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M Bomsel

Publications and source records attributed to M Bomsel.

At least 19 recordsLinked to original sources
PubMed2005

Cyclic FEE peptide increases human gamete fusion and potentiates its RGD-induced inhibition.

BACKGROUND: Alpha6beta1 integrin has been proposed to act as a sperm receptor on the mouse oocyte by interacting with spermatozoon fertilin beta. We investigated, in humans, whether oocyte integrins could act similarly in gamete fusion, using a cyclic peptide containing the putative disintegrin-binding domain of human fertilin beta [cyclic FEE (cFEE)] and RGD peptide. METHODS: Zona-free eggs were inseminated in the absence or presence of peptides. To maintain the membrane protein pattern, the zona pellucida was removed by microdissection. Immunofluorescence and confocal microscopy were used to detect integrin subunits on the oocyte. RESULTS: Unexpectedly, cFEE alone increased human gamete fusion by 94% instead of inhibiting fertilization. Furthermore, cFEE together with RGD potentiated the RGD-induced inhibition of fertilization in a dose-dependent manner. The data suggested the hypothesis of integrin cross-talk, further supported by the co-localization of alpha6beta1 and alphavbeta3 integrins, the putative receptors of cFEE and RGD peptides, respectively. CONCLUSIONS: RGD-sensitive and -insensitive integrins may be associated in a multimolecular complex working as a sperm receptor on the human oocyte membrane. Supplementation of human IVF culture medium with cFEE peptide might improve fertilization rates in ART.

ADAM Proteins↗
PubMed2001

Secretory IgA specific for a conserved epitope on gp41 envelope glycoprotein inhibits epithelial transcytosis of HIV-1.

As one of the initial mucosal transmission pathways of HIV (HIV-1), epithelial cells translocate HIV-1 from apical to basolateral surface by nondegradative transcytosis. Transcytosis is initiated when HIV-1 envelope glycoproteins bind to the epithelial cell membrane. Here we show that the transmembrane gp41 subunit of the viral envelope binds to the epithelial glycosphingolipid galactosyl ceramide (Gal Cer), an alternative receptor for HIV-1, at a site involving the conserved ELDKWA epitope. Disrupting the raft organization of the Gal Cer-containing microdomains at the apical surface inhibited HIV-1 transcytosis. Immunological studies confirmed the critical role of the conserved ELDKWA hexapeptide in HIV-1 transcytosis. Mucosal IgA, but not IgG, from seropositive subjects targeted the conserved peptide, neutralized gp41 binding to Gal Cer, and blocked HIV-1 transcytosis. These results underscore the important role of secretory IgA in designing strategies for mucosal protection against HIV-1 infection.

Adult↗
PubMed2001

Annexin expressions are temporally and spatially regulated during rat hepatocyte differentiation.

Annexin (Anx) 1, 2, 5, and 6 expressions were determined at the transcriptional and translational levels in the rat hepatocytes from gestational day 15 to postnatal day 17. Dramatic shifts were observed in Anx 1 and 2 levels, which peaked at day 1 and gestational day 20, respectively, and reached low levels thereafter. However, Anx 5 and 6 rates were more constant. Prenatal administration of dexamethasone (dex) resulted in a decrease of Anx 1 mRNA levels, and a strong increase in Anx 2 mRNA contents. In adult hepatocytes cultured in the presence of EGF or HGF, Anx 1 and 2 expressions resumed. By immunohistochemistry, Anx 1 was detected only in the cytoplasm of hepatocytes of 1- to 3-day-old rats, Anx 2 and 6 both exhibited a redistribution from the cytoplasm toward the plasma membrane, and Anx 5 was present in the nucleus, cytoplasm, and plasma membrane. Thus, Anx 1, 2, 5, and 6 have individual modes of expression and localization in the differentiating hepatocytes, where they might play unique roles at well defined phases of liver ontogeny.

Animals↗
PubMed2001

Bioelectric recognition assay (BERA).

A novel biosensory method has been developed for the determination of various chemical and biological molecules by assessing their electrophysiological interactions with a group of cells and cell components immobilized in a gel matrix that preserves their 'physiological' functions. The method was applied for the detection of: (i) hepatitis C virus in human blood samples; (ii) plant viruses; and (iii) a herbicide (glyphosate) in aqueous solutions. It was able to rapidly (assay time 3-5 min) and specifically detect the molecules in question at a concentration lower than 100 pg/ml, among other compounds f similar structure. The potential use of BERA biosensors for a rapid and cost-efficient molecule determination without prior knowledge of a specific receptor-molecule interaction is discussed.

Animals↗
PubMed2001

Mouse offspring after microinjection of heated spermatozoa.

