| 2021 | Dietary supplementation of dried plum: a novel strategy to mitigate heat stress in broiler chickens. | J Anim Sci Biotechnol | Heat stress is a significant problem in the poultry industry, causing a severe economic loss due to its detrimental effects on chickens' health and performance. Dried plum (DP) is a good source of minerals, vitamins, antioxidants, and phenolic compounds. Studies have suggested that DP has several health benefits, such as maintaining the body's redox system, immune status, and calcium hemostasis. Based on the health benefits of DP, we hypothesized that the dietary supplementation of DP would alleviate the detrimental effects of heat stress on broiler chickens.To test the hypothesis, day-old broiler chicks (n = 72) were randomly allocated to three treatment groups (n = 24/group): no heat stress (NHS), heat stress (HS), and heat stress with dried plum (HS + DP), and reared under standard conditions. The inclusion of 2.5% DP in the feed of the HS + DP group was made during the treatment period, while birds in other groups were provided with a standard finisher diet. After 21 days, birds in the HS and HS + DP groups were exposed to cyclic heat stress conditions (33 °C for 8 h during daytime) for 3 weeks, while those in the NHS group were reared under normal conditions (22-24 °C). Weekly body weight and feed intake were recorded to calculate the average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). Heat stress significantly decreased the final body weight, ADG, ADFI, and increased FCR compared to the NHS group, whereas dietary supplementation of DP significantly improved these growth performance parameters compared to the HS group. Furthermore, supplementation of DP significantly increased the expression of heat shock protein-related genes (HSF1, HSF3, HSP70, and HSP90), antioxidant-related genes (SOD1, SOD2, GPX1, GPX3, PRDX1, and TXN), tight junction-related genes (CLDN1, and OCLN), and immune-related genes (IL4, MUC2) in the ileum as compared to the HS group. The microbiota analysis showed significant enrichment of Bacillales, Christensenellaceae, Bacillaceae, Peptostreptococcaceae, and Anaerotruncus in heat-stressed birds supplemented with DP as compared to the HS group. Further, DP supplementation also significantly increased the concentration of acetate, propionate, and total VFA in the cecal digesta of the HS + DP group as compared to the HS group.These findings suggest that DP supplementation effectively improved the growth performances and gut health parameters in the heat-stressed birds. Thus, dried plum can be a potential feed supplement to mitigate heat stress in broiler chickens. |
| 2021 | Research Note: Phytobiotics modulate the expression profile of circulating inflammasome and cyto(chemo)kine in whole blood of broilers exposed to cyclic heat stress. | Poult Sci | Heat stress (HS) is a critical concern to the poultry industry as it affects both productivity and well-being. Various managerial and nutritional strategies have been proposed to mitigate the negative effects of HS in chickens, with plant-based additives showing promise. Recently, we reported the positive effect of a phytogenic feed additive (PFA) on growth performance in HS birds. Owing to the antioxidant nature of these compounds, we sought to further explore the effect of PFA on whole blood circulating chemokines, cytokines, and inflammasomes in HS broilers. Broilers (600 males, 1 d) were randomly assigned to 12 environmental chambers, subjected to 2 environmental conditions (12 h cyclic heat stress, HS, 35°C vs. thermoneutral condition [TN], 24°C) and fed 3 diets (control, PFA-C 250 ppm, PFA-C 400 ppm) in a 2 × 3 factorial design. After 21 d of cyclic HS, blood samples were collected for target gene expression analysis. HS upregulated the expression of superoxide dismutase 1 (SOD1) and downregulated glutathione peroxidase-3 (GPX-3), and there was diet × temperature interaction for SOD2, GPX-1, and GPX-3, where gene expression was increased by PFA-C250 during HS but was unchanged for PFA-C400. Plasma total antioxidant capacity (TAC) and malondialdehyde (MDA) content were increased by HS. Gene expression of interleukin-18 (IL-18) was decreased by HS, without further effect of PFA. HS increased tumor necrosis factor α (TNFα), but this effect was mitigated by PFA-C400. C-C motif chemokine ligands 4 and 20 (CCL4 and CCL20) showed a similar pattern to TNFα, with PFA-C400 ameliorating the negative effect of HS. The nucleotide-binding, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome was decreased by HS and further lowered by PFA-C400, but the nucleotide-binding oligomerization domain, leucine-rich repeat, and CARD domain containing 3 (NLRC3) and nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) inflammasomes were increased by PFA under TN conditions, with no effects of HS. Heat shock proteins (HSP) and heat shock factors (HSF) were unaffected by PFA or HS. Together these data indicate that gene expression of circulating inflammatory factors are dysregulated during HS, and supplemental dietary PFA may be protective. |
| 2021 | Functional, biochemical and molecular impact of sediment plumes from deep-sea mining on Mytilus galloprovincialis under hyperbaric conditions. | Environ Res | The deep-sea is the biggest ecosystem in the world and despite the extreme conditions that characterize it, is highly biodiverse and complex. Deep-sea mining has been foreseen as a potential and concerning new stressor, and among the deep-sea mining associated stressors, sediment plumes, likely to be released into the water column as a side effect of mining, can reach habitats within a radius of more than a hundred kilometers. The present study examined the effects of suspended sediments of different grain sizes (63-125 μm, 125-250 μm and 250-500 μm) in the model species Mytilus galloprovincialis, at 4 bar, as a proxy to address the potential effects of sediment plumes, in the water column, with different grain sizes under high pressure conditions. Functional (filtration rate - FR), biochemical (catalase - CAT, glutathione s-transferase - GST, lipid peroxidation - LPO) and molecular (gene expression of [actin (ACTN), glutathione S-transferase alpha (GSTA), superoxide dismutase 2 (SOD2), catalase (CAT), heat shock protein 60 (HSP60), cytochrome c oxidase (COI) and DNA mismatch repair protein (MSH6)]) endpoints were studied in juvenile organisms. The FR decreased significantly for all tested grain size ranges, with a more severe effect for the particles with a diameter between 63 and 125 μm. In addition to the FR, significant changes were also observed for all tested biomarkers. Gene expression was significantly downregulated for CAT and ACTN. Overall, this study demonstrated that the smaller sized particles are the ones leading to more severe effects. Given their high dispersion potential and longer suspension periods under mining operation scenarios, particular attention should be given to the release of sediment plumes that may affect deep-sea environments and the water column. It is, therefore, vital to create standards and guidelines for sustainable mining practices. |
| 2020 | Effects of acute heat stress at different ambient temperature on hepatic redox status in broilers. | Poult Sci | This study aimed to investigate the effects of different acute high ambient temperatures on redox status in liver of broilers. A total of 144 35-day-old Arbor Acres broilers were randomly divided into 4 groups with 6 replicates of 6 birds each and subsequently distributed in different environment chambers for acute heat stress. The temperature of 4 environment chambers were set to 26°C (control), 29°C, 32°C, 35°C for 6 h, respectively. Various indicators were tested to evaluate hepatic redox status. Then, the hallmarks of hepatocellular antioxidant and apoptosis were measured by qRT-PCR and Western Blot. The results showed that with the ambient temperature increase (i) the content of hydrogen peroxide (HO) and protein carbonyl (PC) in the liver of broilers increased significantly (P < 0.05), but the content of malondialdehyde (MDA) and 8-hydroxyguanosine (8-OHdG) was not affected; (ii) the activity of catalase (CAT) and glutathione reductase (GR) increased significantly (P < 0.05). Similarly, the superoxide dismutase (SOD) had an increasing tendency (P = 0.07), and the content of the reduced glutathione (GSH) was also significantly increased (P < 0.05) under high temperature; (iii) the heat shock protein (HSP70), nuclear factor erythroid-2-related factor 2 (Nrf2), and other antioxidant gene (HO-1, NQO1, GCLc, GST, SOD1, SOD2, CAT, Prx3) were upregulated in broilers liver. Moreover, the protein level of HSP70, Nrf2, and Prx3 were also upregulated; (iv) high temperature upregulated the antiapoptotic gene expression (BCL-2); however, the proapoptotic genes (BAK1, caspase-3, and caspase-9) did not change significantly; meanwhile, there was no significant changes in the protein level of caspase-3 and caspase-9. The results of this study indicated that 35-day-old Arbor Acres broilers have a certain tolerance to oxidative stress induced by high ambient temperature. Six hours of acute heat stress-activated Nrf2 signaling pathway. Meanwhile, the expression of related antioxidant genes and proteins is upregulated, consequently resulted in increased antioxidant enzymes activity and GSH. These effects enable the body to scavenge large amounts of reactive oxygen species produced by high temperature and prevent the occurrence of apoptosis. |
| 2020 | The genotoxic effects of mixture of aluminum, arsenic, cadmium, cobalt, and chromium on the gill tissue of adult zebrafish (, Hamilton 1822). | Drug Chem Toxicol | The aim of this study is to investigate the genotoxic effects of mixtures of five metals on zebrafish at two different concentrations; at the permissible maximum contamination levels in drinking water and irrigation waters. The drinking water limits are as follows: 300 µg/L for Aluminum (Al), 10 µg/L for Arsenic (As), 5 µg/L for Cadmium (Cd), 10 µg/L for Cobalt (Co), and 50 µg/L for Chromium (Cr). The irrigation water limits: 5000 µg/L for Al, 100 µg/L for As, 10 µg/L for Cd, 50 µg/L for Co, and 100 µg/L for Cr. The zebrafish underwent chronic exposure for periods of 5, 10, and 20 days. The gene expressions for mitochondrial superoxide dismutase (SOD2), stress-specific receptor protein NCCRP1, the heat shock proteins: Hsp9, Hsp14, Hsp60, Hsp70, DNA repair (XRCC1 and EXO1), and apoptosis (BOK and BAX) were evaluated. It was found that exposure to the low- and high-concentrations of the heavy metal mixtures caused cell stress, an increased expression of the antioxidant genes, and repair proteins. As the duration of exposure was increased, the cells progressed through the apoptotic pathway. This was more evident in the high-concentration exposure groups. The results demonstrated the necessity for a reevaluation of the maximum values of heavy metal and toxic element concentrations as prescribed by the Local Standing Rules of Water Pollution Control Regulation, as well as a reevaluation of the limitations of heavy metal mixture interactions with respect to ecological balance and environmental health.HighlightsThe purpose of this study was to investigate the genotoxic effects of a mixture of Aluminum, Arsenic, Cadmium, Cobalt, Chromium on zebrafish, within drinking water, and irrigation water limits determining the concentration.The zebrafish were exposed to two different concentrations of each metal mixture for 5-, 10-, and 20-day periods. Following exposure, gene expressions of the zebrafish's gill tissues were examined.As a result of the exposure to the metal mixtures, the following occurred: cell stress, increased antioxidant gene activity, and attempts to protect cell viability. However, the cells progressed through the apoptotic pathway after prolonged exposure.The results demonstrated the necessity for a reevaluation of the maximum limits of metal and toxic element concentrations as stated in the Standing Rules of Water Pollution Control Regulation. |
| 2020 | Cadmium elicits alterations in mitochondrial morphology and functionality in C3H10T1/2Cl8 mouse embryonic fibroblasts. | Biochim Biophys Acta Gen Subj | Cadmium is a widespread carcinogen. We previously showed that the administration of low CdCl doses for 24 h to healthy C3H10T1/2Cl8 mouse embryonic fibroblast cell line at the beginning of Cell Transformation Assay (CTA), up regulates genes involved in metal scavenging and antioxidant defense, like metallothioneines, glutathione S-transferases and heat shock proteins. Still, although most cells thrive normally in the following weeks, malignancy is triggered by CdCl and leads to the appearance of foci of transformed cells at the end of the CTA. In this work we aim at elucidating the early metabolic deregulation induced by cadmium, underlying healthy cell transformation into malignant cells.Respiratory metabolism was investigated through Seahorse Agilent assays, while oxidative stress level was assessed through fluorescent probes; DNA damage was evaluated by Comet assay, and mitochondrial morphology was analyzed in confocal microscopy.Results show that the initial response to CdCl involves mitochondria rearrangement into a perinuclear network. However, SOD1 and SOD2 activities are inhibited, leading to increased superoxide anion level, which in turn causes DNA strand breaks. From the metabolic point of view, cells increase their glycolytic flux, while all extra NADH produced is still efficiently reoxidized by mitochondria.Our results confirm previously shown response against cadmium toxicity; new data about glycolytic increase and mitochondrial rearrangements suggest pathways leading to cell transformation.In this work we exploit the widely used, well known CTA, which allows following healthy cells transformation into a malignant phenotype, to understand early events in cadmium-induced carcinogenesis. |
| 2019 | Methane-Rich Saline: A Potential Resuscitation Fluid for Hemorrhagic Shock. | Oxid Med Cell Longev | Hemorrhagic shock is caused by massive blood loss. If the patient is not fully resuscitated in time, this may eventually lead to multiple organ failure and even death. Previous studies on methane-rich saline in animal models showed that it confers resistance against many diseases. In this study, we explored the protective effect of methane-rich saline, used as a resuscitation fluid, in hemorrhagic shock. Hemorrhagic shock was induced in SD rats by bloodletting via intubation of the right femoral artery. The rats were divided into three groups: a sham control group (sham control), a shock group resuscitated by an infusion of autologous blood and an equivalent volume of normal saline (Shock+NS), and a shock group resuscitated by an infusion of autologous blood and an equivalent volume of methane-rich saline (Shock+MRS). Assessment of blood pressure and levels of plasma lactate showed that resuscitation using methane-rich saline (MRS) restored systemic blood pressure and reduced the levels of lactate in the plasma. Meanwhile, lower levels of serum IL-6 and TNF- were also observed in the group resuscitated with MRS. In the heart, liver, and kidney, MRS reduced inflammation and oxidative stress levels. Analysis of organ function via levels of biochemical indicators revealed that the group resuscitated with MRS had reduced serum levels of AST and CK, indicating a potential cardioprotective effect. The expression levels of apoptosis-related proteins, including those of Bcl-2/Bax, and the results of TUNEL-labeling assay indicated that MRS significantly reduced apoptosis in the heart. Methane also had a positive effect on the expression of the PGC-1/SIRT3/SOD2 signaling pathway. Our results showed that MRS can potentially serve as a novel resuscitation fluid because of its anti-inflammatory, antioxidative, and antiapoptotic properties. |
