Induction to tetraploidy in Pacific oysters (Crassostrea gigas)

As an alternative to the use of cytochalasin B(CB), 6-dimethylamino-purine (6-DMAP) and thermal shock (heat shock by increasing the temperature from 25 to 36ºC) could be used to induce tetraploidy in Pacific oyster (Crassostrea gigas) diploids. Induction was performed by applying shocks after elimination of the first polar corpuscle at the end of meiosis I. Ploidy rates were verified using flow cytometry. Tetraploid larvae were obtained using all inductor (6-DMAP, thermal shock and CB) treatments. No difference in the efficiency of tetraploidy induction was noted among 6-DMAP, thermal shock and CB. The number of D-larvae and their yield, determined by calculating the percentage of well-formed D-larvae in relation to the total number of larvae, was similar (p>0.05) among the evaluated induction methods. We suggest that 6-DMAP and thermal shock should be used in tetraploidy induction protocols, thereby avoiding the use of CB, which is a harmful agent for both humans and the environment.(AU)

Letalidad de citocalasina B y otros compuestos aislados del hongo Aspergillus spp. (Trichocomaceae) endófito de Bauhinia guianensis (Fabaceae) / Lethality of cytochalasin B and other compounds isolated from fungus Aspergillus sp. (Trichocomaceae) endophyte of Bauhinia guianensis (Fabaceae)

Los hongos endofíticos son hongos que colonizan los tejidos internos de las plantas; varios compuestos biológicamente activos se han aislado a partir de estos hongos. Existen pocos estudios de compuestos aislados de hongos endófitos de plantas amazónicas. Por lo tanto, este estudio tuvo como objetivo el aislamiento y la identificación estructural de ergosterol (1), peróxido de ergosterol (2), mevalonolactona (3), citocalasina B (4) y citocalasina H (5) a partir de Aspergillus spp. EJC 04, un hongo endofítico de Bauhinia guianensis. La citocalasina B (4) y el derivado diacetato de citocalasina B (4a) mostraron una alta letalidad en el ensayo de Artemia salina. Esta es la primera aparición de citocalasinas en hongos endófitos amazónica de B. guianensis

Endophytic fungi are fungi that colonize internal tissues of plants; several biologically active compounds have been isolated from these fungi. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, this study aimed the isolation and structural identification of ergosterol (1), ergosterol peroxide (2), mevalonolactone (3), cytochalasin B (4) and cytochalasin H (5) from Aspergillus sp. EJC 04, an endophytic fungus from Bauhinia guianensis. The cytochalasin B (4) and the diacetate derivative of cytochalasin B (4a) showed high lethality in the brine shrimp assay. This is the first occurrence of cytochalasins in Amazonian endophytic fungi from B. guianensis

Lethality of cytochalasin B and other compounds isolated from fungus Aspergillus sp. (Trichocomaceae) endophyte of Bauhinia guianensis (Fabaceae).

Endophytic fungi are fungi that colonize internal tissues of plants; several biologically active compounds have been isolated from these fungi. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, this study aimed the isolation and structural identification of ergosterol (1), ergosterol peroxide (2), mevalonolactone (3), cytochalasin B (4) and cytochalasin H (5) from Aspergillus sp. EJC 04, an endophytic fungus from Bauhinia guianensis. The cytochalasin B (4) and the diacetate derivative of cytochalasin B (4a) showed high lethality in the brine shrimp assay. This is the first occurrence of cytochalasins in Amazonian endophytic fungi from B. guianensis.

Dehydroascorbic Acid Promotes Cell Death in Neurons Under Oxidative Stress: a Protective Role for Astrocytes.

