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Biomedical subjects

C Gitler

Publications and source records attributed to C Gitler.

At least 55 records · Page 3Linked to original sources
PubMed1983

Quantitation of Chill-Induced Release of a Tubulin-Like Factor and Its Prevention by Abscisic Acid in Gossypium hirsutum L.

The degree of tubulin polymerization in cotton (Gossypium hirsutum L. cv Acala) cotyledonary tissue was estimated by radioimmunoassay which measured the amount of a tubulin-like factor. It was assumed that the release of this tubulin-like factor indicated depolymerization of microtubules. Exposure to chilling resulted in complete release of the tubulin-like factor. Pretreatment with abscisic acid in the light almost completely prevented the chill-induced release of the tubulin-like factor. Addition of colchicine during the chilling period accelerated the release of the tubulin-like factor. Pretreatment with abscisic acid greatly reduced this effect of colchicine. It is concluded that the destruction of the microtubular network is involved in the development of chilling injury in cotton. Abscisic acid apparently decreased chilling injury by stabilization of the microtubular network.

Journal Article↗
PubMed1983

The malaria merozoite surface: a 140,000 m.w. protein antigenically unrelated to other surface components on Plasmodium knowlesi merozoites.

We previously identified three proteins on the surface of merozoites (140,000, 105,000 and 75,000 m.w.). To determine if 140,000 m.w. protein was related to other surface proteins, we immunized mice with liposomes containing merozoite proteins from the 140,000 m.w. region of the polyacrylamide gel. The immune sera reacted with the surface of viable merozoites and acetone-fixed schizonts by immunofluorescence. The sera immunoprecipitated only the 140,000 m.w. protein from surface-labeled merozoites. We demonstrated that monoclonal antibody 13C11 immunoprecipitated a 250,000 m.w. protein from metabolically labeled schizonts and bound to the merozoite surface. This monoclonal antibody immunoprecipitated the 75,000 and lower m.w. proteins from surface-labeled merozoites but did not bring down the 140,000 m.w. protein. Because the mouse immune sera did not immunoprecipitate the 250,000 m.w. protein from metabolically labeled schizonts or proteins other than the 140,000 m.w. protein from surface-labeled merozoites, we conclude that the 140,000 m.w. protein is unrelated to other merozoite surface antigens identified to date. The mouse immune sera against the 140,000 m.w. protein on the merozoite surface did not immunoprecipitate a 140,000 m.w. protein from metabolically labeled schizonts. Instead, the major protein immunoprecipitated had a m.w. of 144,000. By analogy to the 250,000 m.w. protein and its cleavage products, we propose that the 140,000 m.w. protein on the merozoite surface is a cleavage product of the higher m.w. protein.

Animals↗
PubMed1982

Evidence for the organization of the transmembrane segments of (Na,K)-ATPase based on labeling lipid-embedded and surface domains of the alpha-subunit.

The purpose of this work has been to examine the organization of the intramembranous portion of the alpha-subunit of membrane-bound (Na,K)-ATPase. Covalent labeling of the alpha-subunit and its tryptic fragments from within the lipid bilayer with [125I]iodonaphthylazide was combined with covalent labeling with 32P from [gamma-33P]ATP at the cytoplasmic surface and with [3H]N-(ouabain)-N'-(2-nitro-4-azidophenyl)ethylenediamine from the extra cellular surface. In control experiments using extensive proteolysis and reduced glutathione, it is confirmed that iodonaphthylazide labels segments of the protein within the lipid bilayer. The labeled segments of the alpha-subunit, produced by extensive proteolysis, are selectively extracted by organic solvents. Both at a low and at a high concentration of iodonaphthylazide, about 50% of label added to the medium is covalently attached to protein and lipid. At the low iodonaphthylazide concentration, the NH2-terminal Mr = 46,000 (46K) fragment of the alpha-subunit is preferentially labeled, while at the higher concentration of the 46K fragment, the 78K fragment, and the COOH-terminal 58K fragment are labeled. 32P from [gamma-32P]ATP is incorporated into the 46K fragment while [3H]N-(ouabain)-N'-(2-nitro-4-azido-phenyl)ethylenediamine from the extracellular surface labels all the major fragments, 78K, 58K, and 46K. The data provide evidence for a model of the path of the polypeptide chain with multiple traverses of the alpha-subunit across the bilayer and the NH2-terminal and three trypsin-sensitive bonds exposed at the cytoplasma surface.

Animals↗
PubMed1982

An ion-channel forming protein produced by Entamoeba histolytica.