| 2020 | Ellagic acid prolongs the lifespan of Drosophila melanogaster. | Geroscience | Wild-type Canton-S flies of Drosophila melanogaster were treated with ellagic acid at 100 μM and 200 μM concentrations. Longevity assay showed male flies fed with 200 μM ellagic acid displayed longer mean lifespan and maximum lifespan than control flies. Female flies fed with 200 μM ellagic acid laid less number of eggs than control. The eclosion time was less in female flies fed with 200 μM ellagic acid. Ellagic acid fed female flies performed better than male flies and control flies for heat shock tolerance and starvation stress. Male flies treated with 100 μM ellagic acid recovered faster from cold shock compared with control flies. Male and female flies treated with ellagic acid displayed increased survival following exposure to 5% hydrogen peroxide. Gene expression studies displayed upregulated expressions of CAT, dFOXO, ATG1, and SOD2 in ellagic acid-treated male flies, and upregulated expressions of dFOXO, CAT, and SOD2 in ellagic acid-treated female flies. Results from these studies show the pro-longevity effect of ellagic acid on Drosophila melanogaster. |
| 2019 | Zinc-enriched probiotics enhanced growth performance, antioxidant status, immune function, gene expression, and morphological characteristics of Wistar rats raised under high ambient temperature. | 3 Biotech | The present study was conducted to evaluate the effects of zinc-enriched probiotics (ZnP) on growth performance, antioxidant status, immune function, related gene expression, and morphological characteristics of Wistar rats raised under high heat stress condition during summer. 36, 6-week-old male Wistar rats were randomly divided into three groups; fed with basal diet (control), basal diet with probiotics (P), and basal diet with zinc-enriched probiotics supplementation (ZnP, 100 mg/L), for 40 consecutive days. Blood samples were collected through intracardiac method on the last day of experiment and tissues were collected from liver, heart, and kidneys. The results revealed that both P and ZnP significantly (< 0.05) enhanced growth performance. However, ZnP remarkably increased glutathione content, glutathione peroxidase, and superoxide dismutase activities but reduced malondialdehyde level in serum of the Wistar rats. The concentration of IL-2, IL-6, and IFN-γ was significantly (< 0.05) increased with treatments of P and ZnP compared to control group while IL-10 was significantly (< 0.05) decreased. Additionally, the expression of SOD1, SOD2, MT1, and MT2 genes was significantly (< 0.05) up-regulated with the treatment of ZnP, but Hsp90 and Hsp70 heat shock genes were significantly (< 0.05) down-regulated with the treatment of P and ZnP, respectively. Hematoxylin and Eosin staining showed that both P and ZnP supplementation treatments induced changes in villus height and intestinal wall thickness. In conclusion, zinc-enriched probiotics supplementation can improve the growth performance of Wistar rats under high ambient temperature through enhancing antioxidant status, immune function, related genes expression, and intestinal morphological characteristics. This product may serves as a potential nutritive supplement for Wistar rats under high heat stress conditions. |
| 2019 | FOXO3 on the Road to Longevity: Lessons From SNPs and Chromatin Hubs. | Comput Struct Biotechnol J | Health span is driven by a precise interplay between genes and the environment. Cell response to environmental cues is mediated by signaling cascades and genetic variants that affect gene expression by regulating chromatin plasticity. Indeed, they can promote the interaction of promoters with regulatory elements by forming active chromatin hubs. encodes a transcription factor with a strong impact on aging and age-related phenotypes, as it regulates stress response, therefore affecting lifespan. A significant association has been shown between human longevity and several variants located in intron 2. This haplotype block forms a putative aging chromatin hub in which has a central role, as it modulates the physical connection and activity of neighboring genes involved in age-related processes. Here we describe the role of and its single-nucleotide polymorphisms (SNPs) in healthy aging, with a focus on the enhancer region encompassing the SNP , which upregulates FOXO3 expression and can promote the activity of the aging hub in response to different stress stimuli. protective effect on lifespan may be due to the accessibility of this region to transcription factors promoting its expression. This could in part explain the differences in association with longevity between genders, as its activity in females may be modulated by estrogens through estrogen receptor response elements located in the -encompassing region. Altogether, the molecular mechanisms described here may help establish whether the SNP can be taken advantage of in predictive medicine and define the potential of targeting FOXO3 for age-related diseases. |
| 2019 | Expression and molecular characterization of stress-responsive genes (hsp70 and Mn-sod) and evaluation of antioxidant enzymes (CAT and GPx) in heavy metal exposed freshwater ciliate, Tetmemena sp. | Mol Biol Rep | Response of heavy metals namely cadmium (Cd) and copper (Cu) on the expression of stress responsive genes in the fresh water ciliate, Tetmemena sp. (single cell eukaryote) was studied. Stress responsive genes include heat shock protein genes and genes involved in antioxidant defence system. Quantitative real time PCR (qRT-PCR) was employed to evaluate the effects of Cd and Cu on the expression of cytosolic hsp70 and Mn-sod genes. Increase in the expression of these genes was observed after exposure with the heavy metals. The macronuclear cytosolic hsp70 and Mn-sod (SOD2) genes were also sequenced and characterized using various bioinformatics tools. In antioxidant defence system, the superoxide dismutase (SOD) family is a first line antioxidant enzyme group involved in catalysing reactive oxygen species (ROS) to hydrogen peroxide and molecular oxygen. Influence of Cd and Cu on the activity of SOD has already been reported by our group. Therefore, the enzymatic activities of antioxidant enzymes, catalase (CAT) and glutathione peroxidase (GPx) were studied in the presence of Cd and Cu and there was significant increase in activity of these enzymes in concentration dependent manner. This study suggests that cytosolic hsp70, Mn-sod and the antioxidant enzymes such as CAT and GPx can be used as effective molecular biomarkers for heavy metal toxicity and Tetmemena sp. can be used as potential model for understanding the molecular response to heavy metal contamination in aquatic ecosystems. |
| 2019 | Transport and Recovery of Gilthead Sea Bream ( L.) Sedated With Clove Oil and MS222: Effects on Oxidative Stress Status. | Front Physiol | The use of anesthesia is a common practice in aquaculture to sedate fish and mitigate handling stress. Although the employ of anesthesia is considered beneficial for fish, as it reduces stress and improves welfare, at the same time it may induce hazardous side-effects. The aim of the present study was to investigate the effects of clove oil (CO) and tricaine methanesulfonate (MS222), two of the most used anesthetics, on several oxidative stress related parameters in gilthead sea bream (), as these types of effects of anesthetics have been seldom investigated. To assess these effects, juveniles were placed in a setup of mobile water tanks and were transported during 6 h with either 2.5 mg/L CO or 5 mg/L MS222. After transport, half of the fish were sampled, whereas the remaining fish were transferred to tanks without anesthetics where they were allowed to recover for 18 h before sampling. Changes in the expression levels of several target genes related with the antioxidant response and cell-tissue repair were evaluated in the gills, liver and brain. Those transcripts included glutathione peroxidase 1 (), catalase (), glutathione S-transferase 3 (), glutathione reductase (), superoxide dismutase [Zn] (), heat shock protein-70 (), and metallothionein (). Antioxidant enzymatic activities glutathione S-transferase, GST; catalase, CAT; and glutathione reductase, GR, levels of non-enzymatic antioxidants (non-protein thiols - NPT), and pro-oxidative damage, assessed as lipid peroxidation (LPO), were determined in gills, liver and brain. Acetylcholinesterase activity (AChE) was determined in plasma, gills, brain, muscle and heart as an indicator of neuro-muscular alterations. In plasma, the total antioxidant capacity (TAC) and total oxidative status (TOS) were also measured. Results showed that the use of both anesthetic agents, CO and MS222, interferes with fish antioxidant status. All tested biological matrices displayed alterations in antioxidant endpoints, confirming that these substances, although minimizing the effects of transport stress, may have long term effects on fish defenses. This result is of high relevance to aquaculture considering that the oxidative stress, may increase the susceptibility to different environmental or biotic stress and different types of pathologies. |
| 2019 | Mitophagy regulates mitochondrial network signaling, oxidative stress, and apoptosis during myoblast differentiation. | Autophagy | Macroautophagy/autophagy is a degradative process essential for various cellular processes. We previously demonstrated that autophagy-deficiency causes myoblast apoptosis and impairs myotube formation. In this study, we continued this work with particular emphasis on mitochondrial remodelling and stress/apoptotic signaling. We found increased (p < 0.05) autophagic (e.g., altered LC3B levels, increased ATG7, decreased SQSTM1) and mitophagic (e.g., BNIP3 upregulation, mitochondrial localized GFP-LC3 puncta, and elevated mitochondrial LC3B-II) signaling during myoblast differentiation. shRNA-mediated knockdown of ATG7 (sh) decreased these autophagic and mitophagic responses, while increasing CASP3 activity and ANXA5/annexin V staining in differentiating myoblasts; ultimately resulting in dramatically impaired myogenesis. Further confirming the importance of mitophagy in these responses, CRISPR-Cas9-mediated knockout of () resulted in increased CASP3 activity and DNA fragmentation as well as impaired myoblast differentiation. In addition, sh myoblasts displayed greater endoplasmic reticulum (e.g., increased CAPN activity and HSPA) and mitochondrial (e.g., mPTP formation, reduced mitochondrial membrane potential, elevated mitochondrial 4-HNE) stress. sh and myoblasts also displayed altered mitochondria-associated signaling (e.g., PPARGC1A, DNM1L, OPA1) and protein content (e.g., SLC25A4, VDAC1, CYCS). Moreover, sh myoblasts displayed CYCS and AIFM1 release from mitochondria, and CASP9 activation. Similarly, myoblasts had significantly higher CASP9 activation during differentiation. Importantly, administration of a chemical inhibitor of CASP9 (Ac-LEHD-CHO) or dominant-negative CASP9 (ad-DNCASP9) partially recovered differentiation and myogenesis in sh myoblasts. Together, these data demonstrate an essential role for autophagy in protecting myoblasts from mitochondrial oxidative stress and apoptotic signaling during differentiation, as well as in the regulation of mitochondrial network remodelling and myogenesis. : 3MA: 3-methyladenine; 4-HNE: 4-hydroxynonenal; ACT: actin; AIFM1/AIF: apoptosis-inducing factor, mitochondrion-associated 1; ANXA5: annexin V; ATG7: autophagy related 7; AU: arbitrary units; BAX: BCL2-associated X protein; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; CAPN: calpain; CASP: caspase; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CASP12: caspase 12; CAT: catalase; CQ: chloroquine; CYCS: cytochrome c, somatic; DCF; 2',7'-dichlorofluorescein; DNM1L/DRP1: dynamin 1-like; DM: differentiation media; DMEM: Dulbecco's modified Eagle's medium; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GM: growth media; p-H2AFX: phosphorylated H2A histone family, member X; H2BFM: H2B histone family, member M; HBSS: Hanks balanced salt solution; HSPA/HSP70: heat shock protein family A; JC-1: tetraethylbenzimidazolylcarbocyanine iodide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mPTP: mitochondrial permeability transition pore; MYH: myosin heavy chain; MYOG: myogenin; OPA1: OPA1, mitochondrial dynamin like GTPase; PI: propidium iodide; PINK1: PTEN induced putative kinase 1; PPARGC1A/PGC1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; ROS: reactive oxygen species; SLC25A4/ANT1: solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 4; SOD1: superoxide dismutase 1, soluble; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; VDAC1: voltage-dependent anion channel 1. |
| 2019 | Genetic Polymorphisms in Sepsis and Cardiovascular Disease: Do Similar Risk Genes Suggest Similar Drug Targets? | Chest | Genetic variants are associated with altered clinical outcome of patients with sepsis and cardiovascular diseases. Common gene signaling pathways may be involved in the pathophysiology of these diseases. A better understanding of genetic commonality among these diseases may enable the discovery of important genes, signaling pathways, and therapeutic targets for these diseases. We investigated the common genetic factors by a systematic search of the literature. Twenty-four genes (ADRB2, CD14, FGB, FV, HMOX1, IL1B, IL1RN, IL6, IL10, IL17A, IRAK1, MASP2, MBL, MIR608, MIF, NOD2, PCSK9, PPARG, PROC, SERPINE1, SOD2, SVEP1, TF, TIRAP, TLR1) were extracted as reported genetic variations associated with altered outcome of both sepsis and cardiovascular diseases. Of these genes, the adverse allele (or combinations) was same in nine (ADRB2, FV, HMOX1, IL6, MBL, MIF, NOD2, PCSK9, SERPINE1), and the effect appears to be in the same direction in both sepsis and cardiovascular disease. Shared gene signaling pathways suggest that these are true biological results and could point to overlapping drug targets in sepsis and cardiovascular disease. |
| 2018 | NKT-like cells reveal higher than T lymphocytes expression of cellular protective proteins HSP70 and SOD2 and comparably increased expression of SIRT1 in the oldest seniors. | Folia Histochem Cytobiol | NKT-like cells are "non-classical", "CD1d-independent" NKT cells which represent highly differentiated, conventional T lymphocytes coexpressing several NK (natural killer) associated receptors. They are effector lymphocytes of both innate and adaptive immunity and simultaneously regulatory cells of the adaptive immune system. They reveal large granular lymphocyte morphology and can mediate both MHC-restricted and MHC-unrestricted cytotoxicity, secrete many cytokines and modulate Th1 immune responses. The aim of our study was to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in NKT-like cells compared to T lymphocytes during ageing.The study involved three groups of participants: the oldest seniors (n = 25; aged over 85; mean age 88 ± 0.5 ys), the old (n = 30; aged under 85; mean age 76 ± 0.9 ys) and the young (n = 32; mean age 21 ± 0.3 ys). Whole blood samples were analyzed by flow cytometry to assess the NKT-like (CD3+CD56+) and T (CD3+) cell populations.The group of the oldest seniors differed from the other age groups by much higher percentage of both NKT-like cells and T lymphocytes expressing SIRT1, HSP70 and SOD2. The expression of these proteins correlated positively with the age of the participants. Interestingly, the significantly higher expression of the studied protective proteins; i.e. HSP70 and SOD2 was found in CD3+CD56+ cells compared to CD3+ lymphocytes and this phenomenon concerned all the studied age groups. These differences were not significant regarding the expression of SIRT1; however, the same tendency was noticeable.The analysis of CD3+ and CD3+CD56+ lymphocytes showed the increase in the number of NKT-like cells and decreased number of T cells in the process of ageing. The increased expression of cellular protective proteins SIRT1, HSP70 and SOD2 in NKT-like and T-lymphocytes of the oldest seniors seems to correspond to longevity and the observed correlations may suggest the involvement of these proteins in establishing cellular homeostasis specific for healthy ageing. Furthermore, the higher expression of the protective proteins in NKT-like cells compared to T lymphocytes may indicate their particular role in the interplay between innate and adaptive immunity responses during the process of ageing. |