Ascorbic acid (AA), the reduced form of vitamin C, is incorporated into neurons via the sodium ascorbate co-transporter SVCT2. However, this transporter is not expressed in astrocytes, which take up the oxidized form of vitamin C, dehydroascorbic acid (DHA), via the facilitative hexose transporter GLUT1. Therefore, neuron and astrocyte interactions are thought to mediate vitamin C recycling in the nervous system. Although astrocytes are essential for the antioxidant defense of neurons under oxidative stress, a condition in which a large amount of ROS is generated that may favor the extracellular oxidation of AA and the subsequent neuronal uptake of DHA via GLUT3, potentially increasing oxidative stress in neurons. This study analyzed the effects of oxidative stress and DHA uptake on neuronal cell death in vitro. Different analyses revealed the presence of the DHA transporters GLUT1 and GLUT3 in Neuro2a and HN33.11 cells and in cortical neurons. Kinetic analyses confirmed that all cells analyzed in this study possess functional GLUTs that take up 2-deoxyglucose and DHA. Thus, DHA promotes the death of stressed neuronal cells, which is reversed by incubating the cells with cytochalasin B, an inhibitor of DHA uptake by GLUT1 and GLUT3. Additionally, the presence of glial cells (U87 and astrocytes), which promote DHA recycling, reverses the observed cell death of stressed neurons. Taken together, these results indicate that DHA promotes the death of stressed neurons and that astrocytes are essential for the antioxidative defense of neurons. Thus, the astrocyte-neuron interaction may function as an essential mechanism for vitamin C recycling, participating in the antioxidative defense of the brain.

Chemical and physical methods of triploidy induction in Crassostrea gigas (Thunberg, 1793) / Métodos químico e físico de indução à triploidia em Crassostrea gigas (Thunberg, 1793)

Three methods of triploidy (3N) induction were tested in diploid (2N) oysters Crassostrea gigas: two chemical methods cytochalasin-B (CB) and 6-dimethylaminopurine (6-DMAP), and one physical method with temperature shock. The objective was to evaluate the triploidy induction technology using flow cytometry as a tool to check the results of induction. The experiments were performed in separate and a seawater temperature in the tanks was maintained at 25 C for all experiments. In the experiment I, the efficacy of triploidy induction was evaluated using CB (0.5 mg L-1) and 6-DMAP (390 mols L-1). In the experiment II, the efficiency of triploidy induction was tested using CB (0.5 mg L-1) and 6-DMAP (450 mols L-1). In the experiment III, the efficiency of triploidy induction was evaluated using CB (0.5 mg L-1) and temperature shock (25 to 36 C). In all three experiments, viable triploid larvae were obtained. However, in the experiments I and II (with chemical methods), high mortality of larvae was observed, especially for the treatment CB. From these results, it is suggested the replacement of CB by other methods of triploidy induction, due to its high cost and high toxicity to humans and to the environment.(AU)

Três métodos de indução à triploidia (3N) foram testados em ostras diplóides (2N) (Crassostrea gigas); dois métodos químicos, citocalasina-B (CB) e 6-dimetilaminopurina (6-DMAP), e um método físico, com choque de temperatura. O objetivo foi avaliar a tecnologia de indução à triploidia, utilizando a técnica de citometria de fluxo como ferramenta para verificação dos resultados de indução. Os experimentos foram realizados em separado, sendo que a temperatura da água do mar foi mantida em 25 C em todos os tanques. No experimento I, foi avaliada a eficácia da indução à triploidia com CB (0,5 mg L-1) e 6-DMAP (390 mols L-1). No experimento II, foi testada a eficiência da indução à triploidia com CB (0,5 mg L-1) e 6-DMAP (450 mols L-1). No experimento III, foi avaliada a eficiência da indução à triploidia com CB (0,5 mg L-1) e choque de temperatura (25-36 C). Nos três experimentos, foram obtidas larvas triplóides viáveis. Entretanto, nos experimentos I e II (com métodos químicos), observou-se elevada mortalidade das larvas, especialmente para o tratamento CB. A partir destes resultados, a substituição de CB por outros métodos de indução à triploidia é sugerida, devido ao seu elevado custo e elevada toxicidade para os seres humanos e para o meio ambiente.(AU)

Chemical and physical methods of triploidy induction in Crassostrea gigas (Thunberg, 1793) / Métodos químico e físico de indução à triploidia em Crassostrea gigas (Thunberg, 1793)

