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Biomedical subjects

H Alexandre

Publications and source records attributed to H Alexandre.

At least 19 recordsLinked to original sources
PubMed2003

The thiol reagent, thimerosal, irreversibly inhibits meiosis reinitiation in mouse oocyte when applied during a very early and narrow temporal window: a pharmacological analysis.

The effect of the sulfhydryl reagent, thimerosal (TMS) on meiosis resumption in germinal vesicle (GV)-stage denuded mouse oocytes was studied. It irreversibly inhibits both GV breakdown (GVBD) and the first polar body (pb1) extrusion in concentration- and time-dependent manners, the most striking result being the very early and narrow temporal window during which denuded primary oocytes released from their follicle are susceptible to a pulse of the drug. This inhibition is bypassed by dithiothreitol (DTT) with an efficiency declining with time, while thiosalicylic acid (TA), an analog of TMS devoid of the mercury atom, has no effect on meiosis reinitiation. These results strongly suggest that the inhibitory effect of TMS is a consequence of its sulfhydryl group oxidising activity. The molecular target(s) of this inhibitory oxidation should however be identified. In contrast to DTT, okadaic acid (OA), known to bypass the inhibitory effect of drugs interfering with protein kinase activities, only induces chromatin condensation and GVBD in TMS-pulsed oocytes with a delay of about 8 hr as compared to the control situation. This confirms that a very early thiol oxidation induced by TMS exerts a much more dramatic effect on resumption on meiosis than any pharmacological manipulation of protein kinase activities leading to activation of MPF.

Animals↗
PubMed2003

Effect of taxol and okadaic acid on microtubule dynamics in thimerosal-arrested primary mouse oocytes: a confocal study.

A pulse of thimerosal (TMS), a sulfhydryl reagent, induces an instantaneous, complete and long-lasting microtubule interphasic network disassembly in mouse primary oocytes, correlated with the irreversible inhibition of meiosis reinitiation This inhibition is bypassed by dithiothreitol (DTT) while thiosalicylic acid, an analog of TMS, does induce neither microtubules depolymerisation nor inhibition of reinitiation and resumption of meiosis. This strongly suggests that the dramatic and pleiotropic inhibitory effect of TMS is specifically related to its sulfhydryl group oxidising activity of critical molecules among which tubulin. In contrast to DTT, okadaic acid (OA), known to bypass the inhibitory effect of drugs interfering with protein kinase activities, induces a late chromatin condensation and GVBD in TMS-pulsed oocytes as compared to the control situation, with no significant concomitant microtubule assembly. These cytological features are suggested to be indirectly induced by a late MAPK activation and confirm that a very early thiol oxidation induced by TMS exerts a much more dramatic effect on resumption of meiosis than any pharmacological manipulation of protein kinase activities leading to activation of MPF. Finally, taxol was shown to promote tubulin polymerisation even when microtubules were irreversibly disassembled by thiol oxidation but fails to restore the ability to undergo maturation.

Animals↗
PubMed2001

Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae.

DNA microarrays were used to investigate the expression profile of yeast genes in response to ethanol. Up to 3.1% of the genes encoded in the yeast genome were up-regulated by at least a factor of three after 30 min ethanol stress (7% v/v). Concomitantly, 3.2% of the genes were down-regulated by a factor of three. Of the genes up-regulated in response to ethanol 49.4% belong to the environmental stress response and 14.2% belong to the stress gene family. Our data show that in addition to the previously identified ethanol-induced genes, a very large number of genes involved in ionic homeostasis, heat protection, trehalose synthesis and antioxidant defence also respond to ethanol stress. It appears that a large number of the up-regulated genes are involved in energy metabolism. Thus, 'management' of the energy pool (especially ATP) seems to constitute an ethanol stress response and to involve different mechanisms.

Ethanol↗
PubMed2001

Immunohistochemistry of the golden hamster pituitary during chronic administration of diethylstilbestrol: a quantitative analysis using confocal laser scanning microscopy.

