Search PubMed⌕ Search

Biomedical subjects

P Vassalli

Publications and source records attributed to P Vassalli.

At least 127 records · Page 7Linked to original sources
PubMed1981

Topology of morphologically detectable protein and cholesterol in membranes of polypeptide-secreting cells.

The freeze-fracture morphology of intracellular and plasma membranes in endocrine and exocrine polypeptide-secreting cells has been studied to detect changes while these membranes interact during secretion. A qualitative and quantitative evaluation of intramembrane particles and filipin binding as indicators of protein and cholesterol content of the membranes, respectively, reveals the following changes. From the forming of the maturing pole of the Golgi complex, membranes lose morphologically detectable protein and gain morphologically detectable cholesterol. The protein-poor, cholesterol-rich secretory granule membrane then interacts with a richly particulate plasma membrane in endocrine cells and with a moderately particulate luminal membrane in exocrine cells. The site of interaction between secretory granule and plasma membrane is characterized by a local clearing of intramembrane particles; by contrast, filipin-binding sites revealing cholesterol are present in this area. In exocrine cells, the fused secretory granule, which is initially rich in filipin-cholesterol complexes and poor in particles, appears to lose progressively its filipin labelling to resemble the poorly labelled luminal membrane. These findings, although they cannot be interpreted definitely at present, clearly show impressive changes of membrane structure along the secretory pathway and suggest that a corresponding degree of functional specialization is needed for proper interaction to occur.

Animals↗
PubMed1981

Post-thymic T lymphocyte maturation during ontogenesis.

Peripheral T lymphocytes from newborn (4-6-d-old) mice, isolated from the spleen or lymph nodes, show phenotypic features of immature cortical thymocytes, such as high frequencies of proliferating cells and of peanut lectin-binding cells. These are features of peripheral T cells of recent thymic origin, as shown by in situ labeling of thymocytes and subsequent observation of the migrants to the spleen, which were mainly peanut lectin-binding cells. The function of newborn peripheral T cells was compared, on a per T cell basis, with that of thymocytes and of fully mature peripheral T cells of the adult, using preparations of newborn lymph node cells containing approximately 80% of T lymphocytes. They were strikingly (about 10-fold) less competent than adult T cells in their phytohemagglutinin responsiveness, their capacities to induce a graft vs. host reaction, to proliferate in the mixed lymphocyte reaction, and to help B lymphocytes in a humoral response in vivo and in vitro. In contrast, newborn T lymphocytes were comparable to those of adults in their capacity to generate cytotoxic T lymphocytes. No suppressive effect of newborn T lymphocytes could be demonstrated in several of these assays. These results argue for an asynchronous maturation of two T cell subsets during ontogeny and demonstrate that at least some T lymphocytes leave the thymus as immature T cells resembling cortical thymocytes and further mature at the periphery. Investigation of mice submitted to thymectomy of 5 d of age showed that these incompetent post-thymic T lymphocytes are capable of considerable expansion and maturation in the peripheral lymphoid organs in the absence of a thymic influence.

Animals↗
PubMed1981

Immunosuppressor cells from newborn mouse spleen are macrophages differentiating in vitro from monoblastic precursors.

Newborn mouse spleen, whose cells strongly suppress the in vitro humoral response of adult spleen cells, is essentially a hematopoietic organ. It contains a large percentage of proliferating cells, among which about 50% are erythroblasts (identified by their spectrin content) and about 15% are cells of the myelocytic and monocytic lineage. Lymphoid cells are a minority, with about 20% B and only 1-2% T lymphocytes. After a 4 days, a culture of newborn spleen cells contains 5-10 times more macrophages than that of an adult spleen. Most of these macrophage precursors from the newborn spleen are proliferating cells, partially glass- or plastic-adherent, which differentiate in culture into activated macrophages producing large amounts of plasminogen activator. It is this macrophage excess which is responsible for the immunosuppressive effect of newborn spleen cells in culture, as indicated by (a) the effect of silica particles added to the cultures, which both relieve the suppression and prevent the accumulation of macrophages and (b) the suppression of the humoral response of adult spleen cells when they are cultured on the adherent cells from a newborn but not from an adult spleen. The suppressive effect of macrophages seems to result, at least in part, from the production of prostaglandin, since it can be relieved by indomethacin or aspirin. Suppression is not related to arginine depletion of the medium or to production of an excess of plasminogen activator. T lymphocytes from newborn spleen or lymph nodes have no suppressive capability.

Aging↗
PubMed1981

Heterogeneous distribution of filipin--cholesterol complexes across the cisternae of the Golgi apparatus.