Three methods of triploidy (3N) induction were tested in diploid (2N) oysters Crassostrea gigas: two chemical methods cytochalasin-B (CB) and 6-dimethylaminopurine (6-DMAP), and one physical method with temperature shock. The objective was to evaluate the triploidy induction technology using flow cytometry as a tool to check the results of induction. The experiments were performed in separate and a seawater temperature in the tanks was maintained at 25 C for all experiments. In the experiment I, the efficacy of triploidy induction was evaluated using CB (0.5 mg L-1) and 6-DMAP (390 mols L-1). In the experiment II, the efficiency of triploidy induction was tested using CB (0.5 mg L-1) and 6-DMAP (450 mols L-1). In the experiment III, the efficiency of triploidy induction was evaluated using CB (0.5 mg L-1) and temperature shock (25 to 36 C). In all three experiments, viable triploid larvae were obtained. However, in the experiments I and II (with chemical methods), high mortality of larvae was observed, especially for the treatment CB. From these results, it is suggested the replacement of CB by other methods of triploidy induction, due to its high cost and high toxicity to humans and to the environment.

Três métodos de indução à triploidia (3N) foram testados em ostras diplóides (2N) (Crassostrea gigas); dois métodos químicos, citocalasina-B (CB) e 6-dimetilaminopurina (6-DMAP), e um método físico, com choque de temperatura. O objetivo foi avaliar a tecnologia de indução à triploidia, utilizando a técnica de citometria de fluxo como ferramenta para verificação dos resultados de indução. Os experimentos foram realizados em separado, sendo que a temperatura da água do mar foi mantida em 25 C em todos os tanques. No experimento I, foi avaliada a eficácia da indução à triploidia com CB (0,5 mg L-1) e 6-DMAP (390 mols L-1). No experimento II, foi testada a eficiência da indução à triploidia com CB (0,5 mg L-1) e 6-DMAP (450 mols L-1). No experimento III, foi avaliada a eficiência da indução à triploidia com CB (0,5 mg L-1) e choque de temperatura (25-36 C). Nos três experimentos, foram obtidas larvas triplóides viáveis. Entretanto, nos experimentos I e II (com métodos químicos), observou-se elevada mortalidade das larvas, especialmente para o tratamento CB. A partir destes resultados, a substituição de CB por outros métodos de indução à triploidia é sugerida, devido ao seu elevado custo e elevada toxicidade para os seres humanos e para o meio ambiente.

Cytochalasin B inhibits the proliferation of human glioma U251 cells through cell cycle arrest and apoptosis.

Cytochalasin B (CB) is known to inhibit a number of cancer types, but its effects on gliomas are unknown. We examined the in vitro effects of CB on the proliferation of human glioma U251 cells, as well as determined its mechanism of action. Cell proliferation was determined using CCK-8. The effect of CB on U251 cell morphology was observed under a transmission electron microscope. Cell cycle distribution was assessed using propidium iodine and Giemsa staining, and cell apoptosis was determined by annexin V-fluorescein isothiocyanate/propidium iodide. Cell cycle-related proteins were determined by Western blot. CB effectively inhibited U251 cell proliferation in a dose- and time-dependent manner. The 24, 48, 72, and 96 h IC50 values were 6.41 x 10(-2), 9.76 x 10(-4), 2.57 x 10(-5), and 2.08 x 10(-5) M, respectively. CB increased the proportion of cells in the G2/M phase in a dose-dependent manner, thus increasing the mitotic index and decreasing cdc2 and cyclin B1 protein levels. CB induced morphological changes in the cytoskeleton. Additionally, 10(-5) M CB induced apoptosis in 23.4 ± 0.5% of U251 cells (P < 0.05 vs control group). Caspase-3, -8, and -9 activities were increased after CB treatment. CB inhibited U251 glioma cell proliferation by damaging the microfilament structure. CB also induced glioma cell apoptosis, suggesting that it may be an effective therapeutic agent against gliomas.

Characterization of the Na/HCO3(-) cotransport in human neutrophils.

BACKGROUND: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. METHODS: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. RESULTS: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. CONCLUSION: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.

