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PubMed1986

Ultracryotomy for the study of unfixed membranes.

Plasma membranes from chromaffin cells of bovine adrenal medullae and from chicken macrophages were isolated on a urografin density gradient, frozen and sectioned without previous chemical fixation. Their receptor binding sites were localized by specific labelling. The sections were then post-fixed in the presence of K2Cr2O7 to produce positive staining of the membrane proteins. Chromaffin cell membranes formed single vesicles. The nicotinic acetylcholine receptor (localized using a monoclonal antibody against its cholinergic binding site) was always found in patches on the surface of vesicles, whose profiles corresponded to thickened bilayers. Macrophage membrane vesicles were agglutinated. The mannose receptor (localized using the ligand, mannosylferritin) was randomly distributed within the electron-dense coat of the agglutinated vesicles or on electron-dense caps involved in agglutination. The binding sites of both receptors were intact, as revealed by their being recognized by a monoclonal antibody against their cholinergic binding sites and by the active binding of the mannosylated ligand which was inhibited by mannan. The distribution of the receptors on the vesicles reflected their distribution on the cell surface.

Adrenal Medulla↗
PubMed1983

Differing reactivities of human and guinea-pig complement on haptenized liposomes via the alternative pathway.

Several haptens coupled to dipalmitoylphosphatidylethanolamine (DPPE) were inserted into the liposome membrane with a base composition of an equimolecular mixture of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol). Haptens used were trinitrophenyl (TNP)-DPPE, TNP-aminocaproyl (TNP-Cap)-DPPE, dinitrophenyl (DNP)-DPPE, DNP-aminocaproyl (DNP-Cap)-DPPE, fluoresceinthiocarbamyl (Fl)-DPPE, azobenzenarsonate-tyrosyl (ABA-Tyr)-DPPE, dansyl (DNS)-DPPE, dabsyl (DABS)-DPPE, dithiopyridyl (DTP)-DPPE and maleimidobenzoyl (MB)-DPPE. Reactivity of those haptenized liposomes with complement via the alternative pathway was assessed by release of trapped fluorescent marker from the liposomes following incubation with dilutions of guinea-pig and human sera in a diluent containing MgCl2 and ethyleneglycol-bis(beta-aminoethyl ether)N,N'-tetraacetate (EGTA). In the diluent (Mg-EGTA-GVB), complement activation via the alternative pathway proceeds while that via the classical pathway is inhibited. Fl-liposomes were found to be extremely sensitive to guinea-pig complement, being lysed by guinea pig serum dilutions of up to 1:76 in Mg-EGTA-GVB. Guinea-pig serum could lyse TNP-Cap-liposomes, DNP-Cap-liposomes, TNP-liposomes, DTP-liposomes, MB-liposomes, DNP-liposomes and ABA-Tyr-liposomes, with the reactivity of the liposomes decreasing in this order. However, the only haptenized liposomes sensitive to human serum in Mg-EGTA-GVB were DTP- and MB-liposomes; the other liposomes including Fl-liposomes being unreactive via the alternative pathway in reaction with human complement.

Animals↗
PubMed1978

Immunohistological studies on the distribution of cytochrome b3 reductase in rat liver microsomes.

The distribution of cytochrome b5 reductase in rat liver microsomes purified by density gradient centrifugation was studied using anti-cytochrome b5 reductase/anti-ferritin hybrid antibodies labeled with ferritin. Electron micrographs show that the outer surface and the polar cap of tangential sections of essentially all vesicles derived from endoplasmic reticulum are specifically marked with a few ferritin grains which are not localized in large patches but arranged randomly. This finding was correlated with morphometrical and biochemical measurements. The results, supporting our earlier investigations, suggest that cytochrome b5 reductase may be clustered at the most in very small assemblies consisting of a few molecules which, in turn, are statistically uniformly distributed over the total membrane system.

Animals↗
PubMed1987

CAP Comprehensive Blood Bank Survey--1984.

An average of 2750 laboratories participated in the 1984 Comprehensive Blood Bank Survey of the College of American Pathologists. Results from 16 laboratories were utilized to validate results and assure maintenance of specimen character during shipment. Results of ABO grouping, D typing, special antigen typing, antibody detection, and antibody identification by participants remained good and are discussed. Special ungraded samples were provided as additional challenges. The results of these studies and the responses to supplementary questions are discussed.

