Biomedical subjects

M Geuskens

Publications and source records attributed to M Geuskens.

At least 19 recordsLinked to original sources

PubMed1992

Receptor-mediated uptake and processing of vitamin D-binding protein in human B-lymphoid cells.

Vitamin D-binding protein (DBP), a member of a multigene family including alpha-fetoprotein (AFP) and albumin, is a serum glycoprotein that reversibly binds and transports vitamin D and its metabolites to target cells. In this work, we demonstrate that normal and malignant human B-lymphocytes specifically bind and internalize DBP. Radioiodinated DBP (125I-DBP) was used to follow the uptake of the protein by Raji cells, a human pre-B-lymphoma cell line. Time course studies of DBP uptake by these cells exhibited a saturable profile at both 4 and 37 degrees C. The binding saturation curve obtained by incubating Raji cells at 4 degrees C with different concentrations (1.5 nM to 1.5 microM) of 125I-DBP showed two saturation plateaus; Scatchard analysis showed the presence of two groups of receptor sites with a Kd1 of 2.04 x 10(-7) M (n1 = 42,161 +/- 4,336 sites/cell) and a Kd2 of 1.01 x 10(-6) M (n2 = 198,000 +/- 48,000 sites/cell). After incubation of Raji cells at 37 degrees C with both fluorescein isothiocyanate (FITC) and horseradish peroxidase conjugates, DBP was internalized and could be localized in the cytoplasm. DBP-horseradish peroxidase conjugates were used to follow the uptake and to determine the endocytic pathway of the protein in Raji cells. The initial steps, contrary to those observed for AFP, did not apparently involve coated pits and vesicles. Small vesicles (approximately 50-60 nm) with electron-dense DBP-horseradish peroxidase reaction products were observed that could fuse with large endosomes. These endosomes appeared dispersed in the cytoplasm with some preferential localization in the Golgi centrosphere region. Pulse-chase experiments showed that only 10% of the uptaken protein was released in a nondegraded form. Accordingly, most DBP molecules accumulated in endosomes should be degraded in lysosomes, instead of being recycled back to the surface, as in the case of AFP. Contrary to malignant B-cells (Raji), the uptake ability for DBP of normal quiescent B-lymphocytes was very low. Specific binding and internalization of DBP-FITC by these cells were observed following mitogen-induced activation. Significant values of uptake were obtained at 37 degrees C after 72 h of incubation in the presence of pokeweed mitogen. The binding of DBP-FITC was partially inhibited in the presence of an excess of unlabeled protein. Taken together, the actual results suggest that DBP receptors are constitutively expressed by malignant B-cells and in a transitory form by normal B-lymphocytes undergoing mitogen-induced activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei.

The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.

Adenylyl Cyclases↗

PubMed1992

Activated human T lymphocytes express albumin binding proteins which cross-react with alpha-fetoprotein.

The kinetics of iodinated human serum albumin ([125I]Hu-SA) and alpha-fetoprotein ([125I]Hu-AFP) binding and endocytosis by resting and phytohemagglutinin (PHA)-activated human T lymphocytes were studied comparatively. The binding of both SA and AFP appeared considerably increased upon blastic transformation. SA, like AFP, binds in a saturable way to the surface of PHA-stimulated human T lymphocytes at 4 degrees C and is endocytosed at 37 degrees C. Two saturation plateaus were observed by incubating at 4 degrees C activated T lymphocytes with [125I]Hu-AFP at different concentrations (10 ng-250 micrograms/ml), while only one saturation plateau was obtained by incubating cells with [125I]Hu-SA in the same conditions. Scatchard analysis of binding data revealed two types of binding sites for Hu-AFP and one for Hu-SA. Competition experiments using proteins of human and bovine origin are in favor of the presence on the surface of these cells of a common binding site for AFP and SA. Pulse-chase experiments showed that internalized [125I]SA was released mainly in a degraded form from the cells, in agreement with detection by ultrastructural cytochemistry of peroxidase-conjugated SA in lysosome-like bodies by ultrastructural cytochemistry. This contrasts with the intracellular pathway of AFP, which as previously described (Geuskens, M., et al., Eur. J. Cell Biol. 50, 418-427 (1989)), moves to tubular-vesicular structures in the Golgi region and is recycled for the most part undegraded.

Albumins↗

PubMed1992

Isolation of plasma membranes, Golgi bodies and mitochondria of Xenopus laevis morulae. Identification of plasma membrane proteins.

