Superoxide Reduction Research Articles

Complete Charge Regulation by a Redox Enzyme Upon Single Electron Transfer.

The degree by which metalloproteins partially regulate net charge (Z) upon electron transfer (ET) was recently measured for the first time using "protein charge ladders" of azurin, cytochrome c, and myoglobin [Angew. Chem. Int. Ed. 2018, 57(19), 5364-5368; Angew. Chem. 2018, 130, 5462-5466]. Here, we show that Cu, Zn superoxide dismutase (SOD1) is unique among proteins in its ability to resist changes in net charge upon single ET (e.g., ΔZET(SOD1) =0.05±0.08 per electron, compared to ΔZET(Cyt-c) =1.19±0.02). This total regulation of net charge by SOD1 is attributed to the protonation of the bridging histidine upon copper reduction, yielding redox centers that are isoelectric at both copper oxidation states. Charge regulation by SOD1 would prevent long range coulombic perturbations to residue pKa 's upon ET at copper, allowing SOD1's "electrostatic loop" to attract superoxide with equal affinity (at both redox states of copper) during diffusion-limited reduction and oxidation of superoxide.

Unregulated mTORC1 activation in the liver: hepatic loss of Depdc5 and Tsc1 synergistically produces liver failure and injury

mTORC1 is an important metabolic regulator that responds to insulin/growth factors, nutrients and cellular stresses. DEPDC5 in the GATOR1 complex suppresses mTORC1 in conditions of limited nutrients, while TSC1 in the TSC1/TSC2 complex inhibits mTORC1 during insulin/growth factor deprivation and energetic stresses. Genetic variations in the DEPDC5 gene are associated with increased severity of liver pathologies during chronic hepatitis B and C virus (HBV/HCV) infection, which downregulates TSC1/TSC2. Using mice with liver‐specific Depdc5 deficiency, we examined how Depdc5 affects liver homeostasis, pathophysiology, and its interaction with Tsc1. Depdc5‐deficient mice exhibited enlargement of zone 3 hepatocytes, which progressed into liver damage, inflammation and fibrosis at later age. Double deletion of both Tsc1 and Depdc5 (DKO) produced synergistic mTORC1 activation, which was much more than what was observed from single Tsc1 or Depdc5 knockout mice. DKO mice experienced pronounced and widespread hepatocyte damage, and exhibited externally visible liver failure phenotypes, such as jaundice and systemic growth defects. Through systemic transcriptome analysis, we identified superoxide accumulation and oxidative damage as the main pathogenetic features distinguishing these mice from other mTORC1 models. Indeed, mTORC1 inhibition, as well as superoxide reduction, were sufficient to eliminate most pathological phenotypes we observed in mTORC1‐hyperactivated liver tissues. The current findings provide the genetic evidence supporting that growth factor/insulin‐regulated (Tsc1) and nutrient‐regulated (Depdc5) pathways genetically interact to coordinate mTORC1 activation. In addition, this multi‐pathway method for mTORC1 hyperactivation explains how DEPDC5 perturbations in humans aggravate HBV/HCV pathologies, which inhibit TSC1 and activate mTORC1.Support or Funding InformationR01DK102850, R01DK111465 and R01DK114131 to JHL, F31DK117610 to AH, K01AG061236 to MK, T32AG000114, T32GM008322, P30AG024824, P30AR069620, P30DK034933, P30DK089503 and P30CA046592

AKR1B1-Induced Epithelial-Mesenchymal Transition Mediated by RAGE-Oxidative Stress in Diabetic Cataract Lens.

Purpose: Cataracts are a major cause of visual acuity deterioration in diabetes mellitus (DM) in developed and developing countries. Studies have demonstrated that overproduction of AKR1B1 and receptor for advanced glycation end products (RAGE) plays a major role in the pathogenesis of diabetic cataracts, but it is unclear whether the prevalence of diabetic cataracts is related to epithelial–mesenchymal transition (EMT) in lens epithelial cells. This study aimed to analyze the role of EMT in cataract formation of DM patients. Methods: Immunofluorescence and immunohistochemistry assays were used to estimate AKR1B1, RAGE, AMPK, and EMT levels in epithelial human lens of DM or non-DM cataracts. Results: Immunohistochemical staining demonstrated that pathologic phases and N-cadherin expression levels were significantly higher in epithelial human lens of DM (+) compared to DM (−) cataracts. Immunofluorescent staining showed that AKR1B1 and RAGE were significantly higher in epithelial human lens of DM (+) compared to DM (−) cataracts. Interestingly, acetyl superoxide dismutase 2 (AcSOD2) levels were significantly higher in DM patients’ lens epithelial cells (LECs), whereas AMPKT172 phosphorylation was significantly increased in non-DM patients. This indicates that AMPKT172 might be related to superoxide reduction and diabetic cataract formation. Conclusions: Our results suggest that AKR1B1 overexpression can decrease AMPK activation, thereby increasing AcSOD2 and RAGE-induced EMT in epithelial human lens of DM cataracts. These novel findings suggest that AKR inhibitors may be candidates for the pharmacological prevention of cataracts in patients with DM.