Cytokinesis-block micronucleus assay adapted for analyzing genomic instability of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are multipotent cells used in cell therapy research. One of the problems involving hMSCs is the possibility of genetic instability during in vitro expansion required to obtain a suitable number of cells for clinical applications. The cytokinesis-block micronucleus (CBMN) assay measures genetic instability by analyzing the presence of micronucleus (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) in binucleated cells. The present study describes modifications in the CBMN assay methodology to analyze genetic instability in hMSCs isolated from the umbilical vein and in vitro expanded. The best protocol to achieve binucleated hMSCs with preserved cytoplasm was as follows: cytochalasin B concentration (4.0 µg/mL), use of hypotonic treatment (3 min), and the fixative solution (9 methanol:1 acetic acid). These adaptations were reproduced in three hMSC primary cell cultures and also in XP4PA and A549 cell lines. The frequency of hMSCs treated with mitomycin-C presenting MN was lower than that with other nuclear alterations, indicating that the hMSCs contain mechanisms to avoid a high level of chromosomal breaks. However, a high frequency of cells with NPBs was detected and spontaneous anaphase bridges under normal hMSC in vitro culture were observed. Considering that anaphase bridges are characteristic alterations in tumor cells, the CBMN assay is indicated as an important tool associated with other genetic analyses in order to ensure the safe clinical use of hMSCs in cell therapy.

Genotoxic effects of copper oxide nanoparticles in Neuro 2A cell cultures.

Copper oxide nanoparticles (CuO NPs) are used for their biocide potential however they were also shown to be highly toxic to mammalian cells. Therefore, the effects of CuO NPs should be carefully investigated to determine the most sensitive processes for CuO NP toxicity. In this study, the genotoxicity of CuO NPs was investigated in vitro, using the mouse neuroblastoma cell line Neuro-2A. Genotoxic effects related to DNA fragmentation, DNA methylation and chromosomal damage, as well as lipid peroxidation, were investigated and compared to cytotoxic effects, measured by the mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan. Based on mitochondrial activity, CuO NPs were found to be cytotoxic. At the highest concentration tested (400 mg l⁻¹), 63% of cell viability was found in Neuro-2A cells after 24 h of treatment to CuO NPs. CuO NPs were also found to induce DNA fragmentation, lipid peroxidation and micronucleus formation. The micronucleus assay was the most sensitive to evaluate CuO NP genotoxicity and micronucleus frequency was increased significantly at 12.5 mg l⁻¹ CuO NPs after 24h of treatment. At this concentration, no significant change of cell viability was found using the mitochondrial activity assay. These results highlight the important risk of genotoxic effects of CuO NPs and show that genotoxicity assays are a sensitive approach to evaluate the risk of CuO NP toxicity.

Noncompetitive blocking of human GLUT1 hexose transporter by methylxanthines reveals an exofacial regulatory binding site.

Glucose transporter (GLUT)1 has become an attractive target to block glucose uptake in malignant cells since most cancer cells overexpress GLUT1 and are sensitive to glucose deprivation. Methylxanthines are natural compounds that inhibit glucose uptake; however, the mechanism of inhibition remains unknown. Here, we used a combination of binding and glucose transport kinetic assays to analyze in detail the effects of caffeine, pentoxifylline, and theophylline on hexose transport in human erythrocytes. The displacement of previously bound cytochalasin B revealed a direct interaction between the methylxanthines and GLUT1. Methylxanthines behave as noncompetitive blockers (inhibition constant values of 2-3 mM) in exchange and zero-trans efflux assays, whereas mixed inhibition with a notable uncompetitive component is observed in zero-trans influx assays (inhibition constant values of 5-12 mM). These results indicate that methylxanthines do not bind to either exofacial or endofacial d-glucose-binding sites but instead interact at a different site accessible by the external face of the transporter. Additionally, infinite-cis exit assays (Sen-Widdas assays) showed that only pentoxifylline disturbed d-glucose for binding to the exofacial substrate site. Interestingly, coinhibition assays showed that methylxanthines bind to a common site on the transporter. We concluded that there is a methylxanthine regulatory site on the external surface of the transporter, which is close but distinguishable from the d-glucose external site. Therefore, the methylxanthine moiety may become an attractive framework for the design of novel specific noncompetitive facilitative GLUT inhibitors.

Intranasal administration of aTf protects and repairs the neonatal white matter after a cerebral hypoxic-ischemic event.

