Biomedical subjects

A Pavan

Publications and source records attributed to A Pavan.

At least 37 records · Page 2Linked to original sources

PubMed1995

Monosialoganglioside GM3 induces CD4 internalization in human peripheral blood T lymphocytes.

Gangliosides modulate the expression of CD4 molecules on the cell surface of T lymphocytes. We report here that treatment of human peripheral blood lymphocytes with exogenous monosialoganglioside GM3 induces a rapid down-modulation of the CD4 molecules on the plasma membrane of CD4+ T lymphocytes, as assessed by cytofluorimetric analysis and quantitative immunoelectron microscopy. The CD4 down-modulation was ganglioside-dose dependent and was already evident after 5 min of treatment, reaching the maximum after 20 min. The expression of other surface antigens was not affected by GM3 treatment. The immunoelectron microscopic analysis showed that, following GM3 addition, gold labelled CD4 molecules were rapidly redistributed on the cell surface, clustered and internalized via endocytic pits and vesicles. These results indicate that CD4 down-modulation induced by GM3 occurs through an endocytic mechanism. A persistent low level of CD4 expression on the cell surface up to 24 h after GM3 treatment, compared with a stable expression of either CD4 in untreated cells and CD3 in GM3-treated cells, suggests intracellular degradation of the internalized CD4 molecules.

CD4 Antigens↗

PubMed1995

Enhancing medical database semantics.

Medical Databases deal with dynamic, heterogeneous and fuzzy data. The modeling of such complex domain demands powerful semantic data modeling methodologies. This paper describes GSM-Explorer a Case Tool that allows for the creation of relational databases using semantic data modeling techniques. GSM Explorer fully incorporates the Generic Semantic Data Model-GSM enabling knowledge engineers to model the application domain with the abstraction mechanisms of generalization/specialization, association and aggregation. The tool generates a structure that implements persistent database-objects through the automatic generation of customized SQL ANSI scripts that sustain the semantics defined in the higher lever. This paper emphasizes the system architecture and the mapping of the semantic model into relational tables. The present status of the project and its further developments are discussed in the Conclusions.

Information Systems↗

PubMed1993

Platelet proaggregating activity of human colorectal tumour cell lines does not correlate with the degree of differentiation.

Six human colorectal tumour cell lines with various degrees of differentiation were studied. Each of the cell lines studied showed an in vitro platelet proaggregating activity, represented by the induction of typical aggregation waves. This activity was dose-dependent and probably related to a thrombin-dependent mechanism. However, the degree of cell differentiation did not correlate with the proaggregating activity. In fact, significant differences were observed between the two well differentiated cell lines, while a comparison between well and poorly differentiated cell lines did not reveal any difference. These results were confirmed by the ultrastructural observations, indicating that similar platelet-tumour cell interaction may be found in all the cell lines studied. The present results suggest that platelet proaggregating activity of the human colorectal tumour cell lines under investigation is unrelated to their degree of differentiation.

Blood Platelets↗

PubMed1992

Patching and capping of LFA-1 molecules on human lymphocytes.

The distribution and dynamics of LFA-1 molecules over the surface of human lymphocytes were analysed using immunogold label-fracture and fracture-flip methods. Patching and capping were induced by incubation at 37 degrees C with antibodies directed against the alpha and beta chains respectively of the heterodimeric LFA-1 molecule, and were followed by immunofluorescence. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) to link LFA-1 molecules to the cytoskeleton increased the percentage of capped cells, implying a faster and more efficient process of capping. At all times of clustering or upon phorbol ester treatment, the concentration of LFA-1 in patches and then in caps was not accompanied by a parallel concentration of membrane particles on the freeze-fractured plasma membranes. Our results support the role of the cytoskeleton in regulating the capping phenomenon and in controlling the structural organization of the plasma membranes.

Antibodies↗

PubMed1992

Infection by human herpesvirus 6 (HHV-6) of human lymphoid T cells occurs through an endocytic pathway.