β-glucan alleviates the immunosuppressive effects of oxytetracycline on the non-specific immune responses and resistance against Vibrio alginolyticus infection in Epinephelus fuscoguttatus × Epinephelus lanceolatus hybrids

This study was conducted to examine the combinatory effects of β-glucan and oxytetracycline (OTC) on hybrid giant tiger groupers (Epinephelus fuscoguttatus × Epinephelus lanceolatus). In vitro tests, OTC significantly reduced superoxide anion production and phagocytic activity in primary head kidney leukocytes. However, this suppressive effect was alleviated by co-treatment with β-glucan. Subsequently, feeding trials were performed to investigate the potential immunomodulatory effects of dietary β-glucan alone or in combination with OTC on groupers. A total of 210 healthy groupers (368.00 ± 51.03 g) were divided into six groups. Group 1 was the control group, group 2 (BG) received 5 g β-glucan per kg feed weight, groups 3–5 received 5 g/kg β-glucan in combination with 10, 30, or 50 mg OTC/kg fish weight/day (groups M1, M2, and M3, respectively), and group 6 (O) received 50 mg OTC/kg fish weight/day. Fish were sampled to determine the innate immunity parameters and residual OTC levels in the muscle tissue during a 28-day feeding regimen. Residual OTC levels were considerably higher in groups M3 and O compared with the other groups, and peaked on day 14. This was followed by a slight decrease on day 28, despite a continuous supply of OTC. Notably, fish fed with OTC alone had significantly decreased phagocytic rates and superoxide anion production observed in head kidney leukocytes, as well as poorer protection against Vibrio alginolyticus infection. These immunosuppressive effects were not observed in the fish fed with β-glucan in combination with a lower dose of OTC (group M2). Thus, these data suggest that the combination of dietary β-glucan and OTC exerts synergistic immunostimulating effects that protect groupers from bacterial infection.

Alleviation of Oxidative Stress Induced by 24-Epibrassinolide in Soybean Plants Exposed to Different Manganese Supplies: UpRegulation of Antioxidant Enzymes and Maintenance of Photosynthetic Pigments

Adverse effects promoted by inadequate manganese (Mn) supply (deficiency or toxicity) causes inefficiency of the antioxidant system and degradation of chlorophylls. However, 24-epibrassinolide (EBR) is a natural steroid that exhibits beneficial effects on antioxidant metabolism, chlorophyll levels and stress indicators. Therefore, this research aims to evaluate whether EBR application via spray can alleviate oxidative stress in soybean plants exposed to different Mn concentrations and to determine possible contributions of the antioxidant enzymes and photosynthetic pigments. Experiment followed a completely randomized factorial design with two concentrations of 24-epibrassinolide (0 and 100 nM EBR, described as − EBR and + EBR, respectively) and three Mn doses (0.25, 25 and 2500 µM Mn, described as low, control and high supply of Mn, respectively). Plants treated with low and high concentrations of Mn + EBR exhibit significant increases in all enzymes evaluated (superoxide dismutase, catalase, ascorbate peroxidase and peroxidase). To superoxide dismutase (SOD), EBR spray promoted increments of 77%, 38% and 76% under low, control and high Mn supplementation, respectively, compared to same treatment in absence of EBR. Clearly intense activity is linked to SOD contributed by dismutation of superoxide into hydrogen peroxide, being subsequently decomposed by other enzymes (catalase, ascorbate peroxidase and peroxidase). Concomitantly, plants with Mn deficiency and toxicity sprayed with 100 nM EBR presented maintenance of chlorophylls and carotenoids due to reduction of superoxide and hydrogen peroxide and consequently reduced chloroplast membrane damages as indicated by malondialdehyde levels and electrolyte leakage.

MiR155-5p Inhibits Cell Migration and Oxidative Stress in Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats.

