EFFECTS OF RESVERATROL ON VITRIFIED PORCINE OOCYTES

Vitrified MII porcine oocytes are characterized by reduced developmental competence, associated with the activation of the apoptotic pathway. Resveratrol (R), a polyphenolic compound present in several vegetal sources, has been reported to exert, among all its other biological effects, an antiapoptotic one. The aim of this study was to determine the effects of R (2\u2009\ub5M) on the apoptotic status of porcine oocytes vitrified by Cryotop method, evaluating phosphatidylserine (PS) exteriorization and caspases activation. R was added during IVM (A); 2\u2009h postwarming incubation (B); vitrification/warming and 2\u2009h postwarming incubation (C); all previous phases (D). Data on PS exteriorization showed, in each treated group, a significantly higher (P<0.05) percentage of live nonapoptotic oocytes as compared with CTR; moreover, the percentage of live apoptotic oocytes was significantly (P<0.05) lower in all R-treated groups relative to CTR. The results on caspase activation showed a tendency to an increase of viable oocytes with inactive caspases in B, C, and D, while a significant (P<0.05) increase in A compared to CTR was recorded. These data demonstrate that R supplementation in various phases of IVM and vitrification/warming procedure can modulate the apoptotic process, improving the resistance of porcine oocytes to cryopreservation-induced damage

ITS-rDNA phylogeny of Colletotrichum spp. causal agent of apple glomerella leaf spot.

Several diseases have affected apple production, among them there is Glomerella leaf spot (GLS) caused by Colletotrichum spp. The first report of this disease in apple was in plants nearby citrus orchards in São Paulo State, Brazil. The origin of this disease is still not clear, and studies based on the molecular phylogeny could relate the organisms evolutionarily and characterize possible mechanisms of divergent evolution. The amplification of 5.8S-ITS (Internal Transcribed Spacer) of rDNA of 51 pathogenic Colletotrichum spp. isolates from apples, pineapple guava and citrus produced one fragment of approximately 600 bases pairs (bp) for all the isolates analyzed. The amplified fragments were cleaved with restriction enzymes, and fragments from 90 to 500bp were obtained. The sequencing of this region allowed the generation of a phylogenetic tree, regardless of their hosts, and 5 isolated groups were obtained. From the "in silico" comparison, it was possible to verify a variation from 93 to 100% of similarity between the sequences studied and the Genbank data base. The causal agent of GLS is nearly related (clustered) to isolates of pineapple guava and to the citrus isolates used as control

Physico-chemical analysis of Albian (Lower Cretaceous) amber from San Just (Spain) : implications for palaeoenvironmental and palaeoecological studies

Amber from a Lower Cretaceous outcrop at San Just, located in the Eastern Iberian Peninsula (Escucha Formation, Maestrat Basin), was investigated to evaluate its physico-chemical properties. Thermogravimetric (TG) and Differential Thermogravimetric (DTG) analyses, infra-red spectroscopy, elemental and C-isotope analyses were performed. Physico-chemical differences between the internal light nuclei and the peripheral darker portions of San Just amber can be attributed to processes of diagenetic alteration that preferentially took place in the external amber border colonized by microorganisms (fungi or bacteria) when the resin was still liquid or slightly polymerized. δ13Camber values of different pieces of the same sample, from the nucleus to the external part, are remarkably homogeneous, as are δ13Camber values of the darker peripheral portions and lighter inner parts of the same samples. Hence, neither invasive microorganisms, nor diagenetic alteration changed the bulk isotopic composition of the amber. δ13C values of different amber samples range from -21.1‰ to -24‰, as expected for C3 plant-derived material. C-isotope analysis, coupled to palaeobotanical, TG and DTG data and infra-red spectra, suggests that San Just amber was exuded by only one conifer species, belonging to either the Cheirolepidiaceae or Aracauriaceae, coniferous families probably living under stable palaeoenvironmental and palaeoecological conditions

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 \u3bcm) and EGCG (10, 20 and 60 \u3bcm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 \u3bcm (but not of 60 \u3bcm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 \u3bcm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone

Neuromuscular activity of bothrops fonsecai snake venom in vertebrate preparations

CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOThe neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160μg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160μg/ml, respectively) and attenuated the contractures to 110μM ACh (78-100% inhibition) and 40mM KCl (45-90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200μg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200μg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K(+) were abolished by preincubating venom with commercial antivenom. These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom.The neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160mg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160mg/ml, respectively) and attenuated the contractures to 110mM ACh (78–100% inhibition) and 40mM KCl (45–90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200mg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200mg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K+ were abolished by preincubating venom with commercial antivenom.These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom5615CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORCNPQ – CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOsem informaçãosem informaçã

