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H Alexandre

Publications and source records attributed to H Alexandre.

At least 37 records · Page 2Linked to original sources
PubMed1992

Control of microtubule nucleating activity in the cytoplasm of maturing mouse oocytes.

Taxol, a drug which promotes microtubule assembly, was used to assess the microtubule nucleating activity of pericentriolar material (PCM) in mouse oocytes prevented from undergoing germinal vesicle breakdown (GVBD), compared with oocytes allowed to proceed normally through GVBD and also in nucleate and anucleate oocyte fragments. Both immunofluorescence staining and ultrastructural analysis reveal that taxol induces aster formation in the cortex of oocytes undergoing GVBD, while formation of a continuous sheet of microtubule bundles parallel to the membrane is induced in metabolically GV-arrested oocytes. Since taxol also induces the formation of asters in anucleate as well as in nucleate oocyte fragments, provided they are not treated with activators of protein kinases A or C, it is concluded that microtubule nucleating activity is related to the acquisition of Maturation Promoting Factor (MPF) and does not require mixing between the nucleoplasm and cytoplasm.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Pleiotropic effect of okadaic acid on maturing mouse oocytes.

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Involvement of microtubules and microfilaments in the control of the nuclear movement during maturation of mouse oocyte.

We confirm that the centrifugal migration of the chromosomes in maturing mouse oocytes depends on a microfilament-mediated process. We investigated the role of the cytoskeleton in the germinal vesicle (GV) behavior of oocytes prevented from resuming meiosis by either activators of protein kinase A or activators of protein kinase C. A time-lapse microcinematography study demonstrates that GV immobilization by isobutylmethylxanthine (IBMX) is overcome by colcemid (COL), nocodazole (NOC), and taxol and that cytochalasin D (CCD) reversibly immobilizes the GV of oocytes treated with either IBMX + COL (or NOC) or 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, known to allow a programmed GV cortical translocation. An immunofluorescence analysis shows that the disorganization of a perinuclear microtubule network is the very first cytological clue of maturation. IBMX promotes its persistence while NOC, COL, and taxol induce its immediate disappearance. It is concluded that elements of the cytoplasmic microtubular complex (CMTC) are passively involved in the control of the setting up of a "centrifugal displacement property" (CDP) by counteracting a motive force provided by the microfilament cytoskeleton. Finally, TPA induces a clearcut reorganization instead of a total disorganization of the CMTC. This reorganization is, however, sufficient to allow the microfilaments to drive the GV displacement.

Actin Cytoskeleton↗
PubMed1988

Time-lapse cinematography study of the germinal vesicle behaviour in mouse primary oocytes treated with activators of protein kinases A and C.

A passive erratic movement of the germinal vesicle (GV), already visible in small incompetent oocytes, is followed by an active scalloping of the nuclear membrane soon before GV breakdown (GVBD) in cultured competent oocytes. Maturation can be inhibited by activators of protein kinase A (PK-A) and protein kinase C (PK-C). Our time-lapse cinematography analysis allowed us to describe an unexpected behaviour of the GV when PK-C, but not PK-A, is activated: GV undergoes a displacement toward the cortex according to the same biological clock which triggers the programmed translocation of the spindle in control oocytes. It is concluded that, when oocytes become committed to undergo maturation, the cytoplasm acquires a PK-A-controlled "centrifugal displacement property" which is not restricted to the spindle.

Animals↗
PubMed1988

The influence of phenobarbital and butylated hydroxytoluene on the ploidy rate in rat hepatocarcinogenesis.

The effect of the 'promoters' phenobarbital (PB) and butylated hydroxytoluene (BHT) on the ploidy changes during hepatocarcinogenesis in rats was compared in a densitometric analysis of Feulgen-stained nuclei on paraffin-embedded tissue slices. The triphasic Gerlans protocol for liver-cancer induction was applied. Initiation with a single dose of diethylnitrosamine (DEN), and selection with 2-acetylamino-fluorene (2-AAF) combined with a proliferative stimulus (CCl4 administration), was followed by a treatment with PB or BHT for periods up to 22 weeks. Control animals received no treatment after the initiation and selection procedure. Despite intra- and inter-individual variations, an increase in the amount of 2N nuclei is found in the putative preneoplastic lesions of animals that received initiation and selection (I-S) and 3 weeks basal diet (BD). When the diet is supplemented with PB (after I-S), the increase of diploid nuclei starts earlier. At the time carcinoma arise (22 weeks PB treatment) a decrease in the frequency of 2N nuclei is found. BHT-treated animals which develop no carcinoma within the considered timespan, show a clear increased amount of 2N nuclei in the precancerous lesions only after 14 weeks treatment. It seems that there is a positive correlation between the outgrowth of putative preneoplastic foci and nodules in rat liver and an increase of diploid nuclei in these lesions. PB, as promoter used after initiation and selection, speeds up the development of carcinoma in rat liver, and therefore also the shift to diploidization in these rats starts earlier in comparison with I-S-treated rats. Although BHT does not promote liver carcinogenesis, an increase of diploid nuclei is also observed here during lesion formation. It may, therefore, be concluded that the phenomenon of diploidization is closely linked to and probably necessary for preneoplastic development, but that it is not an absolute indicator for neoplastic transformation.

