Mitochondrial Manganese Superoxide Dismutase Research Articles

Piscidin-1 Induces Apoptosis via Mitochondrial Reactive Oxygen Species-Regulated Mitochondrial Dysfunction in Human Osteosarcoma Cells

Osteosarcoma (OSA) is the most common type of cancer that originates in the bone and usually occurs in young children. OSA patients were treated with neoadjuvant chemotherapy and surgery, and the results were disappointing. Marine antimicrobial peptides (AMPs) have been the focus of antibiotic research because they are resistant to pathogen infection. Piscidin-1 is an AMP from the hybrid striped bass (Morone saxatilis × M. chrysops) and has approximately 22 amino acids. Research has shown that piscidin-1 can inhibit bacterial infections and has antinociception and anti-cancer properties; however, the regulatory effects of piscidin-1 on mitochondrial dysfunction in cancer cells are still unknown. We aimed to identify the effects of piscidin-1 on mitochondrial reactive oxygen species (mtROS) and apoptosis in OSA cells. Our analyses indicated that piscidin-1 has more cytotoxic effects against OSA cells than against lung and ovarian cancer cells; however, it has no effect on non-cancer cells. Piscidin-1 induces apoptosis in OSA cells, regulates mtROS, reduces mitochondrial antioxidant manganese superoxide dismutase and mitochondrial transmembrane potential, and decreases adenosine 5′-triphosphate production, thus leading to mitochondrial dysfunction and apoptosis. The mitochondrial antioxidant, mitoTempo, reduces the apoptosis induced by piscidin-1. Results suggest that piscidin-1 has potential for use in OSA treatment.

Dietary reduced glutathione supplementation can improve growth, antioxidant capacity, and immunity on Chinese mitten crab, Eriocheir sinensis

Eriocheir sinensis is an important aquaculture species in China, and its yield and quality are threatened by oxidative stress caused by deteriorating water conditions. Reduced glutathione (GSH) is an endogenous antioxidant, but whether dietary GSH can increase the resistance of E. sinensis to environmental stress remains unclear. Therefore, in this study, crabs were fed with dietary GSH (0, 300, 600, 900, and 1200 mg/kg diet weight) for up to 10 weeks to determine the effects of different dietary GSH concentrations on growth, antioxidant capacity, and immunity of E. sinensis. The results showed that the weight gain rate and survival rate increased significantly as dietary GSH levels increased from 0 to 900 mg/kg, but decreased at 1200 mg/kg. Compared with the control group, the diet supplemented with 900 mg/kg GSH not only increased the concentration of GSH in the haemolymph and hepatopancreas, but also enhanced the activity of glutathione peroxidase (GSH-Px) (p < 0.05). Diets supplemented with 600 or 900 mg/kg GSH significantly increased the enzymes activities of superoxide dismutase (SOD), lysozyme (LZM), alkaline phosphatase, and acid phosphatase, and significantly decreased the content of malondialdehyde. To understand the changes in the activity of these enzymes further, the expression of related genes was detected. Diets supplemented with 600 or 900 mg/kg GSH significantly upregulated the genes expressions of cytosolic manganese SOD, mitochondrial manganese SOD, copper, zinc-SOD, GSH-Px, LZM, and prophenoloxidase activating factor, and significantly down regulated the expression of Toll-like receptor 1, Toll-like receptor 2, Dorsal, and the myeloid differentiation factor 88. However, a diet supplemented with 1200 mg/kg GSH decreased those positive indicators. Overall, our results demonstrated that an appropriate diet supplemented with 600 or 900 mg/kg GSH enhances antioxidant capacity and immunity, which will enhance the general health of E. sinensis.

Characterization of a Mn-SOD from the desert beetle Microdera punctipennis and its increased resistance to cold stress in E. coli cells.

