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Breast cancer is one of the common tumors occurring in woman and despite treatment, the prognostic is poor. Genistein, a soy isoflavone, has been reported to have chemopreventive\chemotherapeutic potential in multiple tumor types. Here, we investigated the genistein antiproliferative effect in MCF-7 breast cancer, underlying the molecular mechanisms involved in this effect. MCF-7 cancer and CCD1059sK fibroblast cells were treated with estradiol (10 nM) or genistein (0.01-100 µM) for 24, 48, and 72 h and the cell proliferation was investigated by MTT; membrane cell permeability was evaluated by LDH and PI incorporation; apoptosis was investigated by externalization of phosphatidylserine by FACS; and presence of autophagy was detected by LC3A/B immunostaining. The expression of apoptotic proteins and antioxidant enzymes was evaluated by qPCR. The results demonstrate that genistein (100 µM) for 72 h of treatment selectively reduced MCF-7 cell proliferation independent of estrogen receptor activation, while no cytotoxicity was observed in fibroblast cells. Further experiments showed that genistein induced phosphatidylserine externalization and LC3A/B immunopositivity in MCF-7 cells, indicating apoptosis and autophagy cell death. Genistein increased in three times proapoptotic BAX/Bcl-2 ratio and promoted a parallel downregulation of 20 times of antiapoptotic survivin. In addition, genistein promoted a decrease of 5.5, 9.3, and 3.6 times of MnSOD, CuZnSOD, and TrxR mRNA expression, respectively, while the GPx expression was increased by 6.5 times. These results suggest that the antitumor effect of genistein involved the modulation of antioxidant enzyme and apoptotic signaling expression, which resulted in apoptosis and progression of autophagy.

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Neste trabalho foi estudada a correlação entre o perfil proteico do plasma seminal e a motilidade e viabilidade espermática em coelhos submetidos ao tratamento com vetores de expressão contendo o gene da eritropoetina (EPO) e com EPO recombinante humana. Foram identificadas, em coelhos submetidos ao tratamento com vetor de DNA contendo o gene da EPO, duas bandas proteicas associadas a alterações na motilidade espermática - 48kDa à baixa motilidade (P<0,05) e 18kDa à alta motilidade (P<0,05) - e esse fator foi associado a maior viabilidade espermática (P<0,05). Em coelhos submetidos ao tratamento com EPO recombinante, um fator proteico, 63kDa, associou-se à alta motilidade espermática (P<0,05), enquanto dois, 26 e 40kDa, foram associados à alta viabilidade espermática (P<0,05). Esses resultados sugerem que o doping genético pode ocasionar mudanças no perfil proteico do plasma seminal, provocando alterações na motilidade e viabilidade espermática.

In this study the correlation between seminal plasma protein profile and the sperm motility and sperm viability in rabbits submitted to treatment with an expression vector containing EPO gene and with human recombinant EPO was evaluated. In rabbits submitted to treatment with EPO expression vector, two protein bands were associated to sperm motility - 48kDa associated to low motility (P<0.05) and 18kDa to high motility (P<0.05) - and this protein band was also associated to high sperm viability (P<0.05). In rabbits submitted to treatment with human recombinant EPO, a protein factor, 63kDa, was associated to high sperm motility (P<0.05) while two protein factors, 26 and 40kDa, were associated to high sperm viability (P<0.05). These results suggest that gene doping leads to changes in rabbit seminal plasma protein, altering sperm motility and sperm viability.

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The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

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Erythropoietin (EPO) gene therapy can be used for several purposes; however, its effects on reproductive performance are unknown. The aim of this study was to evaluate the toxicological effects of non-viral (EPO) gene transfer on sperm motility, viability, morphology and concentration. Rabbit EPO cDNA was cloned into a pTarget mammalian expression vector. Rabbits were administered with: (1) pTarget/EPO vector, (2) recombinant human EPO (rHuEpo) and (3) saline (control). Both pTarget/EPO and rHuEpo significantly increased (P < 0.05) hematocrit levels 1 week after injection and they remained significantly higher than the control for up to 5 weeks (P < 0.05), showing that both EPO treatments were effective in stimulating the production of red blood cells in rabbits. The EPO gene transfer or rHuEPO administration had no significant effect (P > 0.05) on sperm motility, vigor, viability, concentration or morphology in the testis.