The Golgi apparatus is a key element in the ordered movement of secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane during secretion. It has been shown that cisternae that receive membranes from the reticulum are morphologically similar to the latter and that cisternae liberating secretory granules resemble that plasma membrane. By using an ultrastructural probe for membrane cholesterol, filipin, on freeze-fractured and thin-sectioned exocrine and endocrine pancreatic cells, we have shown that an enrichment in filipin-cholesterol complexes takes places across the stacked cisternae of the Golgi apparatus; the reticulum-related (forming) cisternae are poor in such complexes, but the secretory granule-related (maturing) cisternae contain numerous complexes. Secretory granule membrane is also richly labeled with filipin-cholesterol complexes. The heterogeneous cholesterol distribution in the membranes of the Golgi apparatus, as shown by filipin, emphasizes the polarity of this organelle, in agreement with its role in organizing the traffic of the secretory polypeptides from the rough endoplasmic reticulum to the plasma membrane.

Animals↗
PubMed1981

Structural heterogeneity of endocytic membranes in macrophages as revealed by the cholesterol probe, filipin.

The polyene antibiotic, filipin, by specifically interacting with cholesterol, produces approximately 25-nm protuberances (filipin-sterol complexes) in freeze-fractured membranes, and the addition of filipin to aldehyde fixatives has been recently introduced as a cytochemical technique for the localization of cholesterol in cell membranes. In a previous study we showed that, in fibroblasts filipin-sterol complexes are absent from endocytic coated pits. To establish whether the absence of filipin-sterol complexes is a phenomenon restricted to coated pits or is correlated with endocytosis in general, we applied the filipin probe to cultured mouse peritoneal macrophages, in which different forms of endocytosis take place. The macrophages were incubated with bovine albumin or concanavalin A (Con A) to induce pinocytosis, and with heat-killed straphylococci or opsonized erythrocytes to induce phagocytosis, then fixed in glutaraldehyde/filipin and freeze-fractured. Filipin-sterol complexes were plentiful on the plasma membrane, on the smooth-membrane invaginations and vesicles induced by albumin, on the large endocytic vacuoles induced by Con A, and on the membrane of phagosomes but, in contrast, they were absent from coated pits and vesicles, as well as from coated segments of invagination or vesicles. These results indicate that the membranes involved in different types of endocytosis do not react in the same way with filipin and may, therefore, have a different cholesterol content. This could reflect different mechanisms of formation for the various types of endocytic vesicles.

Animals↗
PubMed1981

Plasma cell endocytosis: is it related to immunoglobulin secretion?

Mouse plasma cells have been exposed to a wide range of soluble and adsorptive macromolecular tracers for 10 min to 4 h to explore the possibility of membrane recycling related to the high secretory rate of these nonregulated secretory cells. Electron microscopic examination showed in all cases labeling of multivesicular and multilamellar bodies and a lesser labeling of smooth-surfaced vesicles. Using cationized ferritin as tracer, an additional very restricted labeling of Golgi cisternae was observed. Comparable labeling patterns were observed when immunoglobulin (Ig) secretion was blocked with the Golgi-specific pertrurbant, monensin, and in the case of poorly differentiated B immunoblasts which secrete little or no Ig. Our observations therefore emphasize that available approaches cannot yet determine whether a mandatory circuit of vesicular traffic couples Ig exocytosis to endocytosis.

Animals↗
PubMed1981

Collaboration between histoincompatible T and B lymphocytes: preferential induction in vivo of antibodies to antigens borne by foreign T cells.

We have compared, in vivo, with nude mice and, in vitro, with cultures of T depleted spleen cells, several components of the humoral response resulting from the helper effect of allo- or xenogeneic T cells on mouse B lymphocytes; the response directed to the histocompatibility antigens (or other surface antigens) borne by foreign T lymphocytes, the effect on the response to an additional unrelated antigen, namely sheep red blood cells, and finally a possible nonspecific polyclonal activation. In vivo, injection of allo- or xenogeneic T lymphocytes into nude mice (with or without SRBC) induced a strong anti-T cell response, manifested by the presence of high titered IgG alloantibodies, without significant enhancement of the response to SRBC. In vitro, addition of xenogeneic T lymphocytes to cultures of T-depleted spleen cells did enhance an anti-T cell and an anti-SRBC response. The preferential induction of an anti-T cell response, observed in vivo, implies a peculiar form of T-B cell collaboration where the B lymphocytes binding to the foreign and activated T cells are selectively activated, suggesting that there is no absolute restriction for specific T-B cell collaboration across the histocompatibility barrier.

