RESUMO
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)
Assuntos
Animais , Purinas , Citocalasina B , Crassostrea , TetraploidiaRESUMO
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
Assuntos
Artemia/efeitos dos fármacos , Aspergillus/imunologia , Citocalasina B/isolamento & purificação , Citocalasina B/análise , Citocalasinas/isolamento & purificação , Bauhinia/microbiologia , Ergosterol/isolamento & purificação , Endófitos/patogenicidadeRESUMO
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.
Assuntos
Artemia/efeitos dos fármacos , Aspergillus/química , Citocalasina B/toxicidade , Citocalasinas/toxicidade , Endófitos/química , Ergosterol/análogos & derivados , Fabaceae/microbiologia , Ácido Mevalônico/análogos & derivados , Acetilação , Animais , Argentina , Aspergillus/isolamento & purificação , Citocalasina B/química , Citocalasina B/isolamento & purificação , Citocalasinas/química , Citocalasinas/isolamento & purificação , Endófitos/isolamento & purificação , Ergosterol/química , Ergosterol/isolamento & purificação , Ergosterol/toxicidade , Dose Letal Mediana , Ácido Mevalônico/química , Ácido Mevalônico/isolamento & purificação , Ácido Mevalônico/toxicidade , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Astrócitos/metabolismo , Ácido Desidroascórbico/farmacologia , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Córtex Cerebral/patologia , Citocalasina B/farmacologia , Desoxiglucose/metabolismo , Feminino , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Humanos , Cinética , Camundongos , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Ratos Sprague-DawleyRESUMO
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)
Assuntos
Animais , Crassostrea/anatomia & histologia , Crassostrea/efeitos dos fármacos , Adenina/administração & dosagem , Citocalasina B/administração & dosagem , TriploidiaRESUMO
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.
Assuntos
Animais , Adenina/administração & dosagem , Citocalasina B/administração & dosagem , Crassostrea/anatomia & histologia , Crassostrea/efeitos dos fármacos , TriploidiaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Citocalasina B/farmacologia , Glioma/metabolismo , Apoptose , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , HumanosRESUMO
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.
Assuntos
Neutrófilos/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Cloreto de Amônio/farmacologia , Benzamidas/farmacologia , Bicarbonatos/farmacologia , Citocalasina B/farmacologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonamidas/farmacologiaRESUMO
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.
Assuntos
Citocinese/efeitos dos fármacos , Instabilidade Genômica , Células-Tronco Mesenquimais/fisiologia , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Núcleo Celular , Forma Celular , Citocalasina B/farmacologia , Feminino , Humanos , Recém-Nascido , Masculino , Testes para Micronúcleos/métodos , Pessoa de Meia-Idade , Cultura Primária de CélulasRESUMO
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⻹), 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⻹ 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.