Study on a 65‐mer peptide mimetic enzyme with GPx and SOD dual function

Excessive reactive oxygen species (ROS) levels are harmful to the body. The peroxidase, GPx, and the superoxide dismutase, SOD, are important antioxidant enzymes for preventing ROS‐induced damage. Se‐CuZn‐65P is an enzyme mimetic with dual GPx and SOD antioxidant function. However, currently, its production is mainly based on the cysteine auxotrophic expression technique, which is inefficient, expensive, and time consuming. In this study, we combined protein engineering and the chemical mutation method to synthesize Se‐CuZn‐65P. The DNA sequence encoding the 65 amino acid peptide with the desired sequence transformations to incorporate the SOD and the GPx catalytic sites was cloned and expressed in a soluble protein expression vector. The protein yield increased up to 152 mg/L, which is 10 times higher than in previous studies. The SOD and GPx activity of Se‐CuZn‐65P was high (1181 U/mg and 753 U/μmol, respectively). The binding constant of glutathione was 5.6 × 10⁴ L·mol^?1, which shows that Se‐CuZn‐65P efficiently catalyzed hydrogen peroxide reduction by glutathione. Mitochondrial damage experiments confirmed the double protective role of the Se‐CuZn‐65P peptide and demonstrated functional synergy between the SOD and the GPx domains, which indicates its potential to be used in the treatment of ROS‐related diseases. Our research may give a new thought to increase the yield of mimic.     

Valproic acid inhibits the proliferation of SHSY5Y neuroblastoma cancer cells by downregulating URG4/URGCP and CCND1 gene expression

Valproic acid (VPA), used for the treatment of epilepsy and bipolar disorder, regulates several signaling pathways in brain cells. The up-regulated gene 4 (URG4/URGCP) is a novel gene located on 7p13. URG4/URGCP stimulates cyclin D1 (CCND1) mRNA expression, and URG4/URGCP silencing diminishes CCND1 mRNA expression in HepG2 cells. This study was performed to investigate the anti-cancer mechanism of action of VPA by analyzing the expression of novel gene URG4/URGCP, CCND1, p21, p53, p65 (RelA), Bax, and Bcl-2 in SHSY5Y neuroblastoma (NB) cancer cells. Cytotoxic effects of VPA in SHSY5Y were noticed in time and dose dependent manner with the IC₅₀ doses within the range of 0.5–10 mM. IC₅₀ doses in the SHSY5Y were detected as 7.5 mM. Expression profiles were determined by semi quantitative RT-PCR and URG4/URGCP protein change by western blot analysis. Our results suggest that VPA induces cell cycle arrest in SHSY5Y due to the decrease in URG4/URGCP, CCND1 gene expression and the increase in p65. To conclude, VPA may be a prospective agent for the treatment of NB as a single agent or in combination with other drugs. Thus, more studies should be designed to find a safe dose with the best effects of VPA.     

Antioxidative enzymes in cultivars of pepper plants with different sensitivity to cadmium

The effect of growing five different cultivars of pepper plants (Capsicum annuum L.) with CdCl₂ concentrations ranging from 0.125 to 0.5 mM on different physiological parameters, and antioxidative enzyme activities of leaves was studied. On the basis of growth parameters, pepper plants were relatively tolerant to cadmium, although metal concentrations higher than 0.125 mM produced a significant inhibition of growth, net photosynthesis, and water use efficiency. Different sensitivities to Cd⁺⁺ ions were observed among cultivars, Abdera being the most resistant to cadmium stress, while Mondo and Herminio were the most sensitive cultivars. Cadmium concentrations of 0.5 mM produced an increase in the activity of glutathione reductase, and guaiacol peroxidase in most cultivars, while catalase and superoxide dismutase (SOD) were slightly depressed. The analysis of the SOD activity pattern by native-PAGE showed the presence in most cultivars of four SODs which were identified as Mn–SOD, Fe–SOD, CuZn–SOD I and CuZn–SOD II. However, the two CuZn–SODs were absent in the Cd-sensitive cv. Herminio. The growth of pepper plants with 0.5 mM cadmium inhibited the activity of CuZn–SODs in all cultivars, while the activity of Mn- and Fe–SOD was enhanced. The activity of NADPH-dehydrogenases (glucose-6-P-dehydrogenase, 6-phosphogluconate dehydrogenase, NADP–isocitrate dehydrogenase and malic enzyme) showed a Cd-dependent enhancement in most cultivars, the highest increase being observed in the tolerant cv. Abdera. These results suggest that in pepper plants the tolerance to Cd toxicity is more dependent on the availability of NADPH than on its antioxidant capacity.     