Migration of vascular smooth muscle cells (VSMCs) is essential for vascular reconstruction in hypertension and several vascular diseases. Our recent study showed that extracellular vesicles derived from vascular adventitial fibroblasts of normal rats inhibited VSMC proliferation by delivering miR155-5p to VSMCs. It is unknown whether miR155-5p inhibits cell migration and oxidative stress in VSMCs of spontaneously hypertensive rats (SHR) and in angiotensin II (Ang II)-treated VSMCs. The purpose of this study was to determine the role of miR155-5p in VSMC migration and its underlying mechanisms. Primary VSMCs were isolated from the aortic media of Wistar-Kyoto rats (WKY) and SHR. Wound healing assay and Boyden chamber assay were used to evaluate VSMC migration. A miR155-5p mimic inhibited, and a miR155-5p inhibitor promoted the migration of VSMC of SHR but had no significant effect on the migration of VSMC of WKY. The miR155-5p mimic inhibited angiotensin-converting enzyme (ACE) mRNA and protein expression in VSMCs. It also reduced superoxide anion production, NAD(P)H oxidase (NOX) activity, as well as NOX2, interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) expression levels in VSMCs of SHR but not in VSMCs of WKY rats. Overexpression of miR155-5p inhibited VSMC migration and superoxide anion and IL-1β production in VSMCs of SHR but had no impact on exogenous Ang II-induced VSMC migration and on superoxide anion and IL-1β production in WKY rats and SHR. These results indicate that miR155-5p inhibits VSMC migration in SHR by suppressing ACE expression and its downstream production of Ang II, superoxide anion, and inflammatory factors. However, miR155-5p had no effects on exogenous Ang II-induced VSMC migration.

Finerenone Reduces Intrinsic Arterial Stiffness in Munich Wistar Frömter Rats, a Genetic Model of Chronic Kidney Disease

Background: Development of albuminuria and arterial stiffness in Munich Wistar Frömter (MWF) rats, a model of chronic kidney disease, is related to alterations in extracellular matrix, increased oxidative stress, and endothelial dysfunction. Finerenone (FIN), a novel, nonsteroidal, potent, and selective mineralocorticoid receptor antagonist, improves endothelial dysfunction through enhancing nitric oxide (NO) bioavailability and decreasing superoxide anion levels due to an upregulation in vascular and renal superoxide dismutase activity. We hypothesize that FIN reduces arterial stiffness in this model associated to the reduction in albuminuria and matrix metalloproteinase (MMP)-2/9 activity. Methods: Twelve-week-old MWF rats with established albuminuria and age-matched normoalbuminuric Wistar (W) rats were treated with FIN (10 mg/kg/day, once-daily oral gavage) or with vehicle (control, C) for 4 weeks. Results: Arterial stiffness was significantly higher in mesenteric arteries (MA) of MWF-C as compared to W-C. FIN treatment significantly lowered β-index, a measure of intrinsic stiffness independent of geometry, in MWF (β_MWF-FIN = 7.7 ± 0.4 vs. β_MWF-C = 9.2 ± 0.5, p < 0.05) positively correlating with urinary albumin excretion. Elastin fenestrae area in the internal elastic lamina of MA from MWF-FIN was significantly larger (+377%, p < 0.05). FIN increased plasma pro-MMP-2 and decreased plasma MMP-2 and MMP-9 activities, correlating with reductions in β-index. MA from MWF-FIN exhibited higher NO bioavailability and reduced superoxide anion levels compared to MWF-C. Conclusion: FIN treatment reduces intrinsic arterial stiffness in MA from MWF rats associated with changes in elastin organization, normalization of MMP-2 and MMP-9 activities, and reduction of oxidative stress. Moreover, reduction of arterial stiffness correlates with reduction in albuminuria.

Effect of pre-storage ascorbic acid and Aloe vera gel coating application on enzymatic browning and quality of lotus root slices.

