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PubMed1997

Nitric oxide inhibits capping in HL-60 cells.

In the present study, we investigated the effect of nitric oxide (NO) on capping, which is associated with the actin polymerization in HL-60 cells (human promyelocytic leukemia cells). We first assessed the effect of NO on the patching and capping by using anti-human LFA-1 monoclonal antibody. Samples were analyzed by a fluorescence microscope. As expected, NO inhibited the percentage of capping dose dependently. We compared the effect of NO on capping with cytochalasin D (CD) and observed that CD also inhibits the capping in HL-60 cells. We next examined the effect of NO on the F-actin content. For assays of F-actin content, the FITC labelled phalloidin was permeabilized and stained in HL-60 cells. The bound fluorescence quantified by flow cytometry using a FACStar. There was a decrease in the F-actin formation in NO treated cells. Taken together, these data indicate that NO inhibits the capping on cellular membrane by decreasing the intracellular F-actin formation in HL-60 cells. We suggest that the formation of capping linked with actin polymerization at the inner leaflet of plasma membrane may be regulated by NO.

Actins↗
PubMed1981

Cell membrane receptor classes delimited through cap formation either with diamide or with membrane mobility agent, A2C.

Receptors on normal human peripheral blood lymphocytes can be divided into two classes by means of the capping response exhibited in the presence of the reagents, diamide or colchicine (microtubule-related) and A2C (microtubule-independent). Diamide and colchicine promote capping of concanavalin A (Con A) receptors. Diamide capping is reversible, while colchicine capping is not reversible under the conditions used. A2C does not promote the capping of Con A receptors. In contrast, diamide and colchicine do not affect the rate at which either anti-immunoglobulin (anti-Ig) or wheat germ agglutinin (WGA) receptors cap, but A2C effectively enhances cap formation for both anti-Ig and WGA receptors. The simplicity of the classification method promises to be of use in the investigation of membrane receptors.

Azo Compounds↗
PubMed1979

Transmembrane communication in cells chronically infected with measles virus.

The transmembrane association of the measles virus hemagglutinin and hemolysin surface proteins with intracellular viral antigens was studied. Rabbit antisera monospecific for measles virus matrix and nucleocapsid proteins and a human antiserum containing specificities for both the hemagglutinin and hemolysin proteins were used to study the co-capping of these proteins in human Lu 106 cell-line, chronically infected with measles virus. Capping of the surface-associated envelope components was accompanied by co-capping of the matrix and nucleocapsid proteins, the latter being localized mainly within the inclusions. This demonstrated transmembrane communication between surface-associated envelope components and the intracellular measles virus matrix and nucleocapsid proteins. The results demonstrated the existence of a linkage between viral inclusions and viral proteins associated with cell membranes. In the presence of cytochalasin B (1--2 micrograms/ml), co-capping of the matrix protein was unchanged or slightly enhanced, whereas co-capping of the nucleocapsid protein decreased, indicating that actin filaments may mediate the communication between viral nucleocapsids and the cell membrane.

Antigens, Surface↗
PubMed1979

Capping of surface immunoglobulin on 'hairy cells' is independent of energy production.

Capping, independent of metabolic energy, of surface immunoglobulin (S-Ig) on 'Hairy cells' from patients with Hairy cell leukaemia (HCL) is described. As controls leukaemic cells from a patient with a prolymphocytic leukaemia (PLL) and blood lymphocytes from healthy individuals were used. The specificity of the energy-independent capping of the HCL-cells as compared to the controls was tested by incubation of the cells at 4 degrees C in the presence of 0.1 M sodium azide with different FITC-labelled ligands. In order to find an explanation for this phenomenon, the influence of cytochalasin B, colchicine and the combination of both drugs on capping of S-Ig and concanavalin (Con-A)-receptors at 37 degrees C was investigated. Furthermore the effect of Con A on S-Ig capping and vice versa was studied. The results show that only S-Ig on HCL cells could form caps at 4 degrees C in the presence of sodium-azide. Cytochalasin B alone induced a strong inhibition of Con A capping on all 3 cell types, whereas S-Ig capping was unaffected. Colchicine alone had practically no effect. Anti-Ig inhibited subsequent patch and cap formation with Con A on both HCL cells and PLL cells, whereas Con A caps and patches were redistributed by anti-Ig on PLL cells, but not on HCL cells. Conversely, Con A could link S-Ig to other receptors, leading to inhibition of S-Ig capping at 4 degrees C on HCL cells and to co-capping of S-Ig at 37 degrees C on both cell types. In addition Con A induced redistribution of S-Ig caps. The combination of co-capping of S-Ig by Con A, followed by redistribution of the caps by FITC-anti-Ig simulated inhibition of S-Ig capping by Con A on PLL cells. The major conclusions are: in some cases inhibition of capping may actually be caused by redistribution of caps; the energy-independent capping cannot be explained by free diffusion of S-Ig in the membrane through lack of any connexion with receptor-mobility regulating systems. It is proposed that the energy requirement of capping is needed to inactivate a specific mechanism,w which restrains receptor mobility and which is non-operative in HCL cells.

