Peroxiredoxin 6 translocates to the plasma membrane of human sperm under oxidative stress during cryopreservation.

Peroxiredoxin 6 (PRDX6) is one antioxidant enzyme which could control the levels of reactive oxygen species and to avoid oxidative damage of sperm. In this study, we aimed to investigate the position change of PRDX6 in human sperm under oxidative stress during cryopreservation. Semen samples were obtained from 98 healthy donors and 27 asthenozoospermic donors. The plasma membrane protein and cytoplasmic protein of sperm samples were extracted and analyzed after cryopreservation. Western blot and immunofluorescence were used to measure the expressions of PRDX6. Liquid chromatography mass spectrometric (LC-MS/MS) analysis was performed to confirm the component of sperm membrane complex. Western blot showed that the detection rate of PRDX6 in plasma membranes with low sperm motility (≤20%) was significantly higher than that with high sperm motility (≥40%). Western blot and Immunofluorescence revealed that cryopreservation and thawing induced the position change of the PRDX6 from cytoplasm to sperm membrane. LC-MS/MS analysis showed that PRDX6, ADP/ATP translocase 4 (ANT4) and glyceraldehyde-3-phosphte dehydrogenase (GAPDHS) were present in the components of membrane complex after cryopreservation. The present study indicated that the presence of PRDX6 in sperm plasma membrane was related to sperm motility. GAPDHS and ANT4 may be involved the position change of the PRDX6 from cytoplasm to sperm membrane under oxidative stress during cryopreservation.

Pretreatment of mice with oligonucleotide prop5 protects them from influenza virus infections.

Influenza A virus is a successful parasite and requires host factors to complete its life cycle. Prop5 is an antisense oligonucleotide, targeting programmed cell death protein 5 (PDCD5). In this study, we tested the antiviral activity of prop5 against mouse-adapted A/FM/1/47 strain of influenza A virus in a mouse model. Prop5 intranasally administered the mice at dosages of 10 and 20 mg/kg/d at 24 h and 30 min before infection, provided 80% and 100% survival rates and prolonged mean survival days in comparison with influenza virus-infected mice (both p < 0.01). Moreover, viral titres in mice pretreated with prop5, at dose of 10 and 20 mg/kg/d, had declined significantly on day two, four, and six post-infection compared with the yields in infected mice (p < 0.05 or p < 0.01); lung index in mice pretreated with prop5 (20 mg/kg/d) had been inhibited on day six post-infection (p < 0.05). Western blotting and immunohistochemistry showed that prop5 could down-regulate the PDCD5 protein expression levels in lung tissues of infected mice. These data indicate that antisense oligonucleotide prop5 is a promising drug for prophylaxis and control influenza virus infections and provides an insight into the host-pathogen interaction.

Cellular microRNA let-7c inhibits M1 protein expression of the H1N1 influenza A virus in infected human lung epithelial cells.

The influenza virus (IV) triggers a series of signalling events inside host cells and induces complex cellular responses. Studies have suggested that host factors play an essential role in IV replication. MicroRNAs (miRNAs) represent a class of small non-coding RNAs that target mRNAs, triggering either translation repression or RNA degradation. Emerging research suggests that host-derived cellular miRNAs are involved in mediating the host-IV interaction. Using miRNA microarrays, we identified several miRNAs aberrantly expressed in IV-infected human lung epithelial cells (A549). Specifically, miR-let-7c was highly up-regulated in IV-infected A549 cells. PITA and miRanda database screening indicated that the let-7c seed sequence is a perfect complementary sequence match to the 3' untranslated region (UTR) of viral gene M1 (+) cRNA, but not to PB2 and PA. As detected by a luciferase reporter system, let-7c directly targeted the 3'-UTR of M1 (+) cRNA, but not PB2 and PA. To experimentally identify the function of cellular let-7c, precursor let-7c was transfected into A549 cells. Let-7c down-regulated IV M1 expression at both the (+) cRNA and protein levels. Furthermore, transfection with a let-7c inhibitor enhanced the expression of M1. Therefore, let-7c may reduce IV replication by degrading M1 (+) cRNA. This is the first report indicating that cellular miRNA regulates IV replication through the degradation of viral gene (+) cRNA by matching the 3'-UTR of the viral cRNA. These findings suggest that let-7c plays a role in protecting host cells from the virus in addition to its known cellular functions.

