Centrosome dynamics during the meiotic progression in the mouse oocyte.
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Animals↗
Biomedical subjects
S Baatout
Publications and source records attributed to S Baatout.
Methods to measure cell proliferation in haematology.
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Animals↗
Thrombopoietic effects and toxicity of interleukin-6 in patients with ovarian cancer before and after chemotherapy: a multicentric placebo-controlled, randomized phase Ib study.
Recombinant human interleukin-6 (IL-6) has previously been shown to increase platelet counts in normal and sublethally irradiated mice, dogs, and primates. To assess its tolerance and efficacy in clinical use, we performed a randomized phase Ib study in patients with ovarian carcinoma. IL-6 was administered during an initial 7-day cycle before any chemotherapy. Beginning 7 days later, six cycles of chemotherapy containing carboplatin were administered every 3 weeks. During chemotherapy cycles 2 to 6, IL-6 was administered from day 4 through day 17 at escalating dose levels from 0.5 to 10 micrograms/kg/d. At each level, three patients received IL-6 and one patient received a placebo. During the prechemotherapy cycle of IL-6, a dose-dependent increase in platelet count was observed from day 12 to 15 and was maximal on day 15 (r = .77; P < .01). The median ploidy of bone marrow megakaryocytes shifted from 16 N to 32 N after 7 days of the initial prechemotherapy IL-6 administration. Dose-dependent increases in C-reactive protein (CRP) and fibrinogen levels were observed on day 8 (P < .0001 for both). A significant decrease in hemoglobin level occured rapidly after initiation of IL-6 therapy and was maximal on day 8 (P < .001). When given after chemotherapy, IL-6 accelerated platelet recovery after chemotherapy cycles 2 to 6. Postponements of scheduled chemotherapy due to thrombocytopenia were less frequent in patients treated with IL-6. No difference in either neutrophils or peripheral blood progenitor assays was observed during or after IL-6 treatment. Toxicity of IL-6 appeared mild and was not dose-limiting up to 10 micrograms/kg/d. Systemic symptoms such as fever, headache, and myalgia were the main side effects and were easily relieved by acetaminophen administration. No biologic toxicity was observed. The data indicate that IL-6 is a well-tolerated cytokine and capable of accelerating platelet recovery in patients receiving chemotherapy.
Acute-Phase Proteins↗
Thrombopoietin. A review.
The regulator of platelet count is believed to be thrombopoietin (mpl ligand). It is thought to act on the megakaryocytic lineage affecting the proliferation of committed cells, the maturation of megakaryocytes and the blood platelet levels by activation of its receptor (the c-mpl). Recently, it has been purified and sequenced. Thrombopoietin offers potential applications for treatment of thrombocytopenia.
Animals↗
Histone H1 kinase activity in one-cell mouse embryos blocked in the G2 phase by X-irradiation.
The activation of the p34cdc2/cyclin B complex is responsible for driving the cell cycle from the G2- to the M-phase. To investigate the effects of irradiation on the activity of the p34cdc2/cyclin B complex in preimplantation embryos, we irradiated one-cell mouse embryos with 2.5 Gy of X-rays at the early pronuclear stage, and measured the fluctuations of histone H1 kinase activity (a biochemical indicator of the kinase activity of the p34cdc2) at different times during the radiation-induced G2-arrest. BALB/c embryos were chosen for these experiments, since earlier results obtained in our laboratory had shown that such a treatment induces a G2-arrest of about 20 hours in more than 90% of the embryos. Our data showed that histone H1 kinase activity of irradiated embryos remained at a very low level during the period of G2-arrest. The level of activity found during late division of the G2-arrested embryos was also significantly lower in comparison with that of control embryos or irradiated embryos dividing without delay. All together, our results suggest that a) low levels of histone H1 kinase activity are sufficient for the division of one-cell embryos, b) there could be a link between the levels of histone H1 kinase activity in mitosis and the health status of the embryo.
Animals↗
Interaction between protein kinase C and actin in megakaryocyte polyploidization.