Molecular mechanisms of <Emphasis Type="Italic">Bombyx batryticatus</Emphasis> ethanol extract inducing gastric cancer SGC-7901 cells apoptosis

Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.     

Selenium and Glutathione Peroxidase Status in Adult Egyptian Patients with Acute Myeloid Leukemia

This study was undertaken to evaluate selenium (Se) and glutathione peroxidase (GPX) status in patients with newly diagnosed acute myeloid leukemia (AML) before and after induction therapy. Twenty-five patients with newly diagnosed AML and 15 healthy age- and sex-matched control subjects were included in this study. Serum Se level by the graphite furnace atomic absorption spectrometric technique and GPX activity by an adaptation of Beutler method was performed for the patients before and after receiving the induction therapy. Serum Se level was significantly lower in patients with AML versus control subjects (63.1?±?8.8 versus 77?±?8.8 µg/L before therapy with a P value <0.01 and 69?±?6.8 versus 77?±?8.8 µg/L after therapy with a P value <0.01).GPX activity was significantly lower in patients with AML versus control subjects (1.6?±?0.4 versus 3.4?±?0.7 µ/g protein pretreatment with a P value <0.01and 1.9?±?0.6 versus 3.4?±?0.7 µ/g protein post induction treatment with P value <0.01).Se level and GPX activity significantly increased in AML patients after treatment. Patients who accomplished complete remission after induction harbored significantly higher Se levels than resistant patients before and after treatment. There was no significant correlation between serum Se level and GPX activity. Decreased Se level and reduced GPX activity in AML patients support the association of carcinogenesis and subnormal Se states.     

Effects of WT1 gene downregulation on apoptosis in porcine fetal fibroblasts

Wilms’ tumor gene 1 (WT1) is located on chromosome 11p13. Besides a role in the development of Wilms’ tumor, specific mutations in the Zn finger region are found in Denys-Drash syndrome and Frasier syndrome, both characterized by urogenital abnormalities, sometimes in combination with Wilms’ tumor. Our past study shows that WT1 is expressed in porcine kidney fibroblasts (PKFs) and swine testis cells (ST cells) and is essential for the maintenance of the development and survival of PKFs and ST cells. But we do not know whether WT1 gene was expressed in porcine fetal fibroblasts or not. To further explore whether WT1 was expressed in porcine fetal fibroblasts (PFFs) and its contribution to cell apoptosis, RT-PCR, immunocytochemical staining, and Western blot were used to detect the expression of WT1, the recombinant plasmids of pLV3-WT1 short hairpin ribonucleic acid (shRNA) were used to downregulate the WT1 gene in porcine fetal fibroblasts, and the role of WT1 in cell proliferation was examined by apoptosis analysis also. Our results indicated that WT1 was expressed in PFFs, the pLV3-WT1 shRNA dramatically reduced the expression of WT1, and downregulation of WT1 directly led to early cell apoptosis by downregulating the expression of antiapoptotic gene Bcl-2 and upregulating the expression of proapoptotic gene Bax in PFFs. Our results demonstrate that WT1 is also essential for the maintenance of the survival of PFFs.     