The effect of ascorbic acid [AA (1%)] and Aloe vera gel [AVG (50%)] coating alone and in combination was investigated on enzymatic browning and quality of lotus root slices during storage at 20±1°C. The combined application of AA and AVG coating delayed surface browning, reduced increase in relative electrolyte leakage (REL) and showed higher overall visual quality (OVQ). Similarly, AA and AVG combined treatment reduced superoxide anion ( ) and hydrogen peroxide (H2 O2 ) production and malondialdehyde (MDA) content, and suppressed peroxidase (POD) and polyphenol oxidase (PPO) activities. In addition, AA and AVG treatment conserved higher AA content, ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase (CAT) enzymes activities along with higher total phenolics and radical scavenging activity. In conclusion, the combined application of AA and AVG coating could be an appropriate treatment to delay surface browning and quality loss of lotus root slices. PRACTICAL APPLICATIONS: Lotus root is an aquatic rhizome vegetable. The fresh-cut slices of lotus roots are prone to post-cut enzymatic browning and quality deterioration during postharvest storage. Browning induced loss of visual quality and microbial infestations are the leading constraints in extending storage and/or shelf life of lotus root slices. Surface browning results in loss of characteristic color eventually leading to significant reduction in market potential and visual quality. However, quality deterioration and development of browning could be delayed with some suitable postharvest treatments. So, the effect of AA and Aloe vera gel based coating was investigated for quality conservation of lotus root slices. The findings of the current work are of global importance in reducing browning and conserving visual quality of lotus root slices in particular and fresh-cut produce in general.

Remifentanil Protects against Lipopolysaccharide-Induced Inflammation through PARP-1/NF-κB Signaling Pathway.

Sepsis is a leading cause of death in patients with severe infection worldwide. Remifentanil is an ultra-short-acting, potent opioid analgesic. In the study, we aimed to investigate the role and underlying mechanism of remifentanil in lipopolysaccharide- (LPS-) induced inflammation in human aortic endothelial cells (HAECs). HAECs were pretreated with phosphate-buffered saline (PBS) or remifentanil (2.5 μM) for 30 min, then stimulated by LPS (10 μg/ml) for another 24 h. Poly(ADP-ribose) polymerase 1 (PARP-1) was inhibited by small interfering RNA (siRNA). Superoxide anion production and DNA damage were analyzed by dihydroethidium (DHE) staining and comet assay. The inducible nitric oxide synthase (iNOS), intercellular adhesion molecule 1 (ICAM-1), PARP-1, poly(ADP-ribose) (PAR), and nuclear factor-kappa B p65 (NF-κB p65) expressions were analyzed by RT-PCR or western blotting analysis. NF-κB p65 nuclear translocation was assessed by immunofluorescence. Compared with the control group, pretreatment with remifentanil significantly reduced superoxide anion production and DNA damage, with downregulation of iNOS, ICAM-1, and PARP-1 expressions as well as PAR expression. Moreover, pretreatment with PARP-1 siRNA or remifentanil inhibited LPS-induced NF-κB p65 expression and nuclear translocation. Remifentanil reduced LPS-induced inflammatory response through PARP-1/NF-κB signaling pathway. Remifentanil might be an optimal choice of analgesia in septic patients.

Serum amyloid A3 confers protection against acute lung injury in Pseudomonas aeruginosa-infected mice.

Pseudomonas aeruginosa is a gram-negative bacterium associated with serious illnesses, including ventilator-associated pneumonia and various sepsis syndromes in humans. Understanding the host immune mechanisms against P. aeruginosa is, therefore, of clinical importance. The present study identified serum amyloid A3 (SAA3) as being highly inducible in mouse bronchial epithelium following P. aeruginosa infection. Genetic deletion of Saa3 rendered mice more susceptible to P. aeruginosa infection with decreased neutrophil superoxide anion production, and ex vivo treatment of mouse neutrophils with recombinant SAA3 restored the ability of neutrophils to produce superoxide anions. The SAA3-deficient mice showed exacerbated inflammatory responses, which was characterized by pronounced neutrophil infiltration, elevated expression of TNF-α, KC/CXCL1, and MIP-2/CXCL2 in bronchoalveolar lavage fluid (BALF), and increased lung microvascular permeability compared with their wild-type littermates. BALF neutrophils from Saa3 knockout mice exhibited reduced superoxide anion production compared with neutrophils from wild-type mice. Adoptive transfer of SAA3-treated neutrophils to Saa3 knockout mice ameliorated P. aeruginosa-induced acute lung injury. These findings demonstrate that SAA3 not only serves as a biomarker for infection and inflammation, but also plays a protective role against P. aeruginosa infection-induced lung injury in part through augmentation of neutrophil bactericidal functions.

Identification and Allelopathy of Green Garlic (Allium sativum L.) Volatiles on Scavenging of Cucumber (Cucumis sativus L.) Reactive Oxygen Species.