Adult↗
PubMed1979

Non-specific effects of avian retrovirus co-incubation on lymphocyte function: abrogation of antigen- and mitogen-induced proliferative responsiveness.

Peripheral blood lymphocytes from chickens bearing tumours induced by avian retroviruses can be stimulated to divide by group-specific antigens present in supernatant fluids of avian retrovirus-infected but not normal chicken embryo fibroblast (CEF) cells. Centrifugation studies revealed that the relevant antigenic activity is non-virion in nature. Indeed, the presence of avian retrovirus particles was found to be inhibitory to the capacity of sensitized lymphocytes to be stimulated in this antigen-driven blastogenesis assay. Similar results were obtained in lymphocyte mitogenesis experiments in which any of peripheral chicken lymphocytes or mouse splenic, lymph node or thymic lymphocytes were co-incubated with either concanavalin A or phytohaemagglutinin in the presence of numerous types of virus particles. This inhibitory effect was not due to infection of lymphocytes by the viruses tested, and was obtained in the case of lymphocyte-virus combinations for which the cells lacked the surface receptors required for viral entry. Virus could be added to lymphocyte cultures as late as 26 h after co-incubation with mitogen, and still inhibit the usual mitogenic response. In addition, co-addition of virus to lymphocytes in the presence of concanavalin A was found to block the capping of ligand-bound receptors which normally ensues. Pre-added virus did not, however, affect the ability of lectins to bind to cells.

Alpharetrovirus↗
PubMed1981

T lymphocytes of young and aged rats. I. Distribution, density, and capping of T antigens.

The distribution, density, and capping of the T cell antigens W3/13, W3/25, and Thy-1 were studied in lymphocytes of young (3 to 4 mo) and aged (greater than or equal to 27 mo) Brown Norway (BN) rats. The total number of W3/13, W3/25, and Thy-1 positive cells and the percentage of Thy-1 positive cells are reduced in the spleens and lymph nodes of aged rats. Analysis of spleen and lymph node cells in a fluorescence-activated cell sorter indicated that with ageing there is a loss of moderately and brightly stained W3/13 and W3/25 positive cells. The density of W3/13, W3/25, and Thy-1 is reduced on spleen and lymph node cells of old rats. The rate of capping of all 3 molecules is diminished on "old" cells. Colchicine treatment of young cells enhanced capping of all 3 molecules and allowed capping of W3/13 by a single ligand. However, capping on "old" lymphocytes was not affected by colchicine treatment. Cytochalasin D inhibited capping to the same extent on young and "old" cells. These results suggest that the membrane composition and cytoskeleton are altered in T lymphocytes of aged rats.

Aging↗
PubMed1990

Thiol proteinase inhibitors reverse the increased protein kinase C down-regulation and concanavalin A cap formation in polymorphonuclear leukocytes from Chediak-Higashi syndrome (beige) mouse.

Protein kinase C (PKC) plays an essential role in intracellular signal transduction for various cell functions, including concanavalin A (Con A)-induced cap formation. This enzyme is known to be proteolysed by calpain, which is a Ca2(+)-dependent thiol proteinase. As reported previously, in polymorphonuclear leukocytes (PMNs) from beige mouse, the model of Chediak-Higashi syndrome, Con A-induced cap formation significantly increased compared with that in normal mouse. However, after pretreatment of beige PMNs with the thiol proteinase inhibitors leupeptin or E-64, the capping decreased to normal levels. Meanwhile, Con A-induced the translocation of PKC from the cytosolic to membrane fraction within 5 min in both mice, which is essential to the activation of this enzyme. However, after the translocation, an abnormal rapid decline in membrane-bound PKC activity was noted in beige mouse PMNs. Both leupeptin and E-64 also corrected the rapid decline in PKC activity observed in the beige mouse. These findings suggest that the normalization of Con A cap formation in beige mouse PMNs by the thiol proteinase inhibitors is associated with the correction of abnormality in PKC activity.

