FSH1 overexpression triggers apoptosis in Saccharomyces cerevisiae.

FSH1 belongs to the family of serine hydrolases in yeast and is homologous to the human ovarian tumor suppressor gene (OVAC2). Our preliminary results showed that cells lacking Fsh1p exhibit an increase in cell growth, and a decrease in the expression of AIF1 and NUC1 (apoptosis responsive genes) when compared to the wild type cells. Growth inhibition of cells overexpressing FSH1 is due to induction of cell death associated with cell death markers typical of mammalian apoptosis namely DNA fragmentation, phosphatidylserine externalization, ROS accumulation, Cytochrome c release, and altered mitochondrial membrane potential. When wild type cells were overexpressed with FSH1 there was up regulation of AIF1 level when compared to control cells suggesting that overexpression of FSH1 regulated cell death in yeast.

Grim-19 expressed by recombinant adenovirus for esophageal neoplasm target therapy.

Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EA) are the two most common types of esophageal cancer, which is the sixth highest cause of cancer­associated mortality and the eighth most common cancer worldwide. Gene associated with retinoid­interferon (IFN)­induced mortality­19 (Grim­19) is reported to be a cell death activator that may be used to define mechanisms involved in IFN­ß­ and retinoic acid­induced cell death and apoptosis in a number of tumor cell lines. The present study constructed a recombinant adenovirus expressing Grim­19 (rAd­Grim­19) and investigated its therapeutic outcomes in ESCC cells and tumor­bearing mice. Grim­19 expression was detected in EC­109 (ESCC) cells by reverse transcription­quantitative polymerase chain reaction and western blot analysis. Tumor cell death and apoptosis induced by rAd­Grim­19 in EC­109 cells were analyzed by flow cytometry. The inhibitory effects of rAd­Grim­19 on EC­109 growth were determined by MTT assays. Furthermore, the therapeutic effects of rAd­Grim­19 were investigated in EC­109­bearing mice. The results demonstrated that Grim­19 mRNA and protein expression was downregulated in EC­109 esophageal carcinoma cells compared with Het­1A normal esophageal epithelial cells. In addition, EC­109 cells exhibited a significant reduction in tumor cell growth in the rAd­Grim­19 group compared with the control groups. Furthermore, rAd­Grim­19 increased EC­109 cell apoptosis compared with the control group. These results indicated that rAd-Grim-19 may regulate tumor cell growth and apoptosis. Additionally, the results demonstrated that rAd­Grim­19 led to beneficial outcomes and prolonged the survival of esophageal tumor­bearing mice. In conclusion, the present study demonstrated that rAd­Grim­19 may have potential as an antitumor agent for esophageal neoplasms and may therefore be beneficial for patients with esophageal neoplasms.

Overexpression of CaAPX Induces Orchestrated Reactive Oxygen Scavenging and Enhances Cold and Heat Tolerances in Tobacco.

Ascorbate peroxidase (APX) acts indispensably in synthesizing L-ascorbate (AsA) which is pivotal to plant stress tolerance by detoxifying reactive oxygen species (ROS). Enhanced activity of APX has been shown to be a key step for genetic engineering of improving plant tolerance. However it needs a deeper understanding on the maintenance of cellular ROS homeostasis in response to stress. In this study, we identified and characterized an APX (CaAPX) gene from Camellia azalea. Quantitative real-time PCR (qRT-PCR) analysis showed that CaAPX was expressed in all tissues and peaked in immature green fruits; the expression levels were significantly upregulated upon cold and hot stresses. Transgenic plants displayed marked enhancements of tolerance under both cold and heat treatments, and plant growth was correlated with CaAPX expression levels. Furthermore, we monitored the activities of several ROS-scavenging enzymes including Cu/Zn-SOD, CAT, DHAR, and MDHAR, and we showed that stress tolerance was synchronized with elevated activities of ROS-scavenging. Moreover, gene expression analysis of ROS-scavenging enzymes revealed a role of CaAPX to orchestrate ROS signaling in response to temperature stresses. Overall, this study presents a comprehensive characterization of cellular response related to CaAPX expression and provides insights to breed crops with high temperature tolerances.