The thermostability of the mammalian sperm genome was previously reported, but no live offspring after conception with heated spermatozoa had yet been obtained. In the present study, mouse spermatozoa were heated at 56 degrees C for 30 min and microinjected into mouse oocytes. Fertilization did not occur unless activation was induced by incubation in a calcium-free medium containing strontium. Under these conditions fertilization and cleavage rates were comparable to those obtained after microinjection of control spermatozoa, but the developmental rate to the blastocyst stage was lower. When transferred to foster mothers, embryos derived from heated sperm developed into phenotypically normal offspring, which grew and reproduced normally. In the mouse, heated spermatozoa can therefore support full embryonic development after microinjection into oocytes.

Animals↗
PubMed2001

Cell-to-cell contact results in a selective translocation of maternal human immunodeficiency virus type 1 quasispecies across a trophoblastic barrier by both transcytosis and infection.

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.

Cell Communication↗
PubMed1999

B7 cosignal potentiates apoptosis of uninfected CD4+ T lymphocytic cell lines primed by HIV envelope proteins.

In lymphoid organs, follicular dendritic cells (FDCs), monocytes, and macrophages are targets for HIV infection and reservoirs for infectious virus. Strikingly, the apoptotic cells in these sites are essentially uninfected CD4+ T lymphocytes, but lie in close proximity to infected cells or FDCs carrying trapped HIV virions. To decipher this apoptotic pathway, we have established a two-step experimental system that reproduces in vitro the HIV envelope protein-mediated apoptosis restricted to uninfected CD4+ T lymphocytic cell lines. In this assay, uninfected CD4+ T cell targets undergo apoptosis following an initial priming step on HeLa cells expressing functional HIV envelope proteins at their plasma membrane and a second and necessary stimulation step via the CD3-TCR complex. The CD4+ T lymphocytic cells susceptible to apoptosis are, in contrast, resistant to cell fusion mediated by HIV envelope protein and express SDF-1. FDCs and macrophages are known to be high B7 expressors. Thus in lymph nodes, the cells that have trapped HIV particles in immune complexes at the plasma membrane present both HIV envelope proteins and B7.1 at their surface. We mimicked this situation in vitro by priming CD4+ T lymphocytes on cells expressing the costimulatory molecule B7 in addition to HIV envelope proteins, and show that it resulted in an acceleration and a twofold increase in apoptosis. Finally, we characterized two enzymes, PI3Kinase and PI-PLC, which are both downstream effectors of the CD4 (HIV envelope protein receptor) and CD28 (B7 receptor) activation pathways, and that participated in the early steps of priming for apoptosis.

Apoptosis↗
PubMed1999

Infectious human immunodeficiency virus can rapidly penetrate a tight human epithelial barrier by transcytosis in a process impaired by mucosal immunoglobulins.

Mucosal surfaces are the main natural site of entry into the body for human immunodeficiency virus (HIV). Herein, an alternative mechanism for virus spread is described. The mechanism, which involves transcytosis of endosome-internalized HIV-particles, was generated by contact of HIV-infected cells with the apical surface of an epithelial cell line. Transcytosed viruses rapidly (in 20-30 min) access the serosal side of the epithelial barrier without infecting the epithelium itself. In turn, transcytosed HIV could infect host submucosal mononucleated target cells, and thus the infection could spread. An investigation was done to determine whether mucosal antibodies could block HIV transcytosis. Both secretory IgA (S-IgA) and IgG that were purified from colostrum from HIV-seropositive women impaired HIV transcytosis, irrespective of the level of the recombinant HIV envelope anti-gp160-specific activities in an ELISA. However, specific S-IgAs were more efficient than IgG. Therefore, mucosal-specific S-IgA to HIV-1 could be relevant to reducing infectivity of HIV-1 in corporeal fluids.

Biological Transport↗
PubMed1998

In adrenocortical tissue, annexins II and VI are attached to clathrin coated vesicles in a calcium-independent manner.

We have previously characterized three populations of clathrin coated vesicles (CCVs) isolated from bovine adrenocortical tissue and designated them as large, medium and small coated vesicles, i.e., LCV, MCV and SCV, respectively. Here, we show that annexins II and VI, two of the annexins involved in membrane traffic, are present in the three populations of CCVs but with different distributions between coat proteins (CP) and lipidic vesicle membrane. Annexin VI is only associated with the membrane, whatever the CCV population. In contrast, annexin II is differently distributed between coat and membrane, depending on the CCV population. Both annexins are bound to membranes in a calcium-independent manner and solubilization studies in Triton X114 (TX114) suggest that they interact poorly with lipids by hydrophobic interactions. Ligand blotting experiments show that both annexins bind to CCV proteins: annexin II to a 200-kDa component in all CCVs and annexin VI to a 100-kDa component in LCV and SCV identified as dynamin, a GTPase essential for endocytic CCV pinching off. Dynamin is tightly associated to annexin VI only in LCVs, the endocytic [transferrin (Tf) positive] vesicles. Our data suggest that annexins II and VI could define specific protein-lipid interaction microdomains that could play a role in the different functions of the CCVs.