We have analyzed by immunoelectron microscopy the early events of binding and internalization of human herpesvirus 6 (HHV-6, strain GS) on a susceptible T-lymphoblastoid cell line, HSB-2. The virions bound to the cell surface at 4 degrees C were tightly associated with the plasma membrane. Gold immunolabeling of the viral envelope proteins was strong and specific. Warming at 37 degrees C for different times showed viral internalization through smooth surfaced pits and vesicles. Fusion events of the virions with the cell plasma membrane were never observed. Gold immunolabeling performed in parallel experiments before or after viral internalization showed: (1) absence of viral envelope proteins on the cell plasma membranes at all times of internalization, again excluding fusion events; (2) entry of the virions with their envelopes. Treatment of the cells with chloroquine, a drug known to affect the endocytic pathway, led to an almost complete inhibition of viral infectivity, suggesting that the endocytosed virions are responsible for a successful infection. Comparable results were obtained using a second strain of HHV-6 (BA92), with biologic and molecular characteristics similar to the prototype strain Z29. The chloroquine inhibition was effective on two different T cell lines (HSB-2 and J-Jhan), as well as on phytohemagglutinin-stimulated peripheral blood mononuclear cells.

Cell Line↗

PubMed1992

Herpes simplex virus envelopment and maturation studied by fracture label.

Herpes simplex virus envelopment and maturation were investigated by thin-section fracture label. The distribution of glycoproteins B and D was analyzed by labeling with antibodies; the precursor and mature forms of the glycoproteins were differentiated by labeling with the lectins concanavalin A (ConA) and wheat germ agglutinin (WGA), respectively. We report that the two glycoproteins were readily detected in the intracellular virion, whether located between the inner and outer nuclear membranes or within cytoplasmic membrane-bound vesicles and in the inner and outer nuclear membranes themselves. The enveloped virion between the inner and outer nuclear membranes labeled with ConA but not with WGA. During the transit to the extracellular space the reactivity of the virion membranes with ConA decreased and that with WGA ensued. The results document that herpes simplex viruses acquire at the inner nuclear membrane an envelope carrying the immature forms of the glycoproteins and that during the transit to the extracellular space the envelope glycoproteins become of the fully processed type.

Animals↗

PubMed1992

Dynamics of transmembrane proteins during Sindbis virus budding.

Label-fracture and immunogold fracture-flip techniques are used to address at the ultrastructural level the dynamics of viral and cellular transmembrane proteins during the budding of Sindbis virus on the plasma membrane of infected cells. Immunolabeling with anti-Sindbis spike antibodies shows that the viral proteins are mostly in clusters, all associated with budding viruses. Ultrastructural observation of the unlabeled freeze-fractured plasma membranes shows that membrane particles aggregate over the budding viruses. These results indicate that the concentration of viral transmembrane proteins gives rise to a parallel concentration of membrane particles. Immunolabeling with anti-CD8 antibodies of cells expressing by transfection the CD8 transmembrane protein and infected with Sindbis virus shows absence of labeling on the particle aggregates over the forming virions. These findings indicate the exclusion of CD8 proteins from the portions of the membrane where budding occurs.

Animals↗

PubMed1991

Size and stability of dipalmitoylphosphatidylcholine/cholesterol unilamellar vesicles are affected by interaction with proteins.

The effect of entrapping the enzyme ascorbate oxidase into dipalmitoylphosphatidylcholine/cholesterol vesicles, was studied by conventional transmission electron microscopy and freeze-fracture. The freeze-fracture technique has definitely demonstrated the unilamellar nature of empty and enzyme-loaded vesicles. Images of freeze-fractured and label-fractured liposomes also indicate that the observed reduction of vesicles volume could be related to the localization of ascorbate oxidase across the membrane. The membrane localization of ascorbate oxidase may explain the oxidation of externally added ascorbate by intact enzyme-loaded liposomes. Finally, the ageing of liposomes appears to be accelerated in the presence of proteins.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1991

Partition of epidermal growth factor receptors on freeze-fractured plasma membranes of A431 cells is affected by the ligand.