Synthesis of (3R)-acetoin and 2,3-butanediol isomers by metabolically engineered Lactococcus lactis.

The potential that lies in harnessing the chemical synthesis capabilities inherent in living organisms is immense. Here we demonstrate how the biosynthetic machinery of Lactococcus lactis, can be diverted to make (3R)-acetoin and the derived 2,3-butanediol isomers meso-(2,3)-butanediol (m-BDO) and (2R,3R)-butanediol (R-BDO). Efficient production of (3R)-acetoin was accomplished using a strain where the competing lactate, acetate and ethanol forming pathways had been blocked. By introducing different alcohol dehydrogenases into this strain, either EcBDH from Enterobacter cloacae or SadB from Achromobacter xylosooxidans, it was possible to achieve high-yield production of m-BDO or R-BDO respectively. To achieve biosustainable production of these chemicals from dairy waste, we transformed the above strains with the lactose plasmid pLP712. This enabled efficient production of (3R)-acetoin, m-BDO and R-BDO from processed whey waste, with titers of 27, 51, and 32 g/L respectively. The corresponding yields obtained were 0.42, 0.47 and 0.40 g/g lactose, which is 82%, 89%, and 76% of maximum theoretical yield respectively. These results clearly demonstrate that L. lactis is an excellent choice as a cell factory for transforming lactose containing dairy waste into value added chemicals.

Potential Application of the Oryza sativa Monodehydroascorbate Reductase Gene (OsMDHAR) to Improve the Stress Tolerance and Fermentative Capacity of Saccharomyces cerevisiae.

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is an important enzyme for ascorbate recycling. To examine whether heterologous expression of MDHAR from Oryza sativa (OsMDHAR) can prevent the deleterious effects of unfavorable growth conditions, we constructed a transgenic yeast strain harboring a recombinant plasmid carrying OsMDHAR (p426GPD::OsMDHAR). OsMDHAR-expressing yeast cells displayed enhanced tolerance to hydrogen peroxide by maintaining redox homoeostasis, proteostasis, and the ascorbate (AsA)-like pool following the accumulation of antioxidant enzymes and molecules, metabolic enzymes, and molecular chaperones and their cofactors, compared to wild-type (WT) cells carrying vector alone. The addition of exogenous AsA or its analogue isoascorbic acid increased the viability of WT and ara2Δ cells under oxidative stress. Furthermore, the survival of OsMDHAR-expressing cells was greater than that of WT cells when cells at mid-log growth phase were exposed to high concentrations of ethanol. High OsMDHAR expression also improved the fermentative capacity of the yeast during glucose-based batch fermentation at a standard cultivation temperature (30°C). The alcohol yield of OsMDHAR-expressing transgenic yeast during fermentation was approximately 25% (0.18 g·g-1) higher than that of WT yeast. Accordingly, OsMDHAR-expressing transgenic yeast showed prolonged survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsMDHAR expression increases tolerance to reactive oxygen species-induced oxidative stress by improving cellular redox homeostasis and improves survival during fermentation, which enhances fermentative capacity.

Capsaicin Inhibited Aggressive Phenotypes through Downregulation of Tumor-Associated NADH Oxidase (tNOX) by POU Domain Transcription Factor POU3F2.

Capsaicin has been reported to preferentially inhibit the activity of tumor-associated NADH oxidase (tNOX), which belongs to a family of growth-related plasma membrane hydroquinone oxidases in cancer/transformed cells. The inhibitory effect of capsaicin on tNOX is associated with cell growth attenuation and apoptosis. However, no previous study has examined the transcriptional regulation of tNOX protein expression. Bioinformatic analysis has indicated that the tNOX promoter sequence harbors a binding motif for POU3F2, which is thought to play important roles in neuronal differentiation, melanocytes growth/differentiation and tumorigenesis. In this study, we found that capsaicin-mediated tNOX downregulation and cell migration inhibition were through POU3F2. The protein expression levels of POU3F2 and tNOX are positively correlated, and that overexpression of POU3F2 (and the corresponding upregulation of tNOX) enhanced the proliferation, migration and invasion in AGS (human gastric carcinoma) cells. In contrast, knockdown of POU3F2 downregulates tNOX, and the cancer phenotypes are affected. These findings not only shed light on the molecular mechanism of the anticancer properties of capsaicin, but also the transcription regulation of tNOX expression that may potentially explain how POU3F2 is associated with tumorigenesis.