Megakaryocytes undergo a peculiar and irreversible program by which they become polyploid through repeated cycles of DNA synthesis without concomitant cell division. In order to study the possible concomitant role of protein kinase C and actin in megakaryocyte polyploidization, three cell lines (DAMI, HEL and K562), expressing some properties of the megakaryocytic lineage and known to differentiate into the megakaryocytic pathway in the presence of phorbol esters, were cultivated in the presence of phorbol myristate acetate alone (PMA, 5 x 10(-9) M, activator of protein kinase C, PKC) or concomitantly with cytochalasin B (2 micrograms/ml, inhibitor of actin polymerization). We have previously shown that DAMI, HEL and K562 cells in which actin polymerization was inhibited by cytochalasin B, acquired megakaryocytic properties in the way that they became polyploid, acquired a megakaryocytic phenotype and arrested proliferation (4). After four days of culture in the presence of PMA and cytochalasin B, the number of polyploid cells (estimated by flow cytometry) increased in comparison with control or PMA-treated cells. However, it was lower than in cytochalasin B-treated cells. Indeed, control cells predominantly diploid (2N) became polyploid with the appearance of 8N, 16N and 32N cells after addition of PMA, cytochalasin B or PMA + cytochalasin B. The endomitotic index (EI, as described in 5) which corresponds to the mean of (¿log2 DNA content expressed in N¿-1) was 0.5 +/- 0.1, 0.7 +/- 0.1 and 0.3 +/- 0.1 in control DAMI, HEL and K562 cells, respectively. The EI increased to 0.9 +/- 0.2; 1.0 +/- 0.2 and 0.4 + 0.1 in cells treated with PMA and to 1.6 +/- 0.3; 1.4 +/- 0, and 0.9 +/- 0.2 when PMA was added concomitantly to cytochalasin B. Total DNA estimated from the cell content and the percentage of cells present at each ploidy stage did not change in cytochalasin B-treated cells in comparison to control conditions. However, treatment of DAMI, HEL and K562 cells with PMA alone or concomitantly with cytochalasin B revealed that the total DNA content significantly decreased in these conditions. At last, treatment of the three cell lines with PMA alone or concomitantly with cytochalasin B for 4 days caused a complete inhibition of proliferation. In conclusion, the concomitant addition of PMA and cytochalasin B to the three cell lines lead to an augmentation of cell ploidy and to a cessation of proliferation. However, we did not observe any synergistic effect of the two compounds. The possible interaction between actin and protein kinase C is discussed in the paper.
Actins↗
Protein content and number of nucleolar organizer regions are enhanced during phorbol ester-induced differentiation of cultured human megakaryocytic cells.
In order to investigate the protein synthesis in megakaryocyte polyploidization, phorbol myristate acetate (PMA, 5 x 10(-9) M), a differentiation marker known to induce megakaryocyte polyploidization, was added to human megakaryocytic cell lines (DAMI, HEL and K562) and the expression of platelet/megakaryocytic integrins, the numbers of nucleolar organizer regions (AgNORs) and the total protein content were estimated. Following exposure of PMA, the expression of the platelet membrane glycoprotein GPIIIa and thrombospondin and transferrin receptors was augmented in the three cell lines. The number of AgNORs shifted from 16.4 +/- 4.3, 24.4 +/- 2.5 and 13.6 +/- 3.1 for unstimulated cells to 20.0 +/- 5.3, 38.7 +/- 7.9 and 16.8 +/- 2.3 for PMA-treated DAMI, HEL and K562 cells, respectively. Furthermore, after treatment with PMA, the numbers of AgNORs clusters or nucleoles increased significantly to 179%, 238% and 154% of controls in DAMI, HEL and K562 cell lines, respectively. Finally, addition of PMA culture for four days, significantly increased the protein contents to 153%, 171% and 254% of controls for DAMI, HEL and K562 cell lines, respectively (p < 0.05 by t-test). In conclusion, the increase in the total protein content and in the number of AgNORs by PMA, suggests that PMA-induced-megakaryocyte polyploidization occurs by enhanced protein production.
Cell Differentiation↗
The G and F contents in megakaryocyte cell lines after stimulation with phorbol myristate acetate.