The aim of this study was to examine by immunohistochemistry the morphologic changes affecting pituitary cell populations in male Syrian hamsters undergoing chronic exposure (3 days to 9 months) to diethylstilbestrol (DES). Cell proliferation in the hypophysis was monitored by the immunohistochemical demonstration of S-phase cells after pulse labeling with 5-bromo-2'-deoxyuridine. Cell proliferation analysis was combined with the identification of different cell populations by immunostaining with antisera raised against hypophyseal hormones. Sections processed for double-label immunofluorescence were examined by confocal microscopy. In the adenohypophysis, the relative surface occupied by gonadotrophs and thyrotrophs decreased rapidly during the first months of treatment while corticotroph and somatotroph populations remained unaffected. Accordingly, the incidence of S-phase cells in these four cell populations was lower than or similar to control values. In contrast, lactotrophs increased gradually during the first month of exposure to DES to reach a maximum value at 2-4 months. At the beginning, this increase was primarily due to hyperplasia but later on it also involved cellular hypertrophy. Somatomammotrophs did not seem to be involved in this model. In the pars intermedia, the labeling index of melanotrophs rose rapidly to reach values 5-6 times higher than controls. After 4 months, neoplasms originating from the pars intermedia were seen invading both the neuro- and the adenohypophysis. At the end of treatment, the pituitary was markedly enlarged resulting from the development of an adenoma of the pars intermedia.

Adrenocorticotropic Hormone↗
PubMed2001

Immunohistochemical analysis of diethylstilbestrol-induced renal tumors in adult male Syrian hamsters: evidence for relationship to peripheral nerve sheath tumors.

Estrogen-induced Syrian hamster kidney tumors (SHKT) are widely used as experimental models for the study of hormonal and renal carcinogenesis. In order to characterize the direction of differentiation of SHKT, kidney sections of diethylstilbestrol (DES)-treated hamsters (1-11 months) were analyzed by immunohistochemistry using a panel of lineage-specific markers. The first tumorous buds found in animals exposed to DES for 4-6 months exhibited prominent S100, Leu-7, and vimentin immunoreactivities. Immunopositivities for neuron-specific enolase, PGP 9.5, desmin, and glial fibrillary acidic protein were mostly detected in medium-sized and large tumors after prolonged exposure to DES (> 6 months). All neoplasms, irrespective of the size and the duration of treatment, appeared negative for cytokeratin, neurofilaments, synaptophysin, and CD99 antibodies. Western blotting confirmed to a large extent the immunohistochemical observations. The systematic analysis of serial kidney sections by confocal microscopy after double immunostaining for S100 and neurofilaments revealed that early neoplastic buds could stem from S100-positive cells associated with nerves bundles. Altogether, these observations suggest that DES-induced SHKT could be related to malignant peripheral nerve sheath tumor and originate from a yet unidentified precursor cell present in the sheath of peripheral nerves.

Animals↗
PubMed2001

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions.

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25 degrees C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l(-1), respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties.

Alcohols↗
PubMed2001

Expression and role of Bcl-2 in rat blastocysts exposed to high D-glucose.

Bcl-2 mRNA expression was detected in rat blastocysts by in situ hybridization. The distribution of mRNA expression was rather heterogenous, with approximately 2% of high-expressing cells. In vitro exposure to 28 mmol/l D-glucose for 24 h resulted in a significant increase in the proportion of these cells compared with control embryos in either 6 mmol/l D-glucose or 28 mmol/l D+L-glucose. Heterogeneity in the expression of Bcl-2 was also observed at the protein level by immunocytochemistry. Exposure to 28 mmol/l D-glucose significantly increased the incidence of chromatin degradation (karyolysis) and nuclear fragmentation (karyorhexis), two nuclear markers of apoptosis in rat blastocysts. When two different antisense oligodeoxynucleotides designed to block Bcl-2 expression were added to 28 mmol/1 D-glucose, the incidence of karyolysis (but not karyorhexis) was increased compared with embryos in 28 mmol/l D-glucose alone. These data suggest that Bcl-2 is involved in the protective response against the induction of karyolysis in blastocysts on their exposure to high concentrations of D-glucose in vitro, whereas karyorhexis appears to result from the activation of an intracellular pathway that is independent of Bcl-2.

Animals↗
PubMed2001

Contribution of the Belgian school of embryology to the concept of neural induction by the organizer.

