Biomedical subjects

I Kahane

Publications and source records attributed to I Kahane.

At least 37 records · Page 2Linked to original sources

PubMed1989

Iron loading modifies the fatty acid composition of cultured rat myocardial cells and liposomal vesicles: effect of ascorbate and alpha-tocopherol on myocardial lipid peroxidation.

Increased generation of free radicals and accelerated lipid peroxidation are important manifestations of iron toxicity. We have studied the effect of iron loading on lipid peroxidation in cultured rat myocardial cells by direct measurement of the fatty acid composition of cellular lipids. Iron loading produced by 24-hour incubation of cultured cells with 0.36 mmol/L ferric ammonium citrate resulted in a moderate reduction in polyunsaturated fatty acids (PUFAs) such as 22:5 and 22:6. A more drastic reduction in PUFAs and an apparent reciprocal increase in the proportion of saturated fatty acids were both obtained after 24 hours of incubation of liposomal vesicles prepared from whole cell lipid extracts with iron at between pH 4.5 and pH 5.5. Reduction of 22:5 and 22:6 was first noticed at 3 hours, and undetectable levels were reached by 12 and 24 hours of incubation. Ascorbate had a biphasic effect on liposomal PUFA levels: at low concentrations (0.057 mmol/L) it enhanced the iron-induced changes in liposomal fatty acid composition, but at higher concentrations (0.57 and 5.7 mmol/L), it inhibited these changes. Unlike ascorbate, alpha-tocopherol (0.023 to 2.3 mmol/L) inhibited the iron-induced reduction in PUFAs in a dose-dependent manner, with complete inhibition of the iron effect at 2.3 mmol/L. These observations underline the particular sensitivity of PUFAs to iron-induced lipid peroxidation. They also illustrate the ability of ascorbate and alpha-tocopherol to modify iron-induced lipid peroxidation. Further studies are required to explore the possible therapeutic implications of these observations in clinical iron overload.

Animals↗

PubMed1988

Purification and characterization of urease from Ureaplasma urealyticum.

The urease from Ureaplasma urealyticum strain T 960 was isolated by the use of affinity chromatography, hydrophobic chromatography and gel filtration. The enzyme was purified by a factor of 155. The urease appeared as a single band of molecular weight (MW) 75,000 using reducing conditions in SDS-polyacrylamide gel electrophoresis. By gel filtration the native MW was determined to be 150,000. Isoelectric focusing showed the presence of two closely migrating enzyme species with a pI of pH 5.1-5.2. These findings show multiple forms of the urease and that these forms are composed of subunits. The electrophoresis experiments also indicate that this enzyme is a major component of the cytoplasm of U. urealyticum. The Km of the purified enzyme was 4.5 mM urea and the specific activity was 33530 mumoles NH3 x min-1 x mg-1. The optimum pH was pH 7-7.5. The urease activity was inhibited by flurofamide, acetohydroxamic acid, N-ethylmaleimide and p-chloromercuribenzoate but not by iodoacetate.

Benzamides↗

PubMed1987

Preliminary X-ray diffraction results on co-crystals of wheat germ agglutinin with a sialoglycopeptide from the red cell receptor glycophorin A.

Diffraction-quality crystals have been obtained for complexes of each of the major wheat germ agglutinin (WGA) isolectins with the tryptic sialoglycopeptide T-5 from the WGA red cell receptor glycophorin A. This octa-glycopeptide possesses a Thr-linked carbohydrate moiety (GalNAc(NeuNAc)-Gal-NeuNAc) with specificity for the WGA binding site. The crystals belong to the orthorhombic space group P2(1)2(1)2 and have unit cell dimensions: a = 112.2 A, b = 51.0 A, c = 63.5 A (isolectin 1); a = 109.0 A, b = 52.3 A, c = 62.4 A (isolectin 2). There are two monomer complexes in each asymmetric unit.

Crystallization↗

PubMed1986

Protective effects of the glutathione redox cycle and vitamin E on cultured fibroblasts infected by Mycoplasma pneumoniae.