| 2018 | CD56bright cells respond to stimulation until very advanced age revealing increased expression of cellular protective proteins SIRT1, HSP70 and SOD2. | Immun Ageing | NK cells are cytotoxic lymphocytes of innate immunity composed of: cytotoxic CD56dim and immunoregulatory CD56bright cells. The study aimed to analyze the expression of cellular protective proteins: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in CD56dim and CD56bright NK cells of the young, seniors aged under 85 ('the old') and seniors aged over 85 ('the oldest'). We studied both non-stimulated NK cells and cells stimulated by IL-2, LPS or PMA with ionomycin. The expression level of proinflammatory cytokines TNF and IFN-γ was also assessed in NK cell subsets and some relationships between the studied parameters were analyzed.CD56bright cells showed sensitivity to most of the applied stimulatory agents until very advanced age in regards to the expression of SIRT1 and intracellular HSP70. On the contrary, CD56dim cells, sensitive to stimulation by most of the stimulatory agents in the young and the old, in the oldest lost this sensitivity and presented rather high, constant expression of SIRT1 and HSP70, resistant to further stimulation. With reference to SOD2 expression, CD56dim cells were insensitive to stimulation in the young, but their sensitivity increased with ageing. CD56bright cells were sensitive to most of the applied agents in the young and the old but in the oldest they responded to all of the stimulatory agents used in the study. Similarly, both NK cell subsets were sensitive to stimulation until very advanced age in regards to the expression of TNF and IFN-γ.CD56bright cells maintained sensitivity to stimulation until very advanced age presenting also an increased expression of SIRT1 and HSP70. CD56dim cells showed a constantly increased expression of these cellular protective proteins in the oldest, insensitive for further stimulation. The oldest, however, did not reveal an increased level of SOD2 expression, but it was significantly elevated in both NK cell subsets after stimulation.The pattern of expression of the studied cellular protective proteins in ageing process revealed the adaptation of NK cells to stress response in the oldest seniors which might accompany the immunosenescence and contribute to the long lifespan of this group of the elderly. |
| 2018 | Dynamic Phosphorylation of the C Terminus of Hsp70 Regulates the Mitochondrial Import of SOD2 and Redox Balance. | Cell Rep | The import of superoxide dismutase-2 (SOD2) into mitochondria is vital for the survival of eukaryotic cells. SOD2 is encoded within the nuclear genome and translocated into mitochondria for activation after translation in the cytosol. The molecular chaperone Hsp70 modulates SOD2 activity by promoting import of SOD2 into mitochondria. In turn, the activity of Hsp70 is controlled by co-chaperones, particularly CHIP, which directs Hsp70-bound proteins for degradation in the proteasomes. We investigated the mechanisms controlling the activity of SOD2 to signal activation and maintain mitochondrial redox balance. We demonstrate that Akt1 binds to and phosphorylates the C terminus of Hsp70 on Serine631, which inhibits CHIP-mediated SOD2 degradation thereby stabilizing and promoting SOD2 import. Conversely, increased mitochondrial-HO formation disrupts Akt1-mediated phosphorylation of Hsp70, and non-phosphorylatable Hsp70 mutants decrease SOD2 import, resulting in mitochondrial oxidative stress. Our findings identify Hsp70 phosphorylation as a physiological mechanism essential for regulation of mitochondrial redox balance. |
| 2018 | Cadmium detoxification induced by salt stress improves cadmium tolerance of multi-stress-tolerant Pichia kudriavzevii. | Environ Pollut | Heavy metal tolerance of microorganisms is the basis of heavy metal removal by growing cells. In this study, a cross-protection effect generated by salt stress significantly enhanced the cadmium tolerance of multi-stress-tolerant Pichia kudriavzevii. Comparative transcriptome analysis using RNA-Seq linked with physiological and biochemical observation was used to elucidate the underlying mechanisms of the improved cadmium tolerance. The expression of cadmium transport related genes (GSTY2, GLR1, GLO2, YCF1 and YOR1), GSH content and GST activity were elevated by salt stress, suggesting enhanced cadmium conjugation and detoxification in yeast cells. The inhibited cadmium uptake by ZRT1 and enhanced cadmium efflux by YOR1 contributed to the decrease in the intracellular cadmium concentration. The improved expression of antioxidant enzyme genes (SOD1, SOD2, SOD6, CAT1 and PRXIID), along with the enhanced activities of antioxidant enzymes (SOD, CAT and POD) resulted in a decrease in cadmium-induced ROS production, protein carbonylation, lipid peroxidation and cell death. The abundant expression of heat shock protein genes (HSP12, HSP10 and SSC1) and genes related to trehalose synthesis (TPS1 and TSL1) induced by salt stress protected yeast cells against complex stress conditions, contributing to the improved cadmium tolerance. These findings will be useful to develop cadmium-tolerant yeasts for cadmium removal by growing cells. |
| 2018 | Stress-induced antioxidant defense and protein chaperone response in the freeze-tolerant wood frog Rana sylvatica. | Cell Stress Chaperones | Freeze tolerance is an adaptive response utilized by the wood frog Rana sylvatica to endure the sub-zero temperatures of winter. Survival of whole body freezing requires wood frogs to trigger cryoprotective mechanisms to deal with potential injuries associated with conversion of 65-70% of total body water into ice, including multiple consequences of ice formation such as cessation of blood flow and cell dehydration caused by water loss into ice masses. To understand how wood frogs defend against these stressors, we measured the expression of proteins known to be involved in the antioxidant defense and protein chaperone stress responses in brain and heart of wood frogs comparing freezing, anoxia, and dehydration stress. Our results showed that most stress proteins were regulated in a tissue- and stress-specific manner. Notably, protein levels of the cytosolic superoxide dismutase (SOD1) were upregulated by 1.37 ± 0.11-fold in frozen brain, whereas the mitochondrial SOD2 isoform rose by 1.38 ± 0.37-fold in the heart during freezing. Catalase protein levels were upregulated by 3.01 ± 0.47-fold in the brain under anoxia stress, but remained unchanged in the heart. Similar context-specific regulatory patterns were also observed for the heat shock protein (Hsp) molecular chaperones. Hsp27 protein was down-regulated in the brain across the three stress conditions, whereas the mitochondrial Hsp60 was upregulated in anoxic brain by 1.73 ± 0.38-fold and by 2.13 ± 0.57-fold in the frozen heart. Overall, our study provides a snapshot of the regulatory expression of stress proteins in wood frogs under harsh environment conditions and shows that they are controlled in a tissue- and stress-specific manner. |
| 2018 | Specific protein carbonylation in human breast cancer tissue compared to adjacent healthy epithelial tissue. | PLoS One | Protein carbonylation is an irreversible post-translational modification induced by severe oxidative stress. Reactive oxygen species (ROS) are constantly produced in cells and play important roles in both cancer progression and cancer suppression. ROS levels can be higher in tumor compared to surrounding healthy tissue but ROS-induced specific protein carbonylation and its unique role in cancer progression or suppression is poorly understood. In this study, we utilized previously validated ELISA and western blot methods to analyze the total and specific protein carbonylation in flash-frozen human breast cancer and matched adjacent healthy tissue to compare relative total, and specific protein carbonylation. Mass spectrometry, two-color western, and immunoprecipitation methods were used to identify and confirm the specifically carbonylated proteins in breast tumor tissue. Superoxide dismutase (SOD) activity was measured as an indicator of antioxidant activity, and LC3-II protein level was analyzed for autophagy by western blot. Findings were further confirmed using the immortalized MDA-MB-231 and MDA-MB-468 breast cancer and MCF-12A noncancerous human epithelial breast cell lines. Our results indicate that tumor tissue has greater total protein carbonylation, lower SOD1 and SOD2 protein levels, lower total SOD activity, and higher LC3-II levels compared to adjacent healthy tissue. We identified and confirmed three specific proteins of interest; filamin A, heat shock protein 90β (HSP90β), and bifunctional glutamate/proline-tRNA ligase (EPRS), that were selectively carbonylated in tumor tissue compared to matched adjacent healthy tissue. Correspondingly, compared to noncancerous MCF-12A epithelial cells, MDA-MB-231 cancer cells exhibited an increase in filamin A and EPRS protein carbonylation, decreased total SOD activity, and increased autophagy, but not increased HSP90β protein carbonylation. Identification of selectively carbonylated proteins and defining their roles in cancer progression may promote the development of targeted therapeutic approaches toward mitigating oxidative damage of these proteins. |
| 2018 | Drosophila larvae fed palm fruit juice (PFJ) delay pupation via expression regulation of hormetic stress response genes linked to ageing and longevity. | Exp Gerontol | Palm fruit juice (PFJ) containing oil palm phenolics is obtained as a by-product from oil palm (Elaeis guineensis) fruit milling. It contains shikimic acid, soluble fibre and various phenolic acids including p-hydroxybenzoic acid and three caffeoylshikimic acid isomers. PFJ has also demonstrated beneficial health properties in various biological models. Increasing concentrations of PFJ and different PFJ fractions were used to assess growth dynamics and possible anti-ageing properties in fruit flies (Drosophila melanogaster) genotype w. Microarray gene expression analysis was performed on whole fruit fly larvae and their fat bodies, after the larvae were fed a control Standard Brandeis Diet (SBD) with or without PFJ. Transcripts from Affymetrix GeneChips were utilised to identify the possible mechanisms involved, with genes having fold changes > |1.30| and p < 0.05 considered differentially expressed. PFJ dose-dependently delayed larval growth and pupation, but not percent eclosion from pupae. Eclosed male fruit flies fed PFJ or its fractions during the larval stage tended to have 20-40% improved survival ratings over controls when allowed to age on the control diet (SBD). Microarray analysis of whole fruit fly larvae revealed that 127 genes were up-regulated, while 67 were down-regulated by PFJ. Functional analysis revealed transport and metabolic processes were up-regulated, while development and morphogenesis processes, including the nutrient-sensing Tor gene, were down-regulated by PFJ, whereas microarray analysis of larval fat bodies found 161 genes were up-regulated, while 84 genes were down-regulated. Genes involved in defence response and determination of adult lifespan, including those encoding various heat shock proteins and the antioxidant enzyme Sod2, were up-regulated, while cell cycle and growth genes were down-regulated. Thus, PFJ supplementation lengthened the growth stages in fruit fly larvae that was reflected in extended ageing of adult flies, suggesting that larval expression of hormetic stress response genes was linked to subsequent ageing and longevity. |
| 2018 | NK cells of the oldest seniors represent constant and resistant to stimulation high expression of cellular protective proteins SIRT1 and HSP70. | Immun Ageing | Natural killer cells (NK cells) are cytotoxic lymphocytes of innate immunity that reveal some immunoregulatory properties, however, their role in the process of ageing is not completely understood. The study aimed to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in human NK cells with reference to the process of ageing. Non-stimulated and stimulated with IL-2, LPS or PMA with ionomycin cells originated from peripheral blood samples of: seniors aged over 85 ('the oldest'; = 25; 88.5 ± 0.5 years, mean ± SEM), seniors aged under 85 ('the old'; = 30; 75.6 ± 0.9 years) and the young ( = 31; 20.9 ± 0.3 years). The relationships between the levels of expression of cellular protective proteins in the studied population were also analyzed. The concentrations of carbonyl groups and 8-isoprostanes, markers of oxidative stress, in both stimulated and non-stimulated cultured NK cells were measured to assess the level of the oxidative stress in the cells.The oldest seniors varied from the other age groups by significantly higher expression of SIRT1 and HSP70 both in non-stimulated and stimulated NK cells. These cells also appeared to be resistant to further stimulations with IL-2, LPS or PMA with ionomycin. Highly positive correlations between SIRT1 and intracellular HSP70 in both stimulated and non-stimulated NK cells were observed. SOD2 presented low expression in non-stimulated cells, whereas its sensitivity to stimulation increased with age of donors. High positive correlations between SOD2 and surface HSP70 were observed. We found that the markers of oxidative stress in NK cells did not change with ageing.The oldest seniors revealed well developed adaptive stress response in NK cells with increased, constant levels of SIRT1 and intracellular HSP70. They presented also very high positive correlations between expression of these cellular protective proteins both in stimulated and non-stimulated cells. These phenomena may contribute to the long lifespan of this group of elderly. Interestingly, in NK cells SOD2 revealed a distinct role in cellular stress response since it showed sensitivity to stimulation increasing with age of participants. These observations provide novel data concerning the role of NK cells in the process of ageing. |
| 2018 | Exosomes from patients with septic shock convey miRNAs related to inflammation and cell cycle regulation: new signaling pathways in sepsis? | Crit Care | Exosomes isolated from plasma of patients with sepsis may induce vascular apoptosis and myocardial dysfunction by mechanisms related to inflammation and oxidative stress. Despite previous studies demonstrating that these vesicles contain genetic material related to cellular communication, their molecular cargo during sepsis is relatively unknown. In this study, we evaluated the presence of microRNAs (miRNAs) and messenger RNAs (mRNAs) related to inflammatory response and redox metabolism in exosomes of patients with septic shock.Blood samples were collected from 24 patients with septic shock at ICU admission and after 7 days of treatment. Twelve healthy volunteers were used as control subjects. Exosomes were isolated by ultracentrifugation, and their miRNA and mRNA content was evaluated by qRT-PCR array.As compared with healthy volunteers, exosomes from patients with sepsis had significant changes in 65 exosomal miRNAs. Twenty-eight miRNAs were differentially expressed, both at enrollment and after 7 days, with similar kinetics (18 miRNAs upregulated and 10 downregulated). At enrollment, 35 differentially expressed miRNAs clustered patients with sepsis according to survival. The pathways enriched by the miRNAs of patients with sepsis compared with control subjects were related mostly to inflammatory response. The comparison of miRNAs from patients with sepsis according to hospital survival demonstrated pathways related mostly to cell cycle regulation. At enrollment, sepsis was associated with significant increases in the expression of mRNAs related to redox metabolism (myeloperoxidase, 64-fold; PRDX3, 2.6-fold; SOD2, 2.2-fold) and redox-responsive genes (FOXM1, 21-fold; SELS, 16-fold; GLRX2, 3.4-fold). The expression of myeloperoxidase mRNA remained elevated after 7 days (65-fold).Exosomes from patients with septic shock convey miRNAs and mRNAs related to pathogenic pathways, including inflammatory response, oxidative stress, and cell cycle regulation. Exosomes may represent a novel mechanism for intercellular communication during sepsis. |