Our previous studies showed that the intracerebral injection of apotransferrin (aTf) attenuates white matter damage and accelerates the remyelination process in a neonatal rat model of cerebral hypoxia-ischemia (HI) injury. However, the intracerebral injection of aTf might not be practical for clinical treatments. Therefore, the development of less invasive techniques capable of delivering aTf to the central nervous system would clearly aid in its effective clinical use. In this work, we have determined whether intranasal (iN) administration of human aTf provides neuroprotection to the neonatal mouse brain following a cerebral hypoxic-ischemic event. Apotransferrin was infused into the naris of neonatal mice and the HI insult was induced by right common carotid artery ligation followed by exposure to low oxygen concentration. Our results showed that aTf was successfully delivered into the neonatal HI brain and detected in the olfactory bulb, forebrain and posterior brain 30 min after inhalation. This treatment successfully reduced white matter damage, neuronal loss and astrogliosis in different brain regions and enhanced the proliferation and survival of oligodendroglial progenitor cells (OPCs) in the subventricular zone and corpus callosum (CC). Additionally, using an in vitro hypoxic model, we demonstrated that aTf prevents oligodendrocyte progenitor cell death by promoting their differentiation. In summary, these data suggest that iN administration of aTf has the potential to be used for clinical treatment to protect myelin and to induce remyelination in demyelinating hypoxic-ischemic events in the neonatal brain.

Acute feedback control of astrocytic glycolysis by lactate.

Neuronal activity is accompanied by a rapid increase in interstitial lactate, which is hypothesized to serve as a fuel for neurons and a signal for local vasodilation. Using FRET microscopy, we report here that the rate of glycolysis in cultured mice astrocytes can be acutely modulated by physiological changes in extracellular lactate. Glycolytic inhibition by lactate was not accompanied by detectable variations in intracellular pH or intracellular ATP and was not dependent of mitochondrial function. Pyruvate was also inhibitory, suggesting that the effect of lactate is not mediated by the NADH/NAD(+) ratio. We propose that lactate serves as a fast negative feedback signal limiting its own production by astrocytes and therefore the amplitude of the lactate surge. The inhibition of glucose usage by lactate was much stronger in resting astrocytes than in K(+)-stimulated astrocytes, which suggests that lactate may also help diverting glucose from resting to active zones.

Glucose increases intracellular free Ca(2+) in tanycytes via ATP released through connexin 43 hemichannels.

The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its neurons interact with specialized ependymal-glial cells, termed tanycytes. The latter express glucose-sensing proteins, including glucose transporter 2, glucokinase, and ATP-sensitive K(+) (K(ATP) ) channels, suggesting their involvement in hypothalamic glucosensing. Here, the transduction mechanism involved in the glucose-induced rise of intracellular free Ca(2+) concentration ([Ca(2+) ](i) ) in cultured ß-tanycytes was examined. Fura-2AM time-lapse fluorescence images revealed that glucose increases the intracellular Ca(2+) signal in a concentration-dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch clamp recordings, through a K(ATP) channel-dependent pathway. Consequently, ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y(1) receptors followed by inositol trisphosphate receptor activation and Ca(2+) release from intracellular stores. The present study identifies the mechanism by which glucose increases [Ca(2+) ](i) in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins in normal tanycytes.

Bovine parthenogenotes produced by inhibition of first or second polar bodies emission.

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission. Only one polar body was inhibited in each strategy in order to keep the diploid chromosome set. In experiment 1, the effect of cytochalasin B on in vitro maturation of bovine oocytes was evaluated. Most oocytes (77%) were arrested at a meiotic stage characterized by the presence of a large internal metaphase plate and absence of polar body. In experiment 2, development of embryos exposed to cytochalasin B during in vitro maturation (CytoB-IVM) or during activation (CytoB-ACT) was compared. Developmental rates did not differ between diploidization strategies, even when three agents were employed to induce activation. Both groups, CytoB-IVM and CytoB-ACT, tended to maintain diploidy. CytoB-IVM parthenogenesis could help to obtain embryos with a higher degree of homology to the oocyte donor.