ABO Blood-Group System↗
PubMed1975

Binding of histamine- and other ligand-conjugated macromolecules to lymphocytes.

The presence of histamine receptors on lymphocyte membranes was investigated using conjugates of histamine and macromolecules tritiated or iodinated with I-125. Histamine-RSA conjugate binds to lymphocytes and causes patching and capping of the bound conjugate. It was found, however, that free histamine did not inhibit the binding of histamine-rabbit serum albumin to mouse lymphocytes, nor did His-RSA interfere with the binding of free histamine. In addition conjugates between RSA and other small molecules, such as ethylamine, ethanolamine, tyramine and glycine, were found to bind to the same sites on lymphocyte membrane as did His-RSA. Ethylamine-RSA like His-RSA when coupled to Sepharose, was capable of removing antibody producing cells from spleen cells of mice immunized against sheep red blood cells. In addition, when spleen cells from such immunized mice were passed through ethylamine or histamine-RSA-Sepharose and the unbound cells were subsequently injected into X-irradiated mice, a 1.8 fold increase in the immunological response was noted. We conclude that the selective binding to lymphocytes of the various ligand-macromolecular conjugates may be due to some general properties of the cell membrane and not to any specific receptors. Nevertheless, these conjugates can be used as a tool to remove selectively antibody producing cells as well as some regulatory cells.

Animals↗
PubMed2006

Dendritic cells transfected with interleukin-12 and tumor-associated antigen messenger RNA induce high avidity cytotoxic T cells.

Dendritic cells (DC) transfected with messenger RNA (mRNA) encoding tumor-associated antigens (TAA) are able to induce potent tumor-specific T-cell responses directed to a broad spectrum of tumor-associated epitopes. The in vitro generation of DC possessing all the features crucial for the induction of type 1 immune responses, such as mature state, migratory potential and interleukin-12 (IL-12p70) production is complicated. Particularly migratory potential is inversely correlated with IL-12p70 production after maturation with prostaglandin E2 (PGE2), which is included in maturation cocktails currently used in most vaccination trials. Here, we show that transfection of PGE2 matured DC with a single mRNA strain encoding for ubiquitin followed by a TAA which was linked to IL-12 by a self-cleaving 2A sequence, produced biological active IL-12p70 and were able to present the transfected TAA up to 72 h after transfection. Furthermore, use of the anti-reverse cap analog for in vitro transcription of the IL-12 mRNA enabled constitutive IL-12p70 production for up to 5 days. These transfected mature DC migrated efficiently towards lymph node derived chemokines. DCs constitutively expressing IL-12p70, generate TAA-specific cytotoxic T cells with an high functional avidity, independent of CD4+ T-cell help.

Antigens, Neoplasm↗
PubMed1997

Role of mast cell- and non-mast cell-derived inflammatory mediators in immunologic induction of synaptic plasticity.

We have previously discovered a long-lasting enhancement of synaptic transmission in mammal autonomic ganglia caused by immunological activation of ganglionic mast cells. Subsequent to mast cell activation, lipid and peptide mediators are released which may modulate synaptic function. In this study we determined whether some mast cell-derived mediators, prostaglandin D2 (PGD2; 1.0 microM), platelet aggregating factor (PAF; 0.3 microM) and U44619 (a thromboxane analogue; 1.0 microM), and also endothelin-1 (ET-1; 0.5 microM) induce synaptic potentiation in the guinea pig superior cervical ganglion (SCG), and compared their effects on synaptic transmission with those induced by a sensitizing antigen, ovalbumin (OVA; 10 micrograms/ml). The experiments were carried out on SCGs isolated from adult male guinea pigs (200-250 g) actively sensitized to OVA, maintained in oxygenated Locke solution at 37 degrees C. Synaptic potentiation was measured through alterations of the integral of the post-ganglionic compound action potential (CAP). All agents tested caused long-term (LTP; duration > or = 30 min) or short-term (STP; < 30 min) potentiation of synaptic efficacy, as measured by the increase in the integral of the post-ganglionic CAP. The magnitude of mediator-induced potentiation was never the same as the antigen-induced long-term potentiation (A-LTP). The agent that best mimicked the antigen was PGD2, which induced a 75% increase in CAP integral for LTP (antigen: 94%) and a 34% increase for STP (antigen: 91%). PAF-, U44619-, and ET-1-induced increases in CAP integral ranged for LTP from 34 to 47%, and for STP from 0 to 26%. These results suggest that the agents investigated may participate in the induction of A-LTP.

