Effects of Zn deficiency, antioxidants, and low-dose radiation on diabetic oxidative damage and cell death in the testis.

Infertility is one of the common complications in diabetic men and mainly due to the loss of germ cells by apoptotic cell death. Although several mechanisms have been proposed to explain the induction of testicular cell death by diabetes, diabetic induction of testicular oxidative stress and damage may be the predominant mechanism responsible for the testicular cell death in diabetes. To explore whether factors that either increase or decrease the testicular oxidative stress and damage will enhance or prevent diabetes-induced testicular cell death, the effect of zinc (Zn) deficiency on diabetes-induced cell death has been examined since Zn was found to play an important role in the protection of testis from oxidative stress and damage. Zn deficiency, induced by its chelator N,N,N,N-Tetrakis(2-pyridylmethyl)-1,2-ethylenediamine, was found to exacerbate diabetes-induced testicular oxidative damage and cell death. In contrast, treatment of diabetic rats with antioxidant N-acetylcysteine or low-dose radiation that can up-regulate endogenous antioxidants significantly attenuated diabetes-induced testicular cell death. These results suggest that diabetes-induced testicular cell death that may eventually cause men's infertility is predominantly mediated by the oxidative stress and damage. To prevent or delay diabetes-caused infertility, diabetic patients should avoid Zn deficiency, and might consider antioxidant supplementation.

Zinc deficiency exacerbates diabetic down-regulation of Akt expression and function in the testis: essential roles of PTEN, PTP1B and TRB3.

Since zinc (Zn) plays an important role in the spermatogenesis and Zn deficiency exacerbated diabetes-induced testicular apoptosis, the present study investigated the effect of Zn deficiency on diabetes-induced testicular Akt-mediated glucose metabolism changes and inflammation. Zn deficiency was induced by chronic treatment of normal and diabetic mice with the Zn chelator N,N,N',N', tetrakis (2-pyridylmethyl) ethylenediaminepentaethylene (TPEN). After diabetes onset induced by streptozotocin, both diabetic and age-matched control mice were given TPEN intraperitoneally for 4 months. Western blotting assay revealed that Akt-mediated glucose metabolism signaling was down-regulated in the diabetic testis and was further decreased in diabetic mice with Zn deficiency, reflected by reduced phosphorylation of both Akt and GSK-3ß and increased phosphorylation of glycogen synthase along with a disarrangement of fatty acid metabolism (increased expression of PPAR-α and decreased adenosine-monophosphate-activated protein kinase phosphorylation). Testicular expressions of plasminogen activator inhibitor-1 and intracellular adhesion molecule-1 as inflammatory factors were increased in the TPEN or diabetes-alone group, but not additive in the group of diabetes with Zn deficiency. A mechanistic study showed that Akt negative regulators phosphatase and tensin homology deleted on chromosome 10 (PTEN), protein tyrosine phosphatases 1B and Tribbles 3 all increased in diabetic testis and further increased in the testis of diabetic mice with Zn deficiency. These studies suggest that Zn deficiency significantly exacerbated diabetic down-regulation of Akt expression and function, most likely by up-regulation of Akt negative regulators. Therefore, prevention of Zn deficiency for diabetic patients is important in order to avoid the exacerbation of diabetic inhibition of glucose metabolism in the testis.

[Establishment and application of nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA].

OBJECTIVE: To establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA in PBMC( peripheral blood monocyte) and MMNC (marrow monocyte). METHODS: Based on the structural differences between HBVcccDNA and HBV rcDNA, two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream were designed. To remove rcDNA, cccDNA was processed by Mung Bean Nuclease,and then amplified by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers. According to the standard preparation, cccDNA levels of specimen were calculated. RESULTS: We have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 5.0 x 10(2) to 3. 9 x 10(7) copies per milliliter. Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative. CONCLUSIONS: The nested real-time fluorescent quantitative PCR method may be applied to detect HBVcccDNA level in PBMC and MMNC. HBVcccDNA can be detected in PBMC and MMNC.

Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA.

BACKGROUND: Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs). METHODS: Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard. RESULTS: The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0 × 10(2) copies/ml to 3.9 × 10(8) copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative. CONCLUSION: The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

Exacerbation of diabetes-induced testicular apoptosis by zinc deficiency is most likely associated with oxidative stress, p38 MAPK activation, and p53 activation in mice.

Since diabetes induces testicular oxidative damage and cell death, and zinc (Zn) plays an important role in the spermatogenesis, the objective of the present study was to define the effects of Zn deficiency on diabetes-induced testicular apoptosis and associated mechanisms. Zn deficiency was induced by chronic treatment of normal and diabetic mice with N,N,N',N'-tetrakis (2-pyridylemethyl) ethylenediamine (TPEN) chelation. After diabetes onset, mice were given intraperitoneally TPEN at 5mg/kg daily for four months, which, like diabetes, induced a significant decrease in testicular Zn level. TUNEL staining revealed that testicular apoptosis was significantly increased along with an increased Bax/Bcl-2 ratio, in diabetic mice and TPEN-treated normal mice. Zn deficiency significantly exacerbated diabetes-induced testicular apoptosis, along with significantly increased oxidative and nitrosative damage and down-regulation of antioxidant Nrf2 expression. Increased oxidative stress was associated with an increase in activation of p38 MAPK and p53 protein in diabetic testis, which was worsened in the testes of diabetic mice with Zn deficiency. Diabetes also induced a significant increase in endoplasmic reticulum stress and associated cell death, which was not affected by Zn deficiency. These results suggest that like diabetes, chronic depletion of Zn with TPEN induces testicular oxidative stress and damage, along with the activation of p38 MAPK and p53 signaling and mitochondria-related apoptotic cell death. Therefore, prevention of Zn deficiency for diabetic patients is important in order to avoid the exacerbation of diabetic effects on testicular cells death.