Insects have developed a complex network of enzymatic antioxidant systems for handling reactive oxygen species (ROS) generated during stress. Superoxide dismutases (SODs) play a determinant role in balancing ROS in insect. However, studies devoted to SODs functions in insects under cold stress are limited. In the present study, we attempted to identify and characterize a mitochondrial manganese SOD (mMn-SOD) from the desert beetle Micordera punctipennis (denoted as MpmMn-SOD) and explore its protective effects on bacteria cells under cold stress. MpmMn-SOD is composed of 202 amino acids with conserved domains required for metal ions binding and enzyme activity. RT-qPCR experiments revealed that the expression of MpmMn-SOD was ubiquitous but tissue-specific and was induced by cold stress. An E. coli (BL21) system was applied to study the function of MpmMn-SOD. The MpmMn-SOD gene was cloned into the prokaryotic expression vector pET-32a to generate a recombinant plasmid pET-32a(MpmMn-SOD). After transformation of the plasmid into E. coli BL21, the fusion protein Trx-His-MpmMn-SOD was overexpressed and identified by SDS-PAGE and Western blotting. Antioxidant activity assay showed that the death zones of the transformed bacteria BL21 (pET32a-mMn-SOD) were smaller in diameter than the control bacteria BL21 (pET32a). Survival curves under −4 °C showed that BL21 (pET32a-mMn-SOD) had significant enhanced cold resistance compared to BL21 (pET32a). Its SOD activity under −4 °C had a significant negative correlation (r = − 0.995) with superoxide anion O2•− content. Accordingly, under cold stress BL21 (pET32a-mMn-SOD) had lower electric conductivity and malondialdehyde (MDA) content than BL21 (pET32a). Taken together, our results showed that cold stress stimulated the expression of MpmMn-SOD in M. punctipennis. The E. coli cells that overexpress MpmMn-SOD increase their resistance to cold stress by scavenging ROS, and mitigate potential cell damage caused by ROS under cold conditions.

Mitochondrial manganese superoxide dismutase from the shrimp Litopenaeus vannamei: Molecular characterization and effect of high temperature, hypoxia and reoxygenation on expression and enzyme activity.

Climate warming has been increasing ocean water temperature and decreasing oxygen concentrations, exposing aquatic organisms to environmental stress conditions. The shrimp Litopenaeus vannamei manages to survive these harsh environmental conditions by enhancing their antioxidant defenses, among other strategies. In this study, we report the mitochondrial manganese superoxide dismutase (mMnSOD) nucleotide and deduced amino acid sequences and its gene expression in L. vannamei tissues. The deduced protein has 220 amino acids with a signal peptide of 20 amino acids. Expression of mMnSOD was analyzed in hepatopancreas, gills and muscle, where gills had highest expression in normoxic conditions. In addition, shrimp were subjected to high temperature, hypoxia and reoxygenation to analyze the effect on the expression of mMnSOD and SOD activity in mitochondria. High temperature and hypoxia showed a synergistic effect in the up-regulation on expression of mMnSOD in gills and hepatopancreas. Moreover, induction in SOD activity was found in the mitochondrial fraction from gills of normoxia at high temperature, probably due to an overproduction of reactive oxygen species caused by an elevated metabolic rate due to the stress temperature. These results suggest that the combined stress conditions of hypoxia and high temperature trigger molecularly the antioxidant response in L. vannamei in a higher degree than only one stressor.

Antioxidant system in implementation of endotoxicosis syndrome in young persons with comorbidity of gastroesophageal reflux disease and autoimmune thyroiditis

Introduction. In recent years, there has been an increase in the prevalence of gastroesophageal reflux disease and autoimmune thyroiditis in young people. This is associated with a hereditary predisposition, viral diseases, which are often recorded in children's and adolescent groups, the unreasonable use of a large number of drugs, as well as the influence of various negative environmental factors, instability of the autonomic nervous system, eating disorders, the presence of bad habits, chronic stress and others. The aim of the work was to investigate the state of total antioxidant status and the content of biomarkers of the second phase of detoxification of xenobiotics - manganese superoxide dismutase and glutathione peroxidase in young people with gastroesophageal reflux disease and autoimmune thyroiditis. Materials and methods. To achieve this goal, 120 patients with a combined course of gastroesophageal reflux disease (GERD) and autoimmune thyroiditis (AIT) were examined, which made up the main group and two comparison groups: with isolated GERD (45 people) and AIT (42 patients). All patients were students of various universities and were between the ages of 18 and 25. Duration of the disease did not exceed 3 years. The total antioxidant status as an indicator of the first phase of defense and the levels of mitochondrial manganese superoxide dismutase and glutathione peroxidase – the second phase of antioxidant defense was determined. Results. The study showed that in all surveyed groups there was a decrease of total antioxidant status, which characterizes the first phase of the antioxidant defense system. Changes in the first phase of antioxidant defense affect the indicators of the second level of the system, which became the basis for studying the activity of mitochondrial manganese superoxide dismutase. Thus, the value of this indicator in all examined individuals exceeded the norm, which confirmed the inclusion of the second controlling phase of antioxidant system into the pathological process. At the same time, an increase inhibition of the glutathione defense link, the second phase of xenobiotic detoxification, were diagnosed. Conclusions. Negative changes occur in the enzyme system of the first line of antioxidant system in patients with GERD and AIT which may be the result of an unfavorable autoimmune background. Control over the work of antioxidant system moves to the second phase of the system, manifested by an increase in the content of mitochondrial manganese superoxide dismutase and inhibition of the activity of glutathione peroxidase in patients with a combination of GERD and AIT.