We have identified a remarkable ion-channel forming material in virulent strains of Entamoeba histolytica that may be responsible for many of the symptoms associated with amoebic dysentery. A polypeptide that we refer to as amoebapore is shed into the growth media and is also found within the amoeba in a high speed sedimentable fraction. Amoebapore has the distinctive property of spontaneously incorporating into lipid bilayers, liposomes, and cells, leading to progressive and irreversible changes in the ion conductance of the target membranes. Exposure of planar lipid bilayers to amoebapore -containing fractions under voltage clamp conditions results in an almost immediate and progressive incorporation of ion channels which continues in an irreversible manner leading to a fall in membrane impedance of up to five orders of magnitude. The ion-channel conductance is moderately cation-selective, voltage dependent, and displays a unit size of 1.6 +/- 0.2 nanoSiemens in 1 M KCl at -10 mV. In the bilayer, the amoebapore -induced conductance exhibits an in situ sensitivity to protease. Amoebapore is mainly concentrated in a fraction sedimenting at 150 000 g. It is insoluble in Triton X-100 but can be dissociated in an active state in 1% SDS. Under these conditions it has an apparent mol. wt. of 13 000 daltons.

Amoeba↗
PubMed1981

Reaction of 5-iodonaphthyl-1-nitrene with the IgE receptor on normal and tumour mast cells.

Mast cells, basophils and a tumour analogue--rat basophilic leukaemia (RBL) cells--have a surface glycoprotein (R epsilon) which specifically binds monomeric immunoglobulin E (IgE), and aggregation of R epsilon causes secretion. When isolated from non-ionic detergent extracts of surface-labelled RBL cells by IgE-specific immunoprecipitation R epsilon appears as a 50,000 (50 K) to 60,000 (60 K) molecular weight (MW) band on electrophoresis in polyacrylamide gels in SDS (SDS-PAGE). Likewise, only a 50 component is observed when the polypeptide that binds IgE is isolated by affinity chromatography in conditions which prevent aggregation of the IgE, even when intrinsically labelled R epsilon is studied. To determine how R epsilon is inserted into the plasma membrane, we reacted RBL cells with the photolysable hydrophobic reagent 5-iodonaphthyl-1-azide (INA), which preferentially labels the intramembranous segments of several intrinsic membrane proteins. We report here that, surprisingly, the label was found, not on the 50 K glycopeptide, but only on a 30 K component which other studies suggest is a subunit of R epsilon (ref 9, 12).

Animals↗
PubMed1981

Liposomes as immunological adjuvants in eliciting antibodies specific to the synthetic polypeptide poly(LTyr, LGlu)-poly(DLAla)--(LLys) with high frequency of site-associated idiotypic determinants.

The antibody response to the synthetic polypeptide, poly(LTyr, LGlu)-poly(DLAla)--poly(LLys), [(T,G)-A--L], injected entrapped in liposomes which served as adjuvant has been analyzed. The liposomes used were composed of phosphatidylcholine, cholesterol, dicetylphosphate and DL alpha-tocopherol (molar ratios as 4:3:0.1:0.5) and therefore, were negatively charged. Since the (T,G)-A--L is also negatively charged, no free complexes were formed. The (T,G)-A--L was found to be entrapped inside the enclosed volume of the liposomes, and no (T,G)-A--L antigenic determinants could be detected on the liposomal membranes. Injection of high-responder C3H.SW (H-2b) mice with (T,G)-A--L-bearing liposomes demonstrated that the i.p. and the i.v. routes of immunization were efficient in eliciting (T, G)-A--L specific antibodies, whereas the i.d. injection led to poor antibody responses. The latter route of immunization is the most effective when (T,G)-A--L is injected in complete Freund's adjuvant (CFA). When low doses (0.1 and 1 microgram) of (T, G)-A--L were used for immunization, the liposomes were better adjuvants than CFA. The effectiveness of the liposomes as immunological adjuvants was also shown in their ability to induce high-potential, primed memory cells. The pattern of low (H-2k,a) and high (H-2b) responsiveness to (T,G)-A--L was retained following immunization with (T,G)-A--L entrapped in liposomes, as tested in two pairs of congenic strains. (T,G)-A--L-specific antibodies induced by injection with 1 microgram antigen entrapped in liposomes bear the (T,G)-A--L site-related idiotypic markers of C3H.SW (Igh-1a) mice in a significantly higher frequency than the homologous idiotypes, namely the antibodies elicited in this strain against (T,G)-A--L in CFA. Thus, liposomes may serve as adjuvants for the production of relatively restricted (T,G)-A--L-specific antibodies of high quality.

