines, which could be applied in tumor-bearing hosts.

Animals↗
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↗