Ginsenoside Rg1 protects rat cardiomyocyte from hypoxia/reoxygenation oxidative injury via antioxidant and intracellular calcium homeostasis.

Ginsenoside Rg1 is a major active ingredient of Panax notoginseng radix which has demonstrated a number of pharmacological actions including a cardioprotective effect in vivo. This study investigated the protective effect and mechanism of ginsenoside Rg1 in cardiomyocytes hypoxia/reoxygenation (H/R) model. Pretreatment with ginsenoside Rg1 (60-120 microM) reduced lactate dehydrogenase release and increased cell viability in a dose-dependent manner. Fluorescence analysis demonstrated ginsenoside Rg1 reduced intracellular ROS and suppressed the intracellular [Ca(2+)] level. Cell lysate detected an increase of T-SOD, CAT, and GSH levels. The myocardial protection of ginsenoside Rg1 during H/R is partially due to its antioxidative effect and intracellular calcium homeostasis.

Proteomic analysis of ionizing radiation-induced proteins at the subcellular level.

Irradiation induces a series of liver diseases. However the molecular mechanisms involving in the process of liver diseases induced by irradiation are still unclear. Subcellular proteomics provides a method to understand regional differences in protein expression levels. With accumulating evidence in the literature that new proteins are implicated in radiation response, in the present study, C57BL/6 mice were treated with irradiation, liver cell homogenates were subfractionated by differential ultracentrifugation into nuclei, mitochondria and cytosol, which were subjected to 2-DE to generate the proteomic maps of these fractions. The differentially expressed proteins in the nuclei, mitochondria and cytosol compartment of liver at 24 and 48 h after exposure to 20 Gy irradiation compared to control were identified by MALDI-TOF MS respectively. Total 37 proteins at 24 h and 29 proteins at 48 h were matched with known proteins after database searching in nuclei, mitochondria and cytosol, respectively, among which nine proteins exhibited changes at both time points. Most of these proteins are involved in antioxidant response, energy metabolism, molecular chaperones and inflammatory response. More antioxidant-associated proteins were induced at 48 h than 24 h. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting further validated 2-DE results of two of these proteins. It is feasible that the differential proteins identified in this study have a biological significance and may provided clues for understanding the mechanism of injury in liver induced by irradiation.

Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents 6-hydroxydopamine induced apoptosis in SH-SY5Y cells.

Oxidative stress caused by dopamine may play an important role in the pathogenesis of Parkinson's disease. Salvianolic acid B is an antioxidant derived from the Chinese herb, Salvia miltiorrhiza. In this study, we investigated the neuroprotective effect of salvianolic acid B against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with salvianolic acid B significantly reduced 6-hydroxydopamine-induced generation of reactive oxygen species, and prevented 6-hydroxydopamine-induced increases in intracellular calcium. Our data demonstrated that 6-hydroxydopamine-induced apoptosis was reversed by salvianolic acid B treatment. Salvianolic acid B reduced the 6-hydroxydopamine-induced increase of caspase-3 activity, and reduced cytochrome C translocation into the cytosol from mitochondria. The 6-hydroxydopamine-induced decrease in the Bcl-x/Bax ratio was prevented by salvianolic acid B. Additionally, salvianolic acid B decreased the activation of extracellular signal-regulated kinase and induced the activation of 6-hydroxydopamine-suppressed protein kinase C. These results indicate that the protective function of salvianolic acid B is dependent upon its antioxidative potential. Our results strongly suggest that salvianolic acid B may be effective in treating neurodegenerative diseases associated with oxidative stress.