Tubuloside A,a phenylethanoid glycoside,alleviates diclofenac induced hepato-nephro oxidative injury via Nrf2/HO-1

The most prominent adverse effects of nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DF) are hepato-renal damage. Natural antioxidants can be preferred as an alternative and/or combination to improve this damage. This present study was conducted to evaluate the protective effect of Tubuloside A (TA) against diclofenac (DF)-induced hepato-renal damage. TA (1 mg/kg, ip) was administered to male Sprague–Dawley rats for 5 days, and DF (50 mg/kg, ip) was administered on Days 4 and 5. Plasma aspartate amino transferase, alanine amino transferase, alkaline phosphatase, blood urea nitrogen and creatinine were measured to evaluate liver and kidney functions. Additionally, oxidative stress parameters (malondialdehyde, glutathione, superoxide dismutase, catalase, and 8-oxo-7,8-dihydro-2′-deoxyguanosine) in blood, liver, and kidney tissues, changes in mRNA expression of genes involved in the Nrf2/HO-1 signalling pathway (Nrf2, HO-1, NQO-1, IL-6, iNOS, Cox-2, TNF-α, IL1-β and NFκB) and apoptotic process (Bcl-2, Cas-3 and Bax) in liver and kidney tissues were determined. Additionally, tissue sections were evaluated histopathologically. Biochemical, histopathological, and molecular results demonstrated the hepato-renal toxic effects of DF, and TA treatment protected the liver and kidney from DF-induced damage. This provides an explanation for the hepato-nephro damage caused by DF and offers new ideas and drug targets together with TA for the prevention and treatment of DF injury.     

Selenium prevents downregulation of antioxidant selenoprotein genes by methylmercury

Selenium (Se) is an essential nutrient required by Se-dependent proteins, termed selenoproteins. The selenoprotein family is small but diverse and includes key proteins in antioxidant, redox signaling, thyroid hormone metabolism, and protein folding pathways. Methylmercury (MeHg) is a toxic environmental contaminant that affects seafood safety. Selenium can reduce MeHg toxicity, but it is unclear how selenoproteins are affected in this interaction. In this study we explored how Se and MeHg interact to affect the mRNA expression of selenoprotein genes in whole zebrafish (Danio rerio) embryos. Embryos were obtained from adult zebrafish fed MeHg with or without elevated Se in a 2×2 factorial design. The embryo mRNA levels of 30 selenoprotein genes were then measured. These genes cover most of the selenoprotein families, including members of the glutathione peroxidase (GPX), thioredoxin reductase, iodothyronine deiodinase, and methionine sulfoxide reductase families, along with selenophosphate synthetase 2 and selenoproteins H, J-P, T, W, sep15, fep15, and fam213aa. GPX enzyme activity and larval locomotor activity were also measured. We found that around one-quarter of the selenoprotein genes were downregulated by elevated MeHg. These downregulated genes were dominated by selenoproteins from antioxidant pathways that are also susceptible to Se-deficiency-induced downregulation. MeHg also decreased GPX activity and induced larval hypoactivity. Elevated Se partially prevented MeHg-induced disruption of selenoprotein gene mRNA levels, GPX activity, and larval locomotor activity. Overall, the MeHg-induced downregulation and subsequent rescue by elevated Se levels of selenogenes regulated by Se status suggest that Se deficiency is a contributing factor to MeHg toxicity.     

Characterization and expression of cytoplasmic copper/zinc superoxide dismutase (CuZn SOD) gene under temperature and hydrogen peroxide (H2O2) in rotifer Brachionus calyciflorus

Superoxide dismutase (SOD, EC 1.15.1.1) is an important antioxidant enzyme that protects organs from damage by reactive oxygen species (ROS). We cloned cDNA encoding SOD activated with copper/zinc (CuZn SOD) from the rotifer Brachionus calyciflorus Pallas. The full-length cDNA of CuZn SOD was 692 bp and had a 465 bp open reading frame encoding 154 amino acids. The deduced amino acid sequence of B. calyciflorus CuZn SOD showed 63.87%, 60.00%, 59.74% and 48.89% similarity with the CuZn SOD of the Ctenopharyn godonidella, Schistosoma japonicum, Drosophila melanogaster and Caenorhabditis elegans, respectively. The phylogenetic tree constructed based on the amino acid sequences of CuZn SODs from B. calyciflorus and other organisms revealed that rotifer is closely related to nematode. Analysis of the expression of CuZn SOD under different temperatures (15, 30 and 37 °C) revealed that its expression was enhanced 4.2-fold (p < 0.001) at 30 °C after 2 h, however, the lower temperature (15 °C) promoted CuZn SOD transiently (4.1-fold, p < 0.001) and then the expression of CuZn SOD decreased to normal level (p > 0.05). When exposed to H₂O₂ (0.1 mM), CuZn SOD, manganese superoxide dismutase (Mn SOD) and catalase (CAT) gene were upregulated, and in addition, the mRNA expression of CuZn SOD gene was induced instantaneously after exposure to vitamin E. It indicates that the CuZn SOD gene would be an important gene in response to oxidative and temperature stress.     

