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Objective To investigate the expression and localization of Aurora kinase A (AURKA) and Aurora kinase B (AURKB) in mouse zygotes during the process of the first mitosis. Methods Quantitative real-time RT-PCR and Western blotting were performed to analyze the expression of AURKA and AURKB. The subcellular location of AURKA and AURKB was studied by confocal microscopy. Results AURKA and AURKB were increasingly expressed from phase G 1 and peaked at phase M. After the entrance into mitosis AURKB became the predominant form both in mRNA and protein levels. The proteins of AURKA and AURKB both distributed in the cytoplasm and were associated with nucleus during the first mitosis of mouse zygotes, with some details in different. Conclusion The expression and localization of Aurora kinases A and B was in a cellcycle regulated manner during the process of the cleavage of mouse zygotes. This discovery will aid in future investigations on their specific roles and molecular mechanisms in the regulation of mammalian early embryonic development.

Objective To investigate the biology function of novel protein related to bovine seminal plasma protein in human testis. Methods Recombination pcDNA3/HBRP was constructed and transfected to HEK293 cell and permanently expression cell line was established. The activity of protein kinase C (PKC) of the cell line was detected by autoradiography method. Results The stable expression cell line of HBRP was obtained. The HBRP inhibited the activity of PKC significantly. Conclusion One of the new functions of novel sperm binding protein in human is the inhibitor action on activity of PKC. It may be involved in the sperm capacitation and acrosome reaction.

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.

To investigate the effects of recombinant human testis sperm binding protein (TSBP) on human sperm motility parameters in vitro.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.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 (P0.05), both forward motile sperm percentage and motility were increased after incubated with recombinant protein at the same concentration for 3 h (P0.05), 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 (P0.05), but no statistically significant difference was observed in sperm motility.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.

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.

To construct an eukaryotic expressing vector of the human novel gene testis sperm binding protein (tsbp), and to express fusion protein and purify the recombinant protein by affinity chromatography.The novel gene tsbp was amplified by PCR from the prokaryotic expressing plasmid pGEX-5X-1/tsbp and an eukaryotic expressing vector pcDNA3.1/myc-His(-)B/tsbp was constructed after DNA recombination. After transfecting HEK293 cells with this recombinant vector via liposome mediation, the expression of the fusion protein was detected by RT-PCR, immunofluorescence and Western blot. Fusion protein His6-tsbp was purified from the cell lysis by immobilized metal affinity chromatography (IMAC) and the efficiency of purification was detected by SDS-PAGE and Western blot.DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pcDNA3.1/myc-His(-)B/tsbp had been constructed successfully. After the recombinant plasmid being transfected into HEK293 cells, RT-PCR verified the expression of tsbp mRNA. The result of immunofluorescence assay was positive and the fusion protein could be detected by Western blot of transfected HEK293 cells. The purified fusion protein could also be detected by SDS-PAGE and Western blot.The novel gene tsbp was successfully cloned, expressed and purified in the form of His6 fusion protein, which is helpful for further study of the function of this testis sperm binding protein.

The purpose of present study is to determine the location of the novel gene hbrp which is related to bovine seminal plasma (BSP) proteins in the human chromosome by the FISH (fluorescent in situ hybridization) technique, and to investigate the relation of HBRP to TPK( tyrosine protein kinase)by genetic engineering. The result is that the novel gene hbrp was successfully localized on human chromosome 19q1.3; HBRP protein obviously inhibited the activity of PKC. It is concluded that the structure of BSP contained two tandemly arranged fibronectin type II(Fn2)--domains which were related to the function of binding protein BSP. Amino acid sequence of HBRP protein with four tandemly arranged Fn2-domains showed that the protein might be binding protein functionly relating to BSP protein. We have concluded that HBRP protein obviously inhibited the activity of TPK. The location of the novel gene on human chromosome 19q1.3 is very important to study the other biological functions of the protein encoded by gene hbrp.

Gene localization is significant in elucidating the interaction between genes,gene and diseases. Using radiation hybrid (RH) technique,we cloned and localized a novel gene,designated human BSP-related protein (HBRP) on 19q13.2 - 13.3, in line with its localization in data bank of overlapping fragment of human genome through bioinformatics method. It is suggested RH is rapid, precise,simple and powerful in gene localization. In addition, we detected the expression and distribution of HBRP in human tissues by RT-PCR. The results showed HBRP was highly expressed in intestine, kidney, liver, spleen, stomach and pancreas, whereas lowly in brain, lung, muscle and heart.

The aim of this study was to observe the expression of mTOR (mammalian target of rapamycin) and its substrates in oral squamous cell carcinoma.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.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.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.