ABSTRACT
Objective To investigate the regulation of blood-testis barrier by Rab13-PKA pathway in rats.Meth-od First, shRNA vector targeting at Rab13 was constructed and then the Rab13 shRNA was transfected into the rat testis by injection.Western blot was used to detect the knock-down effect of Rab13 and the expression of blood-testis barrier ( BTB) constituent proteins.PKA activity was detected by autoradiography and scintillation counting.Further, immunoflu-orescence analysis and phalloidin staining were applied to observe the distribution of occludin and F-actin, respectively. Results The expression level of Rab13 in the testis was reduced by approximately 70%after transfection of Rab13 shRNA as compared with the non-targeted control group ( P<0.01 ) , while the expression of BTB constituent proteins remained unchanged.PKA activity was significantly increased after Rab13 RNAi transfection (P<0.01).The distribution of occlu-din at BTB was remarkably increased after Rab13 RNAi silencing around stage VIII but not at other stages of the seminifer-ous epithelial cycle.The assembly of F-actin at BTB was also intensified in Rab13-silenced testis.Both the changes of dis-tribution of occludin and F-actin induced by Rab13 shRNA were found to be antagonized by the PKA specific inhibitor H89.Conclusions Rab13 can modulate the distribution of occludin and F-actin at the blood-testis barrier in rats by regu-lating PKA activity, which may participate in the regulation of BTB function.
ABSTRACT
Background and purpose:This study investigated the relationship between (S100 calcium-binding protein A4, S100A4) in chronic gastritis, intestinal metaplasia, dysplasia adenomatous, normal tissue tissue samples and expression in gastric cancer and clinical characteristics. Methods:HE staining of the use of gastric specimens taken for histopathological diagnosis;using immunohistochemistry to detect the expression of tissue S100A4 protein;qRT-PCR was used to detect mRNA expression of S100A4 gene;Western Blot detection of S100A4 gene encoding protein. Kaplan-Meier survival curves were used to distinguish and compare survival. Results:S100A4 protein and mRNA expression gradually increased in the following order:normal tissue
ABSTRACT
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.