Effect of selenium on growth and antioxidant capacity of <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. during development of lead-induced oxidative stress

Effects of two selenium concentrations—0.4 and 0.8 mg Se⁶⁺ per kilogram of soil (treatments Se0.4 and Se0.8)—on seedling growth, chlorophyll content (Chl (a + b)), the content of thiobarbituric acidreactive substances (TBARs) indicative of peroxidation rates, and the activities of antioxidant enzymes (ascorbate peroxidase, AsP; glutathione reductase, GR; and guaiacol peroxidase, GPX) were studied in roots and leaves of wheat (Triticum aestivum L., cv. Triso) plants that were exposed for 14 days to oxidative stress induced by 50 and 100 mg Pb²⁺ per kilogram of soil (treatments Pb50 and Pb100, respectively). The pollution of soil with Pb²⁺ inhibited growth, lowered Chl (a + b) content, and intensified peroxidation. The content of TBARs increased by 44 and 72% in leaves and by 25 and 45% in roots for treatments Pb50 and Pb100, respectively. Activities of the antioxidant enzymes GR and GPX were higher in Pb²⁺-treated than in untreated (control) plants. The introduction of Se⁶⁺ into Pb²⁺-free soil (treatment Se0.4) was found to promote growth, stimulate AsP activity by 40% in leaves, and enhance AsP, GR, and GPX activities in roots by 38, 33, and 74%, respectively. The content of TBARs was reduced in Se⁶⁺-treated plants. By contrast, the treatment Se0.8 suppressed growth, elevated TBARs content, and stimulated activities of antioxidant enzymes in roots and leaves. The addition of 0.4 mg Se⁶⁺/kg to Pb²⁺-contaminated soil alleviated the negative influence of lead on plant growth, whereas the addition of 0.8 mg Se⁶⁺/kg aggravated the effect of Pb²⁺ contamination, especially in treatment (Pb100+Se0.8). Thus, the effectiveness of exogenous Se⁶⁺ application on growth and adaptive potential of plants cultivated under optimal pollutant-free conditions and on soils contaminated with lead depended on the concentration of Se⁶⁺ supplemented to soil and on the content of the stressor agent.     

Polymorphism analysis of six selenoprotein genes: support for a selective sweep at the glutathione peroxidase 1 locus (3p21) in Asian populations

Background

There are at least 25 human selenoproteins, each characterized by the incorporation of selenium into the primary sequence as the amino acid selenocysteine. Since many selenoproteins have antioxidant properties, it is plausible that inter-individual differences in selenoprotein expression or activity could influence risk for a range of complex diseases, such as cancer, infectious diseases as well as deleterious responses to oxidative stressors like cigarette smoke. To capture the common genetic variants for 6 important selenoprotein genes (GPX1, GPX2, GPX3, GPX4, TXNRD1, and SEPP1) known to contribute to antioxidant host defenses, a re-sequence analysis was conducted across these genes with particular interest directed at the coding regions, intron-exon borders and flanking untranslated regions (UTR) for each gene in an 102 individual population representative of 4 major ethnic groups found within the United States.

Results

For 5 of the genes there was no strong evidence for selection according to the expectations of the neutral equilibrium model of evolution; however, at the GPX1 locus (3p21) there was evidence for positive selection. Strong confirmatory evidence for recent positive selection at the genomic region 3p21 in Asian populations is provided by data from the International HapMap project.