Garlic and formulations containing allicin are used widely as fungicides in modern agriculture. However, limited reports are available on the allelopathic mechanism of green garlic volatile organic compounds (VOCs) and its component allelochemicals. The aim of this study was to investigate VOCs of green garlic and their effect on scavenging of reactive oxygen species (ROS) in cucumber. In this study, green garlic VOCs were collected by HS-SPME, then analyzed by GS-MS. Their biological activity were verified by bioassays. The results showed that diallyl disulfide (DADS) is the main allelochemical of green garlic VOCs and the DADS content released from green garlic is approximately 0.08 mg/g. On this basis, the allelopathic effects of green garlic VOCs in vivo and 1 mmol/L DADS on scavenging of ROS in cucumber seedlings were further studied. Green garlic VOCs and DADS both reduce superoxide anion and increase the accumulation of hydrogen peroxide of cucumber seedlings. They can also regulate active antioxidant enzymes (SOD, CAT, POD), antioxidant substances (MDA, GSH and ASA) and genes (CscAPX, CsGPX, CsMDAR, CsSOD, CsCAT, CsPOD) responding to oxidative stress in cucumber seedlings.

ETHE1 and MOCS1 deficiencies: Disruption of mitochondrial bioenergetics, dynamics, redox homeostasis and endoplasmic reticulum-mitochondria crosstalk in patient fibroblasts

Ethylmalonic encephalopathy protein 1 (ETHE1) and molybdenum cofactor (MoCo) deficiencies are hereditary disorders that affect the catabolism of sulfur-containing amino acids. ETHE1 deficiency is caused by mutations in the ETHE1 gene, while MoCo deficiency is due to mutations in one of three genes involved in MoCo biosynthesis (MOCS1, MOCS2 and GPHN). Patients with both disorders exhibit abnormalities of the mitochondrial respiratory chain, among other biochemical findings. However, the pathophysiology of the defects has not been elucidated. To characterize cellular derangements, mitochondrial bioenergetics, dynamics, endoplasmic reticulum (ER)-mitochondria communication, superoxide production and apoptosis were evaluated in fibroblasts from four patients with ETHE1 deficiency and one with MOCS1 deficiency. The effect of JP4-039, a promising mitochondrial-targeted antioxidant, was also tested on cells. Our data show that mitochondrial respiration was decreased in all patient cell lines. ATP depletion and increased mitochondrial mass was identified in the same cells, while variable alterations in mitochondrial fusion and fission were seen. High superoxide levels were found in all cells and were decreased by treatment with JP4-039, while the respiratory chain activity was increased by this antioxidant in cells in which it was impaired. The content of VDAC1 and IP3R, proteins involved in ER-mitochondria communication, was decreased, while DDIT3, a marker of ER stress, and apoptosis were increased in all cell lines. These data demonstrate that previously unrecognized broad disturbances of cellular function are involved in the pathophysiology of ETHE1 and MOCS1 deficiencies, and that reduction of mitochondrial superoxide by JP4-039 is a promising strategy for adjuvant therapy of these disorders.

Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access

Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (MnII/MnII, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (FeII/FeII form, 1.32 Å), or prepared aerobically in the photo-reduced FeII/FeII form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.

Short-term pharmacological activation of Nrf2 ameliorates vascular dysfunction in aged rats and in pathological human vasculature. A potential target for therapeutic intervention

Oxidative stress contributes to endothelial dysfunction, a key step in cardiovascular disease development. Ageing-related vascular dysfunction involves defective antioxidant response. Nuclear factor erythroid 2-like-2 (Nrf2), orchestrates cellular response to oxidative stress. We evaluated the impact of Nrf2-activation on endothelium-dependent and H2O2-mediated vasodilations in: aorta (RA), mesenteric artery (RMA), coronary artery (RCA) and corpus cavernosum (RCC) from ageing rats and in human penile arteries (HPRA) and corpus cavernosum (HCC) from erectile dysfunction (ED) patients. Relaxant responses were evaluated in organ chambers and wire myographs. Nrf2 content and heme oxygenase-1 (HO-1) were determined by ELISA. Superoxide and Nrf2 were detected by immunofluorescence. Pharmacological activation of Nrf2 with sulforaphane (SFN) improved NO- and endothelium-derived hyperpolarizing factor-mediated endothelium-dependent vasodilation and H2O2-induced relaxation in vascular beds from aging rats. SFN-induced effects were associated with increased Nrf2 (RMA, RCA) and reduced superoxide detection in RCA. Improvement of vascular function was confirmed in HPRA and HCC from ED patients and mimicked by another Nrf2 activator, oltipraz. Nrf2 increase and superoxide reduction together with HO-1 increase by Nrf2 activation was evidenced in HCC from ED patients. PDE5 inhibitor-induced relaxations of HPRA and HCC from ED patients were enhanced by SFN. Nrf2 short-term pharmacological activation attenuates age-related impairment of endothelium-dependent and reactive oxygen species (ROS)-induced vasodilation in different rat and human vascular territories by upregulation of Nrf2-related signaling and decreased oxidative stress. In ED patients target tissues, Nrf2 potentiates the functional effect of ED conventional pharmacological therapy suggesting potential therapeutic implication.

