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C Gitler

Publications and source records attributed to C Gitler.

At least 37 records · Page 2Linked to original sources
PubMed1987

Entamoeba histolytica: cloning and characterization of actin cDNA.

In order to study gene expression in the human parasite Entamoeba histolytica, a cDNA library of E. histolytica strain 200:NIH was constructed using the phage vector lambda gt10. Three cDNA clones (A, B and C) were selected for further analysis. Each of the three clones hybridized to a distinct mRNA. Two of these mRNAs were translated in vitro after hybrid selection, and yielded distinct translation products. One of these mRNAs, selected by hybridization to clone A, encodes the most abundantly expressed protein in E. histolytica. DNA sequence analysis of this cDNA clone identified the DNA as that encoding actin. The deduced amino acid sequence of E. histolytica actin resembles both cytoplasmic and muscle actins and has an unusual N-terminal glycine residue. We have shown that a family of actin genes is present in E. histolytica. Six different E. histolytica actin clones were obtained from a lambda gt10 genomic library using subcloned cDNA probes. Southern analysis of three different E. histolytica strains (200:NIH, Rhaman, and HM-1:IMSS) revealed at least four different actin genes. Strain HM-1:IMSS, however, differs by the presence of an additional actin gene.

Actins↗
PubMed1987

Selective labeling of proteins in biological systems by photosensitization of 5-iodonaphthalene-1-azide.

The apolar azide of 5-iodonaphthalene-1-azide (Ina) partitions into the lipid bilayer of biological membranes. Upon photolysis at 314 nm, it is rapidly converted into the reactive nitrene, which efficiently attaches covalently to lipid-embedded domains of proteins and, to a lesser extent, to membrane phospholipids. Above 370 nm, Ina absorption is negligible and photolysis at these wavelengths does not occur. However, on addition of the photosensitizing molecule 3-aminopyrene, trifluoperazine, or 8-anilinonaphthalene-1-sulfonate, followed by irradiation at 380 nm, efficient conversion of Ina to reactive species was observed, as measured by [125I]Ina-labeling of membrane proteins and inactivation of the hormonal response of adenylate cyclase. Irradiation at 480 nm in the presence of a fluorescein derivative of n-undecylamine also resulted in a pattern of [125I]Ina-labeled membrane proteins and hormone uncoupling indistinguishable from that obtained following direct photolysis at 314 nm. Photosensitization of the azide molecules is confined to the vicinity of the photosensitizer chromophore. This allowed selective labeling of chromophore-bearing proteins in solution or in membranes. Bovine serum albumin-fluorescein conjugate, in the presence of nonderivatized soluble proteins, was exclusively labeled by [125I]Ina when irradiated at 480 nm, but random labeling occurred on photolysis at 314 nm. Likewise, rhodopsin in rod outer segment membranes from frog retina was exclusively labeled by [125I]Ina upon photosensitization at 380 nm. Random labeling again occurred on direct irradiation at 314 nm. The results suggest that selective labeling in complex biological systems may be achieved by photosensitized activation of azides.

Adenylyl Cyclases↗
PubMed1986

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers.

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.

Affinity Labels↗
PubMed1985

A 3 beta-cholestanyl-containing mono-benzylpenicilloyl oligoamide and peptide suppress anti-benzylpenicilloyl antibody formation in mice.

A long-chain linear mono-benzylpenicilloyl (BPO) oligoamide and a succinoylated mono-BPO decalysine were tested in BALB/c mice for suppression of IgE and IgG1 antibody formation. Both compounds were available with either a free C-terminal end or were C-terminally linked to a hydrophobic 3 beta-cholestanyl residue. Only the sterol-containing derivatives suppressed hapten-specific IgE and IgG1 responses. Substantial suppression was obtained when the compounds were administered before primary or secondary, but not later immunizations. In an adoptive cell transfer experiment, spleen cells from tolerized animals actively suppressed anti-BPO IgE antibody formation of immune spleen cells. This effect was reversed by pretreatment of the tolerized spleen cells with anti-Lyt-2.2 antibody plus complement. The requirement for macrophages in the induction of T suppressor cells was demonstrated by injecting antigen-pulsed macrophages into naive recipients; upon immunization, only mice treated with tolerogen-pulsed macrophages showed suppressed anti-BPO IgE responses. It is suggested that lipid modification of antigens alters their processing and presentation by macrophages in a manner that leads to the induction of T suppressor cells. Injection of the cholestanyl derivatives into passively sensitized guinea pigs elicited anaphylactic reactions. By immune precipitation analysis and molecular weight estimation, these derivatives were shown to form micelles in aqueous solution. Therefore, the anaphylactic response appeared to be due to their behavior as multivalent antigens.