| 2018 | Neuronal-specific impairment of heparan sulfate degradation in Drosophila reveals pathogenic mechanisms for Mucopolysaccharidosis type IIIA. | Exp Neurol | Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder resulting from the deficit of the N-sulfoglucosamine sulfohydrolase (SGSH) enzyme that leads to accumulation of partially-degraded heparan sulfate. MPS IIIA is characterized by severe neurological symptoms, clinically presenting as Sanfilippo syndrome, for which no effective therapy is available. The lysosomal SGSH enzyme is conserved in Drosophila and we have identified increased levels of heparan sulfate in flies with ubiquitous knockdown of SGSH/CG14291. Using neuronal specific knockdown of SGSH/CG14291 we have also observed a higher abundance of Lysotracker-positive puncta as well as increased expression of GFP tagged Ref(2)P supporting disruption to lysosomal function. We have also observed a progressive defect in climbing ability, a hallmark of neurological dysfunction. Genetic screens indicate proteins and pathways that can functionally modify the climbing phenotype, including autophagy-related proteins (Atg1 and Atg18), superoxide dismutase enzymes (Sod1 and Sod2) and heat shock protein (HSPA1). In addition, reducing heparan sulfate biosynthesis by knocking down sulfateless or slalom expression significantly worsens the phenotype; an important observation given that substrate inhibition is being evaluated clinically as a treatment for MPS IIIA. Identifying the cellular pathways that can modify MPS IIIA neuropathology is an essential step in the development of novel therapeutic approaches to prevent and/or ameliorate symptoms in children with Sanfilippo syndrome. |
| 2018 | Co-inoculation of maize with Azospirillum brasilense and Rhizobium tropici as a strategy to mitigate salinity stress. | Funct Plant Biol | Plants are highly affected by salinity, but some plant growth-promoting bacteria (PGPB) may trigger induced systemic tolerance (IST), conferring protection against abiotic stresses. We investigated plant mechanisms under saline stress (170mM NaCl) when maize was singly or co-inoculated with Azospirillum brasilense strains Ab-V5 and Ab-V6 and Rhizobium tropici strain CIAT 899. Under greenhouse conditions, plants responded positively to inoculation and co-inoculation, but with differences between strains. Inoculation affected antioxidant enzymes that detoxify reactive oxygen species (ROS) - ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD) - mainly in leaves. Proline contents in leaves and roots and malondialdehyde (MDA) in leaves - plant-stress-marker molecules - were significantly reduced due to the inoculation, indicating reduced need for the synthesis of these molecules. Significant differences were attributed to inoculation in the expression of genes related to antioxidant activity, in general with upregulation of APX1, CAT1, SOD2 and SOD4 in leaves, and APX2 in roots. Pathogenesis-related genes PR1, prp2, prp4 and heat-shock protein hsp70 were downregulated in leaves and roots, indicating that inoculation with PGPB might reduce the need for this protection. Together the results indicate that inoculation with PGPB might provide protection from the negative effects of saline stress. However, differences were observed between strains, as A. brasilense Ab-V5 did not show salt tolerance, while the best inoculation treatments to mitigate saline stress were with Ab-V6 and co-inoculation with Ab-V6+CIAT 899. Inoculation with these strains may represent an effective strategy to mitigate salinity stress. |
| 2018 | A systematic review of p53 regulation of oxidative stress in skeletal muscle. | Redox Rep | p53 is a tumor suppressor protein involved in regulating a wide array of signaling pathways. The role of p53 in the cell is determined by the type of imposed oxidative stress, its intensity and duration. The last decade of research has unravelled a dual nature in the function of p53 in mediating the oxidative stress burden. However, this is dependent on the specific properties of the applied stress and thus requires further analysis.A systematic review was performed following an electronic search of Pubmed, Google Scholar, and ScienceDirect databases. Articles published in the English language between January 1, 1990 and March 1, 2017 were identified and isolated based on the analysis of p53 in skeletal muscle in both animal and cell culture models.Literature was categorized according to the modality of imposed oxidative stress including exercise, diet modification, exogenous oxidizing agents, tissue manipulation, irradiation, and hypoxia. With low to moderate levels of oxidative stress, p53 is involved in activating pathways that increase time for cell repair, such as cell cycle arrest and autophagy, to enhance cell survival. However, with greater levels of stress intensity and duration, such as with irradiation, hypoxia, and oxidizing agents, the role of p53 switches to facilitate increased cellular stress levels by initiating DNA fragmentation to induce apoptosis, thereby preventing aberrant cell proliferation.Current evidence confirms that p53 acts as a threshold regulator of cellular homeostasis. Therefore, within each modality, the intensity and duration are parameters of the oxidative stressor that must be analyzed to determine the role p53 plays in regulating signaling pathways to maintain cellular health and function in skeletal muscle.Acadl: acyl-CoA dehydrogenase, long chain; Acadm: acyl-CoA dehydrogenase, C-4 to C-12 straight chain; AIF: apoptosis-inducing factor; Akt: protein kinase B (PKB); AMPK: AMP-activated protein kinase; ATF-4: activating transcription factor 4; ATM: ATM serine/threonine kinase; Bax: BCL2 associated X, apoptosis regulator; Bcl-2: B cell Leukemia/Lymphoma 2 apoptosis regulator; Bhlhe40: basic helix-loop-helix family member e40; BH3: Borane; Bim: bcl-2 interacting mediator of cell death; Bok: Bcl-2 related ovarian killer; COX-IV: cytochrome c oxidase IV; cGMP: Cyclic guanosine monophosphate; c-myc: proto-oncogene protein; Cpt1b: carnitine palmitoyltransferase 1B; Dr5: death receptor 5; eNOS: endothelial nitric oxide synthase; ERK: extracellular regulated MAP kinase; Fas: Fas Cell surface death receptor; FDXR: Ferredoxin Reductase; FOXO3a: forkhead box O3; Gadd45a: growth arrest and DNA damage-inducible 45 alpha; GLS2: glutaminase 2; GLUT 1 and 4: glucose transporter 1(endothelial) and 4 (skeletal muscle); GSH: Glutathione; Hes1: hes family bHLH transcription factor 1; Hey1: hes related family bHLH transcription factor with YRPW motif 1; HIFI-α: hypoxia-inducible factor 1, α-subunit; HK2: Hexokinase 2; HSP70: Heat Shock Protein 70; HO: Hydrogen Peroxide; Id2: inhibitor of DNA-binding 2; IGF-1-BP3: Insulin-like growth factor binding protein 3; IL-1β: Interleukin 1 beta; iNOS: inducible nitric oxide synthase; IRS-1: Insulin receptor substrate 1; JNK: c-Jun N-terminal kinases; LY-83583: 6-anilino-5,8-quinolinedione; inhibitor of soluble guanylate cyclase and of cGMP production; Mdm 2/ 4: Mouse double minute 2 homolog (mouse) Mdm4 (humans); mtDNA: mitochondrial DNA; MURF1: Muscle RING-finger protein-1; MyoD: Myogenic differentiation 1; MyoG: myogenin; Nanog: Nanog homeobox; NF-kB: Nuclear factor-κB; NO: nitric oxide; NoxA: phorbol-12-myristate-13-acetate-induced protein 1 (Pmaip1); NRF-1: nuclear respiratory factor 1; Nrf2: Nuclear factor erythroid 2-related factor 2; P21: Cdkn1a cyclin-dependent kinase inhibitor 1A (P21); P38 MAPK: mitogen-activated protein kinases; p53R2: p53 inducible ribonucleotide reductase gene; P66Shc: src homology 2 domain-containing transforming protein C1; PERP: p53 apoptosis effector related to PMP-22; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PGM: phosphoglucomutase; PI3K: Phosphatidylinositol-4,5-bisphosphate 3-kinase; PKCβ: protein kinase c beta; PTEN: phosphatase and tensin homolog; PTIO: 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) has been used as a nitric oxide (NO) scavenger; Puma: The p53 upregulated modulator of apoptosis; PW1: paternally expressed 3 (Peg3); RNS: Reactive nitrogen species; SIRT1: sirtuin 1; SCO2: cytochrome c oxidase assembly protein; SOD2: superoxide dismutase 2; Tfam: transcription factor A mitochondrial; TIGAR: Trp53 induced glycolysis repulatory phosphatase; TNF-a: tumor necrosis factor a; TRAF2: TNF receptor associated factor 2; TRAIL: type II transmembrane protein. |
| 2018 | Melatonin reduces apoptotic cells, SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts. | Reprod Domest Anim | Effects of adding different concentrations of melatonin (10 , 10 and 10 M) to maturation (Experiment 1; Control, IVM + 10 , IVM + 10 , IVM + 10 ) and culture media (Experiment 2; Control, IVC + 10 , IVC + 10 , IVC + 10 ) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10 M) from Experiments 1-2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM + 10 , IVC + 10 , IVM/IVC + 10 ). In Experiment 1, maturated oocytes from Control and IVM + 10 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC + 10 (43.5%; 56.7%) and IVC + 10 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC + 10 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC + 10 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM/IVC + 10 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC + 10 treatment also had a higher mRNA expression of antioxidant gene (SOD2) compared to the Control, as well as the heat shock protein (HSPB1) compared to the IVM + 10 . Reactive oxygen species production was greater in the IVM/IVC + 10 treatment group. In conclusion, the 10 M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality. |
| 2018 | Heat stress prevents the decrease in succinate dehydrogenase activity in the extensor digitorum longus of streptozotocin-induced diabetic rats. | Physiol Res | This study aimed to investigate whether heat stress (HS) prevents a decrease in succinate dehydrogenase (SDH) activity and heat shock protein 60 (HSP60) and superoxide dismutase 2 (SOD2) contents in the extensor digitorum longus of streptozotocin (STZ)-induced diabetic rats. Twelve-week-old male Wistar rats were assigned to one of the four groups (n=6/group): control (Con), HS, diabetes mellitus (DM), and diabetes mellitus and heat stress (DM+HS). Diabetes was induced by the administration of STZ (50 mg/kg). HS was initiated 7 days after STZ treatment and performed at 42 °C for 30 min 5 times a week for 3 weeks. SDH activity was decreased in the DM and DM+HS groups. However, SDH activity was greater in the DM+HS group than in the DM group. Although HSP60 content was lower in the DM group than in the Con group, it was maintained in the DM+HS groups and was higher than that in the DM group. SOD2 content was decreased only in the DM group. These findings suggest that HS prevents the decrease in SDH activity in the skeletal muscle induced by DM. According to this mechanism, the maintenance of SOD2 and HSP60 by HS may suppress the increase in oxidative stress. |
| 2017 | Fusarium infection causes genotoxic disorders and antioxidant-based damages in Orobanche spp. | Microbiol Res | This study aims to evaluate the toxic effects of Fusarium oxysporum on root parasitic weed, Orobanche spp. Comparative genetic and gene expression studies were conducted on uninfected and fungus-infected orobanches. In genetic studies, isolated total DNA was amplified by RAPD PCR. Fragment properties were analysed by GTS test. According to the results, the fragment properties of control and Fusarium infected (experimental) groups varied widely; and it has been observed that Fusarium has genotoxic effects on the DNA of orobanches. In gene expression studies, the expression levels of genes encoding enzymes or proteins were associated with ROS damage and toxic effects, therefore, gene expressions of Mn-superoxide dismutase (SOD), Zn-superoxide dismutase (=SOD2, mitochondrial), glutamine synthetase (GS), heat shock protein gene (HSP70), BAX, Caspase-3 and BCL2 were significantly higher in the experimental group. In the light of obtained data, it was concluded that F. oxysporum (1) caused heavy ROS damage in Orobanche (2) induced significant irrevocable genotoxic effects on the DNA of Orobanche, (3) degraded protein metabolism and synthesis, and finally (4) triggered apoptosis. The results of this study can be a ground for further research on reducing the toxic effects of Fusarium on agricultural products, so that advancements in bio-herbicide technology may provide a sustainable agricultural production. |
| 2017 | Curcumin Upregulates Antioxidant Defense, Lon Protease, and Heat-Shock Protein 70 Under Hyperglycemic Conditions in Human Hepatoma Cells. | J Med Food | Sirtuin 3 (SIRT3) regulates mitochondrial antioxidant (AO) defense and improves mitochondrial disorders. Curcumin protects mitochondria; however, the mechanisms need investigation. We postulated that curcumin increases AO defense under hyperglycemic conditions in HepG2 cells through SIRT3-mediated mechanisms. Cell viability was determined in HepG2 cells cultured with 5 mM glucose, 19.9 mM mannitol, vehicle control, 10 mM glucose, and 30 mM glucose in the absence or presence of curcumin for 24 h. SIRT3, nuclear factor-kappa B (NF-κB), heat-shock protein 70 (Hsp70), and Lon protein expressions were determined using western blot. Transcript levels of SIRT3, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), cAMP response element-binding protein (CREB), glutathione peroxidase 1 (GPx1), and superoxide dismutase 2 (SOD2) were measured by quantitative polymerase chain reaction. Cell viability, SIRT3 protein expression, transcript levels of SIRT3, PGC-1α, CREB, GPx1, and SOD2 and protein expressions of NF-κB, Lon, and Hsp70 were significantly increased in the curcumin-treated hyperglycemic groups. Since curcumin and SIRT3 both improve mitochondrial function and AO defense, SIRT3 may be involved in the protective effects of curcumin. |
| 2017 | Expression of cellular protective proteins SIRT1, HSP70 and SOD2 correlates with age and is significantly higher in NK cells of the oldest seniors. | Immun Ageing | NK cells are key effector lymphocytes of innate immunity provided with constitutive cytolytic activity, however, their role in human ageing is not entirely understood. The study aimed to analyze the expression of proteins involved in cellular stress response sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in non-stimulated NK cells of the oldest seniors ( = 25; aged over 85; mean age 88 years) and compare with NK cells of the old ( = 30; aged under 85; mean age 76 years) and the young ( = 32; mean age 21 years) to find potential relationships between the level of expression of these proteins in NK cells and longevity. The concentration of carbonyl groups and 8-isoprostanes in NK cell lysates reflecting the level of oxidative stress was also measured.The group of the oldest seniors differed from the other age groups by significantly higher percentage of NK cells expressing SIRT1, HSP70 and SOD2. The concentration of both carbonyl groups and 8-isoprostanes in NK cell extracts remained within the normal range in all age groups. The percentage of NK cells with the expression of, respectively, SIRT1, HSP70 and SOD2 correlated positively with age. Some correlations between expression levels of particular protective proteins SIRT1, HSP70 and SOD2 were observed in the study population.The increased expression of cellular protective proteins SIRT1, HSP70 and SOD2 in NK cells of the oldest seniors seems to correspond to longevity and the observed correlations may suggest the involvement of these proteins in establishing NK cell homeostasis specific for healthy ageing process. |
| 2017 | Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function. | J Biol Chem | Stress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at GERAMT Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity and To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu and Arg cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function. |
| 2017 | FABP4/aP2 Regulates Macrophage Redox Signaling and Inflammasome Activation via Control of UCP2. | Mol Cell Biol | Obesity-linked metabolic disease is mechanistically associated with the accumulation of proinflammatory macrophages in adipose tissue, leading to increased reactive oxygen species (ROS) production and chronic low-grade inflammation. Previous work has demonstrated that deletion of the adipocyte fatty acid-binding protein (FABP4/aP2) uncouples obesity from inflammation via upregulation of the uncoupling protein 2 (UCP2). Here, we demonstrate that ablation of FABP4/aP2 regulates systemic redox capacity and reduces cellular protein sulfhydryl oxidation and, in particular, oxidation of mitochondrial protein cysteine residues. Coincident with the loss of FABP4/aP2 is the upregulation of the antioxidants superoxide dismutase (SOD2), catalase, methionine sulfoxide reductase A, and the 20S proteasome subunits PSMB5 and αβ. Reduced mitochondrial protein oxidation in FABP4/aP2 macrophages attenuates the mitochondrial unfolded-protein response (mtUPR) as measured by expression of heat shock protein 60, Clp protease, and Lon peptidase 1. Consistent with a diminished mtUPR, FABP4/aP2 macrophages exhibit reduced expression of cleaved caspase-1 and NLRP3. Secretion of interleukin 1β (IL-1β), in response to inflammasome activation, is ablated in FABP4/aP2 macrophages, as well as in FABP4/aP2 inhibitor-treated cells, but partially rescued in FABP4/aP2-null macrophages when UCP2 is silenced. Collectively, these data offer a novel pathway whereby FABP4/aP2 regulates macrophage redox signaling and inflammasome activation via control of UCP2 expression. |