Exercise Training Attenuates Upregulation of p47(phox) and p67(phox) in Hearts of Diabetic Rats.

Exercise training (ExT) is currently being used as a nonpharmacological strategy to improve cardiac function in diabetic patients. However, the molecular mechanism(s) underlying its beneficial effects remains poorly understood. Oxidative stress is known to play a key role in the pathogenesis of diabetic cardiomyopathy and one of the enzyme systems that produce reactive oxygen species is NADH/NADPH oxidase. The goal of this study was to investigate the effect of streptozotocin- (STZ-) induced diabetes on expression of p47(phox) and p67(phox), key regulatory subunits of NADPH oxidase, in cardiac tissues and determine whether ExT can attenuate these changes. Four weeks after STZ treatment, expression of p47(phox) and p67(phox) increased 2.3-fold and 1.6-fold, respectively, in left ventricles of diabetic rats and these increases were attenuated with three weeks of ExT, initiated 1 week after onset of diabetes. In atrial tissues, there was increased expression of p47(phox) (74%), which was decreased by ExT in diabetic rats. Furthermore, increased collagen III levels in diabetic hearts (52%) were significantly reduced by ExT. Taken together, ExT attenuates the increased expression of p47(phox) and p67(phox) in the hearts of diabetic rats which could be an underlying mechanism for improving intracardiac matrix and thus cardiac function and prevent cardiac remodeling in diabetic cardiomyopathy.

Resveratrol Protects against Titanium Particle-Induced Aseptic Loosening Through Reduction of Oxidative Stress and Inactivation of NF-κB.

Aseptic implant loosening is closely associated with chronic inflammation induced by implant wear debris, and reactive oxygen species (ROS) play an important role in this process. Resveratrol, a plant compound, has been reported to act as an antioxidant in many inflammatory conditions; however, its protective effect and mechanism against wear particle-induced oxidative stress remain unknown. In this study, we evaluated resveratrol's protective effects against wear particle-induced oxidative stress in RAW 264.7 macrophages. At non-toxic concentrations, resveratrol showed dose-dependent inhibition of nitric oxide (NO) production, ROS generation, and lipid peroxidation. It also downregulated the gene expression of oxidative enzymes, including inducible nitric oxide synthase (iNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-1 and NOX-2, and promoted the gene expression and activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx). This protective effect against wear particle-induced oxidative stress was accompanied by a reduction of gene expression and release of tumor necrosis factor-α (TNF-α), and decreased gene expression and phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). These findings demonstrate that resveratrol can inhibit wear particle-induced oxidative stress in macrophages, and may exert its antioxidant effect and protect against aseptic implant loosening.

Expression of GRIM-19 in adenomyosis and its possible role in pathogenesis.