Anaphylaxis↗
PubMed1985

Lethal recognition between Entamoeba histolytica and the host tissues.

Lethal recognition between Entamoeba histolytica and the tissues and defence systems of the host results in a continuous interplay that determines the development of pathological lesions: (i) we have identified several of the steps and mediators utilized by the trophozoites to destroy host cells by contact-mediated cytolysis; (ii) we have established that the alternative complement system represents the main defence available to the host against the invading parasite. The amoebae recognize target cells by means of a lectin specific for N-acetylgalactosamine-containing surface glycoproteins. This recognition appears to activate the amoeba to release, in the area of contact, an attack complex that induces the host cells to undergo cytolysis. The main component of the attack complex is thought to be amoebapore, an ion-channel forming protein that incorporates spontaneously into target cells leading to their depolarization by creating a pathway for ions to flow down their concentration gradient. The known properties of amoebapore are described. The acquisition of complement resistance by the invading trophozoites is essential for their survival within the host and therefore underlies virulence. The resistance to complement killing is not a permanent property of the amoebae. It is lost during axenization and reappears on passage through the host or when the trophozoites are grown axenically in the presence of active complement.

Acetylgalactosamine↗
PubMed1985

Subcellular fractionation of amoebapore and plasma membrane components of Entamoeba histolytica using self-generating Percoll gradients.

Separation and initial characterization of subcellular organelles from Entamoeba histolytica have been achieved by the use of self-generating Percoll gradients. Adequate resolution was obtained within one hour of amoeba homogenization. The ion-channel forming activity, amoebapore, was found associated with highly dense, small diameter particles, that were resolved from the numerous digestive vacuoles, and the plasma membrane. Following iodination of intact trophozoites with lactoperoxidase, the label was found to be incorporated into two distinct sedimentable fractions. The major component contained the near totality of the concanavalin A binding glycoproteins and the 5-iodonaphthalene-1-azide labelled intrinsic membrane proteins. It therefore was identified as the plasma membrane. The minor lactoperoxidase-iodinated component was found in the upper section of the gradient, associated with a particulate fraction nearly devoid of concanavalin A-binding glycoproteins. This fraction appears to represent particulate material on the external surface of the amoeba that is distinct from the plasma membrane. The digestive vesicles, which were identified by the presence of acid phosphatase and beta-D-glucosaminidase activities, appeared in the gradient as one main peak with a shoulder in the region of the plasma membrane. Analysis of the polypeptides and their labelling pattern in each fraction of the gradient are presented. Distinctive characteristics are discussed including the identification of a unique soluble protein highly labelled by 5-iodonaphthalene-1-azide.

Animals↗
PubMed1984

Membrane insertion of prothrombin.

The nature of the interaction of prothrombin with liposomes was examined by labelling with the apolar reagent 5-[125I] iodonaphthyl-1-azide (INA). When liposomes containing phosphatidylcholine were incubated with prothrombin and INA, the amount of incorporated label was greater in the absence of added calcium. Increasing the phosphatidylserine content of the liposomes, irrespective of CA++ resulted in a significant increase in the labelling of prothrombin by INA. Since INA is confined to the lipid core of the bilayer, these results strongly suggest that prothrombin binds to liposomes, and penetrates into the lipid bilayer.

Azides↗
PubMed1984

Cytopathogenicity of Entamoeba histolytica.

The lesions induced in man by Entamoeba histolytica are characterized by massive tissue injury in the absence of major local signs of a host immune response. The amoeba damages surrounding cells preferentially by contact-mediated cytolysis. Recently, a presumptive aetiological factor underlying this process has been identified. It is a protein, amoebapore, capable of spontaneous incorporation into host cell membranes. Therein it induces high conductance ion-channels which rapidly collapse the cellular transmembrane potential and lead to a prelytic state. Amoebapore is present within the amoeba in a highly aggregated state in a small, dense particle. It is shed into the medium in a particulate form by a stimulus-mediated process. Release is enhanced by addition of concanavalin A, lipopolysaccharide or the calcium ionophore A23187. Surface-labelling of intact amoeba, followed by fractionation of the homogenate in self-generating Percoll gradients, identified two labelled fractions, the plasma membrane and a particulate fraction sedimenting in the region of intracellular particulate amoebapore. This latter fraction appears to be material in the process of exocytosis. A highly immunogenic surface lipid has been identified and shown to be involved in the rapid surface redistribution of immune complexes, their shedding and endocytosis. The relevance of these findings to the immunoprophylaxis of amoebiasis is discussed.