Molecular Evolution and Functional Analysis of Rubredoxin-Like Proteins in Plants.

Rubredoxins are a class of iron-containing proteins that play an important role in the reduction of superoxide in some anaerobic bacteria and also act as electron carriers in many biochemical processes. Unlike the more widely studied about rubredoxin proteins in anaerobic bacteria, very few researches about the function of rubredoxins have been proceeded in plants. Previous studies indicated that rubredoxins in A. thaliana may play a critical role in responding to oxidative stress. In order to identify more rubredoxins in plants that maybe have similar functions as the rubredoxin-like protein of A. thaliana, we identified and analyzed plant rubredoxin proteins using bioinformatics-based methods. Totally, 66 candidate rubredoxin proteins were identified based on public databases, exhibiting lengths of 187–360 amino acids with molecular weights of 19.856–37.117 kDa. The results of subcellular localization showed that these candidate rubredoxins were localized to the chloroplast, which might be consistent with the fact that rubredoxins were predominantly expressed in leaves. Analyses of conserved motifs indicated that these candidate rubredoxins contained rubredoxin and PDZ domains. The expression patterns of rubredoxins in glycophyte and halophytic plant under salt/drought stress revealed that rubredoxin is one of the important stress response proteins. Finally, the coexpression network of rubredoxin in Arabidopsis thaliana under abiotic was extracted from ATTED-II to explore the function and regulation relationship of rubredoxin in Arabidopsis thaliana. Our results showed that putative rubredoxin proteins containing PDZ and rubredoxin domains, localized to the chloroplast, may act with other proteins in chloroplast to responses to abiotic stress in higher plants. These findings might provide value inference to promote the development of plant tolerance to some abiotic stresses and other economically important crops.

Abstract 2648: Hypoxia-induced NADPH oxidase 5 (NOX5) promotes tumor growth of head and neck squamous cell carcinoma through HIF-2á signaling

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) has an increased content of reactive oxygen species (ROS). However, clinical trials of antioxidant resulted in no benefit towards HNSCC anti-cancer treatment. NADPH oxidase (NOX) family is found to be the major generator of ROS which might contribute to the failure of antioxidant therapy. For the past 10 years, NOX family have been found with a range of functions in different cancers. Of which, NOX5 is the least well-understood one with limited details of signaling roles. Purpose of the study: To investigate the role of NOX5 in HNSCC. Experimental procedures: Online databases were used to analyze the expression of NOX5 in HNSCC patients. Immunohistochemistry was performed to detect the NOX5 expression level and its co-expression with hypoxia-inducible factors (HIFs) in paraffin-embedded tumor tissues surgically resected from HNSCC patients in Hong Kong. Subcutaneous xenograft model was established using NOX5-silenced HNSCC cells to evaluate the tumor growth potential in vivo. Sulforhodamine B assay and colony formation assay were used to measure the proliferation potential of NOX5-silenced HNSCC cells in vitro. Hypoxia chamber was applied to mimic the hypoxic condition of tumor tissue in vitro. Flow cytometry with Dihydroethidium staining was performed to measure the production of superoxide when silencing or overexpressing NOX5. Superoxide scavenger TEMPOL was applied to test the signaling role of superoxide generated by NOX5. Quantitative PCR and western blot were used to measure the mRNA and protein expression level of NOX5 and HIFs in HNSCC cell lines. Results: Relative mRNA expression of NOX5 was found significantly increased in HNSCC tumor tissues comparing to normal tissues both in Oncomine and TCGA datasets. Interestingly, NOX5 expression was found higher in central areas of tumor nests with the co-expression of HIF-1α and HIF-2α in our HNSCC patients’ samples. Further, NOX5 was upregulated in HNSCC cell lines under hypoxic condition (1% oxygen). Knocking-down of NOX5 impaired HNSCC cell growth both in vivo and in vitro, and this reduction was more significant under hypoxic condition in vitro. Loss of NOX5 resulted in superoxide reduction and HIF-2α impairment but not HIF-1α. Overexpressed NOX5 induced superoxide production and HIF-2α expression, and this induction was attenuated by TEMPOL. Conclusions: Hypoxia-induced NOX5 promotes HNSCC tumor growth by regulating HIF-2α via generating superoxide. NOX5 might serve as a novel therapeutic target for treatment of HNSCC. Citation Format: Siqi Chen, Wei Gao, Jimmy Yu-Wai Chan, Thian-Sze Wong. Hypoxia-induced NADPH oxidase 5 (NOX5) promotes tumor growth of head and neck squamous cell carcinoma through HIF-2á signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2648.