Mutant p53 induces SIRT3/MnSOD axis to moderate ROS production in melanoma cells

The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 isoforms can acquire oncogenic properties referred to as gain-of-function (GOF). In this study, we used wild-type (A375) and mutant p53 (MeWo) melanoma cell lines to assess the regulation of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) by mutant p53. The effects of mutant p53 were evaluated by qPCR, immunoblotting, enzyme activity assay, cell proliferation assay, reactive oxygen species (ROS) assay after cellular transfection. We demonstrate that mutant p53 induces MnSOD expression, which is recovered by the ROS scavenger N-acetyl-l-cysteine. This suggests MnSOD induction as a defense mechanism of melanoma cells to counterbalance the pro-oxidant conditions induced by mutant p53. We also demonstrate that mutant p53 induces the expression of Sirtuin3 (SIRT3), a major mitochondrial NAD+-dependent deacetylase, stimulating MnSOD deacetylation and enzymatic activity. Indeed, the restoration of SIRT3 reverses MnSOD activity decrease by mutant p53 knock-down. Finally, MnSOD knock-down further enhances mutant p53-mediated ROS increase, counteracting mutp53-dependent cell hyperproliferation. This indicates that SIRT3 and MnSOD act to maintain ROS levels controlled to promote cell proliferation and survival, providing new therapeutic opportunities to be further considered for clinical studies in cancer patients bearing mutant TP53 gene.

A prospective study on associations between superoxide dismutase gene polymorphisms and antituberculosis drug-induced liver injury in a Chinese Han population.

Antituberculosis drug-induced liver injury (ATDILI) is increasing globally and, hence, it is crucial to predict its risk in the clinical management of antituberculosis therapy. As a major antioxidant, superoxide dismutase (SOD) is mainly responsible for providing defence against oxidative stress, which is involved in ATDILI. The present study aimed to investigate the associations between polymorphisms in SOD genes, including Cu/ZnSOD (SOD1), mitochondrial manganese SOD (MnSOD or SOD2) and extracellular SOD (SOD3), as well as the susceptibility to ATDILI in a Chinese Han population. In total, 1060 Chinese Han subjects highly suspected to have tuberculosis (TB) were prospectively enrolled from West China Hospital of Sichuan University. Overall, 746 subjects comprising 118 ATDILI and 628 ATD-tolerant TB patients were eligible and were genotyped for seven single-nucleotide polymorphisms in three SOD genes (SOD1: rs4816407 and rs1041740; SOD2: rs4880; SOD3: rs699473, rs2536512, rs2855262 and rs8192290). Logistic regression analysis showed that none of the seven genetic variants in the three SOD genes were significantly associated with susceptibility to ATDILI in the Chinese Han population after Bonferroni correction, except for a potential association for the SOD2 rs4880 A>G (G allele, p=0.190, odds ratio=1.53, 95% confidence interval=1.05-2.23; GG genotype, p=0.155). The promising application of single-nucleotide polymorphisms in the SOD1, SOD2 and SOD3 genes as genetic markers for ATDILI is challenged, and further studies are needed with larger sample sizes and different ethnicities, especially for SOD2 rs4880.

Effects of dietary supplementation with icariin on growth performance, antioxidant capacity and non-specific immunity of Chinese mitten crab (Eriocheir sinensis)

We investigated the effects of icariin (ICA) on growth performance, antioxidant capacity and non-specific immunity in Chinese mitten crab (Eriocheir sinensis). A total of 200 healthy crabs (average weight: 33.58 ± 0.05 g) were randomly assigned to four treatments with five replicates, each with ten individuals per pool. There were four dietary treatments: the control group (fed with the basal diet), the ICA 50 group, the ICA100 group, and the ICA 200 group (fed with the basal diet supplemented with 50, 100, and 200 mg/kg ICA, respectively). These diets were provided for 8 weeks. Results indicated that ICA100 crabs had higher weight gain (WG), specific growth rate (SGR) and survival rate (SR) than the controls. Protein carbonyl content (PCC) and malondialdehyde (MDA) concentrations in the haemolymph and hepatopancreas of ICA100 crabs were significantly lower than in the control group, while the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were significantly higher. The activities of PO, LZM, ACP and AKP were significantly enhanced with ICA supplementation at 50 and 100 mg/kg, yet decreased subsequently at 200 mg/kg. Furthermore, supplementation of 100 mg/kg ICA up-regulated the mRNA expression of prophenoloxidase (proPO), catalase (CAT), mitochondrial manganese superoxide dismutase (mtMnSOD), thioredoxin-1 (Trx1) and peroxiredoxin 6 (Prx6), while the mRNA expression of toll like receptors (TLRs), NF-κB-like transcription factor Relish and lipopolysaccharide-induced TNF-α factor (LITAF) were down-regulated in the hepatopancreas (P < 0.05). These findings indicate that dietary ICA supplementation at an optimum dose of 100 mg/kg may be effective in improving growth performance, antioxidant capability and non-specific immunity of Chinese mitten crab.