| 2016 | Argon Induces Protective Effects in Cardiomyocytes during the Second Window of Preconditioning. | Int J Mol Sci | Increasing evidence indicates that argon has organoprotective properties. So far, the underlying mechanisms remain poorly understood. Therefore, we investigated the effect of argon preconditioning in cardiomyocytes within the first and second window of preconditioning. Primary isolated cardiomyocytes from neonatal rats were subjected to 50% argon for 1 h, and subsequently exposed to a sublethal dosage of hypoxia (<1% O₂) for 5 h either within the first (0-3 h) or second window (24-48 h) of preconditioning. Subsequently, the cell viability and proliferation was measured. The argon-induced effects were assessed by evaluation of mRNA and protein expression after preconditioning. Argon preconditioning did not show any cardioprotective effects in the early window of preconditioning, whereas it leads to a significant increase of cell viability 24 h after preconditioning compared to untreated cells (p = 0.015) independent of proliferation. Argon-preconditioning significantly increased the mRNA expression of heat shock protein (HSP) B1 (HSP27) (p = 0.048), superoxide dismutase 2 (SOD2) (p = 0.001), vascular endothelial growth factor (VEGF) (p < 0.001) and inducible nitric oxide synthase (iNOS) (p = 0.001). No difference was found with respect to activation of pro-survival kinases in the early and late window of preconditioning. The findings provide the first evidence of argon-induced effects on the survival of cardiomyocytes during the second window of preconditioning, which may be mediated through the induction of HSP27, SOD2, VEGF and iNOS. |
| 2016 | Polydatin ameliorates injury to the small intestine induced by hemorrhagic shock via SIRT3 activation-mediated mitochondrial protection. | Expert Opin Ther Targets | Previously, we demonstrated that sirtuin (SIRT)1 plays vital roles in the small intestine (SI), protecting against severe hemorrhagic shock (HS), and that polydatin (PD) can attenuate SI injury via SIRT1 activation.To explore the role of SIRT3 and mitochondria in SI injury after HS, and explore SIRT3 as a therapeutic target of PD in HS.An H2O2-induced model of oxidative stress and an HS model were created in IEC-6 cells and Sprague-Dawley rats, respectively. Protein content and activity of SIRT1/3 and SOD2, acetylated-SOD2 level, and mitochondrial morphology/function were determined.Expression and activity of SIRT1/3 were reduced in SI tissue and IEC-6 cells after HS or oxidative stress, accompanied by an increased acetylated-SOD2 level and damaged mitochondria. Treatment with PD or resveratrol restored SIRT1/3 activity considerably, restored SIRT1/3 expression slightly, and reduced acetylated-SOD2 levels, which lead to elevated SOD2 activity and ameliorated mitochondrial function. The addition of 3-TYP (SIRT3 inhibitor) partially blocked the mitochondrial-protective effects of PD, but did not affect increased SIRT1 activity.The SIRT3-SOD2 signaling pathway is involved in mitochondrial dysfunction induced by HS. PD attenuates mitochondrial dysfunction via activation of the SIRT3-SOD2 pathway, and may be a new approach for HS treatment. |
| 2016 | Targeted proteomics identify metabolism-dependent interactors of yeast cytochrome c peroxidase: implications in stress response and heme trafficking. | Metallomics | Recently we discovered that cytochrome c peroxidase (Ccp1) functions primarily as a mitochondrial H2O2 sensor and heme donor in yeast cells. When cells switch their metabolism from fermentation to respiration mitochondrial H2O2 levels spike, and overoxidation of its polypeptide labilizes Ccp1's heme. A large pool of heme-free Ccp1 exits the mitochondria and enters the nucleus and vacuole. To gain greater insight into the mechanisms of Ccp1's H2O2-sensing and heme-donor functions during the cell's different metabolic states, here we use glutathione-S-transferase (GST) pulldown assays, combined with 1D gel electrophoresis and mass spectrometry to probe for interactors of apo- and holoCcp1 in extracts from 1 d fermenting and 7 d stationary-phase respiring yeast. We identified Ccp1's peroxidase cosubstrate Cyc1 and 28 novel interactors of GST-apoCcp1 and GST-holoCcp1 including mitochondrial superoxide dismutase 2 (Sod2) and cytosolic Sod1, the mitochondrial transporter Pet9, the three yeast isoforms of glyceraldehyde-3-phosphate dehydrogenase (Tdh3/2/1), heat shock proteins including Hsp90 and Hsp70, and the main peroxiredoxin in yeast (Tsa1) as well as its cosubstrate, thioreoxin (Trx1). These new interactors expand the scope of Ccp1's possible roles in stress response and in heme trafficking and suggest several new lines of investigation. Furthermore, our targeted proteomics analysis underscores the limitations of large-scale interactome studies that found only 4 of the 30 Ccp1 interactors isolated here. |
| 2016 | Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension. | Am J Physiol Lung Cell Mol Physiol | Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2 , partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen. |
| 2015 | Cu/Zn-superoxide dismutase and glutathione are involved in response to oxidative stress induced by protein denaturing effect of alachlor in Saccharomyces cerevisiae. | Free Radic Biol Med | Alachlor is a widely used pre-emergent chloroacetanilide herbicide which has been shown to have many harmful ecological and environmental effects. However, the mechanism of alachlor-induced oxidative stress is poorly understood. We found that, in Saccharomyces cerevisiae, the intracellular levels of reactive oxygen species (ROS) including superoxide anions were increased only after long-term exposure to alachlor, suggesting that alachlor is not a pro-oxidant. It is likely that alachlor-induced oxidative stress may result from protein denaturation because alachlor rapidly induced an increased protein aggregation, leading to upregulation of SSA4 and HSP82 genes encoding heat shock proteins (Hsp) of Hsp70 and Hsp90 family, respectively. Although only SOD1 encoding Cu/Zn-superoxide dismutase (SOD), but not SOD2 encoding Mn-SOD, is essential for alachlor tolerance, both SODs play a crucial role in reducing alachlor-induced ROS. We found that, after alachlor exposure, glutathione production was inhibited while its utilization was increased, suggesting the role of glutathione in protecting cells against alachlor, which becomes more important when lacking Cu/Zn-SOD. Based on our results, it seems that alachlor primarily causes damages to cellular macromolecules such as proteins, leading to an induction of endogenous oxidative stress, of which intracellular antioxidant defense systems are required for elimination. |
| 2015 | Polydatin Alleviates Small Intestine Injury during Hemorrhagic Shock as a SIRT1 Activator. | Oxid Med Cell Longev | To evaluate the role of SIRT1 in small intestine damage following severe hemorrhagic shock and to investigate whether polydatin (PD) can activate SIRT1 in shock treatment.The severe hemorrhagic shock model was reproduced in Sprague Dawley rats.Two hours after drug administration, half of the rats were assessed for survival time evaluation and the remainder were used for small intestinal tissue sample collection.Bleeding and swelling appeared in the small intestine with epithelial apoptosis and gut barrier disturbance during hemorrhagic shock. SIRT1 activity and PGC-1α protein expression of the small intestine were decreased, which led to an increase in acetylated SOD2 and decreases in the expression and activity of SOD2, resulting in severe oxidative stress. The decreased SIRT1 activity and expression were partially restored in the PD administration group, which showed reduced intestine injury and longer survival time. Notably, the effect of PD was abolished after the addition of Ex527, a selective inhibitor of SIRT1.The results collectively suggest a role for the SIRT1-PGC-1α-SOD2 axis in small intestine injury following severe hemorrhagic shock and that PD is an effective SIRT1 activator for the shock treatment. |
| 2015 | Polydatin protects hepatocytes against mitochondrial injury in acute severe hemorrhagic shock via SIRT1-SOD2 pathway. | Expert Opin Ther Targets | The aim of the study was to determine whether hepatocyte mitochondrial injury instigates severe shock and to explore effective therapy.Wistar rats were randomly divided into five groups: Control (sham) group, shock + normal saline, shock + cyclosporine A, shock + resveratrol (Res) and shock + polydatin (PD) group. Mitochondrial morphology and function in hepatocytes following treatment were determined.Hepatocytes following severe shock exhibited mitochondrial dysfunction characterized with opening of mitochondrial permeability transition pores, mitochondrial swelling, decreased mitochondrial membrane potential (ΔΨm) and reduced ATP levels. Moreover, severe shock induced oxidative stress with increased lipid peroxidation and reactive oxygen species, decreased SOD2 (Superoxide Dismutase 2) and GSH/GSSG, which resulted in increased lysosomal membrane permeabilization and hepatocyte mitochondrial injury. Additionally, Res and PD restored decreased deacetylase sirtuin1 activity and protein expression in liver tissue following severe shock, suppressed oxidative stress-induced lysosomal unstability and mitochondrial injury by increasing the protein expression of SOD2, and thereby contributed to the prevention of hepatocyte mitochondria dysfunction and liver injury.PD effectively preserved hepatocytes from mitochondrial injury via SIRT1-SOD2 pathway and may be a new approach to treatment of irreversible shock. |
| 2015 | Species-Related Differences in the Proteome of Rat and Human Pancreatic Beta Cells. | J Diabetes Res | The core proteomes of human and rat pancreatic beta cells were compared by label-free LC-MS/MS: this resulted in quantification of relative molar abundances of 707 proteins belonging to functional pathways of intermediary metabolism, protein synthesis, and cytoskeleton. Relative molar abundances were conserved both within and between pathways enabling the selection of a housekeeping network for geometric normalization and the analysis of potentially relevant differential expressions. Human beta cells differed from rat beta cells in their lower level of enzymes involved in glucose sensing (MDH1, PC, and ACLY) and upregulation of lysosomal enzymes. Human cells also expressed more heat shock proteins and radical scavenging systems: apart from SOD2, they expressed high levels of H2O2-scavenger peroxiredoxin 3 (PRDX3), confirmed by microarray, Western blotting, and microscopy. Besides conferring lower susceptibility to oxidative stress to human cells PRDX3 might also play a role in physiological redox regulation as, in rat, its expression was restricted to a beta cell subset with higher metabolic glucose responsiveness. In conclusion, although their core proteomic architecture is conserved, human and rat beta cells differ in their molar expression of key enzymes involved in glucose sensing and redox control. |
| 2015 | Effects of a recombinant complement component C3b functional fragment α2MR (α2-macroglobulin receptor) additive on the immune response of juvenile orange-spotted grouper (Epinephelus coioides) after the exposure to cold shock challenge. | Fish Shellfish Immunol | The effects of Ec-α2MR (Epinephelus coiodes-α2-macroglobulin receptor) on growth performance, enzymatic activity, respiratory burst, MDA level, total antioxidant capacity, DPPH radical scavenging percentage and immune-related gene expressions of the juvenile orange-spotted grouper were evaluated. The commercial diet supplemented with α2MR additive was used to feed the orange-spotted grouper for six weeks. Although a slight increase was observed in the specific growth rate, survival rate and weight gain, no significance was observed among different group. After the feeding trial, the groupers were exposed to cold stress. Respiratory burst activity and MDA level decreased significantly in α2MR additive group by comparing with the control and additive control group, while a sharp increase of ACP activity, ALP activity, total antioxidant capacity and DPPH radial scavenging percentage was observed in α2MR additive group. qRT-PCR analyses confirmed that the up-regulated mRNA expressions of C3, TNF1, TNF2, IL-6, CTL, LysC, SOD1 and SOD2 were observed in α2MR additive group at 20 °C. These results showed that α2MR additive may moderate the immune response in grouper following cold shock challenge. |
| Caffeic acid, a phyto polyphenol mitigates fluoride induced hepatotoxicity in rats: A possible mechanism. | Biofactors | Fluoride induced hepatotoxicity has been extensively studied in both humans and animals. However, the mechanism underlying in the hepatotoxicity of experimental fluorosis remains obscure. The severity of fluoride toxicity was reduced by oral administration of certain plant derived antioxidants. Caffeic acid (CA) a polyphenolic compound has diverse range of pharmacological activity in the biological system. Therefore, the present study was aimed to investigate the protective mechanism of CA, against fluoride induced hepatotoxicity in rats. The rats were treated with 300 ppm of NaF via drinking water ad libitum alone and in combination with CA at a dose of 50 mg/kg daily for 30 days by oral intubation. CA treatment significantly prevented fluoride induced hepatic damage as evident from the histopathological studies and declined levels of serum fluoride and liver marker enzymes. A significant decrease in the levels of enzymatic (SOD2, CAT, GPx, and GSTpi class) and nonenzymatic (GSH and Vitamin C) antioxidants along with increased ROS, lipid peroxidation, protein carbonyl content, and nitrate levels by fluoride were also prevented in these groups. In addition, CA inhibits fluoride induced apoptosis by altering the Bax and caspase-3p20 expressions. Further, fluoride induced upregulation of Nox4, p38α MAPK, Hsp60, and downregulation of Hsp27 are the indicators for the detection of oxidative damage, apoptosis, and mitochondrial stress was also modulated by CA. These findings reveal that CA supplementation has a protective effect against fluoride induced hepatotoxicity in rats. |
| 2014 | [Resveratrol improves intestinal injury in hemorrhagic shock rats by protection of mitochondria and reduction of oxidative stress]. | Zhong Nan Da Xue Xue Bao Yi Xue Ban | To explore whether resveratrol can reduce intestinal damage in hemorrhagic shock rats and the underlying mechanism.A total of 24 Sprague-Dawley rats of specifi c pathogen free (SPF) were randomly divided into a control group(n=8), a resveratrol group (SR group, n=8) and a vehicle group (SS group, n=8). Th e mean arterial pressure was recorded. Two hours aft er hemorrhagic shock, 15 mg/kg resveratrol or 0.3 mL equal volume of vehicle and autologous blood were given, respectively. The intestinal specimens were collected for hematoxylin-eosin (HE) staining and calculated the pathological score. The superoxide dismutase 2 (SOD2) and cytochrome C (Cyt C) protein expression was examined by immunohistochemistry and/or Western blot. ATP level, activities of glutathione peroxidase (GXH-px), catalase (CAT) and SOD were also detected.Two hours after autologous blood transfusion, the mean arterial pressure in the SR group was significantly higher than that in the SS group (P< 0.01). Compared with the SS group, the pathological injury was significantly alleviated and pathological scores were dramatically reduced in the SR group (P< 0.05). The activities of GXH-px, CAT, SOD and the ATP levels in the SR group were significantly higher than those in the SS group (all P< 0.01). Compared with the SS group, the SOD2 expression was significantly higher while the Cyt C expression was dramatically lower in the SR group (both P< 0.01).Resveratrol could alleviate the intestinal injury in hemorrhagic shock rats, which might be associated with its effects on reduction of oxidative stress and protection of mitochondria. |