Albert BRACHET, founder of the Brussels School of embryology, conducted delicate experiments in which he selectively destroyed zones of the grey crescent with heated needles. This allowed him to observe, in 1923, that the median region of the grey crescent of the blastula is a area of spontaneous differentiation and that this "primary self-differentiation centre" organizes the axial organs in anurans. It is thus fair to say that A. BRACHET contributed significantly to the emergence of the organizer concept. Albert DALCQ and Jean PASTEELS, successors of A. BRACHET, trying to solve the problem of the organizer's determination, proposed their famous quantitative theory of embryonic development resulting in the concept of morphogenetic potential, which increases with the CV concentration, a combination of a cortical constituent C and a vegetal substance V. Jean BRACHET, the younger son of A. BRACHET and one of the founding father of molecular biology and embryology, was soon convinced that the organizer owes its inducing power to a chemical substance. Being the first to suggest the role of RNA in protein synthesis, he first imagined that RNA could be the active substance in induction but became convinced afterwards that the inducer must have a proteic nature. His interest in the molecular aspects of induction stimulated research that was to make chemical embryology molecular.

Animals↗
PubMed2001

A history of mammalian embryological research.

Although Reinier DE GRAAF (1641-1673) can be considered the founder of modern reproductive biology, scientific knowledge of mammalian development did not progress significantly until the XlXth century. Determining contributions to this progress were the discovery of the ovum by Karl von BAER (1792-1876), his meticulous observations of the stages of embryogenesis, and, half a century later, the remarkable descriptions made by Edouard VAN BENEDEN (1845-1910) of egg development in rabbits and bats. Yet mammalian embryology remained a purely descriptive discipline until the second half of the XXth century, when a handful of exceptional scientists (notably including John D. BIGGERS, Ralph BRINSTER, Anne McLAREN, and W. WHITTEN) managed to obtain reproducibly the development of mouse eggs in a chemically defined medium and to transfer the eggs to the uterine horns of pseudopregnant females. Around the same time (1959), M.C. CHANG was the first to obtain a mammal (a rabbit) by in vitro fertilisation, thus opening the way to assisted procreation. This was achieved in our species in 1978, by Robert EDWARDS and Patrick STEPTOE. With these feats, mammalian embryology could at last become causal, as A. BRACHET already in 1912 had hoped it would. New concepts soon emerged from the delicate manipulations performed on mouse eggs by scientists such as A. TARKOWSKI, B. MINTZ, J. MULNARD, and R. GARDNER, concepts such as the oustside-inside hypothesis proposed to explain the determination of the ICM and trophectoderm or the clonal theory of cell determination during development. These new ideas were soon to become the focus of intense study. Other investigators, interested in the synthesis and roles of macromolecules, contributed in the late 1960's most of our knowledge on global trends in gene expression during the first stages of development. As for the many unfruitful attempts to obtain artificial parthenogenetic development in mice, these would lead to the discovery of parental genetic imprinting. In the 1950's, Leroy STEVENS and Barry PIERCE made famous a very rare tumour, the teratocarcinoma. This tumour soon became a model for studying mammalian development, adopted by an increasing number of research groups. It became the source of a first generation of pluripotent cells culturable in vitro: embryonal carcinoma (EC) cells. In the 1980's came the next generation: embryonic stem (ES) cells derived from the ICM of blastocysts, whose advent coincided with that of the first transgenic mice. Then came the era of knockout mice and cloning. Scientists now envisage with enthusiasm applications that were unimaginable just a few years ago. Such is the legacy of those few pioneers of the experimental embryology of mammals who, in the late fifties, were striving to make the wish expressed by A. Brachet in 1912 come true at last.

Animals↗
PubMed2000

Identification of a 49-kDa hydrophobic cell wall mannoprotein present in velum yeast which may be implicated in velum formation.