Adjuvants, Immunologic↗
PubMed1981

Dynamics of antibody- and lectin-mediated endocytosis of hapten-containing liposomes by murine macrophages.

The uptake by murine macrophages of liposomes, exhibiting one of a variety of haptenic groups on their surfaces, was greatly enhanced by the addition of an intact antibody or a lectin specific for the incorporated hapten. The uptake of untreated liposomes was slow and linear over long periods, whereas upon addition of the antibody or lectin, over 30-fold increase in the maximal rate of uptake was observed. The process reached a plateau after 90-120 min. The interaction of the antibody- or lectin-treated liposome with the macrophages apparently resulted in an active endocytosis of soluble fluorescent, intraliposomal marker had a granular intracellular pattern in treated cells. The uptake was sensitive to azide and the liposome constituents could not be detected at the cell surface. The size of the liposomes as well as the state of stimulation of the macrophages (thioglycollate stimulated vs. normal) did not seem to have a major effect on the phagocytic process. The time required to reach the plateau in uptake was independent of liposome composition or antibody concentration and is, apparently, an intrinsic property of the cells. The implication of this phenomenon on the dynamics of the relevant macrophage receptors is discussed.

Adsorption↗
PubMed1981

Cell surface labeling of embryonic neural retina cells exposed to low temperature, energy inhibitors, cytochalasin B and colchicin.

Large blebs devoid of receptors for hemocyanin-labeled concanavalin A appear on neural retina cells from 8-day chick embryos exposed to low temperature or to potassium cyanide or 2,4(alpha)-dinitrophenol at room temperature. Labeling with mixed antibodies against a crude retina membrane preparation and with goat anti-rabbit-hemocyanin conjugate showed the same results. Determination of cell ATP content indicated a drop in ATP concentration after exposure to low temperature or to respiration inhibitors. Disruption of microtubules by colchicin inhibited the formation of 'naked' large blebs, whereas cytochalasin B had no such inhibitory effect.

2,4-Dinitrophenol↗
PubMed1978

Red cell membrane glycophorin labeling from within the lipid bilayer.

Human red blood cell membranes were labeled from within the lipid bilayer by the apolar photosensitive reagent, 5-[125I]iodonaphthyl-1-azide. Glycophorin, the major sialoglycoprotein of the red cell membrane, was purified by two different methods; it contained approximately half of the total label incorporated into membrane proteins. The label was confined to the trypsin-insoluble peptide of glycophorin that includes a sequence of 20, mainly apolar, amino acids. These findings provide direct evidence that the labeled segment resides within the membrane in direct contact with the lipid bilayer, and support the suggestion that glycophorin spans the bilayer through its hydrophobic domain.

Azides↗
PubMed1977

Intrinsic proteins of the intestinal microvillus membrane. Iodonaphthylazide labeling studies.

Isolated brush border membranes of the intestinal epithelial cell were labeled with a hydrophobic photoactive compound [125U]iodonaphthylazide. High incorporation of the radioactive naphthylazide was noted for molecular weight bands of 99 000, 86 000, 65 000, 54 000 and 30 000. Minimal labeling occurred in the higher bands of 300 000, 135 000, 125 000 and 17 000. The iodonaphthylazide label was not removed by extensive papain digestion whereas chloramine T iodinated membranes released radioactivity under the same conditions. Neither enzymatic nor transport activities were inhibited by the presence of iodonaphthylazide or the irradiation process. On the basis of the presented data it is concluded that the iodonaphthylazide unspecifically labels those portions of membrane proteins which are inserted into the lipid bilayer matrix.

Alkaline Phosphatase↗
PubMed1977

A simple fluorescent method to determine complement-mediated liposome immune lysis.

A simple inexpective method is described to study the kinetics of complement-mediated immune lysis of liposomes containing sheep red blood cell lipid antigens. It is based on the fact that trapping the fluorescent molecule 1-aminonaphthalene-3,6,8-trisulfonate and the dynamic quencher, alpha, alpha'-dipyridinium p-xylene dibromide within the liposome inner volume results in an extinguished fluorescence signal. On addition of helmolysin plus active complement, liposome lysis occurs. The exit of the fluorophore and quencher and their subsequent dilution in the external volume abolishes the quenching, resulting in a high fluorescence signal. The details of the method are described as well as the initial kinetic results.

1-Naphthylamine↗