Homogenates of Xenopus morulae at the 16-32 cell stage were centrifuged on discontinuous sucrose gradients. Isolated fractions were identified by electron microscopy (EM) as mitochondria, a fraction enriched in Golgi vesicles, and plasma membranes. A special effort was made to prepare plasma membranes free of cytoplasmic contaminants. The resulting purified plasma membranes appeared morphologically identical to plasma membranes in situ. The external surface is covered with a fibrillar coat while vesicles are seen attached to their inner surface. O'Farell's method (1975) was used to obtain protein patterns of the various fractions on 2-dimensional gel electrophoresis. Each fraction displayed a specific pattern. By comparing the different patterns, it was possible to identify a group of proteins as belonging to the plasma membrane fractions. Labelling of cell surface with sulfo-N-hydroxysuccinimido-biotin together with differential extraction of proteins has allowed us to tentatively allocate these proteins in different structures of the plasma membrane fractions. The data presented in this paper corroborate and extend our ultrastructural studies on neogenesis of interblastomeric plasma membranes (Bieliavsky and Geuskens, 1990).

Animals↗

PubMed1992

A study of peritoneal cells from healthy and Schistosoma mansoni-infected mice with special reference to myofibroblasts arising in culture.

Adherent, trypsin-resistant, peritoneal cells from mice with chronic schistosomiasis mansoni, and from control mice, were cultivated in vitro up to 20 days. Fibroblasts regularly appeared, about 6 days after seeding, in cultures of the manyfold more numerous cells from infected mice, concomitantly with a dramatic increase, detected by autoradiography, in the percentage of DNA-replicating cells of the monocyte-macrophage lineage. Peritoneal cells from healthy and from infected mice were fractionated on discontinuous Percoll gradients. Eight cell subsets were harvested in both cases, quantitated, and studied by electron microscopy. Two fractions (2 and 3: 1.041 < densities < 1.060 g/ml) from infected mice were greatly enriched in monoblasts and promonocytes. The cells of the different subsets were seeded separately, trypsin-treated and cultivated in vitro. Cultures of cell fractions 2 and 3 from infected mice contained the majority of the DNA-synthesizing cells and gave regularly rise to fibroblasts. Cultures of the different fractions were used for sequential morphological observations (2-11 days) at the electron microscope level. Early cultures were also used for the ultrastructural detection of the Mac-1 (CD 18/CD 11b) surface antigen by gold immunocytochemistry. A few fibroblasts were rarely observed in cultures of fractions 2 and 3 from control mice, while cells with ultrastructural features of myofibroblasts were regularly observed in cultures of the same fractions harvested from mice with chronic schistosomiasis. Fractions 2 and 3 from infected mice contained a large number of Mac-1 positive monoblasts. The correlations between the presence of monoblasts, DNA replication in cells of the monocyte-macrophage lineage and the appearance of myofibroblasts in cultures of the same fractions derived from infected mice are discussed.

Animals↗

PubMed1991

Activation of an alpha-fetoprotein (AFP)/receptor autocrine loop in HT-29 human colon carcinoma cells.

Immunological and morphological approaches have been used to demonstrate, respectively, alpha-fetoprotein (AFP) synthesis and receptor expression in the HT-29 human colon carcinoma parental cell line. HT-29 cells cultivated in the presence of glucose synthesized and secreted AFP in the medium from 48 to 96 hr after seeding, as revealed by monoclonal antibodies (MAbs) to human AFP. Light microscopic observations of cells incubated with fluoresceinated AFP showed that the protein was specifically bound to the cell surface at 4 degrees C, and was internalized in the cytoplasm at 37 degrees C. At the ultrastructural level, horseradish peroxidase (HRP)-conjugated AFP, as well as HRP-transferrin (Tf) (used as a control), appeared to be internalized via coated pits and vesicles before being delivered to endosomes, from which they were apparently recycled back to the cell surface via small vesicles. Our results suggest that a AFP/receptor autocrine pathway might operate in these cells, be preferentially active at the beginning of the exponential phase of cell culture growth, and contribute to cell proliferation.

Colonic Neoplasms↗

PubMed1991

Receptor-mediated endocytosis and recycling of alpha-fetoprotein in human B-lymphoma and T-leukemia cells.