OBJECTIVE: To study the expression of the gene associated with retinoid-interferon (IFN)-induced mortality 19 (GRIM-19) in the endometrial tissue of patients with adenomyosis and to describe the possible pathogenic mechanisms of this phenomenon. DESIGN: Experimental study using human samples and cell lines. SETTING: University-affiliated hospital. PATIENT(S): Ectopic and eutopic endometrial tissues were obtained from 30 patients with adenomyosis, whereas normal endometrial specimens were obtained from 10 control patients without adenomyosis. INTERVENTION(S): Patients with rapid pathology report-confirmed adenomyosis were recruited, and eutopic and ectopic endometrial tissue samples were collected from patients who had undergone hysterectomies by either the transabdominal or laparoscopic method at Qilu Hospital. Normal endometrial tissue was collected from a group of control patients without adenomyosis. MAIN OUTCOME MEASURE(S): Immunohistochemistry (IHC) was performed to evaluate the expression of GRIM-19, phospho-signal transducer and activator of transcription 3 (Y705) (Y705) (pSTAT3(Y705)), and vascular endothelial growth factor (VEGF) in endometrial tissue samples. The protein levels of GRIM-19, pSTAT3(Y705), STAT3, and VEGF were detected by Western blot. Apoptosis in endometrial specimens was assayed by TUNEL. Immunohistochemistry with an antibody directed against CD34 was performed to detect new blood vessels in the endometrial tissue. GRIM-19 small interfering RNA and a recombinant plasmid carrying GRIM-19 were constructed to evaluate the effects of GRIM-19 on the downstream factors pSTAT3(Y705), STAT3, and VEGF in Ishikawa cells. RESULT(S): The expression of GRIM-19 was down-regulated in the eutopic endometria of patients with adenomyosis compared with the endometria of patients in the control group, and it was further reduced in the endometrial glandular epithelial cells of adenomyotic lesions. Apoptosis was reduced in the eutopic endometrium compared with the control group, and it was significantly reduced in ectopic endometrial tissues. In addition, the ectopic and eutopic endometria of patients with adenomyosis displayed a much higher microvessel density. In the eutopic and ectopic endometria of patients with adenomyosis, the expression levels of pSTAT3(Y705) and VEGF were significantly higher than in the controls. Furthermore, down-regulation of GRIM-19 in Ishikawa cells significantly promoted the activation of both pSTAT3(Y705) and its dependent gene VEGF. CONCLUSION(S): Aberrant expression of GRIM-19 may be associated with adenomyosis through the regulation of apoptosis and angiogenesis.

Melatonin nephroprotective action in Zucker diabetic fatty rats involves its inhibitory effect on NADPH oxidase.

Excessive activity of NADPH oxidase (Nox) is considered to be of importance for the progress of diabetic nephropathy. The aim of the study was to elucidate the effect of melatonin, known for its nephroprotective properties, on Nox activity under diabetic conditions. The experiments were performed on three groups of animals: (i) untreated lean (?/+) Zucker diabetic fatty (ZDF) rats; (ii) untreated obese diabetic (fa/fa) ZDF rats; and (iii) ZDF fa/fa rats treated with melatonin (20 mg/L) in drinking water. Urinary albumin excretion was measured weekly. After 4 wk of the treatment, the following parameters were determined in kidney cortex: Nox activity, expression of subunits of the enzyme, their phosphorylation and subcellular distribution. Histological studies were also performed. Compared to ?/+ controls, ZDF fa/fa rats exhibited increased renal Nox activity, augmented expression of Nox4 and p47(phox) subunits, elevated level of p47(phox) phosphorylation, and enlarged phospho-p47(phox) and p67(phox) content in membrane. Melatonin administration to ZDF fa/fa rats resulted in the improvement of renal functions, as manifested by considerable attenuation of albuminuria and some amelioration of structural abnormalities. The treatment turned out to nearly normalize Nox activity, which was accompanied by considerably lowered expression and diminished membrane distribution of regulatory subunits, that is, phospho-p47(phox) and p67(phox) . Thus, it is concluded that: (i) melatonin beneficial action against diabetic nephropathy involves attenuation of the excessive activity of Nox; and (ii) the mechanism of melatonin inhibitory effect on Nox is based on the mitigation of expression and membrane translocation of its regulatory subunits.

Decreased reactive oxygen species production and NOX1, NOX2, NOX4 expressions contribute to hyporeactivity to phenylephrine in aortas of pregnant SHR.