Animals↗
PubMed1987

Relationship between frequencies of univalents and meiotic segregation in different mouse strains and their hybrids.

Although univalents in diakinesis are often used as an estimator for chromosome mis-segregation during meiosis, no clear-cut relationship was demonstrated between both phenomena. In this study, the frequencies of autosomal and gonosomal univalents in diakinesis were related to the frequencies of aneuploid metaphase-II gonocytes during spermatogenesis and oogenesis of different mouse strains and their hybrids (inbred strains: DBA/2J, C57Bl; outbred strain; Swiss, inbred x outbred hybrids: Swiss X C57Bl, C57Bl X Swiss, inbred X inbred hybrids: DBA/2J X C57Bl, C57Bl X DBA/2J). As far as the frequencies of univalents are concerned, they were shown to be strain-dependent and similar in both sexes. Moreover, there is a high non-disjunction rate of DBA males and PMSG-HCG-primed DBA females. Aneuploidy in metaphase II is also strain-dependent but different in both sexes; in the male, a clear decrease of aneuploidy frequencies is observed as compared to the frequency of univalents. This decrease does not occur in females.

Aneuploidy↗
PubMed1987

Changes in nucleolar transcriptional activity and nuclear DNA content during the first steps of rat hepatocarcinogenesis.

Two complementary genetic parameters were followed in liver parenchymal cells during the first steps of rat hepatocarcinogenesis: the expression of nucleolar genes estimated from their silver stainability and the nuclear DNA content determined after Feulgen staining. Putative preneoplastic lesions as foci and nodules were induced by the triphasic 'Gerlans protocol'. Initiation with a single dose of diethylnitrosamine (DEN) was followed by a selection of initiated cells with 2-acetylaminofluorene (2-AAF) in combination with a single necrogenic dose of CCl4 as a proliferative stimulus. Finally after 1 week of normal diet, the animals were treated or not with phenobarbital (PB) for periods up to 2 months. Serial sections were analysed after silver staining (AgNO3), methyl-green--pyronin staining (Unna-Brachet) and Feulgen staining with densitometric and morphometric methods. Silver staining, which is known to stain an acidic protein associated with rRNA synthesis, increased gradually with the duration of the PB treatment. Morphometry revealed an increase in both nucleolar and nuclear volume; the fraction of nuclei with one nucleolus also increased. These results seem to point towards an increase of nucleolar activity in the early steps of PB promotion. Moreover, this shift cannot be ascribed to an increase of DNA content. Indeed, a parallel study on neighbouring sections stained with Feulgen revealed a shift towards a population of diploid nuclei, in contrast to normal liver cells, which are mostly tetraploid. The observed diploidisation may therefore provide a functional advantage for the expansion of putative preneoplastic cells.

2-Acetylaminofluorene↗
PubMed1986

Carbon source assimilation and fermentation tests: study of 77 animal Pasteurella strains by a microtechnique.

77 strains of Pasteurella from various animal species were tested using the API micromethod for their ability to assimilate 147 carbon substrates and ferment 49 carbohydrates. Typical profiles of P. multocida and P. haemolytica were determined. This micromethod should prove useful in establishing the biotypes of Pasteurella species and in studying their epidemiology and pathogenicity.

Animals↗
PubMed1986

Nosocomial infection and colonization by Klebsiella trevisanii.

During an 18-month period we identified two cases of septicemia and 24 examples of colonization of humans by Klebsiella trevisanii. Organisms were identified using the API 20EC and API 147 assimilation galleries. Of 147 clinical isolates initially identified as K. oxytoca, 18% were found to be K. trevisanii. Tracheal aspirate was the most common source of the organism. An extensive environmental sampling survey in the rooms of 12 colonized patients revealed a possible reservoir of the organism only once (a face cloth).