Molecular characterization of manganese superoxide dismutase (MnSOD) from sterlet Acipenser ruthenus and its responses to Aeromonas hydrophila challenge and hypoxia stress

A novel gene encoding the mitochondrial manganese superoxide dismutase from sterlet Acipenser ruthenus (Ar-MnSOD) was cloned. The full-length cDNA of MnSOD was of 1040 bp with a 672 bp open reading frame encoding 224 amino acids and the deduced amino acid sequence was located in mitochondria. Sequence comparison analysis showed that Ar-MnSOD was highly similar to MnSODs of invertebrates and vertebrates, especially those of freshwater Cyprinidae fishes and mammals. Phylogenetic analysis revealed that Ar-MnSOD was distant from MnSODs of other fishes and belonged to the family of mitochondrial MnSODs (mMnSOD). Consistently, Ar-MnSOD was located in mitochondria. The 3D structure of Ar-MnSOD was predicted and the overall structure was similar to that of MnSODs of humans and the bay scallop Argopecten irradians. In addition, mRNA of Ar-MnSOD was detected to extensively express in all tissues, with the highest level in brain and liver. Spleen and head kidney inoculation of Aeromonas hydrophila led to a significant up-regulation of Ar-MnSOD transcript levels. Also, hypoxia induced a transient increase in transcription of Ar-MnSOD in the gills, but not in the heart and brain, suggesting metabolic depression in these vital organs. The results also implied the anti-hypoxia properties of Ar-MnSOD in the related tissues and proved that Ar-MnSOD was involved in the stress response and (anti) oxidative processes triggered by hypoxia. The results indicated that Ar-MnSOD is induced upon A. hydrophila infection and hypoxia, consistent with its role in host immune and stress-induced anti-oxidative responses.

Nicotinamide mononucleotide alters mitochondrial dynamics by SIRT3-dependent mechanism in male mice.

Nicotinamide adenine dinucleotide (NAD+ ) is a central signaling molecule and enzyme cofactor that is involved in a variety of fundamental biological processes. NAD+ levels decline with age, neurodegenerative conditions, acute brain injury, and in obesity or diabetes. Loss of NAD+ results in impaired mitochondrial and cellular functions. Administration of NAD+ precursor, nicotinamide mononucleotide (NMN), has shown to improve mitochondrial bioenergetics, reverse age-associated physiological decline, and inhibit postischemic NAD+ degradation and cellular death. In this study, we identified a novel link between NAD+ metabolism and mitochondrial dynamics. A single dose (62.5mg/kg) of NMN, administered to male mice, increases hippocampal mitochondria NAD+ pools for up to 24hr posttreatment and drives a sirtuin 3 (SIRT3)-mediated global decrease in mitochondrial protein acetylation. This results in a reduction of hippocampal reactive oxygen species levels via SIRT3-driven deacetylation of mitochondrial manganese superoxide dismutase. Consequently, mitochondria in neurons become less fragmented due to lower interaction of phosphorylated fission protein, dynamin-related protein 1 (pDrp1 [S616]), with mitochondria. In conclusion, manipulation of mitochondrial NAD+ levels by NMN results in metabolic changes that protect mitochondria against reactive oxygen species and excessive fragmentation, offering therapeutic approaches for pathophysiologic stress conditions.

Involvement of a novel protein kinase C (nPKC) in immunocompetence in hemocytes of white shrimp, Litopenaeus vannamei