AIMS: We determined whether decreased reactive oxygen species (ROS) production in the aorta of pregnant spontaneously hypertensive rats (SHR) resulted in increased nitric oxide (NO) bioavailability and hyporeactivity to phenylephrine (PE). MAIN METHODS: Systemic and aortic oxidative stress were measured in pregnant and non-pregnant Wistar rats and SHR. Furthermore, the hypotensive effects of apocynin (30 mg/kg) and Tempol (30 mg/kg) were analyzed. Intact aortic rings of pregnant and non-pregnant rats were stimulated with PE in the absence of or after incubation (30 min) with apocynin (100 µmol/L). The effect of apocynin on the concentrations of NO and ROS were measured in aortic endothelial cells (AEC) using DAF-2DA (10 mmol/L) and DHE (2.5 mmol/L), respectively. Western blotting was performed to analyze eNOS, NOX1, NOX2, NOX4 and SOD expression. ROS production was analyzed by the lucigenin chemiluminescence method. KEY FINDINGS: Aortic oxidative stress and ROS concentration in AEC were reduced in pregnant Wistar rats and SHR, when compared to non-pregnant rats. ROS production and NOX1, NOX2 and NOX4 expression in the aortas were decreased in pregnant SHR, but not in pregnant Wistar rats. Increased eNOS expression in aortas and NO concentration in AEC were observed in pregnant Wistar rats and SHR. Apocynin reduced PE-induced vasoconstriction in the aortas of non-pregnant Wistar rats and SHR, and pregnant Wistar rats, but not in the aortas of pregnant SHR. SIGNIFICANCE: Taken together, these results suggest that ROS production was decreased in the aortas of pregnant SHR and could contribute to higher NO bioavailability and hyporeactivity to PE in the aortas of pregnant SHR.

Regulation of EPS production in Lactobacillus casei LC2W through metabolic engineering.

UNLABELLED: Lactobacillus casei LC2W is an exopolysaccharide(EPS)-producing strain with probiotic effects. The low efficiency and unclear regulation mechanism of EPS biosynthesis have become main constraints for its application in food industry. To investigate the major rate-limiting factors of EPS biosynthesis and to improve its yield, metabolic engineering was applied to this strain. Eight relevant genes related to central metabolism, sugar-nucleotides supply, glycosyltransferase and cofactor engineering were cloned and overexpressed. The results suggested that nox, pfk, rfbB and galT genes were the largest contributors to EPS biosynthesis in this study, which elevated EPS yield by 46·0, 20, 17·4 and 19·6% respectively. Notably, under aerobic condition which was not a suitable condition for lactobacilli to grow in, recombinant strain LC-nox achieved the highest EPS yield of 263·7 mg l(-1) , which was increased by 75% compared to that of the starting strain. The oxygen stress was excluded since the phenomenon was not observed in the control strain under the same condition. Therefore, it was probably that higher NADH oxidase activity led to a decreased NADH availability and reduced lactate concentration, which resulted in the elevation of EPS yield. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributed to the understanding of EPS biosynthesis in Lact. casei through metabolic engineering and provided a starting point for introducing cofactor engineering into this strain. Overexpression of NADH oxidase was found to have a most significant effect on the EPS production. It is the first report that EPS could be accumulated to such a high level under aerobic condition in lactobacilli. Our results provided a novel strategy for the improvement of EPS production in lactic acid bacteria.

GRIM-19 expression is a potent prognostic marker in colorectal cancer.

Retinoid-interferon-induced mortality-19 (GRIM-19), a recently discovered cell death regulatory gene, may function as a tumor suppressor in many human malignancies. However, the expression of GRIM-19 in and its prognostic value for patients with colorectal cancer (CRC) have not been well investigated to date. Here, GRIM-19 expression was measured immunohistochemically in 94 colon samples and by quantitative real-time reverse transcriptase polymerase chain reaction in 15 paired CRC tissues and adjacent normal tissues. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Our results showed that GRIM-19 mRNA and protein levels in adenoma tissues were similar to those in adjacent normal tissues. However, GRIM-19 expression was severely depressed in carcinomas compared to matched normal tissues (P = .000). Additionally, we found GRIM-19 to be located in both the cytoplasm and nucleus in normal tissues but only in the cytoplasm in CRC tissues. Alteration in GRIM-19 expression occurs early in the pathogenesis of CRC; moreover, low GRIM-19 expression was associated with poor tumor differentiation (P = .013), the presence of lymph nodes (P = .000), metastasis to other organs (P = .045) and vascular invasion (P = .010). During a mean period of 40 months follow-up, patients without GRIM-19 had a statistically significantly lower rate of recurrence/metastasis (P < .05) and a shorter overall survival time (P < .01) than the patients with GRIM-19 expression. Taken together, GRIM-19 expression is closely associated with CRC progression and might be a very promising prognostic biomarker for CRC patients.