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Neste trabalho foi estudada a correlação entre o perfil proteico do plasma seminal e a motilidade e viabilidade espermática em coelhos submetidos ao tratamento com vetores de expressão contendo o gene da eritropoetina (EPO) e com EPO recombinante humana. Foram identificadas, em coelhos submetidos ao tratamento com vetor de DNA contendo o gene da EPO, duas bandas proteicas associadas a alterações na motilidade espermática - 48kDa à baixa motilidade (P<0,05) e 18kDa à alta motilidade (P<0,05) - e esse fator foi associado a maior viabilidade espermática (P<0,05). Em coelhos submetidos ao tratamento com EPO recombinante, um fator proteico, 63kDa, associou-se à alta motilidade espermática (P<0,05), enquanto dois, 26 e 40kDa, foram associados à alta viabilidade espermática (P<0,05). Esses resultados sugerem que o doping genético pode ocasionar mudanças no perfil proteico do plasma seminal, provocando alterações na motilidade e viabilidade espermática.(AU)

In this study the correlation between seminal plasma protein profile and the sperm motility and sperm viability in rabbits submitted to treatment with an expression vector containing EPO gene and with human recombinant EPO was evaluated. In rabbits submitted to treatment with EPO expression vector, two protein bands were associated to sperm motility - 48kDa associated to low motility (P<0.05) and 18kDa to high motility (P<0.05) - and this protein band was also associated to high sperm viability (P<0.05). In rabbits submitted to treatment with human recombinant EPO, a protein factor, 63kDa, was associated to high sperm motility (P<0.05) while two protein factors, 26 and 40kDa, were associated to high sperm viability (P<0.05). These results suggest that gene doping leads to changes in rabbit seminal plasma protein, altering sperm motility and sperm viability.(AU)

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To study whether treatment with the beta-blocker atenolol (AT) attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), rats were treated with AT (1.5 mg · kg(-1), intravenously) or saline solution (SS) prior to I (60 minutes), which was produced by occlusion of the superior mesenteric artery, and/or R (120 minutes). After I or I/R, 2-cm jejunal segments were mounted in an organ bath to study neurogenic contractions stimulated by electrical pulses or KCl using a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy analysis. Compared to the sham group, jejunal contractions were similar in the I + AT and the I/R + AT groups, but reduced in the I + SS and the I/R + SS groups. The jejunal enteric nerves were damaged in the I + SS and the I/R + SS groups, but not in the I + AT and the I/R + AT. These results suggest that AT may attenuate intestinal dysfunction caused by I and I/R.

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AIM: To evaluate the effect of four tooth storage temperature-based methods on quality of RNA obtained from cells retrieved from human dental pulps and human pre-dentine. METHODOLOGY: RNA was isolated from dental pulp tissue and from cells retrieved by scraping the pre-dentine of freshly extracted human third molars (n = 15) using TRIzol(®) reagent. Teeth were randomly assigned to the following temperature conditions: immediate RNA isolation after tooth extraction, liquid nitrogen (24 h), -80 °C (24 h), 20 °C (24 h) and 4 °C (6 h). RNA integrity was checked by the density of 28S and 18S ribosomal RNA. RT-PCR was used to analyse the expression of odontoblast makers (DSPP, DMP1 and MEPE) and the housekeeping gene GAPDH. RESULTS: All experimental conditions evaluated preserved RNA integrity. The three odontoblastic markers were amplified from the pulp tissue and from the cells associated with pre-dentine. CONCLUSION: The four storage options allowed RNA isolation for RT-PCR analysis. These findings may facilitate the use of clinically derived human dental pulp and odontoblasts for endodontic research.

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The objective was to evaluate the effect of three cryopreservation methods on the in vitro maturation (IVM) and membrane integrity (MIn) of immature equine oocytes. An open pulled straw (OPS) method, a novel solid surface vitrification (SSV) process, and the addition of a synthetic ice blocker were evaluated. Compared with the control group (N=269), the OPS (N=159) and the SSV (N=202) cryopreservation methods decreased both IVM (50.9 vs. 13.3 and 9.4%, respectively; P<0.001) and MIn (76.6 vs. 31.1 and 33.7%; P<0.001) of immature equine oocytes. However, inclusion of 0.1% ice blocker in the OPS vitrification process increased the rates of both IVM (30.5%; P<0.01) and MIn (45.8%; P<0.05) of the oocytes (N=59). Including 0.1% ice blocker in the SSV process improved the IVM rate (20.9%; P<0.05), whereas MIn remained compromised in this group (N=67). However, increasing the concentration of the ice blocker (to 1.0%) in the cryopreservation methods did not significantly improve rates of IVM. In conclusion, the addition of a synthetic ice blocker (0.1%) to both cryopreservation processes significantly increased rates of both IVM and MIn of immature equine oocytes cryopreserved by OPS.

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The objective was to introduce exogenous DNA into commercially sex-sorted bovine sperm using nanopolymer for transfection. In the first experiment, the optimal concentration and ratio of linear-to-circular plasmid was determined for NanoSMGT in unsorted sperm. A second experiment was conducted to transfect exogenous DNA into sex-sorted sperm. Exogenous DNA uptake occurred in a dose-dependent manner (P < 0.05). The optimal amount of DNA was 10 µg/10(6) cells. The ratios of linear-to-circular plasmid do not influence the uptake by unsorted sperm cells and none of the tested treatments affected sperm motility and viability. Commercially sex-sorted bovine sperm were able to uptake exogenous DNA using nanopolymer; however, both X- and Y-sorted sperm had decreased DNA uptake in comparison to unsorted sperm (P < 0.05). Neither sperm motility nor viability were affected by nanotransfection. In conclusion, nanopolymer efficiently introduced exogenous DNA into commercially sex-sorted bovine sperm; we inferred that these sperm could be used for production of embryos of the desired sex, a technique named NanoSMGT.