Animals↗
PubMed1985

Calcium-dependent association of a protein complex with the lymphocyte plasma membrane: probable identity with calmodulin-calcineurin.

A protein complex is shown to participate in a calcium-dependent association with plasma membranes purified either from pig mesenteric lymph node lymphocytes or from human lymphoblastoid cell lines. Plasma membranes prepared in the presence of calcium possess this complex; those prepared in the absence of calcium (5 mM EGTA) do not. The complex associates itself with the inner cytoplasmic surface of the plasma membrane. This complex is referred to as the "acidic protein band" because of its location during migration upon alkaline-urea gel electrophoresis. The complex dissociates from the plasma membrane during electrophoresis on 8-M urea gels, irrespective of calcium levels during electrophoresis; at intermediate urea concentrations (4-6 M), the complex is not dissociated in the presence of calcium. Upon purification of the acidic protein band, SDS acrylamide gel electrophoresis, immunoblotting, and radioimmunoassay techniques suggest that the acidic protein band is composed of at least four peptides (designated 68K, 59K, 20K, 20K): two of these (68K, 20K) are immunopositive for calcineurin and one (20K) is immunopositive for calmodulin. Immunoblots of urea gels also indicate that the calcineurin heavy chain (68K) can also appear at three different locations on the urea gel. Patches and caps induced in human peripheral blood lymphocytes by fluorescein-conjugated goat anti-human IgG are not coincident with the location of calcineurin, which remains distributed throughout the cell.

Animals↗
PubMed1985

Concanavalin a capping of rhesus monkey polymorphonuclear leukocytes.

The technique of capping, a probe designed to evaluate the fluidity and functional competence of human polymorphonuclear leukocytes (PMNs), has been successfully adapted for rhesus monkey PMNs. The capping characteristics of rhesus monkey PMNs are very similar to those of human PMNs. Utilizing this capping technique as an evaluation tool and with fetal/neonatal rhesus monkey as a functional animal model, the ontogeny of movement and chemotactic characteristics of PMNs can now be studied.

Animals↗
PubMed1980

The contribution of assays for lymphocyte capping and creatine kinase to detection of the Becker-type dystrophy trait.

Members of three unrelated families with the mild Becker type of muscular dystrophy were subjected to lymphocyte capping tests and measurements of serum creatine kinase activity. Both tests correctly identified all nine affected males, but only the capping test was abnormal in seven of eight obligate carriers. The number of capped cells in carriers and affected persons with the Becker-type dystrophy was generally intermediate between those observed for individuals with the Duchenne trait and normal controls, thus potentially aiding in the differential diagnosis between the two myopathies. The lack of sensitivity of measurements of serum creatine kinase activity in identifying carriers is further complicated by the difficulty of establishing reliable reference intervals for this enzyme in 204 healthy controls. Detailed directions for the performance of the capping test are presented.

Adolescent↗
PubMed1985

Polarity of isolated blastomeres from mouse morulae: detection of transcellular ion currents.

Eight- to sixteen-cell stage mouse morulae were dissociated with Ca2+-free medium into blastomeres that were labeled with fluoresceinated-succinylated Con A (FS-Con A) to mark their apical-basal axes. The vibrating probe was then used to map their extracellular current patterns. The average current density around normal blastomeres approached the resolution of the probe system (0.2 microA/cm2) and was undetectable in the majority of blastomeres. Since the current density at the measuring point outside the cell is known to increase with cell size in other systems, enlarged blastomeres were created by fusing together blastomeres of 4-cell stage embryos in 45% polyethylene glycol. Enlarged blastomeres were then aggregated with normal blastomeres using phytohemagglutinin and cultured to the 8- to 16-cell stage to allow them to become polarized. Such aggregates were then dissociated with Ca2+-free medium to recover polarized, enlarged blastomeres. The enlarged blastomeres were 30-65 microns in diameter and 70% of them generated a detectable current; currents were detected around 83% of those blastomeres larger than 40 micron in diameter. The current pattern in these most reliable cases was predominantly inward apical (11/16 or 69%) and outward basal (15/16 or 94%), with lateral currents about three-fold smaller in amplitude than these apical-basal currents. Lateral currents were undetectable in 53% of the cases. Preliminary data suggest that the inward current is carried in part by Na+ influx and is independent of the Na+,K+-ATPase over the short term. Transcellular ion currents were detectable as long as 4 hr after dissociation, and the apical-basal current pattern was usually stable during that time. In contrast, the fluorescent cap of FS-Con A faded within 7-30 min at 35 degrees C but remained stable in 0.1% azide or 1.5 micrograms/ml cytochalasin D. The electrical polarity therefore persisted after the apical cap of Con A fluorescence was no longer visible. We propose that these transcellular ion currents may be involved in the establishment of blastomere polarity and describe a mechanism of action in an "ion current polarization" hypothesis.