Amebiasis↗
PubMed1984

Selective photoinduced uncoupling of the response of adenylate cyclase to gonadotropins by 5-iodonaphthyl 1-azide.

5-Iodonaphthyl 1-azide (INA) has been previously shown to selectively label, on photolysis, only those proteins in contact with the membrane lipids. Low concentrations (less than 10 microM) of INA added to rat ovarian plasma membranes induced, on photoactivation, a selective and complete loss of the response of the adenylate cyclase to stimulation by human chorionic gonadotropin (hCG) or luteinizing hormone (LH). In contrast, this treatment affected neither hCG binding to the receptor nor the stimulation of the enzyme by NaF. That the uncoupling of the receptor from the enzyme by INA occurred within the lipid bilayer can be derived from the finding that the prior presence neither of saturating concentrations of hCG nor of the aqueous nitrene-scavenger glutathione (GSH) prevented this effect. Photolysis at higher concentrations of INA (0.1-1 mM) led to the inhibition of the adenylate cyclase stimulated by fluoride. This effect was totally prevented by glutathione. A similar behavior was obtained with a water-soluble analogue of INA, namely, 5-diazonionapthyl 1-azide (DAN). On photoactivation with 30 microM DAN, the NaF-stimulated adenylate cyclase was inhibited, but this effect was completely prevented by added GSH. At low concentrations where its effects are restricted to the lipid core, INA may represent a useful tool to define receptor coupling with the adenylate cyclase. The capacity of INA at low concentrations to uncouple the hormone receptor from the adenylate cyclase is not restricted to the LH/hCG receptor. Other hormone receptors tested behaved similarly. Therefore, the reported findings appear to represent a general phenomenon.

Adenylyl Cyclases↗
PubMed1984

Lipid antigens derived from erythrocytes infected with Plasmodium berghei.

Lipids were extracted from red blood cells infected with Plasmodium berghei, from the membranes of infected red cells and from free parasites. A radioimmunoassay was used to detect antibodies to these lipids in sera from convalescent and immune rats. Most of the antigenic activity could be attributed to the parasite although some activity was found in lipids isolated from the membranes of infected red blood cells. Absorption studies showed that the binding was specific for malarial lipid antigens. Immune sera showed no cross-reactivity with lipids from red blood cells of non-infected rats. However, sera from non-infected control rats showed low levels of cross-reactivity with the parasitized red cell-derived lipids. Levels of anti-lipid antibodies were directly correlated with the progress of the infection. The highest antibody level occurred when the parasitaemia reached zero. The malarial lipids had no effect on lymphoblast transformation of immune splenocytes in vitro. However, liposomes prepared from either malarial or non-specific lipids caused an increased response to antigen by the blast cells.

Animals↗
PubMed1984

Regenerative capacity of the goldfish visual system is affected by antibodies specific to gangliosides injected intraocularly.

An association between gangliosides and neuronal regeneration in goldfish is demonstrated in the present study. A single intraocular injection of affinity purified anti-GM1 antibodies administered simultaneously with crush injury of the optic nerve, inhibits the regenerating process as expressed by two parameters: protein synthesis in the retina and in vitro sprouting ability from the retina. The retinal level of several gangliosides (such as GD3, GD1a, GD1b and GT1b) is enhanced during regeneration. Although GM1 appears to be a minor retinal ganglioside, antibodies to GM1 exert a marked effect on retinal regenerative process. It is assumed that such antibodies could interact with more abundant retinal gangliosides such as GD1b which shows enhanced biosynthesis during regeneration and which shares a similar disaccharide terminal residue with GM1.

Animals↗
PubMed1983

Diffusion potential cascade. Convenient detection of transferable membrane pores.

