Biomedical subjects

M Geuskens

Publications and source records attributed to M Geuskens.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1984

Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos.

The development of the nucleoli and the sites of rDNA transcription have been studied by high-resolution autoradiography during the cleavage stages of mouse embryos. The appearance of fibrillar centres at the periphery of the fibrillar primary nucleoli has been observed at the 4-cell stage. Several fibrillar centres, interconnected by electron-dense fibrillar strands, form a reticulated region around the fibrillar mass at the 6-to 8-cell stage. After a 10 min pulse with [3H]uridine, only this peripheral network is labelled. At the late morula and at the blastocyst stage, the fibrillar component (nucleolonema) of the reticulated nucleoli is labelled after 10 min [3H]uridine incorporation. When the embryos are reincubated for 2 h in cold medium, the label is localized mainly in the granular component. Fibrillar centres are not labelled. Autoradiograms of in vitro developed embryos pulsed for 2 h with [3H]uridine confirm that the central fibrillar core of the nucleoli of 6-to 8-cell embryos is never labelled. Thus, the fibrillar constituent of this core is not homologous to the fibrillar component of the nucleoli of later stage embryos, which is the site of active rDNA transcription. An interpretation of nucleologenesis during early mouse embryogenesis is proposed.

Animals↗

PubMed1984

Incorporation of alphafetoprotein by the MCF-7 human breast cancer cell line.

The intracellular uptake of alphafetoprotein (AFP) and its fluorescein conjugates, by cultures of the MCF-7 human breast cancer cell line, has been demonstrated using an indirect immuno-peroxidase technique or by direct visualization under fluorescent lighting. The protein was localized in the cytoplasm. Ultrastructural autoradiographs of MCF-7 cells incubated with human 3H-AFP showed protein accumulation in several cytoplasmic organelles, particularly in lipid droplets. Nuclei were free of AFP. A significant species-specificity of AFP internalization was observed in comparative assays with human, mouse, pig and chicken AFP. The incorporation of the protein was prevented by incubation at 0 degrees C or by previous treatment of the cultures with 10nM sodium azide. Attention is paid to the reappearance in a human breast carcinoma cell line of a property associated during ontogenic development with ectodermal derivatives, including the epidermis.

Albumins↗

PubMed1984

Electron microscopy of glycogen in mouse cleaving embryos.

The blastomeres of mouse morulae contain an abundance of beta-glycogen particles detectable cytochemically with good specificity; these particles are dispersed in the hyaloplasm. Attention is drawn to the fact that, with conventional staining techniques, the glycogen of mouse cleaving embryos can easily be confused with ribosomes in the electron microscope.

Animals↗

PubMed1983

Alphafoetoprotein uptake by cloned cell lines derived from a nickel-induced rat rhabdomyosarcoma.

Rat, mouse, pig and chicken alphafoetoproteins (AFP), rat serum albumin and egg albumin, or their fluoresceinated conjugates were added to cultures of several cloned cell lines isolated from a nickel-induced rat rhabdomyosarcoma. The intracellular uptake of assayed proteins was revealed by the indirect immunoperoxidase technique and/or by direct fluorescence microscopy. All the clones examined bound AFP, and all but one internalized the protein. The protein localized in the membrane and the cytoplasm, as well as along straight processes interconnecting cells. Nuclei were always AFP negative. The protein uptake of fluoresceinated conjugates of AFP and serumalbumin was already visible 15 min after incubation and progressed with time to reach a plateau 4-5 h later. Ultrastructural radioautographs of cells incubated with [3H]-AFP (rat) showed protein accumulation in several organelles and particularly in lipid droplets. Parallel to these observations, the intracellular presence of AFP within myofibrillar structures was demonstrated in tongue sections of rat foetuses and neonates. The results presented here provide experimental evidence of the reappearance in cloned cell lines derived from a primary rhabdomyosarcoma of a property pertaining to foetal striated muscle.

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↗

PubMed1981

An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochondrial DNA replication. We therefore conclude that DNA polymerase alpha performs the synchronous bidirectional replication of nuclear DNA and that DNA polymerase gamma, the only DNA polymerase present in the mitochondria, performs the "strand displacement" DNA synthesis of these organelles.

Animals↗

PubMed1979

An ultrastructural study of the effects of wheat germ agglutinin (WGA) on cell cortex organization during the first cleavage of Xenopus laevis eggs. I. Inhibition of furrow formation.

Xenopus laevis fertilized eggs have been treated with wheat germ agglutinin (WGA) before the onset of the first cleavage, at the stripe stage and during groove deepening. The ultrastructure of the animal cortex of the arrested embryos has been compared with that of the same region of control embryos at different stages of first furrow formation and of cytochalasin B-treated embryos. The outer side of the plasma membrane of WGA-treated embryos is covered with a coat which is different from the diffuse material observed in either control or cytochalasin B-treated embryos and which is distributed in patches in the groove region. Narrow indentations of the plasma membrane in the cortex of WGA-treated eggs have been observed, particularly in the blocked or regressed groove. In WGA-treated eggs, a few bundles of microfilaments are located under the plasma membrane at the animal pole, but they are never arrayed in a continuous layer as in the control eggs. In the latter, many microtubules are located in close proximity to the microfilament layer at the beginning of cleavage, but they are only occasionally observed in the same region of WGA-treated eggs. It is concluded that the binding of WGA molecules to their receptors on the surface of the Xenopus zygote interferes with the alignment of microfilaments in the furrow region and provokes the disorganization of the aligned microfilaments once the cleavage has begun. Internalization of portions of the nascent membrane in the groove could play an important part in the arrest of cleavage.

Animals↗

PubMed1979

An ultrastructural study of the effects of wheat germ agglutinin (WGA) on cell cortex organization during the first cleavage of Xenopus laevis eggs. II. Cortical wound healing.

Uncleaved fertilized eggs of Xenopus laevis treated with wheat germ agglutinin (WGA) have been pricked at the animal pole both inside and outside the regressed furrow region. The wounded cortex of both regions has been studied with the electron microscope and compared with the same region of wounded, untreated eggs. In all 3 cases, filaments are organized in an annular zone in the damaged cortex. When the surface is pricked outside the regressed furrow of WGA-treated embryos, bundles of microfilaments radiate from the ring and extend in deep folds which form a 'star' around the wound at the surface of the embryo. However, when the surface is pricked in the new membrane of the regressed furrow, filaments are intermingled with internalized portions of the plasma membrane. It is suggested that, when the surface is pricked outside the furrow region, more filaments are mobilized to counteract the tangential retraction of the membrane which has acquired more rigidity after WGA binding.

Animals↗

PubMed1979

3H-actinomycin D binding to ultrathin section of plastic-embedded Locusta migratoria testicular tubules. Improvement of the technique and further characterization of the reaction.

Semi-thin and ultrathin sections of locust testes have been incubated in 3H-actinomycin D solution and submitted to radioautography. The improved technical conditions described allow the reproducible obtainment of cell radioautographs with a moderate nuclear labelling and a very low nonspecific background which are usable for semi-quantitative results. Extraction with enzymes (DNase, RNase, pronase) or concentrated salt solution have been carried out before 3H-Actinomycin D treatment in order to characterize the reaction. The semi-quantitative results obtained at the light microscope level suggest that, in relation to the structural and chemical changes which occur in chromatin during spermiogenesis, some proteins may be easily hydrolysed in early spermatids. In ultrathin sections of spermatocytes the X chromosome is heavily "stained" with 3H-Actinomycin D, while 3H-uridine is not incorporated into the sex chromatin. These results are discussed in the light of current ideas on the constitution of active chromatin.

Animals↗