Animals↗
PubMed2006

EhPAK2, a novel p21-activated kinase, is required for collagen invasion and capping in Entamoeba histolytica.

p21-activated kinases (PAKs) are a highly conserved family of enzymes that are activated by Rho GTPases. All PAKs contain an N-terminal Cdc42/Rac interacting binding (CRIB) domain, which confers binding to these GTPases, and a C-terminal kinase domain. In addition, some PAKs such as Cla4p, Skm1p and Pak2p contain an N-terminal pleckstrin homology (PH) domain and form a distinct group of PAK proteins involved in cell morphology, cell-cycle and gene transcription. Here, we describe a novel p21-activated kinase, denominated EhPAK2, on the parasitic protozoan Entamoeba histolytica. This is the first reported Entamoeba PAK member that contains a N-terminal PH domain and a highly conserved CRIB domain. EhPAK2 CRIB domain shares 29% of amino acid identity and 53% of amino acid homology with these of DdPAKC from Dictyostelium discoideum and Cla4p from Saccharomyces cerevisiae and binds in vitro and in vivo to EhRacA GTPase. This domain also possesses the conserved residues His123, Phe134 and Trp141, which are important for the interaction with the effector loop and strand beta2 of the GTPase; and the residues Met121 and Phe145, which are specific for the interaction of EhPAK2 with EhRacA. Functional studies of EhPAK2 showed that its C-terminal kinase domain had activity toward myelin basic protein. Cellular studies showed that Entamoeba trophozoites transfected with the vector pExEhNeo/kinase-myc, had a 90% decrease in the ability to invade a collagen matrix as well as severe defects in capping, suggesting the involvement of EhPAK2 in these cellular processes.

Amino Acid Sequence↗
PubMed1988

Membrane Ig-cytoskeletal interactions. I. Flow cytofluorometric and biochemical analysis of membrane IgM-cytoskeletal interactions.

Membrane IgM (mIgM) and mIgD are the receptors for Ag on the surface of B lymphocytes, mIg is soluble in detergent; however, when mIg is cross-linked with anti-Ig, the mIg becomes associated with the cytoskeletal matrix and is rendered detergent-insoluble. By a novel flow cytofluorometric assay and by biochemical analysis, it has been shown that anti-isotype-specific antibodies induce mIgM and mIgD to associate with the cytoskeleton of B lymphocytes in an isotype-specific fashion. The detergent solubility of other prominent B lymphocyte surface proteins, such as class I and class II MHC proteins were unaffected by cross-linking of mIg. A panel of mu-specific mAb was analyzed for their ability to induce mIgM-cytoskeletal association. Although all mAb bound mIgM, only three out of seven rendered mIgM cytoskeletally associated. Further analysis revealed a strict correlation in the capacity of mu-specific mAb to induce capping and to induce the association of mIgM with the cytoskeleton.

Animals↗
PubMed1980

Antibody-induced antigen redistribution on murine mammary tumor cells.

Syngeneic antitumor antibody induced the capping of tumor-associated surface antigens on murine mammary tumor cells. Immunofluorescence staining showed that capping was maximal after 2 approximately 4 hr, and then the capped antigen-antibody complexes disappeared. Radioimmunoassay, however, showed that the quantity of antigen-antibody complexes was the same before and after capping. This redistribution of surface antigens on the tumor cells had no effect on the time course of antibody-dependent macrophage-mediated tumor lysis (ADMC). Inhibition of antigen movement on the cell surface by glutaraldehyde treatment did not inhibit ADMC. These results suggest that syngeneic antitumor antibody induces the capping of surface antigens on murine mammary tumor cells, but that this dynamic redistribution of surface antigens is not correlated with the susceptibility of the cells to ADMC.

Animals↗
PubMed1981

Cytochalasin A inhibits B-lymphocyte capping and activation by antigens.