The kinetics of iodinated human alpha-fetoprotein (AFP) binding and uptake by 2 human neoplastic lymphoid cell lines (CEM and RAJI) have been studied. Three saturation plateaus were obtained by incubating CEM and RAJI cells at 4 degrees C with 125I-AFP at different concentrations. Scatchard analysis suggested the presence of 3 types of receptor site with different affinities and capacities on cells of both lines. AFP binding was inhibited by unlabelled human and bovine AFP, and to a lesser extent by human serum albumin (SAH); no significant competition was observed with human transferrin (Tf) or ovalbumin (Ova). Pulse-chase experiments showed that 125I-AFP was released practically undegraded from the cells. Covalent conjugates of AFP and Tf with horseradish peroxidase (HRP) were used to follow the endocytosis and intracellular pathway of these serum proteins by electron microscopy. Both proteins were observed in coated vesicles, endosomes and a tubular vesicular network localized in the Golgi-centrosphere region. SAH-HRP was internalized to a much lesser extent. Ova-HRP was poorly internalized and was observed in lysosome-like organelles.

Binding, Competitive↗

PubMed1991

Eosinophil granulocytopoiesis in hepatic periovular granulomas during the chronic phase of experimental murine Schistosomiasis mansoni.

We have observed in hepatic periovular granulomas of C3H mice infected with Schistosoma mansoni, in the chronic phase of the disease (12-19 weeks of infection), groups of early precursors and immature eosinophil granulocytes corresponding, at the ultrastructural level, to promyelocytes and myelocytes. Mitosis was also seen in eosinophil myelocytes. These eosinophil myeloid foci were observed in close contact with macrophages and epithelioid cells, and they were surrounded by an extracellular matrix, rich in collagen fibres. These morphological observations give support to the concept of a peripheral proliferation of eosinophils in chronic schistosomiasis, mediated by a factor secreted by macrophages present in granulomas.

Animals↗

PubMed1991

Cytokeratins are exposed on the outer surface of established human mammary carcinoma cells.

Normal human mammary epithelial cells and established tumour cells of the same origin express three to eight cytokeratins, which are distributed throughout the cytoplasm in the form of intermediate filaments. The combined use of the iodogen and the two-dimensional gel electrophoresis methods has allowed us to demonstrate the presence of cytokeratins 8, 18 and 19 on the outer surface of established human mammary carcinoma cells, in particular MCF-7 cells, while they were absent from the surface of normal mammary cells in primary culture. By ultrastructural immunocytochemistry, these cytokeratins were localized on blebs formed by the cell surface. Cytokeratins 8, 18 and 19 were also detected in the culture medium of mammary carcinoma cells.

Antibodies, Monoclonal↗

PubMed1991

A study, by electron microscopy, of the specific uptake of alpha-fetoprotein by mouse embryonic fibroblasts in relation to in vitro aging, and by human mammary epithelial tumour cells in comparison with normal donors' cells.

A covalent conjugate of alpha-foetoprotein (AFP) and horseradish peroxidase (HRP) has been used to follow the internalization pathway of this serum protein in early and late passages of primary cultures of mouse embryonic fibroblasts as well as in a spontaneously immortalized cell line. AFP, as transferrin (Tf) used in parallel as a control, are endocytosed through coated pits and vesicles and move then to endosomes in every case; in cells of the late passages, at least a part of the internalized proteins would be routed to lysosomes. Cells of three different established human mammary cancer lines (MCF-7, Evsa-T, T-47D) internalize AFP-HRP through coated pits and vesicles. Such localization of the conjugate is practically never detected in normal human mammary epithelial cells in primary culture. Taken together, these results are in agreement with the view that AFP receptors are expressed at the surface of proliferating mouse embryonic fibroblasts and human mammary epithelial cancer cells but absent from the surface of normal human mature cells of the same origin.

Animals↗

PubMed1990

Interblastomeric plasma membrane formation during cleavage of Xenopus laevis embryos.

From comparisons by electron microscopy (EM) of organelles isolated by differential centrifugation in sucrose density gradients, and in situ organelles of Xenopus cleaving eggs, we propose a sequence of vesicle transformations leading to the formation and completion of interblastomeric membranes: 1) Mitochondria located under the extremity of the early groove form bulges which detach to form free vesicles. These vesicles grow by progressive assimilation of the material contained in lipid droplets. Finally, they become large glycogen-containing vesicles, the immediate precursors of interblastomeric membranes, and become confluent with the plasma membrane at the upper border of the furrow (Fig. 1, region 1). 2) This process is accompanied by migration of Golgi coated vesicles towards the newly incorporated precursor membranes where they are inserted. At a later stage, smooth endoplasmic reticulum (ser) vesicles are inserted into the growing parallel interblastomeric membranes. These two processes would represent successive protein additions to the previously realized membrane lipidic phase. 3) Besides these events, other large vesicles, possibly formed by coalescence of ser vesicles, transport an abundance of material (precursors of the contractile ring of the furrow?) to the site of the groove in the cortical plasma membrane. They could also play a role in the induction of furrow initiation.