Megakaryocyte polyploidization responds to platelet demand and results from the lack of cytoplasmic separation while the nucleus keeps dividing. In order to investigate the role of actin in the megakaryocyte polyploidization, phorbol myristate acetate (PMA, 5 x 10(-9) M), a differentiation marker known to induce megakaryocyte polyploidization, was added to human megakaryocytic cell lines (DAMI and HEL) and G, F and total actins were estimated by DNase I inhibition. After four days of culture in the presence of PMA, G actin contents in pg per 10(6) cells were 13.0 pg +/- 2.8 and 1.0 pg +/- 0.1 for unstimulated DAMI and HEL cells. F actin contents per 10(6) cells were 5.8 pg +/- 1.5 and 0.1 pg +/- 0.0 for DAMI and HEL cells. Addition of PMA for four days to culture significantly increased G actin contents (235% and 268% of controls) and F actin contents (234% and 394%), for DAMI and HEL cell lines, respectively (p < 0.05 by t-test). In contrast, G/F actin ratio was not affected (p < 0.05 by t-test) by PMA. DAMI cells from each ploidy classes were then sorted on an ELITE Coulter and assayed for actin content. While total actin, G actin and F actin per cell increased in polyploid cells cultured with PMA, there was a reduction in G, F and total actin contents per diploid equivalent when cells became polyploid. In conclusion, megakaryocyte polyploidization of these cell lines is not related to an unbalance between G and F actins but would be rather due at least partly to a defect in total actin production that could lead to a prevention of the formation of the constriction ring in telophase.
Actins↗
Histone H1 kinase activity in ovulated oocytes.
The level of kinase activity of cdkl is known to be high during metaphase of the two meioses. In this experiment, histone H1 kinase activity (which is known to reflect cdk1 activity) was assayed in BALB/c mouse ovulated oocytes at various timepoints after ovulation. Histone H1 kinase activity in ovulated oocytes was stable up to 37 hours after ovulation. After that time, histone H1 kinase activity significantly decreased suggesting that cdkl might be degraded after this period of time if the ovulated oocyte is not fertilised.
Animals↗
Human megakaryocyte polyploidization is associated with a decrease in GPIIIA expression.
Megakaryocytes are platelet forming cells and are characterized by polyploidization, a phenomenon by which nuclear division occurs without corresponding cytoplasmic separation. Among the markers allowing to identify megakaryocytes, glycoprotein (GP) IIIa with GPIb and GPIIb are the most important. Using GPIIIa as a marker to recognize megakaryocytes in the bone marrow, we have estimated GPIIIa expression by flow cytometry in megakaryocyte populations from normal individuals and from patients with chronic myelogenous leukemia, immune thrombocytopenic purpura or polycythemia vera. We showed that the expression of GPIIIa is decreasing during megakaryocyte polyploidization in normal and pathological situations.
Antigens, CD↗
Cytometric methods to analyze radiation effects.
Here, we review the most relevant cytometric methods currently used to analyze ionizing radiation effects. Particular interest has been devoted to the following methods: chromosomal aberrations, micronucleus assay, fluorescence in situ hybridization chromosome painting, comet assay, comet-FISH assay as well as flow cytometry.
Animals↗
Space flight effects on bacterial physiology.
The study of bacterial behavior under space flight conditions is highly important for the early detection of changes in bacterial communities and bacteria with medical, environmental, or life support consequences for survival of the crew in closed space environments. Although many species of prokaryotes have been studied in ground simulation facilities or have been flown in space flights, at present only few hard research data are available to predict the effects of cosmic radiation, microgravity, vibration and hypervelocity on microbial behavior in space flight. The results that are available tend to be fragmentary and often lack a classical, controlled experimental context to interpret them. Thus, many basic questions concerning the effects of space on microbial behavior have yet to be resolved.
Bacteria↗
Physiological changes induced in four bacterial strains following oxidative stress.