Bovine parthenogenotes produced by inhibition of first or second polar bodies emission

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission. Only one polar body was inhibited in each strategy in order to keep the diploid chromosome set. In experiment 1, the effect of cytochalasin B on in vitro maturation of bovine oocytes was evaluated. Most oocytes (77%) were arrested at a meiotic stage characterized by the presence of a large internal metaphase plate and absence of polar body. In experiment 2, development of embryos exposed to cytochalasin B during in vitro maturation (CytoB-IVM) or during activation (CytoB-ACT) was compared. Developmental rates did not differ between diploidization strategies, even when three agents were employed to induce activation. Both groups, CytoB-IVM and CytoB-ACT, tended to maintain diploidy. CytoB-IVM parthenogenesis could help to obtain embryos with a higher degree of homology to the oocyte donor.

Vitrification with glutamine improves maturation rate of vitrified / warmed immature bovine oocytes.

The current study examined the protective effects of l-glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 µg/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. L-glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).

Activation with ionomycin followed by dehydroleucodine and cytochalasin B for the production of parthenogenetic and cloned bovine embryos.

In this work, Dehydroleucodine (DhL) was evaluated as a chemical activator of bovine oocytes and somatic cell nuclear transfer (SCNT) reconstituted embryos. Oocytes were activated with 5 microM Ionomycin (Io) and exposed for 3 h to 1 or 5 microM DhL alone (Io-Dhl1 or Io-DhL5) or combined with Cytochalasin B (Io-DhL1/CB; Io-DhL5/CB). Control groups were Io (Io), Io followed by 1.9 mM 6-Dimethylaminopurine (Io-6DMAP), and embryos produced by in vitro fertilization (IVF). Pronuclear formation and development to blastocysts of activated oocytes were evaluated. Embryos obtained by the DhL concentration that induced the highest blastocyst rates (1 microM) were karyotyped. An additional treatment based in Io-DhL1 plus lengthened (6-h) exposure to CB (Io-DhL1/long CB) was included to improve the proportion of diploid blastomeres. Finally, DhL combined with CB was employed to assist cloning by intracytoplasmic injection of whole cumulus cells. Results showed that DhL induces a pronuclear formation dynamic that was more similar to IVF-produced embryos than DMAP. Development to blastocyst stage was higher after activation with 1 microM DhL than with 5 microM DhL, either for groups combined or not with CB (19.15; 21.74 vs. 6.82; 0%, respectively) (p < 0.05). Io-DhL1 and Io-DhL1/CB treatments induced blastocyst-cleaved embryo ratios not statistically different from those of Io-DMAP (35.85%) and IVF (33.33%) groups (p > 0.05). Io-DhL1/long CB induced higher diploid blastomere rates than Io-Dhl1, Io-DhL1/CB and Io-DMAP (63.8 vs. 36.8; 40 and 31.6%, respectively) (p < 0.05). Moreover, all DhL treatments resulted in polyploidy rates that were lower than Io-DMAP (5.2, 12.0, 10.6, and 31.6%, respectively) (p < 0.05). Io-DhL1/CB and Io-DhL1/long CB induced cloned embryo blastocyst rates that were not significantly different from Io-DMAP (6.1, 9.4, and 18.3%, respectively) (p < 0.05). Our results indicate that Io-DhL1/long CB protocol could be useful for SCNT programs.

Kinetics of transport and phosphorylation of glucose in cancer cells.

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS-30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D-[2-(3)H-glucose] as physiological substrate. Kinetic analysis revealed two components at low- and high-glucose concentrations in both tumor cells. At low glucose and 37 degrees C, the V(max) was 55 +/- 20 and 17.2 +/- 6 nmol (min x mg protein)(-1), whereas the K(m) was 0.52 +/- 0.7 and 9.3 +/- 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC(50) = 0.44 +/- 0.1; K(i) = 0.3 +/- 0.1 microM) and phloretin (IC(50) = 8.7 microM) in AS-30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS-30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS-30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS-30D carcinoma and HeLa cells. The much lower GLUT V(max) and catalytic efficiency (V(max)/K(m)) values in comparison to those of G6P-sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target.

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100 percent; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6 percent, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83 percent, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7 percent ethanol (62 vs 62 percent, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62 percent, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.