NAD(P)(H)-dependent oxidoreductase catalyzes the reduction of ketones or aldehydes to prepare a wide variety of valuable chiral alcohols or amines. However, expensive cofactors are absolutely required for the biocatalytic processes with oxidoreductases, which severely hinder their industrial applications. Consequently, the issue on reducing cofactor costs has become one of the major focuses in the field of biocatalysis. With the substantial development in recent years, a number of strategies have been proposed and implemented to solve the cofactor issues in the oxidoreductase catalyzed biocatalysis, including the establishment of cofactor regeneration system, the improvement of endogenous cofactor availability via metabolic engineering and the development of biomimetic agents to replace cofactors. In this review, recent trends and advances on these strategies are presented, and respective advantages and shortcomings are also discussed with a number of examples.
Alcohols ; metabolism ; Biocatalysis ; Ketones ; metabolism ; Metabolic Engineering ; NADH, NADPH Oxidoreductases ; metabolism ; Oxidation-Reduction

OBJECTIVETo study the expression of gene associated with retinoid-interferon-induced mortality-19(GRIM-19) in preimplantation embryo of mice and explore its role in embryonic development.

METHODSThe protein and mRNA expressions of GRIM-19 in 2-cell, 4-cell, 8-cell, morula, and blastocyst phases of mice preimplantation embryo were detected by Western blot analysis and Real-time polymerase chain reaction (PCR).

RESULTSGRIM-19 was continuously expressed in every stage of preimplantation embryo of mice. Western blot analysis and Real-time PCR demonstrated a gradual increase of GRIM-19 expression from 2-cell, which reached a peak in 8-cell phase and then decreased progressively.

CONCLUSIONSThe expression of GRIM-19 in mouse preimplantation embryos changes as at different developmental phases. GRIM-19 may play an important role during embryonic development.

Animals ; Blastocyst ; drug effects ; metabolism ; Female ; Interferons ; pharmacology ; Mice ; NADH, NADPH Oxidoreductases ; genetics ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; Tretinoin ; pharmacology

OBJECTIVETo investigate the expressions of survivin and GRIM-19 in prostatic cancer tissue and their clinical implications.

METHODSWe detected the expressions of survivin and GRIM-19 in the tissues of normal prostate (NP), benign prostate hyperplasia (BPH) and prostate cancer (PCa) using immunohistochemical staining, RT-PCR and Western blot, and processed the data by SPSS12.

RESULTSThe positive rates of survivin expression were 6.25% , 18.18% and 90.62% in NP, BPH and PCa (P < 0.01), while those of GRIM-19 were 87.50%, 81.82% and 9.37% , respectively (P < 0.01). Semiquantitative RT-PCR and immunohistochemical staining showed that both survivin mRNA and survivin expressions were highly positive in PCa but negative in NP and BPH. Western blot exhibited that the survivin protein was expressed strongly in PCa but weakly in NP and BPH, while the GRIM-19 protein was expressed just contrariwise (P < 0.01).

CONCLUSIONThe expressions of survivin and GRIM-19 may be closely correlated with the pathogenesis of prostate cancer.

Apoptosis Regulatory Proteins ; metabolism ; Case-Control Studies ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Male ; NADH, NADPH Oxidoreductases ; metabolism ; Prostate ; metabolism ; pathology ; Prostatic Neoplasms ; metabolism ; pathology

The study is aimed to explore the molecular mechanism of the treatment of apocynin in dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mice. 5% DSS was used to mimic the UC model, and 2% apocynin was applied to treat the UC mice. HE staining was used for histopathological evaluation. Chemiluminescence technique was used to measure reactive oxygen species (ROS) production, and the rate of consumption of NADPH inhibited by DPI was detected to determine the NADPH oxidases (NOXs) activity. Western blot was applied to identify the level of p38MAPK phosphorylation, Griess reaction assay to analyze NO production, immunoenzymatic method to determine prostaglandin E2 (PGE2) production, real time RT-PCR and Western blot to identify the expression of iNOS and COX2, and enzyme linked immunosorbent assay to detect inflammatory cytokines TNF-α, IL-6, IFN-γ, IL-1β. Rat neutrophils were separated, and then ROS production, NOXs activity, NO and PGE2 production, NOX1 and p-p38MAPK expression were detected. Compared with the UC group, apocynin decreased ROS over-production and NOXs activity (P < 0.01), reduced p38MAPK phosphorylation, inhibited NO, PGE2 and cytokines production (P < 0.01). Apocynin also decreased NOXs activity and ROS over-production (P < 0.01), inhibited p38MAPK phosphorylation and NOX1 expression, and reduced NO and PGE2 production (P < 0.01) in separated neutrophils from UC mice. Therefore, apocynin could relieve inflammation in DSS-induced UC mice through inhibiting NOXs-ROS-p38MAPK signal pathway, and neutrophils play an important role.
Acetophenones ; pharmacology ; Animals ; Colitis, Ulcerative ; chemically induced ; drug therapy ; Cytokines ; metabolism ; Dextran Sulfate ; Inflammation ; drug therapy ; MAP Kinase Signaling System ; Mice ; NADH, NADPH Oxidoreductases ; metabolism ; Neutrophils ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water. In the gut flora, they activate azo pro-drugs, which are used for treatment of inflammatory bowel disease, releasing the active component 5-aminosalycilic acid. The bacterium P. aeruginosa has three azoreductase genes, paAzoR1, paAzoR2 and paAzoR3, which as recombinant enzymes have been shown to have different substrate specificities. The mechanism of azoreduction relies upon tautomerisation of the substrate to the hydrazone form. We report here the characterization of the P. aeruginosa azoreductase enzymes, including determining their thermostability, cofactor preference and kinetic constants against a range of their favoured substrates. The expression levels of these enzymes during growth of P. aeruginosa are altered by the presence of azo substrates. It is shown that enzymes that were originally described as azoreductases, are likely to act as NADH quinone oxidoreductases. The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.
Benzoquinones ; metabolism ; Catalytic Domain ; Enzyme Stability ; Evolution, Molecular ; Flavins ; chemistry ; Hot Temperature ; Kinetics ; Mesalamine ; chemistry ; NAD ; metabolism ; NADH, NADPH Oxidoreductases ; chemistry ; NADP ; metabolism ; Osmolar Concentration ; Oxidation-Reduction ; Phenylhydrazines ; chemistry ; Phylogeny ; Protein Binding ; Pseudomonas aeruginosa ; enzymology ; Spectrophotometry, Ultraviolet