Conclusion

The SNPs and fine haplotype maps described in this report will be valuable resources for future functional studies, for population specific genetic studies designed to comprehensively explore the role of selenoprotein genetic variants in the etiology of various human diseases, and to define the forces responsible for a recent selective sweep in the vicinity of the GPX1 locus.     

Exogenous Selenium Pretreatment Protects Rapeseed Seedlings from Cadmium-Induced Oxidative Stress by Upregulating Antioxidant Defense and Methylglyoxal Detoxification Systems

The protective effect of selenium (Se) on antioxidant defense and methylglyoxal (MG) detoxification systems was investigated in leaves of rapeseed (Brassica napus cv. BINA sharisha 3) seedlings under cadmium (Cd)-induced oxidative stress. Two sets of 11-day-old seedlings were pretreated with both 50 and 100???M Se (Na₂SeO₄, sodium selenate) for 24?h. Two concentrations of CdCl₂ (0.5 and 1.0?mM) were imposed separately or on the Se-pretreated seedlings, which were grown for another 48?h. Cadmium stress at any levels resulted in the substantial increase in malondialdehyde and H₂O₂ levels. The ascorbate (AsA) content of the seedlings decreased significantly upon exposure to Cd stress. The amount of reduced glutathione (GSH) increased only at 0.5?mM CdCl₂, while glutathione disulfide (GSSG) increased at any level of Cd, with concomitant decrease in GSH/GSSG ratio. The activities of ascorbate peroxidase (APX) and glutathione S-transferase (GST) increased significantly with increased concentration of Cd (both at 0.5 and 1.0?mM CdCl₂), while the activities of glutathione reductase (GR) and glutathione peroxidase (GPX) increased only at moderate stress (0.5?mM CdCl₂) and then decreased at 1.0?mM severe stress (1.0?mM CdCl₂). Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon exposure to any levels of Cd. Selenium pretreatment had little effect on the nonenzymatic and enzymatic components of seedlings grown under normal conditions; i.e., they slightly increased the GSH content and the activities of APX, GR, GST, and GPX. On the other hand, Se pretreatment of seedlings under Cd-induced stress showed a synergistic effect; it increased the AsA and GSH contents, the GSH/GSSG ratio, and the activities of APX, MDHAR, DHAR, GR, GPX, CAT, Gly I, and Gly II which ultimately reduced the MDA and H₂O₂ levels. However, in most cases, pretreatment with 50???M Se showed better results compared to pretreatment with 100???M Se. The results indicate that the exogenous application of Se at low concentrations increases the tolerance of plants to Cd-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

Responses to booster hepatitis B vaccination are significantly correlated with genotypes of human leukocyte antigen (HLA)-DPB1 in neonatally vaccinated adolescents

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) near the human leukocyte antigen (HLA)-DP loci that were significantly correlated with outcomes of hepatitis B virus (HBV) infection. We performed a case–control study nested in a well-characterized cohort of booster recipients to assess whether genetic variants of HLA-DPB1 are also associated with response to hepatitis B (HB) vaccination. The cases and controls were 171 and 510 booster recipients whose post-booster titers of antibodies against HBV surface antigen (anti-HBs) were undetectable and detectable, respectively. The HLA-DPB1 genotype was determined using sequence-based techniques. The frequencies of HLA-DPB1 alleles were significantly different between cases and controls (p = 1.7 × 10^?8). The HLA-DPB1 05:01 and 09:01 alleles were significantly more frequent in the cases, and 02:01:02, 02:02, 03:01:01, 04:01:01, and 14:01, were significantly more frequent in the controls. The adjusted odds ratio (OR) of undetectable post-booster anti-HBs titers was significantly correlated with the number of risk alleles (p for trend = 3.8 × 10^?5). For the number of protective alleles, the trend was significantly inversed (p for trend = 1.3 × 10^?5). As compared with subjects with two risk alleles, adjusted OR were 0.34 (95 % confidence interval [CI] 0.21–0.55) and 0.20 (95 % CI 0.08–0.48) for subjects with 1 and 2 protective alleles, respectively. The HLA-DPB1 02:02, 04:01:01, 05:01 and 09:01 alleles were also significantly correlated with the likelihoods of undetectable pre-booster anti-HBs titers. Our results indicated that HLA-DPB1 is significantly correlated with response to booster HB vaccination in adolescent who had received postnatal active HB vaccination. HLA-DBP1 may also determine the long-term persistence of response to HB vaccination.     

