[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was ...[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.展开更多
We investigated the long-lasting effects of early postnatal tactile stimulation （TS） and maternal separation （MS） on the emotional behaviors of adult female rats. A split-litter design was introduced to remove con...We investigated the long-lasting effects of early postnatal tactile stimulation （TS） and maternal separation （MS） on the emotional behaviors of adult female rats. A split-litter design was introduced to remove confusing factors such as maternal disturbance. Pups of the non-tactile stimulation （NTS） group did not receive any handling. Pups subjected to the TS treatment were handled and marked for approximately 30 s daily from postnatal days （PND） 2 - 9 or from PND 10 - 17. Pups subjected to the MS treatment were handled and marked in the same way as the TS pups and then individually placed in a cup with familiar nest bedding for 1 h daily. At the age of 3 months, female rats with different neonatal experiences were employed in the light/dark box test and the one-trial passive avoidance response. Both PND 2 - 9 TS and PND 10 - 17 TS groups exhibited more time spent in the illuminated chamber of the light/dark box, and longer step-through latencies in the passive avoidance response when compared to the NTS group, indicating that early life TS treatment reduced novelty-induced anxious emotion and facilitated the retention of emotional memory in adult female rats. No significant effects were found on any behavioral measures between the MS groups and the TS groups, suggesting that neonatal short-time MS treatment was not intensive enough to alter the emotional behaviors, at least in female rats. Infantile age was not an effective factor for these measures. This result supports the hypothesis that neonatal tactile stimulation and maternal separation lead to different effects on the neural development of postnatal pups.展开更多
Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to ex...Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to explore the relationship between oocyte maturation and GDF-9 gene expression. Preantral follicles of lO-day old mice were isolated from the ovaries and were cultured for 12 days. Oocytes from day 2 （D2）, D4, D6, D8, DIO, D12 cultured in vitro were named the in vitro group and oocytes of day 12 （D12）, D14, D16, D18, D20, D22 grown in vivo were named the in vivo group. Follicle survival, antrum formation and maturation rate were 89.5%, 51.8% and 56.6% respectively in follicles cultured in vitro. After RT-PCR and agarose gel electrophoresis, relative mRNA abundance of GDF-9 was measured in each group of oocytes. At day 8 - 12, the GDF-9 gene expression level of oocytes in vitro was significantly lower than that in vivo （P 〈 0.05）. We conclude that M Ⅱ oocytes can be obtained from in vitro culture of preantral follicles. The GDF- 9 gene expression of oocytes varies at different growth stages in vivo. The low expression of GDF-9 in oocytes cuhured in vitro may be the cause of their low developmental capacity.展开更多
文摘[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.
文摘We investigated the long-lasting effects of early postnatal tactile stimulation （TS） and maternal separation （MS） on the emotional behaviors of adult female rats. A split-litter design was introduced to remove confusing factors such as maternal disturbance. Pups of the non-tactile stimulation （NTS） group did not receive any handling. Pups subjected to the TS treatment were handled and marked for approximately 30 s daily from postnatal days （PND） 2 - 9 or from PND 10 - 17. Pups subjected to the MS treatment were handled and marked in the same way as the TS pups and then individually placed in a cup with familiar nest bedding for 1 h daily. At the age of 3 months, female rats with different neonatal experiences were employed in the light/dark box test and the one-trial passive avoidance response. Both PND 2 - 9 TS and PND 10 - 17 TS groups exhibited more time spent in the illuminated chamber of the light/dark box, and longer step-through latencies in the passive avoidance response when compared to the NTS group, indicating that early life TS treatment reduced novelty-induced anxious emotion and facilitated the retention of emotional memory in adult female rats. No significant effects were found on any behavioral measures between the MS groups and the TS groups, suggesting that neonatal short-time MS treatment was not intensive enough to alter the emotional behaviors, at least in female rats. Infantile age was not an effective factor for these measures. This result supports the hypothesis that neonatal tactile stimulation and maternal separation lead to different effects on the neural development of postnatal pups.
文摘Mice preantral follicles were cultured in vitro for 12 days to achieve metaphase Ⅱ （M Ⅱ ） oocytes. Oocyte growth differentiation factor-9 （GDF-9） gene expression was measured during different growth stages to explore the relationship between oocyte maturation and GDF-9 gene expression. Preantral follicles of lO-day old mice were isolated from the ovaries and were cultured for 12 days. Oocytes from day 2 （D2）, D4, D6, D8, DIO, D12 cultured in vitro were named the in vitro group and oocytes of day 12 （D12）, D14, D16, D18, D20, D22 grown in vivo were named the in vivo group. Follicle survival, antrum formation and maturation rate were 89.5%, 51.8% and 56.6% respectively in follicles cultured in vitro. After RT-PCR and agarose gel electrophoresis, relative mRNA abundance of GDF-9 was measured in each group of oocytes. At day 8 - 12, the GDF-9 gene expression level of oocytes in vitro was significantly lower than that in vivo （P 〈 0.05）. We conclude that M Ⅱ oocytes can be obtained from in vitro culture of preantral follicles. The GDF- 9 gene expression of oocytes varies at different growth stages in vivo. The low expression of GDF-9 in oocytes cuhured in vitro may be the cause of their low developmental capacity.