The role of the glutathione (GSH) redox cycle and vitamin E as antioxidant defense systems was studied in normal human cultured skin fibroblasts infected by virulent Mycoplasma pneumoniae. In cells infected for 20 h, catalase activity was inhibited by 75% and the intracellular GSH decreased to 32% of its normal values. GSH peroxidase and oxidized glutathione (reductase activities in the infected cells were unaffected.) GSSG glutathione in the medium of the infected cells rose in accordance with the intracellular GSH decrease. The observed elevation in GSSG/GSH ratio was attributed to the increase in intracellular H2O2 content in M. pneumoniae-infected cells due to the marked inhibition in their catalase activity. The protective effect of the GSH redox cycle in infected cells was studied by depletion of cellular GSH, prior to their infection with M. pneumoniae, using buthionine sulfoximine (BSO), a selective inhibitor of gamma-glutamyl cysteine synthetase. After 16 h of incubation with BSO, the GSH levels were reduced to 38% of their normal value and recovered to 55% during 24 h after removal of the inhibitor. BSO had no effect on GSH peroxidase and catalase activities in either infected or noninfected cells. The level of malonyldialdehyde (an indicator of membrane lipid peroxidation) in BSO-treated cells infected by M. pneumoniae was 1.8 times higher than in infected controls. Cells enriched with 0.25 and 2.25 micrograms of vitamin E per mg of protein prior to their infection by M. pneumoniae revealed the following: a lesser degree of catalase inhibition, 46 and 30%, respectively, versus 64% in infected control cells that were not supplemented with vitamin E; lower levels of malonyldialdehyde, 55 and 20% increments, respectively, versus a 140% increment in infected controls; higher residual activity of lactate dehydrogenase, 76 and 96%, respectively, versus 58% in infected controls. Our data indicate that the oxidative damage induced in M. pneumoniae-infected cells due to the increase in intracellular levels of H2O2 and O2- is limited by the host cell GSH redox cycle and by supplementation with vitamin E.

Buthionine Sulfoximine↗

PubMed1985

The effect of antibodies to human platelet membrane and cytoplasmic myosins on myosin ATPase activity and platelet aggregation.

Myosins were purified from the membrane fraction and the cytoplasm of human platelets. Polyclonal antibodies to the purified myosins were induced in rabbits. Their effects on the ATPase activity of the purified myosins as well as on the process of platelet aggregation were studied. A strong cross reactivity was found between the two myosins and their respective antibodies by the ELISA technique. It was found that the antibodies preferentially bind to the "head" segment of the myosins, since purified myosin "rod" reacted only weakly with the two kinds of antibodies. The two antimyosin antibodies strongly inhibited the K+(EDTA) ATPase activity of both myosins, as well as the activity of the isolated myosin "heads". The amount of antimembrane myosin antibody required to inhibit the above enzymatic activity was smaller than that of the anticytoplasmatic myosin antibody. Similar results were observed with F(ab)2 fragments of the two kinds of antibodies. No effect of these antibodies or their F(ab)2 fragments was observed on platelet aggregation induced by various agonists, although their inhibitory effect on the platelet myosin ATPase activity was strong.

Adenosine Triphosphatases↗

PubMed1985

Use of virus-attached antibodies or insulin molecules to mediate fusion between Sendai virus envelopes and neuraminidase-treated cells.

Anti-human erythrocyte antibodies or insulin molecules were covalently coupled to the glycoproteins (the hemagglutinin/neuraminidase and the fusion polypeptides) of Sendai virus envelopes with N-succinimidyl 3-(2-pyridyldithio)propionate and succinimidyl 4-(p-maleimidophenyl)butyrate as cross-linking reagents. Reconstituted Sendai virus envelopes, bearing covalently attached anti-human erythrocyte antibodies or insulin molecules, were able to bind to but not fuse with virus receptor depleted human erythrocytes (neuraminidase-treated human erythrocytes). Only coreconstitution of Sendai virus glycoproteins, bearing attached anti-human erythrocyte antibodies or insulin molecules with intact, untreated viral glycoproteins, led to the formation of fusogenic, targeted reconstituted Sendai virus envelopes. Binding and fusion of reconstituted Sendai virus envelopes, bearing anti-human erythrocyte antibodies or insulin molecules, with neuraminidase-treated human erythrocytes were blocked by the monovalent fraction, obtained after papain digestion of immunoglobulins, made of anti-human erythrocyte antibodies or free insulin molecules, respectively. The results of this work demonstrate an active role of the viral binding protein (hemagglutinin/neuraminidase polypeptide) in the virus membrane fusion process and show a novel and efficient method for the construction of targeted, fusogenic Sendai virus envelopes.