Comparative characterization of sweetpotato antioxidant genes from expressed sequence tags of dehydration-treated fibrous roots under different abiotic stress conditions

Drought stress is one of the most adverse conditions for plant growth and productivity. The plant antioxidant system is an important defense mechanism and includes antioxidant enzymes and low-molecular weight antioxidants. Understanding the biochemical and molecular responses to drought is essential for improving plant resistance to water-limited conditions. Previously, we isolated and characterized expressed sequence tags (ESTs) from a full-length enriched cDNA library prepared from fibrous roots of sweetpotato subjected to dehydration stress (Kim et al. in BMB Rep 42:271–276, [5]). In this study, we isolated and characterized 11 sweetpotato antioxidant genes from sweetpotato EST library under various abiotic stress conditions, which included six intracellular CuZn superoxide dismutases (CuZnSOD), ascorbate peroxidase, catalase, glutathione peroxidase (GPX), glutathione-S-transferase, thioredoxin (TRX), and five extracellular peroxidase genes. The expression of almost all the antioxidant genes induced under dehydration treatments occurred in leaves, with the exception of extracellular swPB6, whereas some antioxidant genes showed increased expression levels in the fibrous roots, such as intracellular GPX, TRX, extracellular swPA4, and swPB7 genes. During various abiotic stress treatments in leaves, such as exposure to NaCl, cold, and abscisic acid, several intracellular antioxidant genes were strongly expressed compared with the expression of extracellular antioxidant genes. These results indicated that some intracellular antioxidant genes, especially swAPX1 and CuZnSOD, might be specifically involved in important defense mechanisms against oxidative stress induced by various abiotic stresses including dehydration in sweetpotato plants.     

Long-term hormonal promotion overcomes genetic resistance to mammary cancer

It is well known that ovarian steroids estradiol and progesterone play a vital role in the development of mammary cancer. Here, using the genetically highly resistant Copenhagen rats we demonstrate that sustained exogenous treatment with estradiol and progesterone overcomes genetic resistance to mammary cancer. It has been demonstrated that Copenhagen rats develop preneoplastic lesions upon exposure to carcinogens. However, these preneoplastic lesions fail to progress to ductal carcinomas in situ or overt mammary carcinomas. The preneoplastic lesions eventually decrease in number and are absent by 60 days post-carcinogen treatment. In the present study, we exposed 7-week-old female Copenhagen rats to N-methyl-N-nitrosourea (MNU; 50 mg/kg BW). Immediately after MNU treatment the rats were divided into the following groups: (1) control; (2) 30 mg estradiol 17β; (3) 30 mg progesterone; and (4) 30 mg estradiol 17β plus 30 mg progesterone. All hormone treatments were administered via individual silastic pellets for a period of 9 months post-carcinogen treatment. The control animals displayed a low incidence of mammary cancer (10%). Hormone treatments produced significantly higher incidences of mammary cancer, with estradiol at 50%, progesterone at 65% and estradiol plus progesterone at 90%. Hormone treatment sustained the growth of the lesions induced by MNU by increasing expression of Areg, Bcl-2, Ccnd-1 and Vegf genes, while decreasing expression of Bad, Bax, Casp 3, 8, 9 and p53 genes. Furthermore, hormone treatment increased CCND-1 and PARP proteins levels. The data clearly demonstrates that hormonal environment supports mammary cancer progression by increasing cell proliferation, and angiogenesis while inhibiting apoptosis.     