The fracture immunolabel technique, which permits assessment of the partition of transmembrane proteins with the inner or outer leaflets of the freeze-fractured membrane, was used to analyze the behavior on fracture of epidermal growth factor (EGF) receptors over the plasma membranes of A431 cells. The receptors partition mainly with the outer leaflet of the freeze-fractured plasma membranes, whereas they become associated with the inner leaflet when they are occupied by the ligand. This modified partition is even more evident after receptor clustering induced by incubation with EGF at 37 degrees C. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases the number of receptors over both inner and outer leaflets. An effect similar to that induced by the ligand is obtained when receptor aggregation is achieved using anti-receptor monoclonal antibodies (MAb). The modified partition therefore indicates receptor activation and appears to be a consequence of receptor cross-linking rather than to reflect a conformational change of the receptor molecule. Parallel immunolabeling with anti-phosphotyrosine antibodies of freeze-fractured EGF-treated A431 cells reveals that the receptors, when activated, are associated only with the inner leaflet of the plasma membrane.

Cell Line↗

PubMed1990

Anchorage-dependent surface distribution and partition during freeze-fracture of viral transmembrane glycoproteins.

We have compared in the same cell type the surface distribution and partition in freeze-fractured plasma membranes of Sindbis virus glycoproteins in three different situations: (i) in permanently transformed cells that express the glycoproteins as the only viral product; (ii) in cells in which prebound viruses were forced to fuse with the plasma membrane by low pH treatment; (iii) in virus-infected cells. We report here that the viral proteins expressed on the surface of transfected cells show a uniform and unclustered distribution; conversely, in Sindbis virus-infected cells they appear clustered, regionally distributed, and always associated with budding viruses (i.e., interacting with the nucleocapsid on the cytosolic side of the membrane). Furthermore, the viral proteins expressed on transfected cells or implanted by low pH-mediated fusion partition during freeze-fracture with the exoplasmic faces of the cell plasma membranes, whereas an opposite partition is observed in infected cells. These results strongly suggest that in infected cells the clustering and the partition with the protoplasmic faces of the plasma membrane depend only on the strong "anchorage" of the glycoproteins to the nucleocapsid.

Animals↗

PubMed1990

High-resolution surface views of human lymphocytes during capping of CD4 and HLA antigens as revealed by immunogold fracture-flip.

The surface ultrastructure of lymphocytes during capping of two transmembrane proteins is shown. As seen by fracture-flip the plasma membranes of human lymphocytes are covered by a high density of surface particles. Incubation in 30% glycerol leads to aggregation of these surface particles. Immunogold labelling shows that the transmembrane proteins bearing HLA class I and CD4 antigens are confined to the particle aggregates. These results indicate that surface particles revealed by fracture-flip represent surface protrusions of integral membrane proteins seen as intramembrane particles in freeze-fractured lymphocytes. During capping HLA or CD4 antigens aggregate into progressively larger patches and, finally, into single caps. As revealed by fracture-flip the patches/caps are seen as clearly differentiated raised platforms that are clearly and sharply demarcated relative to contiguous areas of the surface. In non-patched (non-capped) regions, the pattern of distribution and apparent density of surface particles remain unaltered. Immunogold labelling clearly demarcates patches and caps, and shows that virtually no antigen molecules remain dispersed over the non-patched (non-capped) regions. Estimates of the surface density of either HLA or CD4 antigens over the capped areas point to high planar concentrations of the transmembrane proteins that bear these antigens.

Antigens, Surface↗

PubMed1989

Molecular cytochemistry of CD3 and CD4 antigens in human lymphocytes as studied by label-fracture and by fracture-label.

