Development of the beta cells.
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Biomedical subjects
Publications and source records attributed to A Anteunis.
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Transferrin (Tf) and vasoactive intestinal peptide (VIP) were labeled with horseradish peroxidase (HRP) and 125I, respectively. To determine whether two simultaneously incubated ligands are conveyed by the same population of endosomal vesicles in human colon carcinoma cells (HT-29), we used an analysis system derived from the cross-fire method for quantitation of autoradiographic data. This system permitted the collection of data and the statistical calculations required by the double labeling of the cells. HRP-labeled Tf organelles were chosen as reference structures of the endosomal apparatus and taken as the conventional source of the radiolabeling. Our data established from the co-localization hypothesis strongly suggest that after a 30-sec (T 1/2) and a 10-min (T10) internalization at 37 degrees C, VIP and Tf share in major part the same endocytic pathway and even the recycling route to the cell surface. At T10, most of the radiolabeling was located inside the tubulovesicular network, and we also detected slight radiolabeling inside the vesicles recycling Tf. The number of double-labeled endosomes involved in ligand traffic were advantageously observed with our computer-assisted analysis system.
We perfused the pancreas with 125I-labeled vasoactive intestinal peptide (VIP) to follow the concomitant distribution of radioactivity in beta- and acinar cells as a function of time. This distribution was quantitated by computer-assisted analysis of high-resolution video autoradiographs. Density labeling was expressed as normalized specific activity (disintegration density per volume density). Immediately after a 4-min perfusion of 125I-VIP, labeling in beta-cells was mainly concentrated on the cell surface and peripheral tubules and vesicles. After three 30-s pulses of 125I-VIP, separated by intervals of 3.5 min of buffer perfusion, lysosome-like structures were heavily labeled. When VIP internalization was prolonged, labeling was similar to that observed with the 4-min perfusion, indicating a high VIP disposal rate in the lysosome-like structures. In acinar cells, labeling persisted on the surface and the early vacuolar system. We conclude the following: 1) an active endocytotic system, linked to the transport and sorting of a neuromediator, is present in beta-cells; and 2) the differences between the distribution of labeling in acinar and beta-cells suggest that the regulation of VIP internalization is tissue specific.
Desensitization of human carcinoma colonic cells in culture (HT-29) to vasoactive intestinal peptide (VIP) has been reported previously (C. Boissard, J. C. Marie, G. Hejblum, C. Gespach, and G. Rosselin, Cancer Res., 46: 4406-4413, 1986). In the present study, we have determined the ultrastructural localization of VIP and its receptor after exposure of HT-29 cells to VIP monoiodinated on tyrosyl residue 10 together with the molecular forms and the activity of the internalized VIP receptor. Quantitative electron microscope autoradiography showed that after binding at the cell surface, VIP is internalized in heterogeneous endosomes. Cross-linking experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis were performed in different experimental conditions allowing us to selectively obtain cell surface-associated, internalized, or recycled receptors. No detectable alteration of the labeled VIP-receptor complex occurred during the internalization and recycling processes. Furthermore, a loss of the forskolin potentiation of the VIP-induced stimulation of adenylate cyclase was observed after VIP exposure. This feature was time and temperature dependent as was the VIP-induced loss of cell surface receptors, indicating that the internalized VIP receptor is dissociated from the adenylate cyclase.
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The cytotoxic reaction of normal peritoneal mouse cells, containing less than 3% eosinophils, against newborn Trichinella spiralis larvae, in the presence of antibody, was studied using newborn worms less than 2 h of age, or newborn worms that had been maintained in culture at 37 degrees C for 20 h. Newborn worms 2 h old were killed, whereas 20 h newborn worms were not. The cells that initially adhered to the larvae were examined by electron microscopy. Only eosinophils adhered to 2 h newborn worms and only macrophages to 20 h ones. The attached eosinophils degranulated and died after a few hours. The macrophages that adhered to, but did not kill the 20 h newborn worms were morphologically in good state and no lysis of the larvae was observed. These results suggest that different antibody classes are involved in eosinophil and macrophage adherence, and strongly support the hypothesis that eosinophils mediate larval destruction. They also show that rapid changes are taking place after birth in the structure of the larval cuticle and that the age of Trichinella newborn worms is a major factor in the antibody-dependent cellular cytotoxicity reaction.
In resting lymphocytes, three well defined networks are observed and attempts were made to superimpose them upon the networks described in isolated nuclear matrices. These three nuclear structures are seen after DNase and RNase treatment. They are digested by pepsin but their sensitivity to this enzyme is different. The more resistant network corresponds to the outer lamina of the isolated nuclear matrix. The second network is located in the inter-chromatin area. It is more sensitive to pepsin than the lamina and this sensitivity is increased ten-fold when digestion with pepsin is preceded by RNase digestion. This network corresponds to the internal network of isolated nuclear matrices. The third network is located in the intrachromatin area and is the most sensitive to pepsin action.