Complementary (c)DNA encoding novel protein kinase C (PKC) messenger (m)RNA of the white shrimp Litopenaeus vannamei, consisted of 2454-bp cDNA containing an open reading frame (ORF) of 2232 bp, belonging to the novel (n)PKC family of proteins characterized by their containing two phorbol ester/diacylglycerol-binding domains (C1 domain), a C2 domain, and a catalytic domain of the serine/threonine kinase, designated LvnPKC. A comparison of amino acid sequences showed that LvnPKC was closely related to arthropod nPKC. LvnPKC cDNA was detected in all tested tissues with a real-time PCR including the hepatopancreas, gills, muscles, subcuticular epithelium, abdominal nerve, thoracic nerve, brain, the stomach, heart, and especially in hemocytes and the intestines. Moreover, significantly upregulated LvnPKC expression was only observed in the eyestalk, brain, and hepatopancreas of shrimp transferred from 28 °C to 18 °C for 30 min. Induction of LvnPKC expression in hemocytes of L. vannamei injected with Vibrio alginolyticus at 105 cfu shrimp−1 was detected earlier than in those injected with 103 cfu shrimp−1. Shrimp received LvnPKC-dsRNA for 1 days specifically depleted the expression of LvnPKC mRNA in hemocytes compared those of diethylpyrocarbonate water treatment. After that, significantly decreased expressions of lipopolysaccharide - and β-1,3-glucan-binding protein, prophenoloxidase-activating enzyme, peroxinectin, prophenoloxidase I, and prophenoloxidase II in the prophenoloxidase-activating system; lysozyme and cytosolic manganese superoxide dismutase and mitochondrial manganese superoxide dismutase in the antioxidant system were observed. We therefore concluded that LvnPKC is involved in immune defense of L. vannamei exposed to hypothermal stress or infected with V. alginolyticus.

Single-nucleotide polymorphisms linked to body weight revealed in growth selected Macrobrachium rosenbergii

Association of type I single nucleotide polymorphic (SNP) markers with quantitative traits can provide an effective method for detecting genes and functions that are responsible for performance variation in domesticated species. In order to discover novel polymorphisms in candidate genes that could be associated with growth, fragments (175 to 668 bp) from 11 housekeeping, regulatory, and immune response genes of Macrobrachium rosenbergii previously reported to contain 83 SNPs were amplified from genomic DNA of 23 growth selected (cumulative genetic gain of 18%) and 23 unselected individuals and sequenced by Sanger’s method. A total of 45 SNPs were identified from eight genes, of which 20 were novel and 18 were found to be growth associated with allele frequencies > 0.65 in the selected group. Eleven of these were located in exonic regions of which 3 present in crustacean lipocalin (LIPC) and heat shock protein 21 (HSP21) were nonsynonymous. In silico prediction indicates that 2 of the non-synonymous alleles may result in higher stability of the proteins. Of the 5 synonymous growth-associated SNPs, 3 present in phosphoenol pyruvate carboxykinase (PEPCK), cytochrome oxidase 1 (COX1), and HSP70 were a switch to the preferred codon. Seven SNPs were located in the 3′UTRs of lectin 3 and 4 (LEC3, LEC4) and anti-lipopolysaccharide factor 1 (ALF1). Only one altered allele was observed at every locus. No SNPs were found in NaK-ATPase, mitochondrial manganese superoxide dismutase, and tachylectin genes. This is the first such marker association study being reported for M. rosenbergii from India and will be of use in selecting future generations.

Activation of Sirtuin 3 and Maintenance of Mitochondrial Integrity by N-Acetylcysteine Protects Against Bisphenol A-Induced Kidney and Liver Toxicity in Rats.

Mitochondrial impairment ensuing from oxidative imbalance is related to adverse consequences of bisphenol A (BPA), a globally utilized industrial chemical. Recent evidence reveals sirtuin 3 (SIRT3) as a key regulator of mitochondrial homeostasis; however, its role in BPA toxicity remains unidentified. This study explored the potential benefits of N-acetylcysteine (NAC), an effective antioxidant, against BPA toxicity in the kidney and liver, and examined whether SIRT3 was involved in this condition. Male Wistar rats were fed with vehicle, BPA (5, 50 mg/kg), BPA (50 mg/kg) plus NAC (100 mg/kg) and were evaluated after 5 weeks. NAC treatment significantly diminished BPA-induced kidney and liver functional disorders, histopathological alterations, oxidative stress, and apoptosis. The increased mitochondrial reactive oxygen species, the disrupted membrane potential, the swelling, and the impaired mitochondrial fission caused by BPA were also mitigated upon concurrent treatment with NAC. The benefits of NAC were associated with enhanced AMPK-PGC-1α-SIRT3 signaling protein expressions, which led to decreased acetylation of superoxide dismutase 2 (SOD2) and increased expression of mitochondrial antioxidant manganese superoxide dismutase (MnSOD). The findings demonstrate the efficacy of NAC in protecting BPA-induced kidney and liver injury, which, in part, is mediated by activating SIRT3 and improving mitochondrial function, dynamics, and oxidative imbalance.

