Biomedical subjects

A Pavan

Publications and source records attributed to A Pavan.

At least 19 recordsLinked to original sources

PubMed1997

Evidence for the existence of ganglioside-enriched plasma membrane domains in human peripheral lymphocytes.

In human peripheral blood lymphocytes (PBL) monosialoganglioside GM3 appears to be the major ganglioside on the cell plasma membrane. We have analyzed the expression and distribution pattern of GM3 molecules on the lymphocyte plasma membrane by flow cytometry, immunofluorescence, and immunoelectron microscopy, using an anti-GM3 monoclonal antibody. Both CD4+ and CD8+ T lymphocyte subpopulations showed substantial GM3 expression, as determined by thin-layer chromatography and flow cytometric analysis. A clustered distribution of GM3 molecules on the cell surface, revealed by immunofluorescence and immunogold electron microscopy, clearly indicated the presence of GM3 molecule-enriched plasma membrane domains. To better define these domains, we analyzed the ganglioside and protein composition of buoyant low-density Triton-insoluble (LDTI) lymphocyte fractions. The results show that GM3 is enriched approximately 20-fold in LDTI fraction, as compared with total cell lysates. In addition, CD4 and lck molecules are selectively recovered in the same LDTI fraction isolated from human PBL. These findings, together with the observation that anti-CD4 co-immunoprecipitated GM3, support the hypothesis of a possible GM3-CD4 interaction and suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in T-cell activation, antigen recognition, and other dynamic lymphocytic plasma membrane functions.

CD4-Positive T-Lymphocytes↗

PubMed1997

Ultrastructural and phenotypic characterization of CABA I, a new human ovarian cancer cell line.

We have established an ovarian cancer cell line (CABA I) from ascitic fluid obtained from a patient with papillary adenocarcinoma of the ovary prior to drug treatment. The epithelial origin of the cell line was confirmed by morphology and by immunofluorescence analysis using anticytokeratin antibodies. Ultrastructural analysis revealed a very irregular membrane surface and a clear cytoplasm rich in electron-lucent vesicles. CABA I cells grow rapidly in culture (doubling time 18 h) in an anchorage-independent manner. Exogenously added beta-estradiol and epidermal growth factor (EGF) treatments did not influence cell growth rate. FACS analysis to determine the phenotypic profile of tumor-associated antigen, membrane receptor, and adhesion molecule expression indicated that the cell line was positive for different members of the c-erbB family, for alpha 6 and beta 1 integrin receptors, and intensively positive for HLA class I antigens and the folate receptor. Molecular characterization revealed no mutations for c-myc and c-k-ras genes, but did detect an exon 5 mutation in the p53 gene. CABA I cells grew poorly as heterotransplants in nude mice, and tumors showed long latency periods. Because early (15-20) and late (55-60) passage cells maintain the same growth and phenotypic characteristics, the CABA I cell line might provide a good in vitro model system to investigate the cellular and molecular events involved in ovarian carcinogenesis.

Animals↗

PubMed1996

Overexpression of monosialoganglioside GM3 on lymphocyte plasma membrane in patients with HIV infection.

SUMMARY: This study was undertaken to analyze both the GM3 expression on peripheral blood lymphocytes of HIV-infected patients and the relationship between ganglioside content and anti-GM3 reactivity. GM3 expression was determined as a percentage of lipid-bound sialic acid and by cytofluorimetric analysis in 25 AIDS patients, 20 anti-HIV+ asymptomatic subjects, 25 patients with different viral disease, and 25 healthy donors. GM3 distribution was analyzed by immunofluorescence and immunoelectron microscopy. A follow-up study to detect anti-lymphocytic GM3 antibodies was performed in progressive and nonprogressive anti-HIV+ subjects. Lymphocytes from HIV-infected patients showed a significant increase of plasma membrane GM3 content; no difference was found between CD4+ and CD8+ cells. Immunofluorescence and immunoelectron microscopic analysis showed that GM3 was distributed in large clusters over the cell plasma membrane. The follow-up study revealed that the occurrence of anti-lymphocytic GM3 antibodies was significantly higher in patients with progressive disease, compared with asymptomatic non-progressive subjects. These findings revealed that (1) the increased GM3 content in HIV-infected patients is detected at the plasma membrane level, (2) GM3 overexpression is able to induce an increased reactivity with anti-GM3 antibodies, and (3) the appearance of anti-lymphocytic GM3 antibodies in asymptomatic anti-HIV+ subjects could have prognostic relevance for the risk of developing AIDS.

Antibodies, Monoclonal↗

PubMed1996

Intravenous propafenone in paroxysmal atrial fibrillation: a randomized, placebo-controlled, double-blind, multicenter clinical trial. Paroxysmal Atrial Fibrillation Italian Trial (PAFIT)-2 Investigators.

BACKGROUND: Pharmacological conversion of paroxysmal atrial fibrillation is frequently necessary. The aim of this study was to compare intravenous propafenone, a class Ic antiarrhythmic agent, with placebo in paroxysmal atrial fibrillation (AF) of recent onset (< 72 h). PATIENTS AND METHODS: We randomly allocated 75 patients, aged 18 to 70 years, with paroxysmal AF to receive intravenous propafenone (2 mg/kg in 15 min followed by 1 mg/kg in 2 h) or the matching placebo. Patients were followed for 3 h. Exclusion criteria were the presence of one of the following: clinical heart failure, recent acute myocardial infarction, hypotension, atrioventricular block, Wolff-Parkinson-White syndrome, or current treatment with antiarrhythmic agents or digitalis. RESULTS: No sign of heart disease was found in 74.7% of the patients. Echocardiographically determined left atrium diameter was similar in the two groups. Conversion to sinus rhythm occurred in 24 of 41 patients allocated to propafenone and in 10 of 34 patients allocated to placebo (odds ratio 3.2, 95% confidence intervals 1.3-7.9; p < 0.01). Mean conversion time was 34 +/- 29 and 71 +/- 55 min, respectively, for propafenone and placebo. Mean heart rate in nonconverters decreased from 146 to 109 beats/min in patients treated with propafenone while it remained virtually unchanged in those treated with placebo. Only minor side effects were noted. CONCLUSIONS: Intravenous propafenone is an effective therapeutic option for restoring sinus rhythm in patients with paroxysmal AF of recent onset.

Adolescent↗

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↗