Animals↗
PubMed1983

Polypeptides immunologically related to band 3 are present in nucleated somatic cells.

Band 3, the major transmembrane polypeptide of erythrocytes, mediates the exchange of anions (chloride and bicarbonate) across the membrane. We suspected that band 3 was present on nucleated somatic cells as well as erythrocytes because the senescent cell antigen that is immunologically related to band 3 is present on lymphocytes, platelets, adult liver cells, and embryonic kidney cells; and antibodies prepared against the senescent cell antigen isolated from leukocytes react with erythrocyte band 3. For this reason, we examined human fibroblasts, lung cells, neutrophils, mononuclear leukocytes, squamous epithelial (mouth) cells, lung squamous epithelial carcinoma, mouse neuroblastoma cells, and rat hepatocytes for immunoreactive forms of band 3 by using monospecific antibodies to erythrocyte band 3. The results demonstrated that polypeptides sharing common antigenic determinants with erythrocyte band 3 are present in nucleated somatic cells as determined by immunofluorescence, immunoelectron microscopy, and immunoautoradiography. Peptide mapping revealed substantial sequence homology between erythrocyte band 3 and the band 3-like protein of leukocytes. Immunofluorescence studies indicate that the band 3-like proteins in nucleated cells participate in antibody-induced cell surface capping.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1986

Lymphoma Thy-1 glycoprotein is linked to the cytoskeleton via a 4.1-like protein.

In this study we have found that the phosphoprotein doublet of 68,000 and 65,000 daltons (68/65 kD) in mouse T-lymphoma cells shares several structural and functional similarities with erythrocyte band 4.1. Our evidence for identifying the 68/65-kD doublet as a lymphoma 4.1-like protein is as follows: it displays an immunological cross-reactivity with anti-erythrocyte band 4.1 antibody; it exhibits a Svedberg unit of sedimentation coefficient of 4 S; it is phosphorylated in the presence of phorbol ester (phorbol-12-O-tetradecanoylphorbol-13-acetate) and its phosphorylation requires Ca2+; it is phosphorylated primarily at serine residues; and it can bind directly to fodrin (a spectrin-like actin-binding protein). In addition, this lymphoma 4.1-like protein can be both colocalized and coisolated with the major T-lymphocyte-specific glycoprotein, Thy-1 (gp 25). Therefore, all of these results strongly suggest that the lymphoma 4.1-like protein (68/65-kD doublet) may play a pivotal role in linking the Thy-1 (gp 25) glycoprotein to fodrin which, in turn, binds to the actin filaments that are responsible for recruiting Thy-1 antigens into cap structures.

Amino Acids↗
PubMed1978

A sensitive rosetting method for detecting subpopulations of lymphocytes which react with alloantisera.

A procedure is described for directly estimating the proportion of mouse lymphoid cell suspensions which react with alloantisera. The method entails reacting Ig-capped lymphoid cells with alloantisera, and then assessing the uptake of alloantibodies by rosetting the lymphocytes with SRBC coated with sheep IgG specific for mouse Ig. This rosetting procedure was found to be generally more sensitive than the conventional dye exclusion microcytotoxicity test for detecting the binding of alloantibodies to lymphocytes. Furthermore, the rosette method has the advantage that, unlike the complement lysis technique, it has a low and reproducible background and lymphocyte subpopulations which react with alloantisera can be isolated.

Animals↗
PubMed2006

A molecular mechanism for the heparan sulfate dependence of slit-robo signaling.

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.

Amino Acid Sequence↗
PubMed2001

Stability of coagulation proteins in frozen plasma.

This study reports on the frozen stability of all commonly measured coagulation proteins in normal citrated plasma: activated partial thromboplastin time, prothrombin time (%), thrombin time and fibrinogen (Clauss); clotting assays for factors II, V, VII, VIII, IX, X, XI and XII; functional assays for protein C (clotting), protein S (clotting), antithrombin (chromogenic) and plasminogen (chromogenic); and immunological assays for von Willebrand factor and D-dimer. All these factors listed are stable for up to 3 months if frozen at -24 degrees C or lower. At -74 degrees C, all these factors (allowing for 10% variation) were stable for at least 18 months, most were stable for 24 months. The number of proteins showing > 5% variation over baseline after 6 months storage indicates that some decay does occur even at -74 degrees C. There was no clear advantage in snap freezing at -74 degrees C and then storing at -24 degrees C over both freezing and storing at -24 degrees C; therefore, the freezing process itself is not responsible for the loss of stability. The best stability, especially at -24 degrees C, was obtained when small samples (1 ml) were stored in screw-cap tubes with a minimum dead space. The decrease in stability of the coagulation proteins directly correlates with the effect of temperature and time.

