ABSTRACT
The purpose of this study is to reveal the role of Girdin in regulating the aggregation of actin filaments by studying the relationship between PKB/Akt and Girdin. First we used Scansite software (http://scansite.mit.edu) to predict relevant target sites of PKB/Akt on mouse Girdin. To gain insight into the role of phosphorylation of Girdin by PKB/Akt, we assessed the location of phosphorylated Girdin in fertilized eggs by staining with anti-P-Girdin 1 417 Ab. We detected a distinct increase in the fluorescence signal of F-actin and P-Girdin 1 417 after microinjection of Akt WT and myr-Akt. The addition of myr-Akt induced phosphorylation of Girdin in mouse fertilized eggs. In addition, siRNA-mediated Akt-knockdown blocked phosphorylation of Girdin. The distribution of actin filaments was obviously scattered. These results strongly suggest that PKB/Akt could directly phosphorylate Girdin on Ser1 417 and promote its function in mouse fertilized eggs.
Subject(s)
Animals , Mice , Actins , Physiology , Microfilament Proteins , Physiology , Phosphorylation , Proto-Oncogene Proteins c-akt , Physiology , RNA, Small Interfering , Vesicular Transport Proteins , Physiology , ZygoteABSTRACT
<p><b>AIM</b>To explore the effect of Cdc25B overexpression on the development of mouse two-cell embryos.</p><p><b>METHODS</b>The pBSK-Cdc25B was in vitro transcribed into 5'-capped mRNA for microinjection by using mMESSAGE mMACHINE kit. The Cdc25B mRNA was microinjected into mouse embryos at two-cell stage in order to observe the embryonic development and cleavage rate. Using protein kinase activity assay and Western blot to detect the MPF activity as well as the phosphorylation status of Cdc2-Tyr15 in Cdc25B overexpression group respectively.</p><p><b>RESULTS</b>The mouse embryos with Cdc25B overexpression developed to the four-cell stage 48 h after the hCG injection with the percentage of cleavage over 40% compared with the embryos in control groups which still remained at the two-cell stage. Moreover, MPF activity increased significantly after Cdc25B mRNA injection. The phosphorylation status of Cdc2-Tyr15 was coincident with MPF activity.</p><p><b>CONCLUSION</b>The results indicate that Cdc25B overexpression in early mouse two-cell embryos reverses two-cell block and promotes their development into four-cell stage by activating MPF.</p>
Subject(s)
Animals , Female , Male , Mice , Cell Cycle , Cell Division , Embryo, Mammalian , Cell Biology , Embryonic Development , Physiology , Maturation-Promoting Factor , Metabolism , Microinjections , Mitosis , RNA, Messenger , Metabolism , Zygote , Metabolism , cdc25 Phosphatases , PhysiologyABSTRACT
To investigate the function of testis sperm binding protein (TSBP) in sperm capacitation and acrosome reaction, the effect of the recombinant TSBP on the activity of protein kinase A was detected in the transfected cell line. With the use of prokaryotic expressing plasmid pGEX-5X-1/tsbp as template, the novel gene tsbp was amplified by PCR and a eukaryotic expressing vector pcDNA3.1/myc-His(-)B/tsbp was constructed. 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 plamid being transfected into HEK293 cells, Western blot verified the expression of tsbp. Fusion protein His6-TSBP was purified from the cell lysate by immobilized metal-ion affinity chromatography (IMAC). Radioautograph revealed a higher PKA activity in the transfected HEK293 cells than in the control group, which indicates that TSBP can increase the activity of PKA.
Subject(s)
Humans , Male , Acrosome Reaction , Cell Line , Cyclic AMP-Dependent Protein Kinases , Recombinant Proteins , Genetics , Pharmacology , Seminal Plasma Proteins , Genetics , Pharmacology , Sperm CapacitationABSTRACT
In order to meet the requirements of medical teaching contents and curriculum system reform, we adopt the experience ofHarvard University School of Medicine to integrate Biochemistry and Cell Biology into a new one—— "Chemistry and Biology of theCell". We have carried out the integration of the two courses three times. In this new course, repeated contents have been reduced, thelinkage of the two courses has been increased, and the burden of students was lightened,which makes students contact the clinic earlierand enables to improve the students' ability to solve problems.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the content and activity of M-phase promoting factor (MPF) in pleomorphic adenoma, mucoepidermoid carcinoma, buccal carcinoma and normal tissue, in order to evaluate the role of MPF in the development of tumor and the relationship between MPF and malignant degree.</p><p><b>METHODS</b>The content and activity of MPF were assessed by immunobloting and Gollicano method.</p><p><b>RESULTS</b>The cdc2 and cyclinB (two subunits of MPF) were found both in normal and tumor tissues, and their content in tumor was higher than normal tissues. Buccal carcinoma was 64% higher than normal tissues. The activity of MPF in carcinoma was higher than normal tissue and had positive relation with the malignant extent.</p><p><b>CONCLUSIONS</b>The content and activity of MPF in tumor are higher than normal tissue. PKC can activate MPF. These results show PKC may promote tumor proliferation by activating MPF and also, the activity of MPF has some relation with malignant extent.</p>
Subject(s)
Humans , CDC2 Protein Kinase , Cyclin B , Immunoblotting , Maturation-Promoting Factor , Mouth , Chemistry , Mouth Neoplasms , Chemistry , Protein Kinase C , PhysiologyABSTRACT
Objective:To study the mechanism of 149 serine in the abrogation of cdc25b as a meiosis inducer in mouse oocytes.Methods: Immunofluorescence analysis was used to observe the distribution of cdc25b protein in GV stage and G2/M transition oocytes;Fused plasmids of pEGFP-cdc25b-wt,pEGFP-cdc25b-s149a and vector pEGFP-C3 were microinjected into GV mouse oocytes.After culturing for a certain time,the subcellular localization of protein was observed.Results:cdc25b-wt was distributed mainly in cytoplasm in GV stage oocytes and accumulation of protein was observed during G2/M transition while green fluoresent signal of cdc25b-s149a was observed in nuclear and cytoplasm.Conclusion:The results suggest that there existed a shuffling between nuclear and cytoplasm of cdc25b in GV stage oocytes although they seem to be quilt,the phosphorylation of 149 serine could maintain proper distribution of protein by balancing the speed of protein nuclear export and nuclear import.
ABSTRACT
Abstract Objective: To construct the Second half of CILP(C2) MBP fusion protein by sub-cloning technology. Methods: Recombinantfusion proteins, which contain the fragments within the C2 region(designated C2F1, C2F2 and C2F3) of the non-porcine nucleotide pyrophos-phohydrolase-homologous region of CILP, were prepared using pMAL-eHis vector. The recombinat genes are induced by different temperatures(22℃,30℃,37℃ ). Results: Expression using pMAL-eHis system can be induced chemically by adding IPTG. 37℃ temperature prmotes in-soluble inclusion-body formation,but 22℃ temperature can not induce the enough expression of recombinant protein. Onl 30℃ temperaturecan induce enough amount of soluble recombinant protein. The characers of fusion proteins that they carried 6 straight histidines, (His)6, at tbeC-terminus of multiple cloning sites for affinity purification were assessed by sodium dodecy1 sulfate-polyacylamide gel electrophoresis(SDS-PAGE) and Western blot. Nucleotide sequences of the insertion genes were confirmed by dideoxy sequencing. Conclusion: C2F1, C2F2,C2F3-MBP fnsion proteins were constructed successfully.These recombinant proteins may provide important roles in the future study on CILP.