Antioxidant Supplementation Modulates Age-Related Placental Bed Morphology and Reproductive Outcome in Mice.

The number of women who delay their first childbirth is increasing. This demographic shift is an important health issue because advanced maternal age is a risk factor for reproductive capacity loss and the occurrence of placental bed disorders that may lead to placenta abruption, preeclampsia, and placenta insufficiency. A redox imbalance status, resulting from the enhanced production of reactive oxygen species or their deficient neutralization, is proposed to occur in this setting. Thus, uterine redox status was evaluated in young (8- to 12-wk-old) and reproductively aged (38- to 42-wk-old) mice. In addition, it was hypothesized that specific dietary antioxidant supplementation would restore the balance and improve the reproductive outcome of aging female mice. To test this hypothesis, two different antioxidants, the nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor apocynin and the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL), were added to the drinking water of female mice prior to and during pregnancy. Compared to younger females, uteri from reproductively aged nonpregnant mice exhibited areas of endometrial cystic dilation, increased level of NOX1 expression, and enhanced protein carbonylation, especially in the apical surface of the luminal epithelium. Both antioxidants decreased protein carbonylation level in the uterus of reproductively aged mice. When reproductively aged females became pregnant, the litter size was smaller and fetuses were heavier. The change was accompanied by a significant decrease in decidua thickness. Provision of apocynin significantly increased litter size and restored decidua thickness. Reproductively aged mice provided with TEMPOL did not evidence such benefits, but whereas apocynin normalized fetal birth weight, TEMPOL further increased it. These findings emphasize that uterine redox balance is important for reproductive success and suggest that age-related redox imbalance might be compensated by specific antioxidant supplementation.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a type II NADH:quinone oxidoreductase from the human pathogen Staphylococcus aureus.

In recent years, type II NADH dehydrogenases (NDH-IIs) have emerged as potential drug targets for a wide range of human disease causative agents. In this work, the NDH-II enzyme from the Gram-positive human pathogen Staphylococcus aureus was recombinantly expressed in Escherichia coli, purified, crystallized and a crystallographic data set was collected at a wavelength of 0.873 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 81.8, b = 86.0, c = 269.9 Å, contained four monomers per asymmetric unit and diffracted to a resolution of 3.32 Å. A molecular-replacement solution was obtained and model building and refinement are currently under way.

ROS-Induced Nuclear Translocation of Calpain-2 Facilitates Cardiomyocyte Apoptosis in Tail-Suspended Rats.

Isoproterenol (ISO) induced nuclear translocation of calpain-2 which further increased susceptibility of cardiomyocyte apoptosis in tail-suspended rats. The underlying mechanisms remain elusive. In the present study, the results showed that ISO (10 nM) significantly elevated NADPH oxidases (NOXs) activity and NOXs-derived ROS productions which induced nuclear translocation of calpain-2 in cardiomyocytes of tail-suspended rats. In contrast, the inhibition of NADPH oxidase or cleavage of ROS not only reduced ROS productions, but also resisted nuclear translocation of calpain-2 and decreased ISO-induced apoptosis of cardiomyocyte in tail-suspended rats. ISO also increased the constitutive binding between calpain-2 and Ca(2+)/calmodulin-dependent protein kinase II δB (CaMK II δB) in nuclei, concomitant with the promotion of CaMK II δB degradation and subsequent down-regulation of Bcl-2 mRNA expression and the ratio of Bcl-2 to Bax protein in tail-suspended rat cardiomyocytes. These effects of ISO on cardiomyocytes were abolished by a calpain inhibitor PD150606. Inhibition of calpain significantly reduced ISO-induced loss of the mitochondrial membrane potential, cytochrome c release into the cytoplasm, as well as the activation of caspase-3 and caspase-9 in mitochondrial apoptotic pathway. In summary, the above results suggest that ISO increased NOXs-derived ROS which activated nuclear translocation of calpain-2, subsequently nuclear calpain-2 degraded CaMK II δB which reduced the ratio of Bcl-2 to Bax, and finally the mitochondria apoptosis pathway was triggered in tail-suspended rat cardiomyocytes. Therefore, calpain-2 may represent a potentially therapeutic target for prevention of oxidative stress-associated cardiomyocyte apoptosis.