Analysis of velum-forming yeast cell wall components released by beta-1,3-glucanase treatment were compared with those of a non velum-forming yeast. SDS-PAGE electrophoresis and Western blotting with ConA-peroxidase staining of mannoproteins allowed us to identify a 49-kDa mannoprotein present in the cell wall of the velum-forming yeast and hardly visible in the control. The cell wall nature of this protein was confirmed by labelling with the non-permeable sulfosuccinimydiyl-6-(biotinamido)hexanoate reagent. A partial purification of this mannoprotein by anion exchange HPLC followed by surface hydrophobicity determination revealed that the fraction containing the 49-kDa mannoprotein was the most hydrophobic. Since cell surface hydrophobicity plays an important role in aggregate formation, it is likely that this mannoprotein is involved in velum formation.

Biotinylation↗
PubMed2000

Respective roles of protein tyrosine kinases and protein kinases C in the upregulation of beta-catenin distribution, and compaction in mouse preimplantation embryos: a pharmacological approach.

The cellular distribution of beta-catenin was determined by western blotting and laser confocal scanning microscopy in both control and pharmacologically-manipulated mouse preimplantation embryos. Most of the stored maternal beta-catenin is Triton X-100-extractable and distributed throughout the cytoplasm. In 2-cell stage embryos, the remaining molecules are concentrated in regions of cell contact and, to a lesser extent, at non apposed surfaces. Association of beta-catenin with the cortex of non apposed membranes decreases as cleavage proceeds, and is lost at compaction. In contrast to the rapid cross-linking of cell surfaces induced by wheat germ agglutinin, the diacylglyceride-induced compaction-like adhesion of 2- and 4-cell embryos correlates with complete restriction of beta-catenin to the apposing membranes. On the contrary, tyrphostin B46, a specific protein tyrosine kinase inhibitor, fails to induce both premature beta-catenin relocalisation and compaction. In addition, we show that orthovanadate induces a dramatic increase in the level of phosphotyrosine labelling of cell-cell junctions in compacted 8-cell stage embryos without inducing their decompaction. However, most of these orthovanadate tyrosine-phosphorylated proteins are detergent-soluble, while beta-catenin restricted to the apposing membranes is not. In conclusion, our results confirm that diacylglycerol-dependent kinases upregulate both beta-catenin redistribution and compaction, and indicate that neither tyrosine kinases, nor tyrosine phosphatases are critical for the proper onset of compaction which seems, in addition, not causally linked to tyrosine dephosphorylation of beta-catenin.

Animals↗
PubMed2000

Effect of genistein on the temporal coordination of cleavage and compaction in mouse preimplantation embryos.

Initially, we investigated the effect of genistein, an inhibitor of protein tyrosine kinases, on compaction of the mouse embryo since tyrosine phosphorylation of the cadherin-catenins complex was suggested to down-regulate its adhesive function. Genistein prevented cleavage from the 2- to the 4-cell stage in a concentration-dependent manner. The next cleavage is inhibited at all concentrations used. Time course of intercellular flattening is however identical for both control 8-cell embryos and 4-cell arrested embryos. This confirms that compaction takes place according to a biological clock that does not depend on completion of the third cell cycle. Our results also suggest that, since, in contrast to genistein, protein kinases C modulators are known to cause a premature compaction, diacylglycerol-dependent kinases but not protein tyrosine kinases might be upregulators of compaction.

Actins↗
PubMed2000

Effect of genistein alone and in combination with okadaic acid on the cell cycle resumption of mouse oocytes.

Our biopharmacological approach suggests that the now well-documented inhibitory effects of genistein on the maturation of mammalian oocytes do not seem to be related to its effect on tyrosine kinases. Indeed, we show that both tyrphostin B46 and Lavendustin A, two selective inhibitors of protein tyrosine kinases, fail to inhibit meiosis reinitiation. According to recent findings, the G2/M arrest induced by genistein could be due to inhibition of the kinase activity of cdc2. We were therefore mainly interested in dissecting the cytological effects of genistein on mouse primary and secondary oocytes. Genistein exerts the same cytological effects as IBMX on primary oocytes: their germinal vesicle is maintained in a central position, the cytoplasmic microtubule network is stabilized, the central GV immobilization is overcome by demecolcine and they complete normal maturation after their transfer to culture medium. The GV-arresting activity of genistein is also bypassed by OA but combination of both drugs results in a dramatic reorganization of the cytoskeleton leading to a huge membrane bulging, which is quite different to apoptotic-related blebbing. MAP Kinase activation is correlated with meiosis reinitiation. When applied after GVBD has taken place, genistein does not inhibit MAPK activation, metaphase spindle formation and metaphase-to-anaphase transition, but prevents the barrel-shaped MI spindle from undergoing its peripheral migration and the oocytes from extruding their first polar body. It may thus be concluded that the checkpoint control for anaphase onset is unaffected by the drug. On the contrary, our results suggest that spindle anaphase A to spindle anaphase B transition, spindle degradation, mid-body formation and cytokinesis are triggered by a genistein-sensitive mechanism that might be a mid-anaphase checkpoint. Finally, we confirm that genistein induces transition to interphase in metaphase II oocytes but never induces cortical granule exocytosis, the cytoplasmic hallmark of activation.