Label-fracture and fracture-label membrane immunocytochemistry are used to analyze the surface distribution, dynamics and partition on fracture of CD3 and CD4 antigens of human T lymphocytes. Redistribution of the antigens, induced by treatment at 37 degrees C with specific monoclonal antibodies, results in patching and capping of the labeling as observed in label-fractured specimens. Examination of platinum/carbon replicas of freeze-fractured plasma membranes of antibody-treated cells does not reveal recognizable domains of intramembrane particles. However, in cells where the aggregation of intramembrane particles is induced by incubation with glycerol, colloidal gold-labeled CD3 and CD4 molecules are seen confined to particulate domains of the membrane. Therefore, the lack of visible aggregation of intramembrane particles in patched or capped regions of the membrane implies that migration of CD3 and CD4 antigens with concentration in domains of the membrane is achieved contemporaneously with export of other non-capped integral membrane proteins from the same regions, in a process of diffusional equilibrium. Examination of fracture-labeled specimens shows that CD4 molecules partition on fracture with the inner protoplasmic face of the plasma membrane. This partition illustrates the transmembrane attitude of the antigen molecule and is a probable consequence of interaction of the protein with other components of the membrane or with the cytoskeleton.

Antibodies, Monoclonal↗

PubMed1989

Localization of Epstein-Barr virus envelope glycoproteins on the inner nuclear membrane of virus-producing cells.

Epstein-Barr virus-producing cells were used as a model to analyze, with a fracture-immunolabel technique, the distribution, behavior on fracture, and extent of glycosylation of viral transmembrane glycoproteins at the inner nuclear membrane. Surface and fracture immunolabeling with two monoclonal antibodies directed against the carbohydrate or polypeptide portions of the major viral envelope glycoproteins gp350/220 showed the following. (i) The glycoproteins present on the inner and outer nuclear membranes were labeled only with the monoclonal antibody directed against the polypeptide chain, whereas over the surface of virus-producing cells and on mature virions the labeling was dense and uniformly distributed with both monoclonal antibodies. (ii) The glycoproteins were nonuniformly distributed only over the inner nuclear membranes; at the sites of viral budding, the glycoproteins showed a preferential partition with the protoplasmic face. Since fully glycosylated glycoproteins were not present on the nuclear membranes, our observations support the proposed model of herpesvirus maturation. The peculiar distribution and partition on fracture of the envelope glycoproteins on the inner nuclear membrane are similar to those of Sindbis virus envelope glycoproteins on the plasma membrane of infected cells. Therefore, our results suggest that inner nuclear membranes may behave like plasma membranes during viral assembly.

Antigens, Viral↗

PubMed1989

Capping of HLA antigens in human lymphocytes as followed by immunogold label-fracture.

We used immunogold label-fracture to follow the migration of HLA I class and HLA II class antigens during capping as induced by specific monoclonal antibodies. Capping is achieved through a process of clustering and "consolidation" of clusters into larger patches and, finally, a single cap. All receptors appear to cluster from the very start, with no "stray" molecules joining already formed patches. Characterization of exoplasmic and protoplasmic fracture-faces of capping cells fails to reveal any corresponding accumulation of intramembrane particles and/or subtler rugosities. Our results are consistent with the concepts that view the migration of capping molecules as contemporaneous with the efflux of noncapping integral membrane proteins.

Antibodies, Monoclonal↗

PubMed1989

Genetic regulation of macrophage production in response to surface components of Listeria monocytogenes.

Resistance to murine listeriosis requires humoral factors which alter production or delivery of monocytes to infective foci. Production of such factors is under genetic control and may be modulated by bacterial components. Two components from Listeria monocytogenes, monocytosis producing activity (MPA) and immunosuppressive activity (ISA) were used to study monocytopoiesis in mice with known abnormalities in microphage-related functions. Listeria-sensitive A/J mice fail to produce or respond to MPA or an endogenous mediator of monocytosis (EF). These studies provided evidence that a second humoral factor, decreases monocytopoiesis. Sera from A/J mice are more active in this respect and MPA treatment increases the amount of inhibitory factor in A/J mice. A polyclonal B cell activator, ISA, which induces suppressor splenic macrophages, suppresses the anti-SRBC response in resistant B10. A mice but not in A/J mice. ISA induced no change in prostaglandin E2 production by spleen cells of either strain. One interpretation of these findings is that A/J mice after stimulation by ISA or MPA, produce a substance which inhibits development of mononuclear phagocytes.