Animals↗
PubMed1980

Biosynthesis of two forms of IgM heavy chains by normal mouse B lymphocytes. Membrane and secretory IgM.

A study of the biosynthesis of IgM by purified mouse spleen lymphocytes showed that these cells synthesize both 8 S membrane IgM and 19S secretory IgM, which is identical with plasma cell IgM except in its kinetics of processing, assembly, and secretion. The heavy (mu) chains of these two types of lymphocyte IgM differ in their ultimate fate, in processing, isoelectric point, and peptide composition. The separate precursors of the two mu chains have very similar mobilities in sodium dodecyl sulfate polyacrylamide gel electrophoresis, but they can be distinguished by the use of endoglucosaminidase H (endo-H) to remove core sugars, by two-dimensional electrophoresis, and by one-dimensional gel analysis in pulse-chase experiments. CNBr peptide patterns of intracellular "secretory" mu chains of lymphocytes and plasma cells were similar, but membrane mu chains had a COOH-terminal peptide different in structure from that of secreted mu chains, with a higher apparent molecular weight.

Animals↗
PubMed1980

Opposite polarity of filipin-induced deformations in the membrane of condensing vacuoles and zymogen granules.

Filipin binding to membrane sterols induces deformations of the membrane that are detected by freeze-fracture either as 20- to 25-nanometer protuberances or as pits on the fracture faces. By using the filipin probe in pancreatic acinar cells, it was found that the polarity of filipin-induced deformations in the membrane limiting the Golgi condensing vacuoles is opposite that in the membrane limiting the mature zymogen granules. This asymmetry could be due to unequal partitioning of cholesterol between the membrane leaflets in these two compartments during the transformation of the condensing vacuole into the zymogen granule.

Cholesterol↗
PubMed1980

Failure of allogeneic thymocytes to survive in nude mice.

Athymic nude mice or thymectomized mice, irradiated and reconstituted with T-depleted bone marrow cells ("B mice"), were injected with allogeneic or syngeneic thymocytes bearing caryotypically distinct chromosomes. The fate of the thymocytes was investigated after various periods of time using two methods: (a) frequency of the cells bearing the Thy-1 antigen as detected by immunofluorescence with a heteroantiserum among the recipient spleen cells, (b) presence of chromosomally detectable donor cells in phytohemagglutinin-stimulated spleen cell cultures. These two methods indicate that allogeneic thymocytes disappear after about 7 days, semiallogeneic thymocytes after about 20 days, while syngeneic thymocytes, even when injected in 50 times lower number (10(6) cells), are detectable for months, thanks to the sensitivity of the caryotypic method. Allogeneic thymocytes induce the production of high titers of alloantibodies, which were shown to react specifically against their own H-2, a phenomenon interpreted as a "suicidal" allogeneic collaboration. These experiments demonstrate that the failure of allogeneic thymocytes, in contrast to syngeneic thymocytes, to achieve long-term restoration of the immune responsiveness of T-depleted mice is due to a rejection of the foreign thymocytes, and not to a failure of T and B cells to collaborate across the histocompatibility barrier.

Animals↗
PubMed1980

Characterization of mouse thymocyte and peripheral lymphocyte xenoantigens by two-dimensional electrophoresis.

Two-dimensional electrophoresis was used to analyze the surface glycoproteins of murine thymocytes and lymph node cells. Two-dimensional maps of unselected, radioiodinated lymphocyte surface proteins were complex, showing at least 20 different components, but simpler patterns were obtained by using rabbit antibodies directed against the surface proteins of a T lymphoma cell line, to precipitate xenoantigens from lysates of radioiodinated or biosynthetically labeled thymocytes and lymph node cells. These xenoantibodies precipitated 12-13 distinct components from each cell type, of which all but 3 were sialoglycoproteins. Two types of difference between the surface glycoproteins of thymocytes and peripheral lymphocytes could be detected. First, higher mol. wt. glycoproteins and Thy-1 are more acidic in peripheral lymphocytes than in thymocytes, and this difference disappears after neuraminidase treatment. One additional high mol. wt. glycoprotein is also detectable in peripheral lymphocytes, probably reflecting the greater carbohydrate complexity of these molecules, when expressed on such cells. Second, 3 glycoproteins are strongly labeled only on thymocytes, and 3 others only on peripheral lymphocytes. These 6 glycoproteins might represent genuine differentiation antigens.

Animals↗
PubMed1980

Distribution of filipin-cholesterol complexes at sites of exocytosis - a freeze-fracture study of degranulating mast cells.