Animals↗
PubMed1999

Characterization of a coiled coil protein present in the basal body of Trypanosoma brucei.

TBBC (for Trypanosoma brucei basal body component) is a unique gene transcribed in a 4.8 kb mRNA encoding a 1,410 amino acid protein that consists almost entirely of a coiled coil structure. This protein appeared to localize in the basal body, with an accessory presence at the posterior end of the cell, the nucleus and over the flagellum. Since the two other known components of the trypanosome basal body are (gamma)-tubulin and an uncharacterized component termed BBA4 we performed double immunofluorescence experiments with anti-TBBC and either anti-BBA4 or anti-(gamma)-tubulin antibodies. These three components did not colocalize but were very closely associated, BBA4 being the most proximal to the kinetoplast DNA. Anti-TBBC antibodies detected a 170 kDa protein in western blots of total HeLa cell extracts. Moreover, these antibodies stained the centriole of HeLa and COS cells as well as the centriole of mouse spermatozoa, indicating that a TBBC-like centriolar component has been conserved during the evolution of eukaryotes.

Amino Acid Sequence↗
PubMed1998

A study of the temporary adhesion of the podia in the sea star asterias rubens (Echinodermata, asteroidea) through their footprints

Sea stars are able to make firm but temporary attachments to various substrata owing to secretions released by their podia. A duo-glandular model has been proposed in which an adhesive material is released by two types of non-ciliated secretory (NCS1 and NCS2) cells and a de-adhesive material is released by ciliated secretory (CS) cells. The chemical composition of these materials and the way in which they function have been investigated by studying the adhesive footprints left by the asteroids each time they adhere to a substratum. The footprints of Asterias rubens consist of a sponge-like material deposited as a thin layer on the substratum. Inorganic residues apart, this material is made up mainly of proteins and carbohydrates. The protein moiety contains significant amounts of both charged (especially acidic) and uncharged polar residues as well as half-cystine. The carbohydrate moiety is also acidic, comprising both uronic acids and sulphate groups. Polyclonal antibodies have been raised against footprint material and were used to locate the origin of footprint constituents in the podia. Extensive immunoreactivity was detected in the secretory granules of both NCS1 and NCS2 cells, suggesting that their secretions together make up the bulk of the adhesive material. No immunoreactivity was detected in the secretory granules of CS cells, and the only other structure strongly labelled was the outermost layer of the cuticle, the fuzzy coat. This pattern of immunoreactivity suggests that the secretions of CS cells are not incorporated into the footprints, but instead might function to jettison the fuzzy coat, thereby allowing the podium to detach.

Journal Article↗
PubMed1994

Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata.

The lipid composition of a strain of each of two yeasts, Saccharomyces cerevisiae and Kloeckera apiculata, with different ethanol tolerances, was determined for cells grown with or without added ethanol. An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance. The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed. We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol:phospholipid and sterol:protein ratios and an increase in unsaturation index.

Bacterial Proteins↗
PubMed1994

Ethanol adaptation mechanisms in Saccharomyces cerevisiae.

Determination of the membrane lipid composition of Saccharomyces cerevisiae revealed an increase in the unsaturation index, qualitative and quantitative changes in sterol content and an alteration of the activity of the plasma membrane ATPase when cells were pre-adapted to ethanol. All these changes may constitute different adaptation mechanisms which allow the cell to cope with ethanol stress. The importance of the lipid environment on the plasma membrane ATPase activity is also discussed.

Adaptation, Physiological↗