| 2015 | Effect of 593C>T GPx1 SNP alone and in synergy with 47C>T SOD2 SNP on the outcome of critically ill patients. | Cytokine | During critical illness and sepsis there is severe antioxidant depletion, and this scenario raises the critical ill patient's mortality risk. Glutathione peroxidase (GPx) is one of the first endogenous antioxidant defense enzymes, and it works cooperatively with superoxide dismutase (SOD) and catalase (CAT) to detoxify free radicals from the cellular environment. Genetic studies are important to understand the complexity of human oxidative stress and how the organism responds to an extreme situation such as critically care conditions. Previous studies with a GPx1 single nucleotide polymorphism (593C>T SNP; rs1050450; protein variant in GPx1: Pro198Leu) showed 593T carriers and 593TT homozygotes present higher risk to develop different diseases. We assessed the relationship of the genotype distribution of GPx1 SNP in critically ill patients with their conditions (organ dysfunction, sepsis, and septic shock) and their outcome. We monitored 626 critically ill patients daily from the ICU (intensive care unit) admission to their discharge from hospital, or death. Our study revealed a significant association between 593TT GPx1 genotype and mortality; the mortality rate was higher in homozygous 593TT GPx1 (N=94) when compared with the group of subjects with genotypes 593CT or 593CC GPx1 (N=532) (52% vs. 38%, P=0.009; OR=1.79; 95% CI=1.13-2.85). Evaluating the subgroup of 293 ICU patients with sepsis, a pooled analysis including two genetic variants GPx1 and SOD2 (47C>T SNP, rs4880; protein variant in MnSOD: Ala-9Val) showed a significant difference in relation to progression to septic shock. The frequency of septic shock among septic patients with 593T GPx1 and 47C SOD2 alleles (N=122) was higher when compared with septic patients carrying other settings of genotypes (N=174) (78% vs. 66%; P=0.028; OR=1.81; 95% CI=1.03-3.18). Accepting the previously reported functional effects of these two SNPs on GPx1 and SOD2 gene expressions and, consequently, on GPx1 and MnSOD enzyme activities, we believe our results may be considered as an important contribution for the understanding of oxidative imbalance during the critical ill. |
| 2015 | Effects of vitrification on the expression of pluripotency, apoptotic and stress genes in in vitro-produced porcine blastocysts. | Reprod Fertil Dev | The aims of the present study were to: (1) evaluate the effect of vitrification and warming on quality parameters and expression levels of pluripotency, apoptotic and stress genes in in vitro-produced (IVP) porcine blastocysts; and (ii) determine the correlation between these parameters. To this end, total cell number, DNA fragmentation, peroxide levels and the relative transcript abundance of BCL-2 associated X protein (BAX), BCL2-like 1 (BCL2L1), heat shock protein 70 (HSPA1A), POU class 5 homeobox 1 (POU5F1), superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) were analysed in fresh and vitrified IVP blastocysts. The results suggest that vitrification procedures have no effect on total cell number and gene expression of BAX, BCL2L1, SOD1 and SOD2 or the BAX:BCL2L1 ratio. Nevertheless, a significant increase in DNA fragmentation (2.9±0.4% vs 11.9±2.0%) and peroxide levels (80.4±2.6 vs 97.2±3.1) were seen in vitrified compared with Day 7 fresh blastocysts. In addition, after blastocyst vitrification, relative transcript abundance was downregulated for POU5F1 and upregulated for HSPA1A. Finally, there was a significant correlation of POU5F1 and HSPA1A with DNA fragmentation (POU5F1, r=-0.561; HSPA1A, r=0.604) and peroxide levels (POU5F1, r=-0.590; HSPA1A, r=0.621). In conclusion, under the conditions of the present study, vitrification and warming of IVP porcine blastocysts resulted in altered expression of POU5F1 and HSPA1A, but had no effect on BAX, BCL2L1, SOD1 and SOD2 expression. |
| 2014 | Responses to ROS inducer agents in zebrafish cell line: differences between copper and UV-B radiation. | Fish Physiol Biochem | Fish are commonly exposed to environmental pollutants, which in turns could induce an oxidative stress. So, it is important to understand the effects and the responses elicited by these toxicants in fish species, being fish cell lines important tools for this purpose. Thus, the aim of the present study was to compare the effects of copper and UV-B radiation exposure on zebrafish hepatocytes (ZFL lineage) in terms of reactive oxygen species (ROS) levels, sulfhydril groups content and mRNA levels of important genes related to cellular response to toxic agents. Exposure of ZFL cells to UV-B radiation (23.3 mJ/cm(2)) significantly increased levels of intracellular ROS and mRNA of both superoxide dismutase isoforms (sod1 and sod2), three glutathione S-transferase isoforms (gstα, gstµ and gstπ) and a heat shock protein (hsp70). However, no changes in nonprotein sulfhydryl groups (NP-SH) content, as well as in the mRNA levels of genes related to glutathione (GSH) synthesis and recycling, were observed. Contrary to this, copper exposure (20 mg/L) diminished NP-SH content and increased the levels of mRNA of genes related to GSH synthesis (gclc and gs). Moreover, copper exposure increases the mRNA levels of some genes related to antioxidant defenses (gpx and gstπ), biotransformation reactions (cyp1a1) and protein repair (hsp70). In conclusion, these results demonstrated that both toxicants could increase ROS levels in ZFL cell line, but the responses are different, which could be related to activation of different signaling pathways. |
| 2014 | Characterization of adipocyte stress response pathways during hibernation in thirteen-lined ground squirrels. | Mol Cell Biochem | To avoid the harsh conditions of winter climates, hibernating mammals undergo a systematic depression of physiological function by reducing their metabolic rate. During this process, hibernators are exposed to significant stresses (e.g., low body temperature, ischemia-reperfusion) that must be dealt with appropriately to avoid irreversible tissue damage. Consequently, we investigated the contribution of stress-responsive antioxidant enzymes, heat shock proteins, signal transduction pathways (e.g., mitogen-activated protein kinases, MAPK), and transcription factors for their role in conferring tolerance to stress in the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus). Using a combination of multiplex protein panels and traditional immunoblotting procedures, we have focused on these stress factors in brown adipose tissue (BAT) and white adipose tissue (WAT) over cycles of torpor-arousal since they provide the means for heat production as a result of non-shivering thermogenesis and the mobilization of critical energy reserves, respectively. We show the differential and tissue-specific regulation of stress factors including a unified upregulation of the antioxidant enzyme Thioredoxin 1 in both tissues, an upregulation of superoxide dismutase (SOD1 and SOD2) in WAT, and an increase in heat shock proteins during the transitory periods of the torpor-arousal cycle (HSP90α in BAT and HSP60 in WAT). Additionally, an upregulation of the active form of ERK1/2 and p38 in BAT and select transcription factors (e.g., CREB-1 and ELK-1) in both tissues were identified. These data provide us with greater insight into the molecular mechanisms responsible for this animal's natural stress tolerance and outline molecular signatures which define stress resistance. |
| 2014 | Lifelong training preserves some redox-regulated adaptive responses after an acute exercise stimulus in aged human skeletal muscle. | Free Radic Biol Med | Several redox-regulated responses to an acute exercise bout fail in aged animal skeletal muscle, including the ability to upregulate the expression of antioxidant defense enzymes and heat shock proteins (HSPs). These findings are generally derived from studies on sedentary rodent models and thus may be related to reduced physical activity and/or intraspecies differences as opposed to aging per se. This study, therefore, aimed to determine the influence of age and training status on the expression of HSPs, antioxidant enzymes, and NO synthase isoenzymes in quiescent and exercised human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis before and 3 days after an acute high-intensity-interval exercise bout in young trained, young untrained, old trained, and old untrained subjects. Levels of HSP72, PRX5, and eNOS were significantly higher in quiescent muscle of older compared with younger subjects, irrespective of training status. 3-NT levels were elevated in muscles of the old untrained but not the old trained state, suggesting that lifelong training may reduce age-related macromolecule damage. SOD1, CAT, and HSP27 levels were not significantly different between groups. HSP27 content was upregulated in all groups studied postexercise. HSP72 content was upregulated to a greater extent in muscle of trained compared with untrained subjects postexercise, irrespective of age. In contrast to every other group, old untrained subjects failed to upregulate CAT postexercise. Aging was associated with a failure to upregulate SOD2 and a downregulation of PRX5 in muscle postexercise, irrespective of training status. In conclusion, lifelong training is unable to fully prevent the progression toward a more stressed muscular state as evidenced by increased HSP72, PRX5, and eNOS protein levels in quiescent muscle. Moreover, lifelong training preserves some (e.g., CAT) but not all (e.g., SOD2, HSP72, PRX5) of the adaptive redox-regulated responses after an acute exercise bout. Collectively, these data support many but not all of the findings from previous animal studies and suggest parallel aging effects in humans and mice at rest and after exercise that are not modulated by training status in human skeletal muscle. |
| 2014 | Cadmium (Cd(2+)) exposure differentially elicits both cell proliferation and cell death related responses in SK-RC-45. | Toxicol In Vitro | Cadmium (Cd(2+)) is a major nephrotoxic environmental pollutant, affecting mostly proximal convoluted tubule (PCT) cells of the mammalian kidney, while conditionally Cd(2+) could also elicit protective responses with great variety and variability in different systems. The present study was designed to evaluate the molecular mechanism of Cd(2+) toxicity on human PCT derived Renal Cell Carcinoma (RCC), SK-RC-45 and compare its responses with normal human PCT derived cell line, NKE. Exposure of SK-RC-45 cells with different concentrations of CdCl2 (e.g. 0, 10 and 20μM) in serum free medium for 24h generate considerable amount of ROS, accompanied with decreased cell viability and alternations in the cellular and nuclear morphologies, heat shock responses and GCLC mediated protective responses. Also phosphatidylserine externalization, augmentation in the level of caspase-3, PARP, BAD, Apaf1 and cleaved caspase-9 along with decreased expression of Bcl2 and release of cytochrome c confirmed that, Cd(2+) dose dependently induces solely intrinsic pathway of apoptosis in SK-RC-45, independent of JNK. Furthermore, the non-toxic concentration (10μM) of Cd(2+) induced nuclear translocation of Nrf2 and increased expression in the level of HO-1 enzyme suggesting that at the milder concentration, Cd(2+) induces protective signaling pathways. On the other hand, exposure of NKE to different concentrations of CdCl2 (e.g. 0, 10, 20, 30 and 50μM) under the same conditions elevate stronger heat shock and SOD2 mediated protective responses. In contrary to the RCC PCT, the normal PCT derived cell follows JNK dependent and extrinsic pathways of apoptosis. Cumulatively, these results suggest that Cd(2+) exposure dose dependently elicit both cell proliferative and cell death related responses in SK-RC-45 cells and is differentially regulated with respect to normal kidney epithelia derived NKE cells. |
| 2013 | CYP1A1, GCLC, AGT, AGTR1 gene-gene interactions in community-acquired pneumonia pulmonary complications. | Mol Biol Rep | This study was conducted to establish the possible contribution of functional gene polymorphisms in detoxification/oxidative stress and vascular remodeling pathways to community-acquired pneumonia (CAP) susceptibility in the case-control study (350 CAP patients, 432 control subjects) and to predisposition to the development of CAP complications in the prospective study. All subjects were genotyped for 16 polymorphic variants in the 14 genes of xenobiotics detoxification CYP1A1, AhR, GSTM1, GSTT1, ABCB1, redox-status SOD2, CAT, GCLC, and vascular homeostasis ACE, AGT, AGTR1, NOS3, MTHFR, VEGFα. Risk of pulmonary complications (PC) in the single locus analysis was associated with CYP1A1, GCLC and AGTR1 genes. Extra PC (toxic shock syndrome and myocarditis) were not associated with these genes. We evaluated gene-gene interactions using multi-factor dimensionality reduction, and cumulative gene risk score approaches. The final model which included >5 risk alleles in the CYP1A1 (rs2606345, rs4646903, rs1048943), GCLC, AGT, and AGTR1 genes was associated with pleuritis, empyema, acute respiratory distress syndrome, all PC and acute respiratory failure (ARF). We considered CYP1A1, GCLC, AGT, AGTR1 gene set using Set Distiller mode implemented in GeneDecks for discovering gene-set relations via the degree of sharing descriptors within a given gene set. N-acetylcysteine and oxygen were defined by Set Distiller as the best descriptors for the gene set associated in the present study with PC and ARF. Results of the study are in line with literature data and suggest that genetically determined oxidative stress exacerbation may contribute to the progression of lung inflammation. |
| 2013 | Heat stress and sudden infant death syndrome--stress gene expression after exposure to moderate heat stress. | Forensic Sci Int | The aim of the present study was to investigate stress gene expression in cultured primary fibroblasts established from Achilles tendons collected during autopsies from sudden infant death syndrome (SIDS) cases, and age-matched controls (infants dying in a traumatic event). Expression of 4 stress responsive genes, HSPA1B, HSPD1, HMOX1, and SOD2, was studied by quantitative reverse transcriptase PCR analysis of RNA purified from cells cultured under standard or various thermal stress conditions. The expression of all 4 genes was highly influenced by thermal stress in both SIDS and control cells. High interpersonal variance found in the SIDS group indicated that they represented a more heterogeneous group than controls. The SIDS group responded to thermal stress with a higher expression of the HSPA1B and HSPD1 genes compared to the control group, whereas no significant difference was observed in the expression of SOD2 and HMOX1 between the two groups. The differences were related to the heat shock treatment as none of the genes were expressed significantly different in SIDS at base levels at 37 °C. SOD2 and HMOX1 were up regulated in both groups, for SOD2 though the expression was lower in SIDS at all time points measured, and may be less related to heat stress. Being found dead in the prone position (a known risk factor for SIDS) was related to a lower HSPA1B up-regulation in SIDS compared to SIDS found on their side or back. The study demonstrates the potential usefulness of gene expression studies using cultured fibroblasts established from deceased individuals as a tool for molecular and pathological investigations in forensic and biomedical sciences. |