Aged↗
PubMed1985

Involvement of DNA-polymerase activities in mouse-blastocyst differentiation in vitro.

Full-grown blastocysts were cultured for 70-92 h in Dulbecco's modified Eagle's medium supplemented with fetal calf serum. They were treated with (1) aphidicolin, a specific inhibitor of eucaryotic DNA polymerase alpha, (2) dideoxythymidine (d2Thd), the precursor of dideoxythymidine triphosphate (d2TTP), a specific inhibitor of DNA polymerase beta and (3) d2TTP itself. Cytophotometric measurements of both the DNA content and the nuclear areas were performed. The results show firstly that trophectoderm differentiation into primary giant trophoblast cells is already irreversibly programmed at the blastocyst stage and does not depend on any DNA replication cycle from that stage onwards. Secondly, the typical enlargement of the trophoblastic nuclei, which occurs at the peri-implantation period, is not related to a proportional increase in DNA content. Thirdly, the onset and progress of DNA endoreduplication in trophoblastic cells is carried out by DNA polymerase alpha during the first part of gestation instead of DNA polymerase beta, as has previously been shown to be the case for mid-gestation rat trophoblast 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

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↗
PubMed1982

[Effect of specific inhibition of DNA replication by aphidicolin on primary differentiation of preimplantation mouse embryos].

Aphidicolin inhibits cleavage at every stage of preimplantation of Mouse embryos without affecting compaction. Blastulation is initiated only if the treatment is applied at the late 8-cell stage. It is concluded that the S phase of the 4th cell cycle might be critical for the commitment of the embryo for blastocyst formation. The 4th cycle thus corresponds to a "quantal cell cycle". Moreover, aphidicolin induces premature cavitation which occurs even when the embryos have not yet initiated cytokinesis.

Animals↗
PubMed1982

Studies on differentiation without cleavage in Chaetopterus. Requirement for a definite number of DNA replication cycles shown by aphidicolin pulses.

Aphidicolin, a powerful inhibitor of nuclear DNA replication, has been used to establish the level of polyploidisation required for the realization of the late morphogenetic events (segregation, pseudogastrulation and ciliation) of differentiation without cleavage in Chaetopterus-activated eggs. A parallel has been drawn between cytophotometric estimations of the DNA content and cytoplasmic differentiation in eggs treated with aphidicolin under different experimental conditions: either pulses with aphidicolin were followed by culture in sea water or the eggs were treated with aphidicolin after development had taken place in sea water for various lengths of time. The results suggest that a 'quantal' monasterial cycle might take place 3 h after activation, corresponding presumably to the fourth or fifth replication cycle. Moreover, early DNA replication seems to be more important for morphogenesis than late DNA replication.

Animals↗
PubMed1981

Studies on differentiation without cleavage in Chaetopterus: effects of inhibition of DNA synthesis with aphidicolin.

The effects of aphidicolin - a powerful inhibitor of DNA polymerase alpha and of DNA replication - on normal development and on differentiation without cleavage of Chaetopterus eggs have been studied with cytological, cytochemical, and biochemical methods. The experiments show that the initial period of pseudocleavage can take place in the absence of nuclear DNA synthesis, but further development (segregation, hatching, ciliation) requires DNA synthesis. However ciliated unicellular larvae can be obtained under conditions where the DNA content of the embryos in only 40% of the controls. In fertilized eggs, aphidicolin immediately stops cleavage. The significance of these results is discussed.

Animals↗
PubMed1980

Studies on differentiation without cleavage in Chaetopterus variopedatus: requirements for nucleic acid and protein synthesis.

Activation of unfertilized Chaetopterus eggs by treatment with an excess of KCl may lead to the production of unicellular ciliated larvae (Lillie's differentiation without cleavage). The effects of a number of inhibitors of protein (puromycin, cycloheximide, emetin), RNA (actinomycin D), and DNA (hydroxyurea) synthesis of differentiation without cleavage and on normal development have been studied in Chaetopterus. Incorporation of radioactive leucine, uridine, and thymine has been followed by biochemical methods and by autoradiography. The DNA content of the large polyploid nucleus has been estimated by cytophotometry. The initial pseudocleavage period of differentiation without cleavage is characterized by a burst in DNA and protein synthesis; the inhibitors have little or no effect on this burst and on pseudocleavage itself. Protein and DNA synthesis levels off during the following phase (segregation), but the inhibitors become more effective. RNA synthesis is almost linear for 20 h. These results are compared with those obtained on eggs from other species.

Animals↗