A valinomycin-mediated K+ diffusion potential across the membrane of multilamellar liposomes is stable for longer than 30 min and can be collapsed by a nonselective channel such as gramicidin. The kinetics of the potential collapse are complex but can be qualitatively broken down into a series of processes involving (1) binding of the gramicidin to the outer membrane, (2) dimerization to form a functional channel, (3) the flow of ions through the channel, (4) the establishment of a new diffusion potential on the next bilayer within the multilamellar liposome, and (5) the dissociation of gramicidin from the outer bilayer into the adjacent internal aqueous space. These processes are then repeated, in turn, for all the internal bilayers until the K+ concentration gradient (and membrane potential) is completely dissipated. Process 5 appears to be rate limiting at high gramicidin concentrations, but ion flux, process 3, becomes slower at low gramicidin concentrations where the collapse of the K+ gradient displays voltage dependence. Of course the rates of these processes can also be manipulated by changing the composition or size of the liposome and by varying the ion concentrations. Since the diffusion potential can be conveniently monitored with a voltage-sensitive fluorescent dye, 3,3'-diethylthiodicarbocyanine iodide [diS-C2-(5)], a simple method for the detection and partial characterization of membrane pores emerges from this investigation.

Dimyristoylphosphatidylcholine↗
PubMed1983

The selective detection of cell surface determinants by means of antibodies and acetylated avidin attached to highly fluorescent polymer microspheres.

Procedures are described for the synthesis of 500 A-diameter polymer microspheres containing a novel fluorescent cross-linking agent. These microspheres have very high fluorophore concentration without quenching of the fluorescence and show very low nonspecific interaction with cells. When monoclonal anti-Thy-1.2 is attached to the fluorescent microspheres, specific binding results in 10(4) spheres being attached per thymocyte while non-specific binding is less than 1%. Similar values are obtained for an indirect staining procedure. The high non-specific binding of cationic avidin to negative cell surfaces is shown to be decreased to negligible levels by acetylation of the amine groups of the protein without decreasing its high-affinity binding to biotin. The use of acetyl-avidin (pI = 6.7) directly, or when attached to fluorescent microspheres, resulted in a highly selective detection of biotinyl groups on the erythrocyte or lymphocyte cell surface. Attachment of biotinyl groups to the hinge carbohydrates of antibodies did not affect their specificity. It allowed their detection by means of microspheres-acetyl-avidin conjugates.

Acetylation↗
PubMed1983

Molecular events in the processing of avidin by antigen-presenting cells (APC). II. Identical processing by APC of H-2 high- and low-responder mouse strains.

The differences between the immune response (Ir) phenotypes of H-2 gene-controlled high- and low-responder mice have been attributed to events occurring at the interaction between antigen-presenting cells (APC) and lymphocytes. To investigate this interaction we undertook a study of molecular events in the processing of avidin, a molecule whose uniquely strong affinity for binding to biotin renders it traceable at very low concentrations, and a molecule to which the T-lymphocyte immune response is controlled by Ir genes. In this paper we describe the generation of processed avidin by APC and characterize it biochemically and immunologically. We found that APC of H-2 genetically high- and low-responder mice were indistinguishable in their capacity to generate immunogenic processed avidin (PA). Immunogenic PA differed from native avidin in size and carbohydrate moieties, but preserved its capacity to bind biotin, and was 1000-fold more efficient than NA as an immunogen for primed T lymphocytes. Primed T lymphocytes appeared to recognize PA that was conformationally intact. Highly immunogenic PA was not H-2 restricted. Thus, differences in the Ir phenotype of the response to avidin could not be attributed to determinant selection by APC.

Animals↗
PubMed1983

Molecular events in the processing of avidin by antigen-presenting cells (APC). III. Activation of T-lymphocyte lines and H-2 restriction are mediated by processed avidin associated with I-region gene products.

We investigated the interaction between processed avidin (PA) and avidin-specific lines of T lymphocytes free of resident antigen-presenting cells (APC). We found that PA was able to replace the requirement for APC in the T-lymphocyte proliferative assay, only if the PA was associated with an Ia-positive moiety (IPM) supplied by the APC. In addition to supplying a necessary signal for a proliferative response to PA, IPM imposed H-2 restriction on the PA molecule. The association between PA and IPM was reversible and the two moieties could be physically separated and recombined. The results support a conclusion that major histocompatibility restriction of the interaction between T lymphocytes and APC is due to the association between processed antigen and an APC element containing I-region products.

Animals↗