| 2013 | Upregulation of the mitochondrial Lon Protease allows adaptation to acute oxidative stress but dysregulation is associated with chronic stress, disease, and aging. | Redox Biol | The elimination of oxidatively modified proteins is a crucial process in maintaining cellular homeostasis, especially during stress. Mitochondria are protein-dense, high traffic compartments, whose polypeptides are constantly exposed to superoxide, hydrogen peroxide, and other reactive species, generated by 'electron leakage' from the respiratory chain. The level of oxidative stress to mitochondrial proteins is not constant, but instead varies greatly with numerous metabolic and environmental factors. Oxidized mitochondrial proteins must be removed rapidly (by proteolytic degradation) or they will aggregate, cross-link, and cause toxicity. The Lon Protease is a key enzyme in the degradation of oxidized proteins within the mitochondrial matrix. Under conditions of acute stress Lon is highly inducible, possibly with the oxidant acting as the signal inducer, thereby providing increased protection. It seems that under chronic stress conditions, however, Lon levels actually decline. Lon levels also decline with age and with senescence, and senescent cells even lose the ability to induce Lon during acute stress. We propose that the regulation of Lon is biphasic, in that it is up-regulated during transient stress and down-regulated during chronic stress and aging, and we suggest that the loss of Lon responsiveness may be a significant factor in aging, and in age-related diseases. |
| 2013 | Blast exposure causes early and persistent aberrant phospho- and cleaved-tau expression in a murine model of mild blast-induced traumatic brain injury. | J Alzheimers Dis | Mild traumatic brain injury (mTBI) is considered the 'signature injury' of combat veterans that have served during the wars in Iraq and Afghanistan. This prevalence of mTBI is due in part to the common exposure to high explosive blasts in combat zones. In addition to the threats of blunt impact trauma caused by flying objects and the head itself being propelled against objects, the primary blast overpressure (BOP) generated by high explosives is capable of injuring the brain. Compared to other means of causing TBI, the pathophysiology of mild-to-moderate BOP is less well understood. To study the consequences of BOP exposure in mice, we employed a well-established approach using a compressed gas-driven shock tube that recapitulates battlefield-relevant open-field BOP. We found that 24 hours post-blast a single mild BOP provoked elevation of multiple phospho- and cleaved-tau species in neurons, as well as elevating manganese superoxide-dismutase (MnSOD or SOD2) levels, a cellular response to oxidative stress. In hippocampus, aberrant tau species persisted for at least 30 days post-exposure, while SOD2 levels returned to sham control levels. These findings suggest that elevated phospho- and cleaved-tau species may be among the initiating pathologic processes induced by mild blast exposure. These findings may have important implications for efforts to prevent blast-induced insults to the brain from progressing into long-term neurodegenerative disease processes. |
| 2013 | A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients. | Proteome Sci | We report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals.Five cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera.This protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals. |
| 2013 | Klotho gene polymorphism of rs3752472 is associated with the risk of urinary calculi in the population of Han nationality in Eastern China. | Gene | The incidence of urolithiasis has considerably increased throughout the world in the last two decades. Clinical researches have showed an association between oxidative stress and stone formation. Emerging evidence indicated a novel function for klotho protein in anti-oxidative stress. In this study, we aimed at investigating a possible relationship between klotho gene polymorphisms and the risk of calcium oxalate urolithiasis in the population of Han nationality in Eastern China.Klotho gene polymorphisms rs3752472 in exon3, rs650439 in intron 4 and rs577912 in intron 1 were investigated in 426 patients with calcium oxalate stones compared with 282 age-matched healthy volunteers with no history of stone formation, using TaqMan SNP Genotyping Assays.Significant differences were found between rs3752472 and the risk of nephrolithiasis as CC genotype of rs3752472 klotho polymorphism had almost 2-fold increased stone risk compared with the heterozygote genotype CT and homozygous genotype TT(95% CI=1.013-2.255, OR=1.512,p=0.043).Our results showed that the rs3752472 polymorphism of klotho gene is associated with the risk of calcium oxalate urolithiasis and may act as a risk factor during stone formation in our study population. |
| 2013 | Polymorphisms in oxidative stress-related genes and mortality in breast cancer patients--potential differential effects by radiotherapy? | Breast | We assessed whether variants in 22 oxidative stress-related genes are associated with mortality of breast cancer patients and whether the associations differ according to radiotherapy. Using a prospective cohort of 1348 postmenopausal breast cancer patients, we estimated hazard ratios (HR) and 95% confidence intervals (CI) for 109 single nucleotide polymorphisms (SNPs) using Cox proportional hazards regression. Validation of results was attempted using two Scandinavian studies. Eleven SNPs in MT2A, NFE2L2, NQO1, PRDX1, and PRDX6 were significantly associated with overall mortality after a median follow-up of 5.7 years. Three SNPs in NQO1 (rs2917667) and in PRDX6 (rs7314, rs4916362) were consistently associated with increased risk of dying across all three study populations (pooled: HRNQO1_rs2917667 1.20, 95% CI 1.00-1.44, p = 0.051; HRPRDX6_rs7314 1.16, 95% CI 1.00-1.35, p = 0.056, HRPRDX6_rs4916362 1.14 95% CI 1.00-1.32, p = 0.062). Potential effect modification by radiotherapy was found for CAT_rs769218. In conclusion, genetic variants in NQO1 and PRDX6 may modify breast cancer prognosis. |
| 2013 | Higher frequency of septic shock in septic patients with the 47C allele (rs4880) of the SOD2 gene. | Gene | To analyze the effect of the two different versions of the manganese superoxide dismutase gene (SOD2) on sepsis. The SOD2 gene presents the 47C>T single nucleotide polymorphism (SNP; ID: rs4880) which produces MnSOD with different activities. The -9Val MnSOD (47T allele) is less efficient than the -9Ala version (47C allele). During sepsis there are abundance of ROS, high SOD2 expression and excess of H(2)O(2) synthesis. High concentrations of H(2)O(2) could affect the sepsis scenario and/or the sepsis outcome.We determined the 47C>T single nucleotide polymorphism (SNP) frequencies in 529 critically ill patients with or without sepsis, facing outcome. To collect information on population frequencies, we obtained a pilot 47C>T genotypic and allelic frequencies in a random group of 139 healthy subjects.We compared the 47C allele carriers (47CC+47CT genotypes) with 47TT homozygotes and noticed a significant association between 47C allele carriers and septic shock in septic patients (P=0.025). With an adjusted binary multivariate logistic regression, incorporating 47C>T SNP and the main clinical predictors, we showed high SOFA scores [P<0.001, OR=9.107 (95% CI=5.319-15.592)] and 47C allele [P=0.011, OR=2.125 (95% CI=1.190-3.794)] were significantly associated with septic shock outcome. With this information we presented a hypothesis suggesting that this negative outcome from sepsis is possibly explained by effects on cellular stress caused by 47C allele.In our population there was a significant higher frequency of septic shock in septic patients with the 47C allele of the SOD2 gene. This higher 47C allele frequency in septic patients with negative outcome could be explained by effects of higher activity MnSOD on cellular stress during the sepsis. |
| 2013 | Dissecting molecular stress networks: identifying nodes of divergence between life-history phenotypes. | Mol Ecol | The complex molecular network that underlies physiological stress response is comprised of nodes (proteins, metabolites, mRNAs, etc.) whose connections span cells, tissues and organs. Variable nodes are points in the network upon which natural selection may act. Thus, identifying variable nodes will reveal how this molecular stress network may evolve among populations in different habitats and how it might impact life-history evolution. Here, we use physiological and genetic assays to test whether laboratory-born juveniles from natural populations of garter snakes (Thamnophis elegans), which have diverged in their life-history phenotypes, vary concomitantly at candidate nodes of the stress response network, (i) under unstressed conditions and (ii) in response to an induced stress. We found that two common measures of stress (plasma corticosterone and liver gene expression of heat shock proteins) increased under stress in both life-history phenotypes. In contrast, the phenotypes diverged at four nodes both under unstressed conditions and in response to stress: circulating levels of reactive oxygen species (superoxide, H(2)O(2)); liver gene expression of GPX1 and erythrocyte DNA damage. Additionally, allele frequencies for SOD2 diverge from neutral markers, suggesting diversifying selection on SOD2 alleles. This study supports the hypothesis that these life-history phenotypes have diverged at the molecular level in how they respond to stress, particularly in nodes regulating oxidative stress. Furthermore, the differences between the life-history phenotypes were more pronounced in females. We discuss the responses to stress in the context of the associated life-history phenotype and the evolutionary pressures thought to be responsible for divergence between the phenotypes. |
| 2012 | Exposure of Wistar rats to 24-h psycho-social stress alters gene expression in the inferior colliculus. | Neurosci Lett | Recently, we have demonstrated that the exposure of Wistar rats to psycho-social stress results in a transient auditory hypersensitivity. Here, to learn more about modifications occurring in auditory brainstem, we have analyzed gene expression pattern in inferior colliculus using quantitative RT-PCR. As targets, we have chosen genes associated with: neural activity (FBJ osteosarcoma viral oncogene, cFos), hypoxia (nitric oxide synthase inducible, iNos; superoxide dismutase 2, Sod2), neuroprotection (nerve growth factor beta, Ngfb; heat shock factor 1, Hsf1; heat shock protein 70, Hsp70) and inflammation (tumor necrosis factor alpha, Tnfa; tumor necrosis factor alpha receptor, Tnfar; substance P, Sp; cyclooxygenase 2, Cox2). We found that the expression of all genes was modified following stress, as compared to the controls. Immediately after stress, the number of transcripts encoding iNos, Sod2, Hsf1, Ngfb, Tnfa, Tnfar and Sp was significantly increased, suggesting possible modulation during exposure to stressor. Interestingly, we found that expression of Hsf1 and Ngfb at this particular time was left-right asymmetrical: there were more transcripts of both genes found in the left colliculi, as compared to the right colliculi. Three hours post-stress, iNos, Hsf1, Tnfa and Tnfar were still upregulated, Sod2, Ngfb and Sp went back to baseline and Cox2 was upregulated. Six hours post-stress, cFos mRNA became downregulated. The number of Hsp70 mRNA increased 24h post-stress. Except for the reduced number of cFos transcripts, expression of all other genes tested reached the baseline seven days post-stress. Presented results corroborate the concept of auditory system responding to the psycho-social stress. Post-stress changes in the IC gene expression could likely indicate shift from allostasis to homeostasis in the auditory brainstem. |
| 2012 | Proteome profiling of tolbutamide-treated rat primary hepatocytes using nano LC-MS/MS and label-free protein quantitation. | Electrophoresis | Tolbutamide is used as a first line oral antihyperglycemic drug for type 2 diabetes. One side effect of this drug, hepatotoxicity, is well recognized; however, the precise mechanisms underlying tolbutamide-induced hepatotoxicity remain unclear. In this respect, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxicity induced by this drug. In this study, we aimed to identify molecular pathways based on proteins responding to cellular toxicity in tolbutamide-treated primary hepatocytes, using nano UPLC-MS/MS analysis. Rat primary hepatocytes were treated with an IC(20) concentration for 24 h to study the hepatotoxic effects of tolbutamide. For high-throughput label-free quantitation, tryptic-digested peptides of proteins from cell lysates were analyzed using LC-MS/MS and quantitated using the IDEAL-Q software, in which several parameters, such as assisted sequence, elution time, and mass-to-charge ratio were included. We quantified a total of 330 distinct proteins from the tolbutamide-treated hepatocytes and identified 55 upregulated and 82 downregulated proteins with expression changes. Among these differentially expressed proteins, we focused mainly on the 18 upregulated proteins belonging to xenobiotic cytochrome P450 (CYP), drug metabolism/detoxification, oxidative stress/antioxidant response, and cell damage pathway. CYP2D1, CYP2C11, UDP-glucuronosyltransferase 2B (UGT2B), superoxide dismutase 2 (SOD2), 60 kDa heat shock protein (HSPD1), heat shock protein 90 (HSP90), and catalase (CAT) were confirmed by Western blot analysis. In addition, various xenobiotic CYP proteins upregulated in the tolbutamide-treated group, CYP2D1, CYP2C13, and CYP2C11 were confirmed by reverse transcriptase-PCR analysis. Our results offer important new insights into the molecular mechanisms of tolbutamide-induced hepatotoxicity. |
| 2012 | PPAR-γ activator pioglitazone prevents age-related atrial fibrillation susceptibility by improving antioxidant capacity and reducing apoptosis in a rat model. | J Cardiovasc Electrophysiol | The in vivo role of peroxisome proliferator-activated receptor (PPAR)-γ, an essential transcriptional mediator of lipid and glucose metabolism, in atrial fibrillation (AF) remains to be fully elucidated. We investigated the effects of pioglitazone, a PPAR-γ activator, in an in vivo AF rat model.We studied 3 groups of Wistar rats: young group, 3-month-old rats treated with vehicle; aged group, 9-month-old rats treated with vehicle; and aged+Pio group, 9-month-old rats treated with pioglitazone. After 4-week treatment, AF duration induced by 30-second burst pacing, gene and protein expressions, and atrial structural changes were compared between the 3 groups. Atrial oxidant reducing activity was measured by electron spin resonance method. AF duration was markedly prolonged in the aged group but significantly shortened in the aged+Pio group. Age-induced decrease in free radical reducing activity was reversed by pioglitazone. Gene and protein expression levels of antioxidant molecules Sod2 (MnSOD) and Hspa1a (heat shock 70 protein) were significantly enhanced, and p22(phox) and gp91(phox), two NADPH oxidase subunits, were significantly decreased in aged+Pio rats. Pioglitazone treatment significantly increased phosphorylated (p-) Akt but significantly reduced p-ERK1/2 and p-JNK. Pioglitazone significantly restored p-Bad and reduced cleaved caspase-3 and -9, indicating that pioglitazone prevented age-related enhancement of apoptotic signaling. Microscopic analysis revealed suppression of age-related histological changes (interstitial fibrosis and apoptosis) by pioglitazone.Pioglitazone inhibited age-related arrhythmogenic atrial remodeling and AF perpetuation by improving antioxidant capacity and inhibiting the mitochondrial apoptotic signaling pathway. PPAR-γ activators could become a novel upstream therapy for age-related AF. |
| 2011 | Manganese SOD mimics are effective against heat stress in a mutant fission yeast deficient in mitochondrial superoxide dismutase. | Biol Trace Elem Res | Previous studies revealed a close connection between heat shock and manganese-dependent superoxide dismutase (SOD2) in eukaryotes. This paper shows that SOD mimics based on manganese complexes caused an increase in thermotolerance for a mutant fission yeast deficient in mitochondrial superoxide dismutase. Manganese compounds used for tests are SOD mimics, from two different classes: salen manganese (EUK-8) and Mn porphyrin (Mn(III)TE-2-PyP(5+)). The tests were conducted using a Schizosaccharomyces pombe model, comparing the viability of two strains at chronic heat stress (37°C)--a wild type versus a strain with the mitochondrial superoxide dismutase gene deleted [SOD2(-)]. The presence of massive free radical species in S. pombe SOD2(-) was demonstrated using a luminol-enhanced chemiluminescence test derived from a menadione-mediated survival protocol.Survival tests revealed that the SOD2-deleted S. pombe is about 100 times more sensitive to heat stress than the wild-type strain. This survival deficit can be corrected by EUK-8 and Mn(III)TE-2-PyP(5+) to almost the same degree but not by manganese chloride II (MnCl(2)). Using a simple spot assay for viability testing, this new model proved to be an easy alternative for the initial estimation of manganese SOD mimics efficiency. |