Chronic Stress Combined with a Fructose Diet Reduces Hypothalamic Insulin Signaling and Antioxidative Defense in Female Rats

Background: Increased fructose consumption and chronic exposure to stress have been associated with the development of obesity and insulin resistance. In the hypothalamus, a crossroad of stress responses and energy balance, insulin and glucocorticoids regulate the expression of orexigenic neuropeptides, neuropeptide Y (NPY) and agouti-related protein (AgRP), and anorexigenic neuropeptides, proopio­melanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Objectives: We investigated whether chronic stress and fructose diet disrupt these hormonal signaling pathways and appetite control in the hypothalamus, contributing to the development of insulin resistance and obesity. Potential roles of hypothalamic inflammation and oxidative stress in the development of insulin resistance were also analyzed. Methods: Insulin, glucocorticoid, and leptin signaling, expression of orexigenic and anorexigenic neuropeptides, and antioxidative and inflammatory statuses in the whole hypothalamus of fructose-fed female rats exposed to unpredictable stress for 9 weeks were analyzed using quantitative PCR and Western blotting. Results: Chronic stress combined with a fructose-enriched diet reduced protein content and stimulatory phosphorylation of Akt kinase, and elevated 11β-hydroxysteroid dehydrogenase 1 and glucocorticoid receptor expression, while alterations in appetite regulation (NPY, AgRP, POMC, CART, leptin receptor, and SOCS3 expression) were not observed. The expression of antioxidative defense enzymes (mitochondrial manganese superoxide dismutase 2, glutathione reductase, and catalase) and proinflammatory cytokines (IL-1β, IL-6, and TNFα) was reduced. Conclusions: Our results underline the combination of long-term stress exposure and fructose overconsumption as more detrimental for hypothalamic function than for either of the factors separately, as it enhanced glucocorticoid and impaired insulin signaling, antioxidative ­defense, and inflammatory responses of this homeostasis- regulating center.

140 - Increased expression and activity of Sod2 promotes transcoelomic metastasis in ovarian cancer

Ovarian cancer is the leading cause of death from gynecologic malignancy due to asymptomatic progression of the disease to advanced stages. Patients with widespread metastases are characterized by a large volume of ascites in the peritoneal cavity within which floating cancer cell populations are found in malignant cases. Metastasis of ovarian cancer occurs when cancer cells escape the primary tumor and adapt to survive under stress conditions associated with anchorage independence in the ascites. We demonstrate that ovarian cancer cells overcome matrix detachment-induced oxidative stress by upregulating expression and activity of the mitochondrial antioxidant enzyme Manganese Superoxide Dismutase 2 (Sod2), and demonstrate that increased expression of Sod2 in anchorage independence is necessary for survival of ovarian cancer cell in vitro and for successful omental metastasis in vivo. Our data reveal a multi-modal regulation of Sod2 in anchorage independence, that includes transcriptional, translational and post-translational regulation at different stages following matrix detachment. In early anchorage independence, a rapid increase in Sod2 protein translation is observed within 30 minutes after cell detachment, without change in mRNA expression. Concomitantly, Sod2 activity is rapidly increased following matrix detachment in a Sirtuin-3 (SIRT3)-dependent manner. We demonstrate that SIRT3, a mitochondrial NAD+ deacetylase, which activates Sod2 by deacetylating key lysine residues including K68, is also necessary for anchorage independent survival and metastasis in vivo. This early post-transcriptional/post-translational regulation of Sod2 is followed by increased mRNA transcription and protein expression of Sod2 within 24 hours of anchorage independence. Concomitantly, we observe increased expression of the stress-response transcription factor Nrf2, a master regulator of antioxidant genes, including Sod2. Collectively, our data suggest that ovarian cancer cells exhibit a multi-modal regulatory system to ensure rapid and sustained expression and activity of Sod2 in response to anchorage independence, which provides protection from mitochondrial oxidative stress associated with transcoelomic metastasis.

Daily zeaxanthin supplementation prevents atrophy of the retinal pigment epithelium (RPE) in a mouse model of mitochondrial oxidative stress.

