The protective effect of vitamin E against oxidative damage caused by formaldehyde in the testes of adult rats

Aim： To investigate the effect of formaldehyde （FA） on testes and the protective effect of vitamin E （VE） against oxidative damage by FA in the testes of adult rats. Methods： Thirty rats were randomly divided into three groups： （1） control; （2） FA treatment group （FAt）; and （3） FAt ＋ VE group. FAt and FAt ＋ VE groups were exposed to FA by inhalation at a concentration of 10 mg/m^3 for 2 weeks. In addition, FAt ＋ VE group were orally administered VE during the 2-week FA treatment. After the treatment, the histopathological and biochemical changes in testes, as well as the quantity and quality of sperm, were observed. Results： The testicular weight, the quantity and quality of sperm, the activities of superoxide dismutase （SOD）, glutathione peroxidase （GSH-Px） and glutathione （GSH） were significantly decreased whereas the level of malondialdehyde （MDA） was significantly increased in testes of rats in FAt group compared with those in the control group. VE treatment restored these parameters in FAt ＋ VE group. In addition, microscopy with hematoxylin-eosin （HE） staining showed that seminiferous tubules atrophied, seminiferous epithelial cells disintegrated and shed in rats in FAt group and VE treatment significantly improved the testicular structure in FAt ＋ VE group. Conclusion： FA destroys the testicular structure and function in adult rats by inducing oxidative stress, and this damage could be partially reversed by VE.

NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line

AIM:To construct the recombinant lentivirus expression plasmid,pLenti6/V5-NT4 p53(N15)-antennapedia(Ant),and study its effect on HepG2 cells.METHODS:Plasmid pLenti6/V5-NT4 p53(N15)-Ant was constructed incorporating the following functional regions,including signal peptide sequence and pro-region of neurotrophin 4,N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein,Ant.Hepatocellular carcinoma(HepG2)cells were used for transfection.3-[4,5-dimethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide(MTT)assay,lactate dehydrogenase(LDH)release assay,transmission electron microscopy(TEM)and flow cytometric analysis(FCM)were employed to investigate the effects of LV-NT4(Si)-p53(N15)-Ant in vitro on HepG2 cells.In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4(Si)-p53(N15)-Ant on tumor growth in nude mice.RESULTS:LV-NT4(Si)-p53(N15)-Ant significantly suppressed the growth of HepG2 cells.MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4(Si)-p53(N15)-Ant than by LV-EGFP.The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%,respectively,48 h after infection with LV-NT4(Si)-p53(N15)-Ant,and was 33.9% and 95.8%,respectively,72 h after infection with LV-NT4(Si)-p53(N15)-Ant(P < 0.01).Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4(Si)-p53(N15)-Ant,but no signif icant changes in HepG2 cells infected with LV-EGFP.Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4(Si)-p53(N15)-Ant,with degraded membranes,resulting in necrosis.LDH release from HepG2 cells was analyzed at 24,48,72 and 96 h after infection with LV-NT4(Si)-p53(N15)-Ant and LV-EGFP,which showed that LDH release was signif icantly higher in LV-NT4(Si)-p53(N15)-Ant treatment group(682 IU/L)than in control group(45 IU/L,P < 0.01).The longer the time was after infection,the bigger the difference was in LDH release.FCM analysis showed that LV-NT4(Si)-p53(N15)-Ant could induce two different kinds of cell death:necrosis and apoptosis,with apoptosis being the minor type and necrosis being the main type,suggesting that LV-NT4(Si)-p53(N15)-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis.The in vivo study showed that LV-NT4(Si)-p53(N15)-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight.

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility

Aim： To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 （USP26） gene and its involvement in idiopathic male infertility in China. Methods： Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. Results： Of 41 infertile men, 9 （22.0%, P = 0.01） had changes in USP26 gene on the X chromosome. A compound mutation （364insACA; 460G→A） was detected in 8 patients （19.5%, P = 0.01） and a 1044T→A substitution was found in 1 patient （2.4%, P 〉 0.05）. All three variations led to changes in the coding amino acids. Two substitutions predict some changes： 460G→ A changes a valine into an isoleucine, and 1044T → A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. Conclusion： The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

The expression of Usp26 gene in mouse testis and brain

Deubiquitinating enzymes （DUBs） play an important role in ubiquitin-dependent processes as negative regulators of protein ubiquitination. Ubiquitin-specific protease 26 （USP26） is a member of this family. The expression of Usp26 in mammalian testis and in other tissues has yet to be fully elucidated. To study the expression of Usp26 mRNA and protein in various murine tissues, reverse transcription （RT）-PCR and immunohistochemistry analyses were carried out. The RT-PCR analysis showed that the Usp26 transcript was expressed in all of the tested tissues. USP26 protein localization was examined by immunohistochemistry, and it was shown that USP26 was not detectable at 20 days postpartum, with the expression restricted to the cytoplasm of condensing spermatids （steps 9-16）, Leydig cells and nerve fibers in the brain. In addition, the USP26 protein was detected at moderate levels in myocardial ceils, the corpus of epidydimis, epithelium of the renal tubules and the seminal gland of postnatal day 35 mice. Its spatial and temporal expression pattern suggests that Usp26 may play an important role in development or function of the testis and brain. Further research into these possibilities is in progress.