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Este estudo buscou clonar o cDNA do sbGnRH, identificar sua expressão em diferentes tecidos do linguado, bem como avaliar possíveis diferenças no RNA mensageiro (RNAm) desse gene no cérebro de linguados machos juvenis e adultos. Por meio da RT-PCR, demonstrou-se pela primeira vez, a clonagem da região codificadora do sbGnRH contendo 297 nucleotídeos do cérebro do linguado. A expressão do sbGnRH foi detectada em vários tecidos periféricos. Foram detectados níveis mais elevados de RNAm do sbGnRH no hipotálamo dos animais adultos. Estes resultados sugerem que o sbGnRH está envolvido na puberdade do linguado.

The objectives of this study were to clone sbGnRH cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain sbGnRH expression in juvenile and adult males. Using RT-PCR the cloning of a 297 nucleotides coding region of sbGnRH from Brazilian flounder brain was demonstrated for the first time. Expression of sbGnRH was detected in several peripheral tissues. Brain gene expression in the adult flounder was higher than those found in juvenile. These results suggest that sbGnRH is involved on the Brazilian flounder puberty.

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Este estudo buscou clonar o cDNA do sbGnRH, identificar sua expressão em diferentes tecidos do linguado, bem como avaliar possíveis diferenças no RNA mensageiro (RNAm) desse gene no cérebro de linguados machos juvenis e adultos. Por meio da RT-PCR, demonstrou-se pela primeira vez, a clonagem da região codificadora do sbGnRH contendo 297 nucleotídeos do cérebro do linguado. A expressão do sbGnRH foi detectada em vários tecidos periféricos. Foram detectados níveis mais elevados de RNAm do sbGnRH no hipotálamo dos animais adultos. Estes resultados sugerem que o sbGnRH está envolvido na puberdade do linguado.(AU)

The objectives of this study were to clone sbGnRH cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain sbGnRH expression in juvenile and adult males. Using RT-PCR the cloning of a 297 nucleotides coding region of sbGnRH from Brazilian flounder brain was demonstrated for the first time. Expression of sbGnRH was detected in several peripheral tissues. Brain gene expression in the adult flounder was higher than those found in juvenile. These results suggest that sbGnRH is involved on the Brazilian flounder puberty.(AU)

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Neste estudo, identificaram-se polipeptídeos associados à integridade da membrana plasmática (IMP) de espermatozóides suínos após o processo de congelamento/descongelamento. Por meio do perfil protéico do plasma seminal em SDS-PAGE, observou-se a presença de nove bandas polipeptídicas com pesos moleculares que variaram de 11,97 a 122,52kDa. Detectou-se que uma banda de 26,58kDa esteve associada à baixa IMP (<55 por cento). Não foi verificada associação entre as outras bandas e a IMP. Conclui-se que o fator polipeptídico de 26,58kDa está associado à baixa integridade da membrana plasmática do espermatozóide suíno após o congelamento/descongelamento.

Polypeptides associate to membrane integrity (MI) of swine spermatozoa submitted to freezing and thawing were identified. The protein profile of seminal plasma analyzed by SDS-PAGE allowed the identification of nine polypeptide bands with molecular weight ranging from 11.97 to 122.52kDa. One 26.58kDa band was associated with reduced MI (<55 percent). No associations among other bands and MI were observed. The 26.58kDa factor is associated with reduction of membrane integrity of swine spermatozoa after freezing and thawing.

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Neste estudo, identificaram-se polipeptídeos associados à integridade da membrana plasmática (IMP) de espermatozóides suínos após o processo de congelamento/descongelamento. Por meio do perfil protéico do plasma seminal em SDS-PAGE, observou-se a presença de nove bandas polipeptídicas com pesos moleculares que variaram de 11,97 a 122,52kDa. Detectou-se que uma banda de 26,58kDa esteve associada à baixa IMP (<55 por cento). Não foi verificada associação entre as outras bandas e a IMP. Conclui-se que o fator polipeptídico de 26,58kDa está associado à baixa integridade da membrana plasmática do espermatozóide suíno após o congelamento/descongelamento.(AU)

Polypeptides associate to membrane integrity (MI) of swine spermatozoa submitted to freezing and thawing were identified. The protein profile of seminal plasma analyzed by SDS-PAGE allowed the identification of nine polypeptide bands with molecular weight ranging from 11.97 to 122.52kDa. One 26.58kDa band was associated with reduced MI (<55 percent). No associations among other bands and MI were observed. The 26.58kDa factor is associated with reduction of membrane integrity of swine spermatozoa after freezing and thawing.(AU)

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