OBJECTIVETo investigate the significance of NADPH quinine oxidoreductase 1 (NQO1) protein overexpression on prognostic evaluation of head and neck squamous cell carcinoma (HNSCC).

METHODSNQO1 protein was detected in 162 of HNSCC, 45 cases of adjacent nontumor tissues and 26 samples of normal head and neck epithelia using EnVision immunohistochemical. Correlation between NQO1 overexpression and patients prognosis was also analyzed.

RESULTSThe positive rate and strongly positive rate of NQO1 protein were 84.0% (136/162) and 69.8% (113/162) in HNSCC, respectively, and both of which were significantly higher than either those in adjacent nontumor tissues and normal head and neck epithelia (both P < 0.01). NQO1 expression was significantly correlated with the clinical stage, pT and chemoradiotherapy of HNSCC (P < 0.01). Kaplan-Meier survival analysis showed that overall survival and disease-free survival rates were significantly higher in HNSCC patients with high level NQO1 expression than that those with low level of NQO1 expression (Log-rank = 6.625 , P = 0.010;Log-rank = 6.234 , P = 0.013). Additional analysis by Cox proportional hazard regression model showed that high level of NQO1 expression was an independent hazard predictor for overall survival of patients with HNSCC (Wald = 6.626, P = 0.008).

CONCLUSIONSNQO1 expression level is closely correlated with the progression and prognosis of patients with HNSCC. High level of NQO1 expression may be used as an important indicator for patients with poor prognostic HNSCC.

Breast ; enzymology ; Carcinoma, Squamous Cell ; enzymology ; mortality ; pathology ; Disease-Free Survival ; Female ; Head and Neck Neoplasms ; enzymology ; mortality ; pathology ; Humans ; Kaplan-Meier Estimate ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; NADH, NADPH Oxidoreductases ; metabolism ; Prognosis ; Proportional Hazards Models

The purpose of this study was to investigate the age-related NADPH oxidase (arNOX) activity in patients with age-related knee osteoarthritis (OA). Serum and cartilage arNOX activities were determined using an oxidized ferricytochrome C reduction assay. Full-thickness knee joint cartilages obtained through total knee replacement surgery were graded according to the Outerbridge (OB) classification. Radiographic severity of OA was determined on Knee X-rays according to the Kellgren-Lawrence (K/L) grading system. Cartilage beta-galactosidase, HIF-1alpha, and GLUT-1 expression levels were evaluated as markers for tissue senescence, hypoxia, and glycolysis. Higher arNOX activities occurred with higher levels of cartilage beta-galactosidase, HIF-1alpha, and GLUT-1 (P = 0.002). arNOX activity in cartilages with surface defects (OB grade II, III) was higher than in those without the defects (OB grade 0, I) (P = 0.012). Cartilage arNOX activity showed a positive correlation with serum arNOX activity (r = -0.577, P = 0.023). Serum arNOX activity was significantly higher in the OA subgroup with bilateral ROA than in the OA with no or unilateral ROA (2.449 +/- 0.81, 2.022 +/- 0.251 nM/mL, respectively, P = 0.019). The results of this study demonstrate that OA itself is not a cause to increase arNOX activities, however, arNOX hyperactivity is related to a high degree of cartilage degradation, and a high grade and extent of ROA in age-related OA.
Biomarkers/metabolism ; Cartilage Diseases/*enzymology ; Cartilage, Articular/*enzymology ; Enzyme Activation ; Female ; Humans ; Male ; Middle Aged ; NADH, NADPH Oxidoreductases ; Osteoarthritis, Knee/*diagnosis/*enzymology ; Osteoporosis/*diagnosis/*enzymology ; Reproducibility of Results ; Sensitivity and Specificity ; Statistics as Topic

OBJECTIVETo analysis the effects of water deficit on the transcript level of SOD, APX, DHAR and MDHAR genes in Scutellaria baicalensis.