Gene therapy of thyroid cancer via retrovirally-driven combined expression of human interleukin-2 and herpes simplex virus thymidine kinase

OBJECTIVE AND DESIGN: Based on our clinical experience with combined gene therapy of glioblastoma, we developed a retroviral vector expressing two therapeutic genes (i.e. thymidine kinase of herpes simplex virus, HSV-TK, and interleukin-2, IL-2) and evaluated its efficiency in vitro and in vivo. METHODS: Expression of therapeutic genes in transduced thyroid carcinoma cells was analyzed by real-time RT-PCR. Ganciclovir sensitivity of infected cells was assessed in vitro in thyroid carcinoma cell lines and in vivo in nude mice bearing xenografted thyroid cancers. The combined effect of IL-2/HSV-TK was compared with the effect of IL-2 alone. RESULTS: Expression of therapeutic genes was higher in differentiated than in anaplastic thyroid carcinoma cells. Ganciclovir treatment led to dose- and time-dependent killing of transduced cells in vitro. A bystander effect was demonstrated by using mixtures of infected and non-infected cells. In vivo studies showed a significant reduction of growth and the presence of an inflammatory infiltrate in transduced thyroid tumors expressing IL-2 alone, as compared with non-infected tumors. By using the retroviral vector expressing IL-2/HSV-TK, treatment with ganciclovir led to complete eradication of anaplastic tumors and a >80% reduction of the size of differentiated thyroid carcinomas. Histological analysis of tumor specimens showed extensive necrosis and inflammatory cell infiltrates. The combination of IL-2/HSV-TK plus ganciclovir was significantly more efficient than IL-2 alone in eradicating tumor masses. The bystander effect was also obtained in vivo. CONCLUSIONS: These findings demonstrate the feasibility and efficiency of a combined immunomodulating and suicide gene therapy approach for thyroid carcinomas

Solid state and solution fine tuning of the linear and nonlinear optical properties of (2-pyrene-1-yl-vinyl)pyridine by protonation&#8211;deprotonation reactions

The unexpected and acido-triggered reversible luminescence and nonlinear optical properties of (2-pyrene-1-yl-vinyl)pyridine, a simple and highly transparent chromophore, are studied both in solution and in the solid state. Remarkably, for the first time the acidomodulation of the NLO response of a poled thin film is reported

Chromosome analysis in Pseudopaludicola (Anura, Leiuperidae), with description of sex chromosomes XX/XY in P-saltica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Taxonomic changes have frequently occurred in the anuran genus Pseudopaludicola as a consequence of high morphological similarity among its species. The present work reports karyotypic analysis of three Pseudopaludicola species sampled in their type locality and four Pseudopaludicola populations from distinct localities, aiming at contributing to the systematics of this genus. Chromosomes were stained with Giemsa or submitted to the silver staining (Ag-NOR) and C-banding techniques. The karyotype was 2n=22 in P. mineira, Pseudopaludicola sp. and two populations of P. saltica. The chromosome pair 8 was heteromorphic in P. saltica, characterizing a XX/XY sex-determination system with telocentric X and submetacentric Y. Highly similar karyotypes with 2n=18 chromosomes were observed in P. canga, P. aff. canga from Barreirinhas, State of Maranhao, Uberlandia, State of Minas Gerais and Icem, State Sao Paulo. The high similarity among the karyotypes 2n=18 suggested that the populations of P. aff. canga belong to the group 'pusilla', the same group of P. canga. The data demonstrated also that P. aff. canga from Barreirinhas (northeast region) is cytogenetically identical to P. canga with regarding the NOR site position in pair 3 and the presence of a heterochromatic block in the pair 2, whereas P. aff. canga from Uberlandia and Icem (southeast) had the NOR in the pair 9. Moreover, the cytogenetic data discriminated P. mineira and Pseudopaludicola sp. from the previously analyzed species with 22 chromosomes, and suggested that Pseudopaludicola sp. is an undescribed species. Sexual heteromorphic chromosomes are firstly reported in Pseudopaludicola and the data indicated the need of an extensive taxonomic review in this genus.14724352Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Ensino de Sao Paulo (FUNDESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [6/60055-0