Adrenal Cortex↗
PubMed1998

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342.

Dictyostelium discoideum cells are highly resistant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size > 21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism.

Animals↗
PubMed1998

Intracellular neutralization of HIV transcytosis across tight epithelial barriers by anti-HIV envelope protein dIgA or IgM.

Human immunodeficiency virus, generated during contact between HIV-infected cells and the apical surface of an epithelial cell, can cross a tight epithelial barrier by transcytosis. We show that transcytosis of primary HIV isolates is blocked by dimeric IgA or IgM against HIV envelope proteins. Neutralization occurs intracellularly within the apical recycling endosome, and immune complexes are specifically recycled to the mucosal surface. One epitope involved in neutralization is a conserved sequence of the gp41 HIV envelope protein subunit. Finally, transcytosis also occurs across functional human mucosal tissue in a process inhibited by a serosal internalization of IgM against the HIV envelope protein. These results suggest that induction of mucosal immunity to HIV envelope proteins may impair the transcytotic route of HIV mucosal transmission.

Antigen-Antibody Complex↗
PubMed1998

Human gamete fusion can bypass beta1 integrin requirement.

Since alpha6beta1 integrin has been shown to function as a sperm adhesion receptor in the mouse, we investigated the potential role of beta1 integrin in the gamete fusion process in humans. The expression of beta1 integrin was morphologically analysed by indirect immunofluorescence and confocal microscopy. A homogeneous and intense staining was detected at the plasma membrane, and in some subcortical vesicles of germinal vesicle stage oocytes (GV). Beta1 almost disappeared from oolemma and cytoplasm of metaphase I (MI) oocytes, but was re-expressed as asymmetrical patches at the plasma membrane of metaphase II stage oocytes (MII). A functional fusion assay based on Hoechst or calcein-AM dye transfer from one gamete to the other showed that maturing oocytes were able to fuse with an increasing number of spermatozoa (11-22 from GV to MII respectively), and that fused spermatozoa co-localized with beta1 integrin patches. Human gamete fusion was only partially inhibited either by RGD-containing peptide (GRGDTP), or by blocking anti-human beta1 integrin monoclonal antibody (DE9), with a maximum of 50% inhibition. Despite the combined addition of GRGDTP and blocking mouse anti-human beta1 integrin DE9 in the assay, a complete inhibition of fusion could not be achieved. A mouse polyclonal antibody raised against human oocyte membranes was more potent in inhibiting the fusion. Since beta1 integrin expression at the plasma membrane was not correlated to oocyte fusibility, and since it was only partially inhibited by DE9 and/or RGD peptide, we suggest that human gamete fusion can bypass the beta1 requirement. Beta1 integrin certainly participates in human gamete fusion by acting in co-operation with multiple integrin/disintegrin couples or another cofactor, not yet identified.

Animals↗
PubMed1997

High-level ability of secretory IgA to block HIV type 1 transcytosis: contrasting secretory IgA and IgG responses to glycoprotein 160.

The IgG and secretory IgA (S-IgA) responses to the HIV-1 envelope (gp160 antigen) were analyzed in the colostrum (Col) and in the cervicovaginal fluid (CVF) of HIV-l-infected women. We show IgG antibodies (Abs) to the recombinant gp160 to be predominant as compared with the corresponding S-IgA isotype. The low level of the S-IgA response cannot be related to a general disturbance of the mucosal-associated Iymphoid tissue (MALT) because the level of a current Ab to a caries-associated antigen from Streptococcus sobrinus was in the normal range in these secretions. The major subclass of IgA to gp160 was of the alpha1 isotype both in Col and in CVF. However, the specific activities of S-IgA1 and S-IgA2 were different when expressed as the ratio of the anti-gp160 related to total Ig of each subclass. Indeed, the specific activity of the S-IgA2 was predominant over S-IgA1 in the Col, whereas the reciprocal results were found in CVF, showing a subcompartmentalization of these secretions. The ability of S-IgA and IgG to block one of the pathways involved in the HIV-1 penetration across mucosa, i.e., transcytosis through epithelial cells, was evaluated using a functional in vitro assay. Both S-IgA and IgG Abs impaired virus transcytosis, irrespective of the level of antigp160 specific activities. However, specific S-IgA was more efficient than IgG. These features suggest that mucosal specific S-IgA to HIV-1 could be relevant in decreasing infectivity of HIV-1 in corporal fluids.

Adult↗
PubMed1997

Modifications of the human oocyte plasma membrane protein pattern during preovulatory maturation.