Animals↗

PubMed1989

Schistosomiasis and in vitro transdifferentiation of murine peritoneal macrophages into fibroblastic cells.

We developed a method for avoiding contamination by fibroblasts when cultures of peritoneal cells are initiated. Macrophages were identified by immunogold detection [light microscope, transmission (TEM) and scanning (SEM) electron microscopes] of membrane antigens (Mac-1+, Thy-1,2-), non-specific esterase activity and ultrastructural features (TEM). As compared with controls, the yield of peritoneal macrophages was 2- and 12-fold higher, respectively, in acutely and chronically infected mice. In all, 30 "chronic", 18 "acute" and 18 control cultures were followed up. At a given cell-density seeding, the decline of control, "acute" and "chronic" cultures starts at about day 10, 15, and 27, respectively. In "chronic" cultures only, fibroblast-like cells appear from day 6 onwards; their number increases with time. Cells showing characters intermediary between macrophages and fibroblasts were observed. We suggest that fibroblast-like cells result from the in vitro transdifferentiation of a limited number of in vivo committed macrophages.

Animals↗

PubMed1989

Partial reversion of conditional transformation correlates with a decrease in the sensitivity of rat cells to killing by the parvovirus minute virus of mice but not in their capacity for virus production: effect of a temperature-sensitive v-src oncogene.

The cytolytic effect of the autonomous parvovirus minute virus of mice, prototype strain (MVMp), was studied in cultures of ts 339/NRK rat cells that display a temperature-sensitive transformed phenotype as a result of their transformation with a Rous sarcoma virus strain matured in the v-src oncogene. A shift from restrictive (39.5 degrees C) to permissive (34.5 degrees C) temperature was associated with a marked sensitization of these cells to killing by MVMp. In contrast, ts 339/NRK cell derivatives supertransformed with a wild-type src oncogene were sensitive to MVMp at both temperatures, suggesting that the expression of a functional oncogene product may determine, at least in part, the extent of the parvoviral cytopathic effect. Although ts 339/NRK cells were quite resistant to parvoviral attack at 39.5 degrees C, they were similarly proficient in MVMp uptake, viral DNA and protein synthesis, and infectious particle production at both permissive and restrictive temperatures. Consistently, electron microscopic examination of infected ts 339/NRK cultures incubated at 39.5 degrees C revealed the presence, in the majority of the cells, of numerous full and empty virions that were predominantly located in autophagic-type vacuoles. Thus, in this system, the reversion of transformed and MVMp-sensitive phenotypes appears to correlate with the setting up of a noncytocidal mode of parvovirus production. These results raise the possibility that the physiological state of host cells may affect their susceptibility to parvoviruses by modulating not only their capacity for virus replication but also cellular processes controlling the cytopathic effect of viral products.

Animals↗

PubMed1989

Morphological characterization of the pathway of endocytosis and intracellular processing of transferrin and alpha-fetoprotein in human T lymphocytes stimulated with phytohemagglutinin (PHA).

Covalent conjugates of transferrin (Tf) and alpha-fetoprotein (AFP) with horseradish peroxidase (HRP) have been used to follow, at the ultrastructural level, the uptake and the intracellular pathway of these proteins in peripheral blood human lymphocytes stimulated by phytohemagglutinin (PHA) to blast formation. Both proteins enter specifically the cells via vesicles (60-70 nm in diameter) and endosomes. They are then observed in multivesicular bodies and tubular vesicular elements in the Golgi region. AFP is thus found in the same subcellular compartments as Tf and is probably also recycled, as most of the 125I-labeled protein leaves the cells undegraded. Unstimulated lymphocytes do not internalize significantly AFP-HRP. The uptake of a noncovalent conjugate of AFP-HRP and [3H]-arachidonic acid [3H-(20:4)] is usually poor, at 37 degrees C, in unstimulated lymphocytes as well as, at 4 degrees C, in lymphocytes stimulated for 72 h. Stimulated lymphocytes incubated at 37 degrees C with the radioactive conjugate show a heavy labeling of cell organelles and more particularly of lipid droplets. AFP could regulate the intracellular delivery of fatty acid molecules.