Downregulation of gene expression and activity of GRIM-19 affects mouse oocyte viability, maturation, embryo development and implantation.

PURPOSE: To investigate the expression of GRIM-19 (Gene associated with retinoid-interferon-induced mortality 19) in mouse oocytes and preimplantation embryos, and to study the effect of GRIM-19 on the developmental competence of mouse oocytes and embryos. METHODS: GRIM-19 was evaluated at both mRNA and protein levels. The expression of GRIM-19 gene was downregulated in mouse oocytes cultured in vitro by specific small interfering RNA (siRNA) injection, while the activity of GRIM-19 was decreased by microinjection of a GRIM-19 antibody into the cytoplasm of germinal vesicle (GV) oocytes. Oocytes matured in vitro were then fertilized by intracytoplasmic sperm injection (ICSI), followed by observation and evaluation of fertilization rate, cleavage rate, blastocyst formation rate and implantation rate. RESULTS: GRIM-19 is expressed throughout oocyte maturation and preimplantation embryo development stages. GRIM-19 was localized primarily in the cytoplasm of all cells examined. Downregulation of gene expression and activity of GRIM-19 resulted in decreased oocyte viability, potency of oocyte maturation, embryo development and implantation. CONCLUSIONS: GRIM-19 may play important roles in mouse oogenesis and early embryonic development and implantation.

Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging.

Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)-cSrc-phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression.

Molecular cloning, computational and expression analysis of anthocyanidin reductase in tea (Camellia sinensis).

Tea [Camellia sinensis (L.) O. Kuntze] is one of the most popular non-alcoholic beverages rich in phenolic compounds, which includes epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) and catechin (C). Anthocyanidin reductase (ANR) is responsible for catechin biosynthesis in plants, and analysis of its protein sequences and structures will be valuable for further research in the field. We have screened our dormant bud-specific complementary DNA (cDNA) library and reported 1,322-bp cDNA encoding CsANR. Analysis of the sequence revealed the presence of 1,011-bp open reading frame with coding capacity for a polypeptide of 337 amino acids, flanked by 1,123- and 196-bp 5' and 3' untranslated regions, respectively. Theoretical molecular weight (MW) and isoelectric point (pI) of the deduced ANR protein were predicted (using ProtParam) to be 36.4 kDa and 6.54. For the first time, we have reported 3D model of ANR from C. sinensis. Quality of the predicted model was analysed with PROCHECK analysis. Molecular docking of modelled ANR revealed similar binding pockets for both substrates and products. Expression analyses of CsANR and accumulation pattern of catechins were observed to be varied with developmental age of tissue and seasonal condition. Variation in accumulation pattern of catechins and its fractions was found to be correlated with expression pattern of ANR.

Glutathione transferase omega 1 is required for the lipopolysaccharide-stimulated induction of NADPH oxidase 1 and the production of reactive oxygen species in macrophages.

Bacterial lipopolysaccharide (LPS) stimulation of macrophages and inflammation via the Toll-like receptor 4 (TLR4) signaling pathway through NF-κΒ generates reactive oxygen species (ROS) and proinflammatory cytokines such as IL-1ß, IL-6, and TNFα. Because glutathione transferase Omega 1-1 (GSTO1-1) can catalyze redox reactions such as the deglutathionylation of proteins and has also been implicated in the release of IL-1ß we investigated its role in the development of LPS-mediated inflammation. Our data show that shRNA knockdown of GSTO1-1 in macrophage-like J774.1A cells blocks the expression of NADPH oxidase 1 and the generation of ROS after LPS stimulation. Similar results were obtained with a GSTO1-1 inhibitor. To maintain high ROS levels during an inflammatory response, LPS stimulation causes the suppression of enzymes such as catalase and glutathione peroxidase that protect against oxidative stress. The knockdown of GSTO1-1 also attenuates this response. Our data indicate that GSTO1-1 needs to be catalytically active and mediates its effects on the LPS/TLR4 inflammatory pathway upstream of NF-κΒ. These data suggest that GSTO1-1 is a novel target for anti-inflammatory intervention.