Regulation of MAP kinase-mediated endothelial dysfunction in hyperglycemia via arginase I and eNOS dysregulation

Emerging evidence suggests that arginase contributes to endothelial dysfunction in diabetes. Intracellular signaling pathways, which interplay between arginase and eNOS enzyme activity leading to the development of endothelial dysfunction in hyperglycemia are not fully understood. Here, we analyzed the possible involvement of hyperglycemia (HG) induced arginase expression in eNOS protein regulation and activity and also the impact of arginase inhibition on eNOS activity. Furthermore, the roles of p38 MAPK and Erk1/2 phosphorylation in upregulation of arginase expression and eNOS dysregulation in endothelial cells (ECs) under hyperglycemia were evaluated. Protein analysis showed a concurrent increase in arginase I expression and decrease in eNOS expression and phosphorylation at Ser1177 under HG conditions. There was no simultaneous change in phosphorylation of eNOS at Thr495 in HG. Arginase inhibition prevented increased arginase activity, restored impaired NO bioavailability and reduced superoxide anion generation. Inhibition of MAP-kinases demonstrated that, unlike Erk1/2, p38 MAPK is an upstream activator in a signaling cascade leading to increased arginase I in HG conditions. P38 MAPK protein expression and phosphorylation were increased in response to HG. In the presence of a p38 MAPK inhibitor, HG-induced arginase expression was blunted. Although Erk1/2 was activated in HG, increased arginase expression was not blocked by co-treatment with an Erk1/2 inhibitor. Activation of both, p38 MAPK and Erk1/2 in HG, induced a downregulation in eNOS activity. Hence, applying MAPK inhibitors increased eNOS phosphorylation in HG.In conclusion, these findings demonstrate contributions of arginase I in the development of endothelial cell dysfunction under HG conditions via impaired eNOS regulation, which maybe mediated by p38 MAPK.

Repurposing of the Nootropic Drug Vinpocetine as an Analgesic and Anti-Inflammatory Agent: Evidence in a Mouse Model of Superoxide Anion-Triggered Inflammation.

Clinically active drugs for the treatment of acute pain have their prescription limited due to the significant side effects they induce. An increase in reactive oxygen species (ROS) has been linked to several conditions, including inflammation and pain processing. Therefore, new or repurposed drugs with the ability of reducing ROS-triggered responses are promising candidates for analgesic drugs. Vinpocetine is a clinically used nootropic drug with antioxidant, anti-inflammatory, and analgesic properties. However, the effects of vinpocetine have not been investigated in a model with a direct relationship between ROS, inflammation, and pain. Based on that, we aimed to investigate the effects of vinpocetine in a model of superoxide anion-induced pain and inflammation using potassium superoxide (KO2) as a superoxide anion donor to trigger inflammation and pain. In the KO2 model, vinpocetine dose-dependently reduced pain-like behaviors (spontaneous pain and hyperalgesia), paw edema, and neutrophil and mononuclear cell recruitment to the paw skin (assessed by H&E staining, fluorescence, and enzymatic assays) and to the peritoneal cavity. Vinpocetine also restored tissue endogenous antioxidant ability and Nrf2 and Ho-1 mRNA expression and reduced superoxide anion production and gp91phox mRNA expression. We also observed the inhibition of IκBα degradation by vinpocetine, which demonstrates a reduction in the activation of NF-κB explaining the diminished production of IL-33, IL-1β, and TNF-α. Collectively, our data show that vinpocetine alleviates pain and inflammation induced by KO2, which is a mouse model with a direct role of ROS in triggering pain and other inflammatory phenomena. Thus, the results suggest the repurposing of vinpocetine as an anti-inflammatory and analgesic drug.