The distribution of membrane cholesterol at sites of exocytosis was studied with the cytochemical probe, filipin, in freeze-fractured rat peritoneal mast cells. When degranulation was induced in mast cells by polymyxin B, the membrane of all secretory granules, as well as the plasma membrane, including bulged areas signaling incipient exocytosis, appeared heavily labeled with filipin-sterol complexes (recognizable as 25-nm protuberances). This is evidence that cholesterol depletion is not an absolute requirement for membrane fusion.

Animals↗
PubMed1980

High molecular weight surface glycoproteins of murine lymphocytes.

High m.w. glycoproteins in the range of 170 and 210K represent major cell surface components of murine lymphocytes. The expression of such glycoproteins has been characterized for thymocytes and peripheral T and B lymphocytes in BALB/c mice using surface and biosynthetic labeling, nonionic detergent extraction, and immunoprecipitation with antibodies directed against antigenic determinants expressed on the cell surface of a murine T cell lymphoma. Immunoprecipitates were then resolved by 1-dimensional and 2-dimensional gel electrophoresis. Two groups of antigenically distinct glycoproteins have been defined: 1) a 170K component present on thymocytes and peripheral T lymphocytes but undetectable on B lymphocytes; this glycoprotein has been termed T 170 to suggest its potential use for distinguishing T from B lymphocytes; 2) a higher m.w. glycoprotein bearing antigenic determinants common to T and B cells but expressed as one 180K glycoprotein on thymocytes, 2 glycoproteins of 180 and 190K on peripheral T lymphocytes, and as one 210K glycoprotein on B lymphocytes. The relationships of these glycoproteins with the other known lymphocyte surface markers of high m.w. is discussed.

Animals↗
PubMed1979

Evidence for hydrophobic region within heavy chains of mouse B lymphocyte membrane-bound IgM.

The gel filtration behavior, in the presence of detergents, of membrane-bound IgM from normal mouse spleen B lymphocytes was compared to that of secretory IgM from mouse plasma cells. The proteins were labeled either by surface radioiodination or biosynthetically with radioactive amino acids. Cell lysates were fractionated on calibrated Sepharose 6B columns in the presence of the detergents Nonidet P-40 or deoxycholate. Eluted fractions were immunoprecipitated and the reduced or unreduced precipitates were analyzed by sodium dodecyl sulfate gel electrophoresis followed by radioautography. Surface (125)I-labeled 8S IgM exhibited a gel filtration pattern in Nonidet P-40 corresponding to much higher apparent molecular weight than that of secretory 8S IgM, a difference that almost disappeared when gel filtration was performed in the presence of deoxycholate, which forms much smaller micelles than does Nonidet P-40. Biosynthetically labeled lymphocytes contain two types of IgM molecules differing in their gel filtration behavior and fate: one identical to secretory 8S IgM of plasma cells and secreted in the medium during chase periods, and the other identical to surface (125)I-labeled IgM and remaining cell-associated. Because the surface-bound 8S IgM was not found to be associated with other labeled molecules, it is likely that the detergent-binding behavior of surface IgM is due to a hydrophobic segment carried by these Ig molecules. That lymphocytes synthesize two types of mu chains was also shown by the use of tunicamycin, an inhibitor of glycosylation. In its presence, two unglycosylated mu chains were observed: one identical in size to that made by tunicamycin-treated plasma cells, and the second slightly larger. Gel filtration in Nonidet P-40 of the cell lysates of tunicamycin-treated lymphocytes showed that the nonsecretory 8S IgM contains this second type of mu chains, whereas the IgM molecules of the secretory type contain plasma cell-like mu chains. It is suggested that membrane IgM mu chains contain a hydrophobic segment which is responsible for its association to the membrane.

Animals↗
PubMed1979

Absence of filipin-sterol complexes from large coated pits on the surface of culture cells.

Monolayer cultures of normal or transformed fibroblasts and of liver cells fixed in a glutaraldehyde solution containing 300 microM filipin, a sterol-specific polyene antibiotic, were freeze-fractured to study the distribution of cholesterol within their plasma membranes. Filipin-sterol complexes, recognizable as 25- to 30-nm protuberances scattered in the fracture face of plasma membrane, were absent from invaginations corresponding to large, bristle-coated pits (and possibly also from small, flask-shaped invaginations). These results suggest that invaginating regions on the cell surface are specialized plasma membrane domains with a lower cholesterol content than the surrounding membrane. The localized change in membrane fluidity due to the low cholesterol concentration could play a role in endocytosis.

Animals↗