Antibodies↗

PubMed1985

Immunohistochemical studies with antibodies to myosins from the cytoplasm and membrane fraction of human blood platelets.

Antibodies were raised to myosins extracted from the cytoplasm and solubilized membranes of human blood platelets. Both antibodies had similar titers as shown by enzyme-immunoassay and bound to the same sites as shown by immunohistochemistry. They were specific for cytoplasmic myosins (e.g., in human white blood cells, platelets and fibroblasts and rat endothelial cells). They showed no crossreaction with human or rat smooth muscle.

Animals↗

PubMed1985

Human ciliated epithelial cells from nasal polyps as an experimental model for Mycoplasma pneumoniae infection.

Human ciliated epithelial cells derived from nasal polyps and cultured in a monolayer were studied as an experimental model for Mycoplasma pneumoniae infection. Scanning electron microscopy revealed two types of cultured epithelial cells: one which was covered by microvilli only and another which had microvilli and actively beating cilia. M. pneumoniae adhered to both types of cells, and the adherence followed saturation kinetics as a function of time. Infection of the cells for 20 h resulted in 75% inhibition of their intracellular catalase activity and a 3.5-fold increase in their malonyldialdehyde levels compared with noninfected controls. This indicates the presence of cellular oxidative damage due to M. pneumoniae infection. It is suggested that human nasal ciliated epithelial cells may serve as a representative model for studying M. pneumoniae in relation to its natural host.

Adhesiveness↗

PubMed1985

Detection of Mycoplasma pneumoniae adhesin (P1) in the nonhemadsorbing population of virulent Mycoplasma pneumoniae.

Mycoplasma pneumoniae organisms possessing a hemadsorbing-negative (HA-) phenotype comprise more than 50% of the population of virulent M. pneumoniae cultures. Monoclonal antibody to P1, the major adhesin of M. pneumoniae reacts with this HA- mycoplasma fraction based upon radioimmunoprecipitation and immunoblotting. Demonstration of P1 in the entire mycoplasma population suggests that topological organization of this adhesin in the membrane or the physiological state of the mycoplasmas may determine hemadsorbing capabilities.

Adhesins, Bacterial↗

PubMed1985

Detection of the major adhesin P1 in triton shells of virulent Mycoplasma pneumoniae.

Filamentous structures designated Triton shells were obtained from virulent Mycoplasma pneumoniae by treatment with Triton X-100. Monoclonal antibodies directed against M. pneumoniae were used in conjunction with radioimmunoprecipitation and Western blotting to detect immunologically reactive polypeptides in Triton shells. The major adhesin, protein P1, was associated with these structures.

Adhesins, Bacterial↗

PubMed1984

Characterization of the ATPase activities of myosins isolated from the membrane and the cytoplasmic fractions of human platelets.

Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.

Actins↗

PubMed1984

Multiphasic interactions of Mycoplasma pulmonis with erythrocytes defined by adherence and hemagglutination.

The mechanism(s) of interaction between Mycoplasma pulmonis and eucaryotic cells was studied by adherence to and hemagglutination of erythrocytes. Simple and complex carbohydrates and glycoproteins were unable to inhibit either adherence or hemagglutination, indicating that neither was a lectin activity. Both interactions appeared to be hydrophobic due to their requirement for salt and their sensitivity to temperature. Hemagglutination, but not adherence, was inhibited by both trypsin and glutaraldehyde treatment of the mycoplasma, suggesting that adherence and hemagglutination are qualitatively different. The erythrocyte receptor sites for the two activities were also separable since hemagglutination, but not adherence, required trypsinization of erythrocytes. The hemagglutinin was shown to be an integral mycoplasma component and not a broth contaminant. Once removed, hemagglutinating activity could not be replenished by incubation in serum or broth at 4 degrees C, but could be regenerated during protein synthesis under nonreplicative conditions. Thus, a mycoplasma membrane protein was detected which was capable of interacting with opposing membrane surfaces through hydrophobic interactions. Consequently, a multiphasic model of M. pulmonis-eucaryotic cell interactions was proposed.