Understanding and Modulating Metalloenzymes with Unnatural Amino Acids, Non-Native Metal Ions, and Non-Native Metallocofactors.

Metalloproteins set the gold standard for performing important functions, including catalyzing demanding reactions under mild conditions. Designing artificial metalloenzymes (ArMs) to catalyze abiological reactions has been a major endeavor for many years, but most ArM activities are far below those of native enzymes, making them unsuitable for most pratical applications. A critical step to advance the field is to fundamentally understand what it takes to not only confer but also fine-tune ArM activities so they match those of native enzymes. Indeed, only once we can freely modulate ArM activity to rival (or surpass!) natural enzymes can the potential of ArMs be fully realized. A key to unlocking ArM potential is the observation that one metal primary coordination sphere can display a range of functions and levels of activity, leading to the realization that secondary coordination sphere (SCS) interactions are critically important. However, SCS interactions are numerous, long-range, and weak, making them very difficult to reproduce in ArMs. Furthermore, natural enzymes are tied to a small set of biologically available functional moieties from canonical amino acids and physiologically available metal ions and metallocofactors, severely limiting the chemical space available to probe and tune ArMs. In this Account, we summarize the use of unnatural amino acids (UAAs) and non-native metal ions and metallocofactors by our group and our collaborators to probe and modulate ArM functions. We incorporated isostructural UAAs in a type 1 copper (T1Cu) protein azurin to provide conclusive evidence that axial ligand hydrophobicity is a major determinant of T1Cu redunction potential ( E°'). Closely related work from other groups are also discussed. We also probed the role of protein backbone interactions that cannot be altered by standard mutagenesis by replacing the peptide bond with an ester linkage. We used insight gained from these studies to tune the E°' of azurin across the entire physiological range, the broadest range ever achieved in a single metalloprotein. Introducing UAA analogues of Tyr into ArM models of heme-copper oxidase (HCO) revealed a linear relationship between p Ka, E°', and activity. We also substituted non-native hemes and non-native metal ions for their native equivalents in these models to resolve several issues that were intractable in native HCOs and the closely related nitric oxide reductases, such as their roles in modulating substrate affinity, electron transfer rate, and activity. We incorporated abiological cofactors such as ferrocene and Mn(salen) into azurin and myoglobin, respectively, to stabilize these inorganic and organometallic compounds in water, confer abiological functions, tune their E°' and activity through SCS interactions, and show that the approach to metallocofactor anchoring and orientation can tune enantioselectivity and alter function. Replacing Cu in azurin with non-native Fe or Ni can impart novel activities, such as superoxide reduction and C-C bond formation. While progress was made, we have identified only a small fraction of the interactions that can be generally applied to ArMs to fine-tune their functions. Because SCS interactions are subtle and heavily interconnected, it has been difficult to characterize their effects quantitatively. It is vital to develop spectroscopic and computational techniques to detect and quantify their effects in both resting states and catalytic intermediates.

&amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt; Evaluation of the Potential Antioxidant of &amp;lt;i&amp;gt;Bidens segetum&amp;lt;/i&amp;gt; Mart. ex Colla (Asteraceae) in Melanocyte and Melanoma Cells

Bidens segetum Martius ex Colla known as the “picão do mato”, is an herbaceous plant that occurs in the Cerrado biome of some Brazilian states. Among the species of Bidens, we highlight B. pilosa known as “picão preto”, of which several activities are reported as antioxidant and antibacterial. Ethanolic extract from Bidens segetum (EEBs) showed an-tioxidant potential when analyzed by free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and antifungal activity against Cladosporium cladosporiodes and C. sphareospermum fungi. PFFR3.3 subfraction from EEBs has 81.5% of 5-O caffeoylquinic acid (5-CQA) and potential antioxidant (DPPH). However, PFFR3.3 did not decrease superoxide anion in metastatic melanoma cells by dihydroeth-idium assay (DHE). PP4 subfraction is a mixture of polyacetylenes that has antifungal (Cladosporium) and antioxidant activity, since reduced superoxide anion amount in melanoma cells after 5 min of treatment. However, no dose-response and time-response curve were observed, not even with the authentic standard (5-CQA). Complementary chemical studies will be performed to confirm the polyacetylenes and 5-CQA structures present in the EEBs from B. segetum and new methodologies should be performed to confirm the antioxidant activity of these com-pounds and the effects on melanocytes and melanomas.