METHODThree-month-old S. baicalensis was in glasshouse under water deficit stress, and the transcript level of SOD, APX, DHAR and MDHAR genes were analysis utilized semi-quantitative RT-PCR.

RESULTCompared with the control group, a significant decline of the transcriptional level of APX gene was observed at 70 days after water deficit. The transcript level of DHAR gene was reduced at 30 and 50 days after water deficit. And MDHARI gene was significant declined at 50 days.

CONCLUSIONAsA which is an important antioxidant plays a major role in hydrogen peroxide clear system under water deficit, and maybe have an antagonistic effect to the accumulation of baicalein.

Ascorbate Peroxidases ; genetics ; metabolism ; Ascorbic Acid ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Gene Expression Regulation, Plant ; drug effects ; Hydrogen Peroxide ; metabolism ; NADH, NADPH Oxidoreductases ; genetics ; metabolism ; Oxidoreductases ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Scutellaria baicalensis ; enzymology ; genetics ; metabolism ; Superoxide Dismutase ; genetics ; metabolism ; Time Factors ; Water ; metabolism ; pharmacology

In vascular smooth muscle cells, reactive oxygen species (ROS) were known to mediate platelet-derived growth factor (PDGF)-induced cell proliferation and NADH/NADPH oxidase is the major source of ROS. NADH/NADPH oxidase is controlled by rac1 in non-phagocytic cells. In this study, we examined whether the inhibition of rac1 by adenoviral-mediated gene transfer of a dominant negative rac1 gene product (Ad.N17rac1) could reduce the proliferation of rat aortic vascular smooth muscle cells (RASMC) stimulated by PDGF via decreasing intracellular ROS. RASMC were stimulated by PDGF (80 ng/mL) with or without N-acetylcysteine 1 mM or infected with 100 mutiplicity of infection of Ad.N17rac1. Intracellular ROS levels were measured at 12 hr using carboxyl-2', 7'-dichlorodi-hydrofluorescein diacetate confocal microscopy. At 72 hr, cellular proliferation was evaluated by cell number counting and XTT assay. Compared with control, ROS levels were increased by 2-folds by PDGF. NAC and Ad.N17rac1 inhibited PDGF-induced increase of ROS by 77% and 65%, respectively. Cell number was increased by PDGF by 1.6-folds compared with control. NAC and Ad.N17rac1 inhibited PDGF-induced cellular growth by 45% and 87%, respectively. XTT assay also showed similar results. We concluded that inhibition of rac1 in RASMCs could reduce intracellular ROS levels and cellular proliferation induced by PDGF.
Adenoviridae/genetics ; Animal ; Aorta, Thoracic/cytology ; Cell Division/drug effects/physiology ; Cells, Cultured ; Gene Expression/physiology ; Gene Transfer Techniques ; Multienzyme Complexes/antagonists & inhibitors ; Muscle, Smooth, Vascular/*cytology/*metabolism ; NADH, NADPH Oxidoreductases/antagonists & inhibitors ; NADPH Oxidase/antagonists & inhibitors ; Platelet-Derived Growth Factor/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/*metabolism ; rac1 GTP-Binding Protein/*genetics/metabolism

Endotoxic responses to bacterial lipopolysaccharide (LPS) are triggered by Toll-like receptor 4 (TLR4) and involve the production of inflammatory mediators, including interleukin-6 (IL-6), by macrophages. The detailed mechanism of IL-6 production by macrophages in response to LPS has remained unclear, however. We now show that LPS induces IL-6 synthesis in mouse peritoneal macrophages via the leukotriene B4 receptor BLT2. Our results suggest that TLR4-MyD88 signaling functions upstream of BLT2 and that the generation of reactive oxygen species (ROS) by NADPH oxidase 1 (Nox1) and consequent activation of the transcription factor nuclear factor (NF)-kappaB function downstream of BLT2 in this response. These results suggest that a TLR4-MyD88-BLT2-Nox1-ROS-NF-kappaB pathway contributes to the synthesis of IL-6 in LPS-stimulated mouse macrophages.
Animals ; Cell Line ; Interleukin-6/*biosynthesis ; Leukotriene B4/metabolism ; Ligands ; Lipopolysaccharides/immunology ; Macrophages/immunology/*metabolism ; Macrophages, Peritoneal/immunology/metabolism ; Mice ; Myeloid Differentiation Factor 88/*metabolism ; NADH, NADPH Oxidoreductases/metabolism ; NF-kappa B/metabolism ; Reactive Oxygen Species/metabolism ; Receptors, Leukotriene B4/*metabolism ; Signal Transduction