Cytochalasin B (CB) has been shown to be a potent depressant of the antigen-induced clone expansion and terminal differentiation of mouse B-lymphocytes to antibody-forming cells. This effect could be the result of the microfilament-disrupting effect of CB with subsequent inhibition of antigen-sIg complex redistribution, a series of events which seems to be necessary for B-lymphocyte activation. CB is not very active in depressing capping and will inhibit glucose transport. To further investigate the mechanism of action of cytochalasins, the effect of cytochalasin A (CA) on cap formation and plaque-forming cell generation was studied, since CA is less inhibitory of glucose transport and more inhibitory of cap formation. The results presented here indicate that complexes of anti-Ig-sIg will be prevented from capping by as little as 1 microgram of CA, a quantity sufficient to depress markedly the generation of plaque-forming cells to SRBC in culture. These results further confirm our conclusion that the depression of B-lymphocyte activation may be related to the depression of cap formation. It also strongly suggested that inhibition of glucose transport can be regraded as a negligible factor in this depression.

Animals↗
PubMed1987

Temperature-mediated processes in teleost immunity: the effects of temperature on membrane immunoglobulin capping on channel catfish B lymphocytes.

1. In order to better understand ligand-induced redistribution of membrane receptors and lymphocyte activation in ectothermic vertebrates, flow cytometry was used to monitor the effects of both in vivo acclimation temperature and in vitro assay temperatures on the kinetics of monoclonal antibody-induced membrane immunoglobulin (mIg) capping on channel catfish lymphocytes. 2. It was observed that the kinetics of mIg capping were dependent on in vitro assay temperatures, in vivo acclimation temperatures, and the length of time of in vivo acclimation. In the latter situation in vivo acclimation of fish to 27, 22 and 17 degrees C was considered complete after 3 weeks, while acclimation to 12 degrees C required a minimum of 5 weeks. 3. The energies of activation required for mIg capping ranged from 33 to 24 kcal/mol; lower energies of activation were observed with lower temperature acclimation. 4. It was also noted that the lower energies of activation were associated with concomitant decreases in cellular phospholipid saturated/unsaturated fatty acid ratios. 5. It appears that channel catfish B cell mIg capping, presumably a requisite for immune function, can be significantly affected by environmental temperatures; most likely such effects are attributable to changes in plasma membrane viscosities.

Acclimatization↗
PubMed1996

Fatty acid unsaturation increases expression and capping of murine lymphocyte CD44 and CD45.

We studied the effect of incubating murine lymphocytes with cis-unsaturated fatty acids on expression and capping of CD44 and CD45. Lymphocytes were incubated with stearic (18:0) or oleic (18:1 omega-9) acid bound to bovine serum albumin (BSA). After incubation with rat anti-CD44 or anti-CD45 monoclonal antibodies and then with fluorescent-labeled anti-rat antibody, mean fluorescence intensity (FI) was measured by using flow cytometry. Capping was measured after warning and fixation in paraformaldehyde. Steady-state fluorescence anisotropy (rs) was measured after the cells had been incubated with trimethylammoniumdiphenylhexatriene. Incubation with oleic acid, but not stearic acid or BSA alone, was associated with an increase in FI of CD44. Expression of CD45, however, was increased by both stearic and oleic acids to the same degree over BSA controls. CD44 and CD45 capping were both increased by incubation with oleic acid. Rs was decreased in cells incubated with oleic acid, suggesting an increase in membrane fluidity. We conclude that incubation with oleic acid increases expression of CD44 and increases capping of both CD44 and CD45. These findings were confirmed in feeding experiments, in which rs was reduced and CD44 capping increased by polyunsaturated fatty acid diets.

Animals↗
PubMedDate not supplied

Construction, expression and recognition of an H-2 molecule lacking its carboxyl terminus.

A mouse major histocompatibility antigen (H-2) gene, encoding a novel H-2Ld molecule lacking its intracytoplasmic domain, has been constructed and introduced into mouse L-cells. The novel H-2 molecule is found on the surface of the transfected cells at the same level as L-cells transfected with the native H-2Ld gene. Allo- and influenza-specific cytotoxic T lymphocytes can recognize the truncated H-2 gene product nearly as efficiently as the normal H-2Ld gene product. However, vesicular stomatitis virus-specific cytotoxic T lymphocytes recognize the truncated H-2Ld molecule less efficiently than the complete H-2Ld product. The rate of capping of the truncated H-2Ld molecule was investigated and found to be the same as that of the complete H-2Ld gene product.

Amino Acid Sequence↗