Animals↗

PubMed1984

Role of superoxide anion in host cell injury induced by mycoplasma pneumoniae infection. A study in normal and trisomy 21 cells.

The role of Mycoplasma pneumoniae-generated superoxide and hydrogen peroxide in inducing host cell injury was studied in normal and trisomy 21 human cells. As a result of M. pneumoniae infection, catalase activity in infected normal skin fibroblasts and ciliated epithelial cells decreased by 74-77% as compared with uninfected controls. Addition of superoxide dismutase to the infected cultured cells totally prevented the inhibition whereas addition of catalase or catalytically inactivated superoxide dismutase had no protective effect. Trisomy 21 erythrocytes and cultured skin fibroblasts in which CuZn-superoxide dismutase content is 50% greater than in normal cells were infected by M. pneumoniae. The inhibition of catalase activity in these cells was 7-33% and 0-20.5%, respectively, as compared with 65-72% and 48-68% inhibition in normal infected controls. Following M. pneumoniae infection, the levels of malonyldialdehyde, an indicator for membrane lipid peroxidation were raised in trisomy 21 cultured fibroblasts by 10-32% while in normal cells malonyldialdehyde increased by 140-870%. Externally added superoxide dismutase, but not catalase, reduced the extent of lipid peroxidation in normal infected cells. Lactate dehydrogenase release from normal infected cells was time correlated with the increase in their malonyldialdehyde formation. It is suggested that superoxide generated during M. pneumoniae infection is involved in the inhibition of host cell catalase activity. The inactivation of this cellular antioxidative defense mechanism results in progressive oxidative damage to the M. pneumoniae-infected cells.

Catalase↗

PubMed1984

In vitro studies on the mechanism of adherence and pathogenicity of mycoplasmas.

Most pathogenic mycoplasmas adhere to the cytoplasmic membrane of host cells in an avid, almost irreversible way. This interaction seems hydrophobic in nature and may be induced in the area of contact when the surface proteins are cleared away by electrostatic forces. In several mycoplasmas (e.g., Mycoplasma pneumoniae, M. gallisepticum and M. genitalium) the initial steps of adherence are reversible interactions occurring between mycoplasma adhesins that recognize specific sialoglycoconjugates on the host cell membrane. In M. pneumoniae the major adhesin (P1) is an integral membrane protein of about 165 kDa. About 10% of the P1 molecules are linked to the cytoskeleton elements. Part of the proteins in these cytoskeletal elements are phosphorylated, which may explain the anchorage and possible mobilization of P1 to the tip structure, as was indicated by immunohistochemical electron microscopy. With the close contact caused by attachment of the mycoplasmas to the host cells, their pathogenicity can be expressed. Studies with M. pneumoniae on a variety of human cells in culture indicated that superoxide anions are generated during the infection. They drastically inhibit the catalase activity of the host cells. Addition of exogenous superoxide dismutase or increasing its level endogenously minimize the inhibition of catalase. With much of the catalase inhibited, oxidation of cell components occurs, among which are membrane lipids as indicated by elevated malonyldialdehyde levels in infected cells. These may lead to membrane leakage and to the cytopathology of mycoplasma infection.

Adhesins, Bacterial↗

PubMed1983

Structural properties of myosin from the particulate fraction of human blood platelets.

Fractionation of human blood platelets has revealed that myosin, a contractile and mechanochemical protein, is present in both the soluble and particulate fraction. The aim of this study was to elucidate whether platelets contain more than one myosin isoform, especially in view of the fact that in other cellular systems (cardiac muscle, amoeba) several myosin isoenzymes were found. The particulate fraction was solubilized by Triton X-100, and the myosin was purified by the same procedure used for the cytoplasmic myosin. The final preparation contained, in addition to myosin, a 130-kDa polypeptide, which was observed also in myosin preparations obtained from the soluble fraction. The electrophoretic mobilities of the two myosins were identical under both dissociating and nondissociating conditions. Comparison of the molecular structure of the heavy chain of the two myosins by limited proteolysis with Staphylococcus aureus V8 protease showed that the proteolytic fragments of the two myosins were rather similar, with only minor alterations in the quantitative distribution of the products. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the map of the particulate myosin, as compared to its soluble counterpart. According to the two-dimensional peptide map, the 130-kDa polypeptide seems to be a proteolytic fragment of the myosin heavy chain and most probably the rod portion of the molecule. The observed minor variations in the structure of myosins isolated from the soluble and the fractions of human platelets may reflect differences in their respective physiological functions.