Exogenous l-ascorbic acid regulates the antioxidant system to increase the regeneration of damaged mycelia and induce the development of fruiting bodies in Hypsizygus marmoreus

Hypsizygus marmoreus is an important commercial edible fungus, but the lack of basic studies on this fungus has hindered further development of its commercial value. In this study, we found that the treatment of damaged vegetative mycelia with 1 mM l-ascorbic acid (ASA) significantly increased the antioxidant enzyme activities (GPX, GR, CAT and SOD) and antioxidant contents (GSH and ASA) and reduced the ROS levels (H₂O₂ and O₂^-) in mechanically damaged mycelia. Additionally, this treatment increased mycelial biomass. At the reproductive stage, our results demonstrated that the treatment of damaged H. marmoreus mycelia with 2.24 mM ASA significantly increased the antioxidant enzyme activities (GPX, GR, GST, TRXR and CAT), endogenous ASA contents and GSH/GSSG ratios in different developmental stages and significantly decreased the MDA and H₂O₂ contents. Furthermore, this study showed that the expression levels of the antioxidant enzyme genes were consistent with the enzyme activities. Damaged mycelia treated with ASA regenerated 2–3 d earlier than the control group and showed significantly enhanced fruiting body production. These results suggested that exogenous ASA regulated mycelia intracellular ASA content to increase mycelial antioxidant abilities, induce the regeneration of damaged mycelia and regulate the development of fruiting bodies in H. marmoreus.     

Supranutritional selenium intake from enriched milk casein impairs hepatic insulin sensitivity via attenuated IRS/PI3K/AKT signaling and decreased PGC-1α expression in male Sprague–Dawley rats

Selenium (Se)-enriched milk provides antioxidant benefits and has therapeutic potential against cancer. However, both antidiabetic and prodiabetic effects have been attributed to Se. Our objective was to evaluate the effect of Se-enriched milk casein on insulin sensitivity in rats when given at the requirement of 0.25 ppm Se and supranutritionally on both low- and high-fat diets. Two hundred sixteen male Sprague–Dawley rats were fed low- or high-fat diets containing one, two or eight times the Se requirement in a randomized block design. After 7 weeks, 72 rats were subjected to the hyperinsulinemic–euglycemic clamp with [3-³H]glucose infusion to estimate glucose fluxes. Tissues were collected from the remaining 144 rats 8 min after ip saline or insulin injection. During hyperinsulinemic–euglycemic clamps, glucose infusion rate was 22% lower (P=.058), and endogenous glucose production was 76% higher (P=.054) when Se content increased from one to eight times the requirement on low-fat diets, indicating impaired hepatic insulin sensitivity. Se also decreased the ability for insulin to stimulate Akt phosphorylation at Thr308. Hepatic oxidation state and expression of selenoprotein P and glutathione peroxidase-1 were unaffected while expression of insulin receptor substrate (IRS)-1 and−2 and PPARγ coactivator-1α (PGC-1α) decreased with supranutritional Se and high-fat intake. In addition, hepatic expression of regulatory and catalytic subunits of phosphatidylinositol 3-kinase (PI3K) decreased with supranutritional intake of Se. Se intake from enriched casein up to eight times the requirement impairs hepatic insulin sensitivity in a mechanism similar to fat feeding, via attenuated IRS/PI3K/Akt signaling and decreased PGC-1α expression.     

Nitric Oxide Induces Cell Death by Regulating Anti-Apoptotic BCL-2 Family Members

Nitric oxide (NO) activates the intrinsic apoptotic pathway to induce cell death. However, the mechanism by which this pathway is activated in cells exposed to NO is not known. Here we report that BAX and BAK are activated by NO and that cytochrome c is released from the mitochondria. Cells deficient in Bax and Bak or Caspase-9 are completely protected from NO-induced cell death. The individual loss of the BH3-only proteins, Bim, Bid, Puma, Bad or Noxa, or Bid knockdown in Bim^−/−/Puma^−/− MEFs, does not prevent NO-induced cell death. Our data show that the anti-apoptotic protein MCL-1 undergoes ASK1-JNK1 mediated degradation upon exposure to NO, and that cells deficient in either Ask1 or Jnk1 are protected against NO-induced cell death. NO can inhibit the mitochondrial electron transport chain resulting in an increase in superoxide generation and peroxynitrite formation. However, scavengers of ROS or peroxynitrite do not prevent NO-induced cell death. Collectively, these data indicate that NO degrades MCL-1 through the ASK1-JNK1 axis to induce BAX/BAK-dependent cell death.