Oxidative damage is implicated in the pathogenesis of age-related macular degeneration (AMD). The dry form of AMD (geographic atrophy) is characterized by loss of RPE, photoreceptors, and macular pigments. The cumulative effects of oxidative stress impact mitochondrial function in RPE. In Sod2flox/floxVMD2-cre mice, the RPE specific deletion of Sod2, the gene for mitochondrial manganese superoxide dismutase (MnSOD), leads to elevated oxidative stress in retina and RPE, and causes changes in the RPE and underlying Bruch’s membrane that share some features of AMD. This study tested the hypothesis that zeaxanthin supplementation would reduce oxidative stress and preserve RPE structure and function in these mice. Zeaxanthin in retina/RPE/choroid and liver was quantified by LC/MS, retinal function and structure were evaluated by electroretinogram (ERG) and spectral domain optical coherence tomography (SD-OCT), and antioxidant gene expression was measured by RT-PCR. After one month of supplementation, zeaxanthin levels were 5-fold higher in the retina/RPE/choroid and 12-fold higher in liver than in unsupplemented control mice. After four months of supplementation, amplitudes of the ERG a-wave (function of rod photoreceptors) and b-wave (function of the inner retina) were not different in supplemented and control mice. In contrast, the c-wave amplitude (a measure of RPE function) was 28% higher in supplemented mice than in control mice. Higher RPE/choroid expression of antioxidant genes (Cat, Gstm1, Hmox1, Nqo1) and scaffolding protein Sqstm1 were found in supplemented mice than in unsupplemented controls. Reduced nitrotyrosine content in the RPE/choroid was demonstrated by ELISA. Preliminary assessment of retinal ultrastructure indicated that supplementation supported better preservation of RPE structure with more compact basal infoldings and intact mitochondria. We conclude that daily zeaxanthin supplementation protected RPE cells from mitochondrial oxidative stress associated with deficiency in the MnSOD and thereby improved RPE function early in the disease course.

Dietary fructooligosaccharide can mitigate the negative effects of immunity on Chinese mitten crab fed a high level of plant protein diet

An 8-week feeding trial was carried out under controlled condition to evaluate the effect of dietary fructooligosaccharide (FOS) on growth performance, whole body composition, antioxidant status and immunity of crabs fed high levels of plant protein diets. Thus, six experimental diets were formulated (designated as F0P50, F0P60, F0P70, F0.2P50, F0.2P60 and F0.2P70), which contain two FOS levels (0 or 0.2%) and three plant protein levels (50, 60, or 70%) according to a 2 × 3 factorial design. The results showed that weight gain increased significantly as dietary plant protein level decreased from 70% to 50%. At 50% plant protein level, the addition of 0.2% FOS can significantly elevate weight gain (WG) (P < 0.05). The highest value in survival rate was observed in crabs fed F0.2P50 and F0.2P60 diet. Crabs fed F0.2P50 diet showed significantly higher crude protein content (P < 0.05) compared with those in other groups, but there were no significant differences in the contents of moisture, crude lipid and ash among all groups (P > 0.05). Catalase (CAT) activity in crabs fed F0.2P50 increased significantly (P < 0.05) compared with crabs fed F0P60, F0P70, F0.2P60 and F0.2P70, but malondialdehyde (MDA) concentrations decreased significantly (P < 0.05). Meanwhile, nitric oxide (NO) concentration, acid phosphatase (ACP) and alkaline phosphatase (AKP) activities of crabs fed 0.2% FOS diets increased significantly (P < 0.05) compared with crabs fed 0% FOS diets. The expressions of prophenoloxidase (propo) was significantly (P < 0.05) affected only by dietary plant protein levels with the highest values observed in 50% plant protein diet, whereas the opposite was true for Myeloid differentiation factor 88 (myd88). The mRNA expressions of mitochondrial manganese superoxide dismutase (mtmnsod), lipopolysaccharide-induced TNF-α factor (litaf) and toll like receptors (tlrs) were significantly affected (P < 0.05) by both FOS and plant protein levels. The cytosolic manganese superoxide dismutase (cytmnsod) mRNA expressions in F0.2P50 and F0.2P60 groups were significantly higher than those in F0P70 and F0.2P70 groups. The results in this study indicated that supplementation with 0.2% FOS can enhance growth performance in crabs fed lower plant protein diets and as well improve immunity in those fed with higher plant protein diets.

Identification and Molecular Characterisation of a Novel Manganese Superoxide Dismutase Gene from Flesh Fly Larvae,Sarcophaga argyrostoma(Diptera: Sarcophagidae)

The mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) plays a vital role in defence against reactive oxygen species (ROS) in eukaryotic mitochondria. This study was conducted to characterise SOD as an antioxidant gene from haemolymph of third larval instar of the flesh fly, Sarcophaga argyrostoma after challenging with hydrogen peroxide. Mn-SaSOD of S. argyrostoma has a single open reading frame (orf ) of 765 nucleotides that encode a polypeptide of 254 amino acids. Subsequent analyses of Mn-SOD gene sequences revealed that the identified Mn-SaSOD gene in this study shared homology with similar genes from other insect species. In addition, four manganese ion-binding sites (metalbinding sites) and Mn-Fe SOD signature were identified within the orf of Mn-SaSOD. One putative transcription factor-binding site (TATT) within the 5′-flanking region was predicted. This finding suggests that Mn-SaSOD might be involved in environmental stress responses and could be used in the production of novel antioxidants to compensate for the increasing demand of natural antioxidants instead of the chemical synthetic ones.