Animals↗

PubMed1988

Nonrandom distribution of epidermal growth factor receptors on the plasma membrane of human A431 cells.

The localization of epidermal growth factor (EGF) receptors over the plasma membranes of human epidermoid carcinoma A431 cells was analyzed at the electron microscopic level using surface replica techniques and conventional thin sections, in combination with immunocytochemistry. Immunolabeling was performed using two distinct monoclonal antibodies directed against the extracellular portion of the receptor, followed by protein A-colloidal gold conjugates. Unexpectedly, with the first monoclonal antibody used, the distribution of the receptors in both unfixed and glutaraldehyde-fixed cells was clearly regionalized, showing a preferential localization of the immunolabeling at the cell periphery as well as over the areas rich in microvilli and in coated and uncoated pits. A similar pattern of distribution was observed also with the other monoclonal antibody, but only when the cells were fixed with glutaraldehyde before immunolabeling. Treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate modifies this distribution, inducing a more disperse pattern. Our observations suggest that a minor group of EGF receptors, which may represent the high-affinity receptors, presents a regional distribution, similar to that described for typical recycling receptors.

Carcinoma, Squamous Cell↗

PubMed1988

Wheat germ agglutinin fracture-label of Golgi apparatus membranes in proliferating cells.

The thin section fracture-label technique has been recently used to analyze the distribution and compartmentalization of fully glycosylated components on intracellular membranes. Labelling with the lectin wheat germ agglutinin over the freeze-fractured membranes of Golgi apparatus in various secretory and non-secretory cells as well as in human peripheral lymphocytes was always very weak or absent even over the trans-most cisternae. In order to investigate if the labelling density may reflect the cellular activity in membrane biogenesis, we used in this study wheat germ agglutinin fracture-label of rapidly proliferating cells and mitogen-activated lymphocytes. Labelling over the fractured cisternae of the medial and trans portions of the Golgi apparatus was intense. Treatment with cycloheximide of proliferating cells induced a drastic reduction of the labelling over the Golgi cisternae.

Cell Division↗

PubMed1987

Regional distribution of Sindbis virus glycoproteins on the plasma membrane of infected baby hamster kidney cells.

Sindbis virus-infected baby hamster kidney (BHK) cells were analysed in surface replicas or conventional thin sections after specific immunolabelling with antiviral glycoprotein antibodies in conjunction with colloidal gold-conjugated protein A. Newly synthesized viral glycoproteins were detected, beginning 1 1/2 h after infection, while the virus maturation started 3 h after infection. The glycoproteins appeared to be inserted on the plasma membrane in large spots located mainly in the central area of the cells: no clustering of the labelling was detected. Later, the glycoproteins appeared to arrange linearly in regions in the medial portion of the cells. No labelling was found in the peripheral area or on the cell edges. A drastic change in the surface labelling was detected following the commencement of virus maturation: gold particles were organized mostly in small clusters, each labelling a budding virus. Very few glycoproteins appeared not to be involved in budding figures. The maturation of the virus was clearly regionalized, but during this time it also involved the peripheral area and the cell edges; preferential budding in narrow cellular processes was often observed. It appeared thus that either isolated glycoproteins soon after infection, or clustered glycoproteins at later times, are strictly regionalized on the plasma membrane: however, the early post-infection distribution is clearly different from that seen later during virus maturation. Our experiments support the concept of discrete plasma membrane domains even in cells that do not display distinct specialization of their surface.

Animals↗