Blood Coagulation↗
PubMed1979

Redistribution and modulation of Gross murine leukemia virus antigens induced by specific antibodies.

Gross murine leukemia virus (G-MuLV)-induced rat leukemia cells in tissue culture replicate G-MuLV, express strong virus-associated membrane antigenicity, and are consistently killed by specific antibodies and complement in cytotoxicity tests. To explore the effect of specific antibodies, rat anti-G-MuLV antisera were added to the cultures of leukemia cells for variable periods of time. Redistribution of virus particles as well as of membrane virus antigens in the form of polar patches and caps was observed by electron microscopy, indirect immunofluorescence, and immunoelectron microscopy. Substantial decreases in cytotoxicity indexes accompanied these changes. The antigen modulation induced by anti-G-MuLV antibodies in vitro paralleled similar changes obtained in vivo by transplanttion of leukemia cells in rats with high anti-G-MuLV antibody titers. The importance of antigen modulation in this system resides in its direct relationship with the malignant potential of the leukemia cells.

AKR murine leukemia virus↗
PubMed1975

Association of beta-2-microglobulin and HL-A in the cell membrane. A general property in human cells?

To study whether the recently reported close association of beta-2-microglobulin and HL-A determinants is restricted to lymphocyte membranes or might be a more general property, experiments were performed where normal and malignant human cells were tested for coaggregation-capping of the two determinants. The normal cells were fibroblasts and the epithelial cells were from skin. The turmor cell lines were HeLa clone S3 carcinoma cells and National Collection of Type Cultures strain 3075, which is a line of malignant human skin cells. The cells were double-stained with fluorescein-labelled conjugates to demonstrate the reaction of anti-HL-A and anti-beta-2-microglobulin with the respective cell membrane components. Aggregation and capping of the HL-A and beta-2-microglobulin determinants in the cell membrane were always observed after incubation at 37 C for 30 min. The red and green fluorescence were always superimposed when anti-beta-2-microglobulin was used in the first (aggregating) incubation step. This demonstrates that the HL-A and beta-2-microglobulin determinants are also associated in the cell membrane of cells other than lymphocytes.

Animals↗
PubMed1976

[Hairy cell leukemia. Characterization of the leukemic cell: surface immunoglobulins, Fc-receptors and stimulation by mitogens (author's transl)].

In four patients with hairy cell leukemia the expression of surface immunoglobulins and Fc-receptors on the leukemic cells as well as the stimulation of the leukemic cells by various T- and B-cell mitogens was studied. Using a combined cytochemical radioautographic method the tartrate resistant isoenzyme of the acid phosphatase and immunological markers could be demonstrated simultaneously on single cells. Surface immunoglobulins were detected by 125I-labelled (Fab')2-fragments of monospecific anti-heavy-chain-antibodies. In two patients only mu- and delta-determinants were found on the isoenzyme-positive hairy cells; in one patient 86% and in the other 44% of these cells carried both heavy chains on the same cell. Another patient showed both gamma- and delta-chains on the isoenzyme-positive cells and the fourth patient gamma-chains only. When the hairy cells of one patient were cultured in vitro in human serum-free medium for 14 days, mu- and delta-determinants were found just as on freshly isolated cells suggesting that hairy cells synthesize immunoglobulins. By indirect immunofluorescence the surface-Ig on hairy cells was shown to be capped very rapidly at room temperature. The concentrated surface-Ig was found over that cytoplasmic area where most of the ingested latex particles were located. In addition, using 125I-labelled aggregated human IgG (M.W. 5-15 X 10(6)), Fc-receptors were found on virtually all of the isoenzyme-containing hairy cells in all four patients. Furthermore, a distinct, low-degree stimulation of the isoenzyme-positive hairy cells could be demonstrated when the cells were cultured in the presence of pokeweed mitogen or Lima-bean lectin (B- and T-cell-mitogens), whereas no stimulation was observed by phytohemagglutinin and concanavalin A (T-cell-mitogens). In three patients studied, isoenzyme-positive hairy cells were negative with regard to rosette formation with sheep erythrocytes.