Using quantitative electron microscopic autoradiography we demonstrated the existence of specific VIP receptors in B cells of the pancreatic islets. The presence of those receptors strongly suggests that the neuromediator can induce insulin release by direct stimulation of B cells.
The effect of the activity of macrophages on the antibody-dependent cytotoxicity against Trichinella spiralis newborn larvae was studied in vitro. Macrophages present in peritoneal exudates from mice genetically selected for high and low antibody production (HL and LL, respectively) showed an inverse cytotoxic effect. Cells from HL mice were ineffective, whereas cells from LL mice had a very high killing capacity. Ultrastructural studies of cells after incubations of up to 36 h supported these observations. Furthermore, peritoneal macrophages from congenitally athymic (nu/nu) mice showed a higher killing potential than cells from thymus-bearing littermates (+/nu) mice. The activity of the latter cells could be increased by in vitro pretreatment of the mice with Calmette-Guérin bacillus, a well-known macrophage stimulating agent. The results indicate that macrophages, although not the only effector cells, may play an important role in the defence against T. spiralis newborn larvae.
Cells from the axial organ of the starfish Asterias rubens were fractionated into two populations, adherent and non-adherent to nylon wool. In both populations the ultrastructural study revealed the presence of cells resembling the lymphocytes of the vertebrates, as well as phagocytic, peroxidase positive cells. The lymphocyte-like cells in the non-adherent population (average diameter 4 mu) have a high nucleo-cytoplasmatic ratio and are morphologically similar to Th lymphocytes while the adherent cells (average diameter 5.5 mu) are more similar to Bm lymphocytes. These observations are in line with the hypothesis that there exist, in the starfish, a primitive immune system with characteristics resembling those of the immune system of vertebrates.
The ultrastructural changes in the spatial organization of nucleolar DNA in lymphocytes during phytohemagglutinin (PHA) stimulation was studied in guinea pigs by means of oxidized diaminobenzidine (DAB) at low pH as a differentially contrasting stain for nucleic acids and by the use of reconstruction of serial sections. The extended DNA filaments situated inside the fibrillar area originate from a large aggregation of heterochromatin, which is closely associated with the nucleolus, and from the perinucleolar shell of condensed chromatin. It is suggested that these two distinct regions of chromatin might be associated with different functions.
Three-dimensional reconstructions show that the nucleoli from L 929 cells are associated with one or several large aggregates of chromatin displaying a honeycomb-like structure. The form and the number of both nucleoli and honeycomb structures vary as the cells emerge from the resting state and enter exponential growth. Quantitative data show that the number of honeycomb structures decreases as the number of nucleoli diminishes; both numerical regressions are significant. In addition, the nucleoli and the honeycomb structures enlarge when the cells enter the exponential growth phase. In resting cells the number of honeycomb structures is correlated to the number of nucleoli. Therefore we conclude that the large nucleolar mass of condensed chromatin, which in L 929 cells displays a honeycomb structure, contains a portion of the nucleolar organizing region.
The nucleolar lesions provoked by the action of ribonuclease (RNase) on living chick embryo fibroblasts were studied by means of microcinematographic analysis and at the ultrastructural level using oxidized diaminobenzidine as a differentially contrasting stain for nucleic acids. This study has shown that the induction of nucleolar dispersion by RNase was only the beginning of a series of discrete steps. The following sequences are described: dispersion of the nucleolus into fragments, their reassembly, and the emission of spherules which appear of chromatin origin. At that step nucleoli are typically segregated. The alteration of the nucleolar associated chromatin seemed to be primordial in these processes. Moreover, the large mass of heterochromatin intimately associated with the nucleolus and which has been considered to be a part of the nucleolar organizer region apparently plays a chief part in the reassembly of the nucleolar fragments into a segregated nucleolus. Ribonuclease is compared to other drugs known to act on nucleolar DNA.
A method using sucrose gradient centrifugation is described for the purification of plasma membranes of guinea pig peritoneal macrophages. The subcellular fractions obtained have been submitted to a biochemical and ultrastructural analysis. Two plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I, have been assayed at the same time as markers for other subcellular organelles, DNA (nuclei), succinic dehydrogenase (mitochondria), inosine diphosphatase (endoplasmic reticulum), and acid phosphatase (lysosomes). The exposure of the plasma membranes to a low concentration of digitonin allowed us to obtain their high purification. They are only contaminated by 2-3% of other cell components present in the macrophages homogenate. The representative ultrastructural technique used has confirmed the purity of the plasma membranes isolated.
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The organization of nucleolar DNA in interphase nuclei of somatic cells was studied at the ultrastructural level using oxidized DAB as a nucleic acid stain. Some finely filamentous networks of DNA-containing structures were observed within the nucleolar fibrillar component. They originated from the perinucleolar shell of condensed chromatin and from a chromatinic area with a honeycomb like structure. The latter was significantly associated with nucleoli and is believed to be a part of the nucleolar organizer region.
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