In order to study the behaviour and resistance of bacteria under extreme conditions, physiological changes associated with oxidative stress were monitored using flow cytometry. The study was conducted to assess the maintenance of membrane integrity and potential as well as the esterase activity, the intracellular pH and the production of superoxide anions in four bacterial strains (Ralstonia metallidurans, Escherichia coli, Shewanella oneidensis and Deinococcus radiodurans). The strains were chosen for their potential usefulness in bioremediation. Suspensions of R. metallidurans, E. coli, S. oneidensis and D. radiodurans were submitted to 1 h oxidative stress (H2O2 at various concentrations from 0 to 880 mM). Cell membrane permeability (propidium iodide) and potential (rhodamine-123, 3,3'-dihexyloxacarbocyanine iodide), intracellular esterase activity (fluorescein diacetate), intracellular reactive oxygen species concentration (hydroethidine) and intracellular pH (carboxyflurorescein diacetate succinimidyl ester (5(6)) were monitored to evaluate the physiological state and the overall fitness of individual bacterial cells under oxidative stress. The four bacterial strains exhibited varying sensitivities towards H2O2. However, for all bacterial strains, some physiological damage could already be observed from 13.25 mM H2O2 onwards, in particular with regard to their membrane permeability. Depending on the bacterial strains, moderate to high physiological damage could be observed between 13.25 mM and 220 mM H2O2. Membrane potential, esterase activity, intracellular pH and production of superoxide anion production were considerably modified at high H2O2 concentrations in all four strains. In conclusion, we show that a range of significant physiological alterations occurs when bacteria are challenged with H2O2 and fluorescent staining methods coupled with flow cytometry are useful for monitoring the changes induced not only by oxidative stress but also by other stresses like temperature, radiation, pressure, pH, etc....
Bacteria↗
Endothelial differentiation using Matrigel (review).
Different extracellular matrices have helped in the study of angiogenesis. One, Matrigel (an extracellular matrix prepared from Engelbreth-Holm-Swarm murine sarcoma), induces endothelial cells to differentiate into blood capillaries. This interpretative review attempts to discuss the recent advances concerning the study of endothelial differentiation with Matrigel in terms of scientific relevance.
Amino Acid Sequence↗
Matrigel: a useful tool to study endothelial differentiation.
Matrigel is a matrix of a mouse basement membrane neoplasm. It represents a complex mixture of basement membrane proteins including laminin, type IV collagen, entactin/nitrogen and proteoheparan sulfate, but it also contains growth factors. Matrigel induces endothelial cells to differentiate as evidenced by both the morphologic changes and by the reduction in proliferation and, therefore, offers a convenient model to study biochemical and molecular events associated with angiogenesis. Further, Matrigel permits to study the roles of the extracellular matrix in angiogenesis.
Amino Acid Sequence↗
Permeafix: a useful tool to detect antigens and DNA in flow cytometry.
A new product called Permeafix has been developed for gentle fixation of cells and permeability of their membranes. This product is useful for the staining of intracellular antigens and parallel quantification of DNA content simultaneously with cell surface staining.
Antigens↗
Inhibition of actin polymerization by cytochalasin B induces polyploidization and increases the number of nucleolar organizer regions in human megakaryocyte cell lines.