Molecular Characterization of a Mitochondrial Manganese Superoxide Dismutase From Chilo suppressalis (Lepidoptera: Crambidae).

In insects, superoxide dismutases (SODs) play a critical role in the scavenging of harmful reactive oxygen species (ROS) and protecting against oxidative stress induced by various environmental stresses. The Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is an economically important insect pest of rice crops. In this study, a mitochondrial manganese SOD (Cs-mMnSOD) gene was characterized in C. suppressalis. The deduced Cs-mMnSOD protein has typical highly conserved features of mitochondrial manganese SODs, including four manganese binding residues, the signature DVWEHAYY peptide, and a mitochondrial-targeting sequence at the N-terminus. Transcription of Cs-mMnSOD was detectable at all developmental stages, but highest in pupae. Furthermore, the mRNA level of Cs-mMnSOD was strongly upregulated (more than twofold increase) following exposure to low and high temperatures (4, 30 and 35°C), insecticides (chlorpyrifos and chlorantraniliprole), and chemical reagents (cumene hydroperoxide, paraquat, H2O2 and CdCl2), but slightly elevated (less than twofold increase) in response to 8°C, abamectin and CuSO4. Additionally, the Cs-mMnSOD transcription results were consistent with the enzymatic activity data of the protein product. Purified recombinant Cs-mMnSOD protein expressed in Escherichia coli displayed SOD activity and thermostability. Furthermore, E. coli cells overexpressing Cs-mMnSOD exhibited long-term resistance to the oxidative inducers cumene hydroperoxide and paraquat. Our findings indicate that Cs-mMnSOD plays an important role in protecting C. suppressalis against oxidative damage.

Kinin B1 Receptor Knockdown Prevents DOCA‐Salt Hypertension by Modulating Mitochondrial Oxidative Stress in the Brain

The imbalance between reactive oxygen species (ROS) production and anti‐oxidant capacity in the central nervous system results in oxidative stress and is implicated in the pathogenesis of hypertension. Deoxycorticosterone acetate (DOCA)‐salt hypertension is associated with increased expression of the pro‐inflammatory kinin B1 receptor (B1R) in the brain. We previously reported that DOCA‐salt hypertension is attenuated in global B1R knockout (B1RKO) mice. However, the functional role of B1R in inducing oxidative stress within the brain during neurogenic hypertension has not been fully studied. In this study, we tested the hypothesis that the beneficial effects of B1R knockdown are mediated by reduction in mitochondrial oxidative stress in the brain. DOCA‐salt treatment (1mg/g body wt. DOCA, 1% saline in drinking water for 3 weeks) resulted in significantly lower blood pressure (BP; telemetry) level in B1RKO mice (121 ±2 mmHg, n=12) compared to wild‐type controls (WT) (138 ±3 mmHg, n=12). DOCA‐salt treatment resulted in decreased catalase (79.1 ±5.6 vs. 150.3 ±10.4 mmol/min/ml, p&lt;0.01), total superoxide dismutase (SOD, 4.19 ±0.3 vs. 5.97 ±0.3 U/ml, p&lt;0.01), and the mitochondrial antioxidant manganese SOD (0.67 ±0.09 vs. 1.58 ±0.05 U/ml, p&lt;0.01) activities in hypothalamic homogenates of WT mice, which were prevented in B1RKO mice (134.8 ±4.6 mmol/min/ml, 5.89 ±0.1 U/ml, and 1.68 ±0.22 U/ml, respectively, p&lt;0.01). WT mice treated with DOCA‐salt showed increased oxidative stress as indicated by increased total ROS levels in the hypothalamus as measured using electron paramagnetic resonance spectrometer. This increase in ROS production was not observed in B1RKO mice treated with DOCA‐salt. Furthermore, DOCA‐salt hypertension resulted in decreased phosphorylation of eNOS‐ser1177 in the PVN (35 ±5 % decrease, n=9, p&lt;0.05 vs WT), which was prevented in B1RKO mice. In addition, the gene expression of PPAR Gamma coactivator (PGC) 1‐Alpha and Uncoupling protein‐2 (UCP‐2), two important genes involved in mitochondrial function, were decreased in the PVN of DOCA‐salt treated WT mice but not in B1RKO mice. Together, these data provide evidence that kinin B1R knockdown reduces mitochondrial oxidative stress, improves antioxidant and nitric oxide signaling, and there by attenuates the development of DOCA‐salt hypertension.Support or Funding InformationThe American Heart Association (15SDG25720021)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.