ABSTRACT
To further test whether protein kinase A (PKA) can affect the mitotic cell cycle, one-cell stage mouse embryos at S phase (22 h after hCG injection) were incubated in M16 medium containing various concentrations of H-89, a PKA inhibitor. With increasing concentrations of H-89 (0-50 μmol/L), the G(2) phase of eggs was decreased and the cleavage rate was accelerated. A concentration of 40 μmol/L H-89 led to all of the mouse eggs entering the M phase of mitosis. Furthermore, to study the role of PKA in regulating the phosphorylation status of S149 and S321 sites of cell division cycle 25B (CDC25B) on one-cell stage fertilized mouse eggs, pBSK-CDC25B-WT, pBSK-CDC25B-S149A, pBSK-CDC25B-S321A and pBSK-CDC25B-S149A/S321A were transcribed into mRNAs in vitro, then mRNAs were microinjected into S phase of mouse fertilized eggs and cultured in M16 medium pretreated with H-89. Then, the cleavage of fertilized eggs, maturation promoting factor (MPF) activity and phosphorylation status of CDC2-Tyr15 were observed. In the presence of 40 μmol/L H-89, the cleavage rate of fertilized eggs in CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups was significantly higher than that in the control groups, and the peak of MPF activity appeared in the CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups earlier than that in the control groups. CDC2-Tyr15 phosphorylation state was consistent with MPF activity. In conclusion, the present study suggests that PKA regulates the early development of mouse embryos by phosphorylation of S149 and S321 of CDC25B, which plays an important role in the regulation of G(2)/M transition in the mitotic cell cycle of fertilized mouse eggs.
Subject(s)
Animals , Female , Male , Mice , Cyclic AMP-Dependent Protein Kinases , Genetics , Physiology , Embryonic Development , Physiology , Microinjections , Mitosis , Phosphorylation , Serine , Genetics , Metabolism , Zygote , Cell Biology , cdc25 Phosphatases , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of recombinant human testis sperm binding protein (TSBP) on human sperm motility parameters in vitro.</p><p><b>METHODS</b>Sperm specimens obtained from 22 healthy fertile men were prepared by the Percoll gradient-centrifugation technique. The sperm suspension was incubated with recombinant His6-TSBP at the concentration of 0.01 mg/ml or 0.1 mg/ml at 37 degrees C for 1 or 3 hours in vitro. The combination of the recombinant protein and sperm membrane was determined by Western blot, and the sperm motility parameters were analyzed by computer-aided sperm analysis (CASA). The same procedure was performed for 12 asthenospermia patients.</p><p><b>RESULTS</b>In the 22 healthy volunteers, the percentage of forward motile sperm was increased after incubated with 0.1 mg/ml recombinant protein for 1 h (P < 0.05), both forward motile sperm percentage and motility were increased after incubated with recombinant protein at the same concentration for 3 h (P < 0.05), but no effect was observed after incubation with 0.01 mg/ml recombinant protein. In the 12 asthenospermia patients, the forward motile sperm percentage was increased after incubated with 0.1 mg/ml recombinant protein for 3 h (P < 0.05), but no statistically significant difference was observed in sperm motility.</p><p><b>CONCLUSION</b>Recombinant His6-TSBP at the concentration of 0.1 mg/ml can increase sperm motility in healthy fertile men and the forward motile sperm percentage in both healthy fertile men and asthenospermia patients in vitro.</p>
Subject(s)
Adult , Humans , Male , Infertility, Male , Metabolism , Recombinant Proteins , Pharmacology , Seminal Plasma Proteins , Pharmacology , Sperm MotilityABSTRACT
To investigate the effect of Protein kinase B on the expression and location of p21 in mouse early development. Immunopreciptation technology was used to detect the localization of p21 and Western blotting was used to analyze the expression of p21 after microinjecting mRNA of WT-PKB, myt-PKB and PKB-KD to mouse eggs. There was no obvious difference between the three kinds of mRNA in the expression of p21. But the cell localization altered. The p21 retain in cytoplasm after microjecting myt-PKB. In mouse fertilized egg PKB/Akt controls the cell cycle by changing the cell localization of p21.
Subject(s)
Animals , Female , Male , Mice , Cell Nucleus , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cytoplasm , Metabolism , Fluorescent Antibody Technique , Microinjections , Mutation , Proto-Oncogene Proteins c-akt , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Time Factors , Zygote , Cell Biology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>The aim of this study was to observe the expression of mTOR (mammalian target of rapamycin) and its substrates in oral squamous cell carcinoma.</p><p><b>METHODS</b>mTOR and its substrates alpha1, alpha2, beta1, beta2 isoforms of p70 S6 kinase (p70S6k) and 4EBP1 were examined by means of RT-PCR, Western-blot test.</p><p><b>RESULTS</b>The result of RT-PCR showed that in poorly differentiated tissue, the expression level of mTOR and its substrates alpha1, alpha2, beta1, beta2 isoforms of p70S6k increased obviously, while that of 4EBP1 decreased, while that in well differentiated tissue was second to it, the normal oral tissue was the last. The expression of Western-blot was the same as the RT-PCR.</p><p><b>CONCLUSION</b>The expression of mTOR and its substrates differs in different types of oral squamous cell carcinoma. The result suggests that mTOR, p70S6K and 4EBP1 might play important roles in oral squamous cell carcinoma. It may be an important target protein to treat tumor in the future.</p>