Megakaryocyte polyploidization is an advantageous and regulated mechanism which leads to an increase in platelet production. In megakaryocyte cell lines, polyploidization can be obtained by using cytochalasin B, an inhibitor of the actin polymerization. The Nucleolar Organizer Regions (AgNORs) are parts of nucleolar DNA transcribed into ribosomal RNA. They are detected by silver staining technique and their number is proportional to protein synthesis. In order to estimate protein synthesis in polyploidizing megakaryocytes, AgNORs were measured in three cell lines with megakaryocyte properties (DAMI, HEL and K562) after a 4-day culture in the presence or absence of cytochalasin B, an inhibitor of the actin polymerization. The mean number of AgNORs per cell was 16.4 +/- 4.3 (m +/- SEM); 24.4 +/- 2.5 and 13.6 +/- 3.1 for DAMI, HEL and K-562 cell lines, respectively. The addition of cytochalasin B (2 micrograms/ml) increased significantly the number of AgNORs per cell (DAMI: 437%, HEL: 384% and K-562: 345% of controls, p < 0.05 by t-test). Moreover, the numbers of nucleoles per cell after addition of cytochalasin B were augmented significantly (DAMI: 258%, HEL: 271% and K-562: 264% of controls, p < 0.05 by t-test). The total protein content estimated by Bradford's method increased significantly to 938%, 326% and 388% of controls in DAMI, HEL and K562, respectively (p < 0.05 by t-test) in cells where actin was inhibited by cytochalasin B. In the presence of cytochalasin B, the endomitotic index (EI) [mean of (log2 DNA content expressed in N) 1] measured by flow cytometry increased to 368%, 207% and 538%, for DAMI, HEL and K-562 cell lines, respectively (p < 0.05 by t-test) after treatment with cytochalasin B. In contrast, the number of AgNORs per unit of DNA (EI) and the total protein content per unit of DNA did not change for DAMI, HEL and K-562 cell lines (p < 0.05 by t-test) after treatment with cytochalasin B. In conclusion, the increase in the number of the Nucleolar Organizer Regions by an agent known to stimulate polyploidization of megakaryocytic cell lines suggests that polyploidization occurs by enhanced protein production proportionally to DNA synthesis.
Actins↗
Inhibition of tubulin polymerization in megakaryocyte cell lines leads to polyploidization which affects the metabolism of actin.
Megakaryocyte polyploidization responds to platelet demand and results from the lack of cytoplasmic separation while the nucleus keeps dividing. In normal telophase, the plane of the actin constriction ring is determined by the tubulin spindle. In order to investigate the role of tubulin in the megakaryocyte polyploidization, two cell lines with megakaryocyte properties (DAMI and HEL) were incubated for 4 days in the presence or absence of colchicine (10 ng/ml), an inhibitor of the tubulin spindle. As compared to control conditions, cell cultured in the presence of colchicine reveal an augmentation of cell size, the apparition of multilobed nuclei and an increase in the cytoplasm basophilia, suggesting a megakaryocyte morphology. Furthermore, when cells are cultured in the presence of colchicine, diameters measured by morphometry augment from 17.4 microns +/- 1.7 to 34.5 microns +/- 2.0 and from 27.3 microns +/- 0.3 to 40.2 microns +/- 0.6 for DAMI and HEL cell lines, respectively (p < 0.05 by t-test). After four days of culture in the presence of colchicine, cells undergo arrest proliferation. Ploidy measured by flow cytometry, shows that control cells predominantly diploid (2N) become polyploid with the appearance of 8N, 16N and 32N cells after addition of colchicine. Moreover, the endomitotic index ¿mean of (log2 DNA content expressed in N)-l¿ increases significantly from 0.5 +/- 0.1 to 1.2 +/- 0.1 and from 0.6 +/- 0.1 to 1.4 +/- 0.0 after treatment with colchicine for the DAMI and HEL cell lines, respectively. To identify the nature of the molecules involved in this phenomenon, both forms of actin (monomeric, G- and polymerized, F-) were evaluated by a DNase I inhibition assay. G-actin contents in pg per 10(6) cells are 13.0 pg +/- 2.8 (m +/- SEM) and 1.0 pg +/- 0.1 for unstimulated DAMI and HEL cells. F-actin contents per 10(6) cells are 5.8 pg +/- 1.5 and 0.1 pg +/- 0.0 for DAMI and HEL cells. The addition of colchicine for four days of culture significantly increased the G-actin content (251% and 475% of controls) and F-actin content (170% and 619% of controls) for DAMI and HEL cell lines, respectively. In contrast, the G/F-actin ratio was not affected by colchicine. DAMI cells from each ploidy class were then sorted on an ELITE Coulter and assayed for actin content. While total actin, G-actin and F-actin per cell were augmented in polyploid cells cultured with colchicine, there was a reduction in G-, F- and total actin contents per diploid equivalent when cells become polyploid. In conclusion, these data suggest that inhibition of the tubulin spindle by colchicine induces polyploidization of megakaryocytes by a reduction of both forms of actin, possibly by preventing the actin constriction ring in the telophase.
Actins↗