The plasma membrane protein pattern of human oocytes was established using a highly sensitive nonisotopic technique. Unfertilized, 2-day-old metaphase II (MII) and immature oocytes were biotinylated at 4 degrees C and zona pellucida were mechanically removed. Proteins were resolved by 7% SDS PAGE, and electrotransferred to PVDF membranes. Plasma membrane proteins were selectively detected by streptavidin-horseradish peroxidase (HRP) and enhanced chemoluminescence (ECL). Thirteen biotinylated polypeptide bands were identified in MII oocyte plasma membrane (126, 110, 98, 90, 77, 71, 64, 61, 58, 54, 50, 48, 44 kD). During maturation, the total amount of membrane proteins decreased dramatically from the germinal vesicle (GV) to the MII stage oocytes, while the relative proportion of the 71 kD band increased from 9.9% to 13.1% and 27.4% in GV, metaphase I, and MII stage oocytes, respectively. Improvement of the detection technique permitted to establish the protein profile of a single oocyte loaded per lane (n = 12). Five to ten polypeptides were identified, indicating a great polymorphism of the plasma protein pattern, even for oocytes from the same cohort. Hamster and mouse oocyte plasma membrane protein patterns were also investigated with the same technique. Both presented 15 bands, 12 of which had a molecular weight similar to those from the human oocytes. In conclusion, the protein pattern in the human plasma membrane appears qualitatively limited to 13 species, and quantitatively, their amount decreases during oocyte preovulatory maturation. A great polymorphism from one oocyte to another was detected. The protein pattern is highly conserved between human, hamster, and mouse oocytes. This very sensitive technique will allow further studies on the functional significance of this protein pattern.

Animals↗
PubMed1997

Transcytosis of infectious human immunodeficiency virus across a tight human epithelial cell line barrier.

Contact between various epithelial cell lines and HIV chronically infected mononuclear cell lines results in a massive and rapid budding of HIV virions toward the epithelium followed by their internalization into epithelial endosome-like structures. Here it is shown that as early as 30 minutes after apical contact, primary virus isolates generated from primary peripheral blood leukocytes from HIV-infected patients can cross an epithelial cell line barrier using transcytosis, the characteristic epithelial transcellular vesicular pathway. As the next step in the spread of infection, transcytosed HIV particles can productively infect mononuclear cells located at the basolateral side of the epithelial barrier. These observations suggest an alternative, rapid and efficient mechanism for transmission of HIV across an intact epithelial barrier.

Antibodies, Monoclonal↗
PubMed1997

Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery.

After reaching early endosomes by receptor-mediated endocytosis, diphtheria toxin (DT) molecules have two possible fates. A large pool enters the degradative pathway whereas a few molecules become cytotoxic by translocating their catalytic fragment A (DTA) into the cytosol. Impairment of DT degradation by microtubule depolymerization does not block DT cytotoxicity. Therefore, DTA membrane translocation into the cytosol occurs from an endocytic compartment located upstream of late endosomes. Comparisons between early endosomes and endocytic carrier vesicles in a cell-free translocation assay have demonstrated that early endosomes are the earliest endocytic compartment from which DTA translocates. DTA translocation is ATP-dependent, requires early endosomal acidification, and is increased by the addition of cytosol. Cytosol-dependent DTA translocation is GTP gamma S-insensitive but is blocked by anti-beta COP antibodies.

Animals↗
PubMed1996

Specific lipid protein interactions characterize 3 populations of clathrin coated vesicles involved in the LDL receptor traffic.

We have previously isolated 3 different populations of clathrin coated vesicles (CCV) involved in the LDL-receptor traffic in bovine adrenal cortex. We now show that each CCV type contains the transferrin-R and the CI-MPR, therefore, they provide a good model for studying the membrane organization that may govern their targeting in one of the biosynthetic, endocytic and/or recycling pathways. Transferrin--prototype of recylcing ligand--, and alpha adaptin, dynamin and the 110 kDa phosphatidylinositol-3-kinase subunit--of the trafficking machinery--were mainly detected in only 2 of the vesicle populations which could be involved in the endocytic/recycling pathway. The third population which contained larger amounts of gamma adaptin and do not carry transferrin could be involved in the biosynthetic pathway. The vesicle lipid pattern and the saturation of their fatty acyl chains were analyzed and confirmed these results. The nature of the interactions between vesicle components was then determined using several classes of detergents. Only non ionic ones could solubilize the LDL-R in a complex with either alpha or gamma adaptin. In contrast, they dissociated clathrin or beta-beta' adaptins. Taken together these results prompt us to suggest an integrated model for targeting in membrane traffic. Besides specific targeting signals carried by cargo proteins and recognized by proteins from the coat and the cytosolic trafficking machinery, lipids would play a key modulatory role. At each step in the membrane traffic, the proteins which carry multiple targeting signals would interact transiently with a specific set of lipids. This would result in the exposure of the appropriate targeting signals which could now become recognized by the proper targeting machinery.

Adrenal Cortex↗