Effects of rs35100176 polymorphism in IPO8 promoter on gene expression / 中国病理生理杂志

AIM:To investigate the effect of rs 35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 ( IPO8) gene on its mRNA expression .METHODS:A 342-bp fragment of IPO8 gene promoter con-taining the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced .The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples .The PCR products were sequenced and inserted into the luciferase reporter vector pGL 3-Basic.Recombinant vectors were trans-fected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system.The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells.RESULTS:The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT, CCT/-and -/-, and the gene frequencies were 18.37%, 55.10% and 26.53%, respectively.The re-combinant expression vectors pGL 3-3N Insertion and pGL3-3N Deletion were successfully constructed .The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL 3-3N Deletion.Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO 8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote .CONCLUSION: The CCT 3-nucleotide insertion variant decreases the pro-moter activity of IPO8, thus affecting the gene expression .

Preparation and identification of scFv and bsFv against transferrin receptor / 华中科技大学学报（医学）（英德文版）

To obtain single chain variable fragment (scFv) and bivalent single chain variable fragment (bsFv) against transferrin receptor, up-stream and down-stream primers were designed according to the complementary sequences of FR1 region of variable heavy (VH) and FR4 of variable light (VL), respectively, which contained inter-linker G4S and the restriction endonuclease SfiI, AscI and NotI. Two pieces of scFv fragments were first amplified through PCR and then inserted into plasmid pAB1, which could express scFv protein once induced by IPTG in the host bacteria. To express scFv and bsFv, E. coli TG1 was cultured in LB broth and was induced by IPTG. The restriction enzyme digestion map and DNA sequencing demonstrated that scFv and bsFv genes were successfully inserted into the expression plasmid. SDS-PAGE and Western blotting revealed the protein band at 35kD and 60kD, which were consistent with the molecular weight of scFv and bsFv respectively. Flow cytometry showed that scFv and bsFv harbored the specific binding activity with TfR expressed in various tumor cells, and the avidity of bsFv was higher than that of the parent scFv.

Effect of Dihydroartemisinin on the Ultrastructure of Trichomonas vaginalis Trophozoites in vitro / 中国寄生虫学与寄生虫病杂志

Objective To observe the effect of dihydroartemisinin(DHA) on the ultrastructure of Trichomonas vaginalis cultured in vitro.Methods The trophozoites of T.vaginalis were cultivated with liver extract solution medium containing 1 mg/ml dihydroartemisinin,and were then observed by scanning and transmission electron microscopes after the treatment for 2.5-4.0 h.Results The cell membranes of the trophozoites treated with DHA were damaged considerably.The surface of T.vaginalis showed deepening folds,hollow,and cracks.The nuclear membrane appeared fractures.There were a few of crevices in the nucleus and cytoplasm.Disordered and dilated endoplasmic reticulum,injured and deformed hydrogenosomes were found.Cytoplasm of the damaged parasites spilled over from torn place.After the cell membrane was peeled off,the nuclei,hydrogenosomes and pelta were exposed,which finally resulted in the death of the denatured parasites.Conclusion Dihydroartemisinin can destroy membrane structure and organelles of Trichomonas vaginalis trophozoites,which leads to decomposition and necrosis of the parasites.

Effect of clonidine on intraocular pressure and its central adrenergic mechanism / 中国药理学通报

AIM To investigate the effect of clonidine on intraocular pressure(IOP) and the possible role in which ? adrenergic mechanism plays. METHODS The change on IOP was observed following clonidine administered via three different routes: (1)clonidine topically administered to eyes; (2)clonidine intracerebroventricularly injected (icv)or topically administered after yohimbine or prazosin icv; (3)microinjection of clonidine into locus coeruleus(LC). RESULTS Clonidine decreased IOP significantly, ? 2 adrenoceptor antagonist, but not ? 1 adrenoceptor antagonist, can reverse the decreasing effect on IOP caused by clonidine icv and administered topically. CONCLUSION Clonidine administered both topically or intracranially can decrease IOP;? 2 adrenoceptor in central nervous system is involved in this effect.