Endocytosis↗

PubMed1986

Ultrastructural studies of the intracellular translocation of endocytosed alpha-foetoprotein (AFP) by cytochemistry and of the uptake of 3H-arachidonic acid bound to AFP by autoradiography in rat rhabdomyosarcoma cells.

A covalent conjugate of alpha-foetoprotein (AFP) and horseradish peroxidase (HRP) has been used to follow, at the ultrastructural level, the pathway of AFP uptake and translocation in a rat rhabdomyosarcoma cell line. The cells were incubated for several times at 4 degrees C and/or 37 degrees C, and fixed. AFP-HRP was found to enter the cells via coated pits and receptosomes and to move to tubular elements of the trans-reticular portion of the Golgi. Some observations suggest that AFP can be recycled back to the cell surface. On the other hand, the cells were incubated with a noncovalent conjugate of AFP and 3H-arachidonic acid [3H-(20:4)], and the uptake of the fatty acid molecules studied by ultrastructural autoradiography. The cytoplasmic labeling, very low after an incubation in the presence of [3H-(20:4)]-AFP for 2 hours at 4 degrees C, increased rapidly after transfer of the cells for 5 minutes to 37 degrees C. These observations support the hypothesis that AFP plays a role in the intracellular delivery of polyunsaturated fatty acids.

Animals↗

PubMed1986

Fibronectin localization and endocytosis in early and late mouse embryonic fibroblasts in primary culture: a study by light and electron microscopic immunocytochemistry.

In spreading fibroblasts, strong endocytosis of exogenous fluorescent fibronectin (FN) was observed from the beginning of their attachment to the substratum. In early fibroblasts, the internalized FN was localized both in the peripheral ruffles and in the perinuclear cytoplasm; in late fibroblasts, whose spreading was slower, FN uptake was not detected in the ruffles. In growing cultures, supracellular FN fibres, detected by direct fluorescence microscopy or by the indirect peroxidase-anti-peroxidase (PAP) complex technique, were scarce on early cells, but very numerous on the upper face and on the filopodia of late cells. At the ultrastructural level, FN, localized with the immuno-gold staining method, was found associated with fibres of the extracellular matrix and the upper face of the cells. FN was endocytosed via smooth vesicles and we suggest that the internalization process is slower in the late cells. In confluent early cultures, an extended network of pericellular FN was observed as usual. The pericellular FN of late grouped cells was present as a few coarse fibres connected with some of the cell surface threads.

Animals↗

PubMed1986

Cationized ferritin binding and internalization during in vitro aging of mouse embryonic fibroblasts.

The anionic surface sites of mouse embryo fibroblasts in primary culture, aging in vitro, have been studied using cationized ferritin (CF) as a marker. We have not observed significant differences between early and late cells with respect to CF binding; the labelling is uneven and patchy in both cases. Due to the increase of their surface area with the mitotic age, the total amount of negative charges, per cell, must however be much higher in the late passage cells. Virtually no differences in the rates of CF endocytosis via coated pits, with subsequent delivery to large vacuoles and lysosomes, have again been observed, but there was a cisternal formation from apposed plasma membranes in the late cells. New findings with regard to previous studies with CF concern the capping of the labelled surface components, followed by phagocytosis of the cap material, and the organization of the extracellular matrix in a rather regular network at the periphery of the cells.

Animals↗

PubMed1982

Subcellular immunolocalization of alphafetoprotein (AFP) in cell lines established from Morris hepatoma 7777 and 8994. Lack of effect of dexamethasone on the ultrastructural detection of AFP in the 8994 cells.

The subcellular localization of alphafetoprotein (AFP) has been studied in two cell lines (McA-RH 7777 and McA-RH 8994) derived from Morris hepatomas 7777 and 8994, using immunoperoxidase labelling techniques. After fixation of the cells in a low concentration of glutaraldehyde, a hot saponin treatment was used to increase the permeability of their membranes without destroying their morphology. The perinuclear spaces, the lamellae of the endoplasmic reticulum and the saccules of the Golgi apparatus contain electron-dense precipitates; 90% of the 7777 cells and 55% of the 8994 cells in exponential phase of growth are AFP-positive. Dexamethasone, a synthetic glucocorticoid, increases AFP production by the 8994 cells without raising the number of AFP-positive cells.

Animals↗