Actomyosin↗

PubMed1983

Distribution of actin, myosin and actin binding protein in platelets of patients with hyperlipoproteinemia.

Significant anomalies in the quantity and relative distribution of the contractile proteins actin, myosin and actin binding protein (ABP) were observed in platelets obtained from patients with hyperbetalipoproteinemia (type IIa) and in patients with hypertriglyceridemia (type IV). Changes were observed in unfractionated platelets, (increased ABP in type IIa patients and increased actin in both type IIa and type IV) in isolated platelet membranes (increased ABP and actin in type IV, and increased myosin in type IIa) and in the KCl extract of platelets (increased actin in type IV and increased myosin in type IIa). The myosin ATPase specific activity was increased in platelets of type IV patients. No changes were observed in the concentrations and distribution of membrane glycoproteins in the platelets of these patients. The above anomalies in the contractile proteins might be relevant to the known functional anomalies of the platelets of patients with hyperlipoproteinemias.

Actins↗

PubMed1983

Inhibition of host cell catalase by Mycoplasma pneumoniae: a possible mechanism for cell injury.

This study demonstrates that viable Mycoplasma pneumoniae cells inhibit catalase activity in several types of intact human cells as well as in solution. Human erythrocyte catalase was inhibited up to 72%, and the inhibition of catalase in human cultured skin fibroblasts, lung carcinoma epithelial cells, and ciliated epithelial cells from human nasal polyps ranged between 75 and 80%. UV light-killed mycoplasmas failed to inhibit catalase activity both in intact cells and in vitro. After M. pneumoniae infection of human cultured skin fibroblasts, the level of malonyldialdehyde, an indicator for membrane lipid peroxidation, was 3.5 times higher than in control fibroblasts. Virulent M. pneumoniae completely inhibited catalase activity in solution, whereas the nonvirulent strains had a lesser ability to inhibit catalase activity. These findings suggest that as a result of host cell catalase inhibition by M. pneumoniae, the toxicity of the hydrogen peroxide generated by the microorganism and the affected cell is enhanced, thereby inducing host cell damage.

Catalase↗

PubMed1983

Role of internal domains of glycophorin in Plasmodium falciparum invasion of human erythrocytes.

Human erythrocyte glycophorin, a putative receptor to Plasmodium falciparum malaria parasites, was studied in terms of its structural domains involved in mediating invasion. These domains were isolated from purified glycophorin A and from supernatants and membranes obtained from protease-treated erythrocytes. They were tested for invasion blocking capacity by using an in vitro assay system. The role of carbohydrate-rich domains was assessed with the following compounds: (i) sialoglycopeptides released by proteases either from whole cells or isolated glycophorin A; (ii) the sialoglycoproteins fetuin and alpha 1 acid glycoprotein and the N-acetylglucosamine-rich ovomucoid; and (iii) the saccharides N-acetylneuraminlactose, N-acetylglucosamine, and free sialic acid. With the exception of N-acetylglucosamine, all of the compounds failed to block invasion. The role of carbohydrate-poor domains of glycophorin was assessed with peptides isolated from membranes of proteolyzed cells and with the hydrophobic fragment of glycophorin A. Glycophorin and the derived hydrophobic peptides formed high-molecular-weight aggregates in physiological solutions. They all inhibited invasion to a comparable extent. The inhibitory potency of glycophorin A increased by sixfold after reconstitution into egg lecithin vesicles. The observations reported here underscore the role played by the hydrophobic domain in the glycophorin-mediated blockage of invasion. They also suggest that in the interactions between P. falciparum merozoites and the erythrocyte membrane, the exposed glycosylated domains of glycophorins provide the initial but rather weak binding sites, whereas the internal domains of the molecules provide the more stable attachment sites for merozoites.

Acetylglucosamine↗