Acid Phosphatase↗
PubMed1983

Polarization of NK cell cytoskeleton upon conjugation with sensitive target cells.

We studied the cytoskeletal changes in natural killer (NK) cells during conjugate formation, i.e., when NK cells make contact with sensitive vs resistant target cells. F-actin and vinculin were seen to polarize at the contact sites upon conjugation with sensitive K562 cells, whereas in conjugates with resistant Raji target cells such an orientation was an infrequent finding. Myosin and two other cytoskeletal proteins, spectrin and vimentin, on the other hand, showed a random distribution in conjugating NK cells regardless of the target cell type. Hence the cytoskeletal redistribution associated with conjugation seems to be different from the receptor capping phenomenon, which is accompanied by clustering of actin, myosin, vimentin, and spectrin. On the basis of these results it seems probable that the lytic conjugate formation in NK-mediated cytotoxicity is associated with the formation of a specific type of junction that involves actin and vinculin. This cytoskeletal reorganization precedes and could be a prerequisite for the polarization of the cellular secretory apparatus and may be functionally responsible for the required cytokinetic movements.

Actins↗
PubMed1988

Isolation, structure and protein composition of the perforatorium of rat spermatozoa.

Heads of spermatozoa were sonically separated from tails and treated in 1 N NaOH until the perforatoria were partially detached from the nucleus. Their complete detachment was then assured by repeatedly passing the suspension through a 22-gauge needle. The perforatoria were then separated from nuclei on sucrose gradients and the purity of the fraction was verified by electron microscopy. The isolated perforatoria were denatured and used to raise antibodies or run on polycrylamide gels. Such gels revealed many polypeptide bands, six of which were most prominent (Mr approximately 13,000, 13,400, 16,000, 33,000, 35,000, and 43,000). Of these, the 16,000 Mr polypeptide was major. Anti-perforatorium serum reacted with the perforatoria of fixed spermatozoa, with a substance found between the plasmalemma and the outer acrosomal membrane of the acrosomal head cap and with the inner component of the ventral spur, but not with the postacrosomal dense lamina. This observation indicated that the perforatorium and dense lamina, although structurally continuous to form the perinuclear theca, are biochemically distinct. On Western blots, the anti-perforatorium serum reacted with the prominent polypeptides of the perforatorium and cross-reacted with some less prominent polypeptides of the fibrous sheath (FS) and outer dense fibers (ODF), most notably with a 16,000 Mr polypeptide found in both. Likewise anti-FS or anti-ODF serum cross-reacted with some major and minor polypeptides of the perforatorium, again most notably with a major 16,000 Mr polypeptide. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. These results demonstrate that the perforatorium is composed of several polypeptides, is immunologically distinct from the postacrosomal dense lamina, may be immunologically similar to a substance found between the plasmalemma and outer acrosomal membrane and to a substance found on the inner aspect of the ventral spur, has antigenic determinants in common with the FS and ODF, and may share a 16,000 Mr polypeptide with these two cytoskeletal structures of the flagellum.

Animals↗
PubMed1989

Laminin-induced capping and receptor expression at cell surface in a rat rhabdomyosarcoma cell line: involvement in cell adhesion and migration on laminin substrates.

The present study reveals the dynamic distribution of membrane laminin receptors induced by laminin binding in a rat rhabdomyosarcoma cell line RMS S4. The treatment of the cells with soluble laminin did not modify cell adhesion to laminin-coated substrates in in vitro attachment assays. Fluorescent labeling of membrane-bound laminin revealed that occupied receptors were induced to cluster and cap. New free membrane binding sites were made evident after capping of bound laminin by a double labeling technique. Cytochalasin D (CD) treatment prevented the capping process. The adhesion of CD-treated cells to laminin-coated substrates was inhibited by cell preincubation with soluble laminin. Cycloheximide treatment had no effect on the ability of RMS S4 cells to adhere to adsorbed laminin after preincubation in the presence of soluble laminin. These results taken as a whole suggest that free receptors may arise from an intracellular pool that could be maintained by membrane receptor recycling. Since capping and motility seem related events, migration of RMS S4 cells on laminin was studied in the agarose drop assay. Immobilized laminin stimulated basic cell motility by more than 200%. E8 laminin fragment retained partially the motility stimulating property of laminin while P1 pepsinic fragment had no effect. The presence of constantly available receptors at the cell surface could be determinant in the ability of cells to migrate on laminin substrates.

Animals↗