| 2012 | Training alters the skeletal muscle antioxidative capacity in non-insulin-dependent type 2 diabetic men. | Scand J Med Sci Sports | The present study analyzes the oxidative stress situation in the skeletal muscle of overweight/obese men suffering from non-insulin-dependent type 2 diabetes mellitus [T2DM, n=16, years=61±7, body mass index (BMI)=31±4 kg/m(2) ] and BMI-matched non-diabetic male control subjects (CON, n=7, years=53±6, BMI=30±4 kg/m(2) ). Furthermore, it investigates whether physical training can alter the skeletal muscle antioxidative capacity of T2DM patients at rest. Molecule content analyses (immunohistochemical stainings) of 8-iso-prostaglandin-F2α (8-Iso-PGF), superoxide dismutase-2 (SOD2), glutathione peroxidase-1 (GPX1), peroxiredoxin isoforms (PRDX 1-6) and heat-shock-protein-70 (HSP70) were performed in biopsies taken from the vastus lateralis muscle. Under basal conditions, 8-Iso-PGF was significantly decreased in T2DM patients (-35.7%), whereas PRDX2 and PRDX6 were significantly increased relative to CON (+82.6%; +82.3%). Differences were neither observed in SOD2 nor in GPX1 or PRDX1, 3, 4, 5 density. Regular physical activity (moderate endurance or resistance training twice a week for 3 months) did not alter PRDX1, 2, 3, 4, 6 in the skeletal muscle of T2DM patients, but significantly increased SOD2 (+65.9%), GPX1 (+62.4%), PRDX5 (+37.5%), and HSP70 (+48.5%). Overweight/obese men with non-insulin-dependent T2DM exhibit up-regulated cytosolic peroxiredoxin contents relative to BMI-matched controls. Regular training further up-regulates cytosolic and mitochondrial antioxidative enzymes in T2DM patients and improves their cellular protection systems. This may contribute to a retardation of the disease's progression. |
| 2011 | Zinc pyrithione impairs zinc homeostasis and upregulates stress response gene expression in reconstructed human epidermis. | Biometals | Zinc ion homeostasis plays an important role in human cutaneous biology where it is involved in epidermal differentiation and barrier function, inflammatory and antimicrobial regulation, and wound healing. Zinc-based compounds designed for topical delivery therefore represent an important class of cutaneous therapeutics. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in over-the-counter topical antimicrobials, and has also been examined as an investigational therapeutic targeting psoriasis and UVB-induced epidermal hyperplasia. Recently, we have demonstrated that cultured primary human skin keratinocytes display an exquisite sensitivity to nanomolar ZnPT concentrations causing induction of heat shock response gene expression and poly(ADP-ribose) polymerase (PARP)-dependent cell death (Cell Stress Chaperones 15:309-322, 2010). Here we demonstrate that ZnPT causes rapid accumulation of intracellular zinc in primary keratinocytes as observed by quantitative fluorescence microscopy and inductively coupled plasma mass spectrometry (ICP-MS), and that PARP activation, energy crisis, and genomic impairment are all antagonized by zinc chelation. In epidermal reconstructs (EpiDerm™) exposed to topical ZnPT (0.1-2% in Vanicream™), ICP-MS demonstrated rapid zinc accumulation, and expression array analysis demonstrated upregulation of stress response genes encoding metallothionein-2A (MT2A), heat shock proteins (HSPA6, HSPA1A, HSPB5, HSPA1L, DNAJA1, HSPH1, HSPD1, HSPE1), antioxidants (SOD2, GSTM3, HMOX1), and the cell cycle inhibitor p21 (CDKN1A). IHC analysis of ZnPT-treated EpiDerm™ confirmed upregulation of Hsp70 and TUNEL-positivity. Taken together our data demonstrate that ZnPT impairs zinc ion homeostasis and upregulates stress response gene expression in primary keratinocytes and reconstructed human epidermis, activities that may underlie therapeutic and toxicological effects of this topical drug. |
| 2011 | Protective effect of quercetin, EGCG, catechin and betaine against oxidative stress induced by ethanol in vitro. | Exp Mol Pathol | There is a need for a nontoxic antioxidant agent to be identified which will prevent alcoholic liver disease (ALD) in alcoholic patients. We tested 4 candidate agents: quercetin, EGCG, catechin and betaine, all of which occur naturally in food. HepG2 cells overexpressing CYP2E1 were subjected to arachidonic acid, iron and 100mM ethanol with or without the antioxidant agent. All the agents prevented oxidative stress and MDA/4HNE formation induced by ethanol, except for EGCG. Catechin prevented CYP2E1 induction by ethanol. All the agents tended to down-regulate the ethanol-induced increased expression of glutathionine peroxidase 4 (GPX4). All the agents, except catechin, tended to reduce the expression of SOD2 induced by ethanol. Heat shock protein 70 was up-regulated by ethanol alone and betaine tended to prevent this. All 4 agents down-regulated the expression of Gadd45b in the presence of ethanol, which could explain the mechanism of DNA demethylation associated with the up-regulation of the gene expression observed in experimental ALD. In conclusion, the in vitro model of oxidative stress induced by ethanol provided evidence that all 4 agents tested prevented some aspect of liver cell injury caused by ethanol. |
| 2011 | Manganese superoxide dismutase dimorphism relationship with severity and prognosis in cardiogenic shock due to dilated cardiomyopathy. | Free Radic Res | The aim was to determine (a) Ala-16Val-SOD2 dimorphisms; (b) allelic frequency and phenotype of a common Pro-Leu polymorphism in GPx1, in a cohort of patients with a cardiogenic shock (CS) due to dilated cardiomyopathy without acute coronary syndrome. Consecutive patients with de novo CS that worsened a dilated (DCM) or ischemic (ICM) cardiomyopathy. Congenital heart disease, pacemaker and other shock aetiologies were excluded. To determine oxidative stress (OS), this study evaluated lipid peroxidation, protein oxidation and erythrocyte GPx, SOD and catalase activities. Ala16Val-SOD2 (dbSNP: rs4880) and Pro198Leu-GPx1 (dbSNP: rs1050450) polymorphisms were studied by allelic discrimination using fluorogenic probes and the 5'nuclease (TaqMan) assay. Twenty-four patients (with ICM (n = 8) or DCM (n = 16), age = 57.5 ± 10.7 years, LVEF = 25.3 ± 8.5%, NT-proBNP levels = 8540 ± 1703 ng/L) were included during a 15 month follow-up. OS parameters were significantly higher in patients than in controls. Distribution of MnSOD genotypes was 47% Val/Val-variant, 29.5% Ala/Val and 23.5% Ala/Ala-variants. Severity of CS was more important in patients with Val/Val-variant and can be put in parallel with NT-proBNP levels (Val/Val-variant: 11 310 ± 3875 ng/L vs Ala/Ala-variant: 6486 ± 1375 ng/L and Ala/Val-variant: 6004 ± 2228 ng/L; p < 0.05) and hemodynamic support duration (144.6 vs Ala/Val-variant: 108.8 h and Ala/Ala-variant: 52.5 h; p < 0.05) with a positive correlation (Spearman rho = 0.72, p < 0.05). Moreover, Val/Val-variant significantly influenced the mortality (Spearman rho = 0.67, p < 0.05), but not the morbidity (p = 0.3). Distribution of GPx genotypes was 64% Pro/Pro, 18% Pro/Leu and 18% Leu/Leu. GPx-variants influenced neither GPx activities nor cardiac events. In conclusion, CS was associated with markers of increased OS. GPx polymorphism did not influence the GPx activity. Only the Val-encoding MnSOD allele was significantly correlated with the severity and prognosis of CS. |
| 2010 | Biological effects of inorganic arsenic on primary cultures of rat astrocytes. | Int J Mol Med | It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO2) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 microM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 microM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed. Since arsenic is not known to be a direct-acting genotoxic agent, we investigated the possibility that its effects are due, in astrocytes as well, to ROS formation, as already described for other cell types. However, FACS analysis after CM-H2DCFDA staining did not evidence any significant increase of ROS production while, on the contrary, at the highest arsenite concentrations used, ROS production decreased. Concordantly, we found that, if most cells in the culture are still alive (i.e. up to 10 microM arsenite), they show a treatment-dependent increase in the concentration of SOD1. On the other hand, SOD2 concentration did not change. Finally, we found that astrocytes also synthesize PIPPin, an RNA-binding protein, the concentration of which was recently reported to change in response to stress induced by cadmium. Here we also report that, in cells exposed to high doses of arsenite, an anti-PIPPin antibody-positive faster migrating protein appears. |
| 2010 | The neurogenic basic helix-loop-helix transcription factor NeuroD6 confers tolerance to oxidative stress by triggering an antioxidant response and sustaining the mitochondrial biomass. | ASN Neuro | Preserving mitochondrial mass, bioenergetic functions and ROS (reactive oxygen species) homoeostasis is key to neuronal differentiation and survival, as mitochondria produce most of the energy in the form of ATP to execute and maintain these cellular processes. In view of our previous studies showing that NeuroD6 promotes neuronal differentiation and survival on trophic factor withdrawal, combined with its ability to stimulate the mitochondrial biomass and to trigger comprehensive antiapoptotic and molecular chaperone responses, we investigated whether NeuroD6 could concomitantly modulate the mitochondrial biomass and ROS homoeostasis on oxidative stress mediated by serum deprivation. In the present study, we report a novel role of NeuroD6 as a regulator of ROS homoeostasis, resulting in enhanced tolerance to oxidative stress. Using a combination of flow cytometry, confocal fluorescence microscopy and mitochondrial fractionation, we found that NeuroD6 sustains mitochondrial mass, intracellular ATP levels and expression of specific subunits of respiratory complexes upon oxidative stress triggered by withdrawal of trophic factors. NeuroD6 also maintains the expression of nuclear-encoded transcription factors, known to regulate mitochondrial biogenesis, such as PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator-1alpha), Tfam (transcription factor A, mitochondrial) and NRF-1 (nuclear respiratory factor-1). Finally, NeuroD6 triggers a comprehensive antioxidant response to endow PC12-ND6 cells with intracellular ROS scavenging capacity. The NeuroD6 effect is not limited to the classic induction of the ROS-scavenging enzymes, such as SOD2 (superoxide dismutase 2), GPx1 (glutathione peroxidase 1) and PRDX5 (peroxiredoxin 5), but also to the recently identified powerful ROS suppressors PGC-1alpha, PINK1 (phosphatase and tensin homologue-induced kinase 1) and SIRT1. Thus our collective results support the concept that the NeuroD6-PGC-1alpha-SIRT1 neuroprotective axis may be critical in co-ordinating the mitochondrial biomass with the antioxidant reserve to confer tolerance to oxidative stress. |
| 2010 | Astrocyte targeted overexpression of Hsp72 or SOD2 reduces neuronal vulnerability to forebrain ischemia. | Glia | Brief forebrain ischemia is a model of the delayed hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation. Previous studies demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes occurs hours to days before delayed neuronal death. In this study we tested the strategy of directing protection to astrocytes to protect neighboring neurons from forebrain ischemia. Two well-studied protective proteins, heat shock protein 72 (Hsp72) or superoxide dismutase 2 (SOD2), were genetically targeted for expression in astrocytes using the astrocyte-specific human glial fibrillary acidic protein (GFAP) promoter. The expression constructs were injected stereotacticly immediately above the hippocampal CA1 region on one side of the rat brain two days prior to forebrain ischemia. Cell type specific expression was confirmed by double label immunohistochemistry. When the expression constructs were injected two days before transient forebrain ischemia, the loss of CA1 hippocampal neurons observed seven days later was significantly reduced on the injected side compared with controls. This neuroprotection was associated with significantly better preservation of astrocyte glutamate transporter-1 immunoreactivity at 5-h reperfusion and reduced oxidative stress. Improving the resistance of astrocytes to ischemic stress by targeting either the cytosolic or mitochondrial compartment was thus associated with preservation of CA1 neurons following forebrain ischemia. Targeting astrocytes is a promising strategy for neuronal preservation following cardiac arrest and resuscitation. |
| 2009 | Changes in the cellular proteins of pulmonary alveolar macrophage infected with porcine reproductive and respiratory syndrome virus by proteomics analysis. | J Proteome Res | Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect and replicate preferentially in pulmonary alveolar macrophages (PAMs). We applied proteomic approaches to investigate the change in cellular proteins of PAMs infected with PRRSV in vitro. A total of 23 cellular proteins with significant alteration in different courses postinfection were identified. These proteins could be classified into the functions associated with morphogenesis, protein synthesis, metabolism, and stress response and ubiquitin-proteasome pathway. Of the altered proteins, two proteins, heat shock 27 kDa protein (HSP27) and superoxide dismutase 2 (SOD2), involved in stress response or ubiquitin-proteasome pathway were observed to be up-regulated. Our study is the first attempt to analyze the cellular protein profile of PRRSV-infected PAMs by proteomics and provide valuable information for better understanding the function alterations of PAMs induced by PRRSV infection. |
| 2008 | Identification of tumor necrosis factor signaling-related proteins during Epstein-Barr virus-induced B cell transformation. | Acta Virol | Epstein-Barr virus (EBV) infection in vitro transforms primary B cells into continuously proliferating lymphoblastoid cell lines (LCLs) that have been widely used as a genomic resource for variety of immunological and genetic studies. However, the biochemical and biological characteristics that distinguish LCLs from the B cells have not been thoroughly investigated. Our proteomic approach showed that EBV infection induced changes in the profiles of tumor necrosis factor (TNF) signaling-related proteins in LCLs including heat shock protein family members TNF receptor-associated protein 1 (TRAP-1), heat shock 70-kDa protein 9 (HSPA9)) and superoxide dismutase 2 (SOD2). In addition, our literature co-occurrence study placed TNF at the center of a gene cluster network of differentially expressed proteins in LCLs. This study suggested that deregulation of TNF signaling pathway could contribute to the cellular transformation and immortalization of the EBV-infected B cells. |
| 2008 | Differences in the protein expression in limbal versus central human corneal epithelium--a search for stem cell markers. | Exp Eye Res | In the search for potential limbal stem cell protein markers, the purpose of this study was to characterize differences in protein expression between human central and limbal corneal epithelium by a proteomic approach using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) combined with mass spectrometry (LC-MS/MS). The results were subsequently confirmed by Western blotting and immunohistochemistry. We detected more than 1000 protein spots in each gel. Thirty-two spots were significantly over-expressed in the central part and 70 spots were significantly over-expressed in the limbal part. We identified 25 different proteins. Among these 11 proteins representing different cellular locations and functions were selected for further investigations. Most interestingly, superoxide dismutase 2 (SOD2), was expressed in clusters of cells in the basal limbal epithelium. Heat shock protein 70 protein 1 (HSP70.1) and annexin I were highly abundant in limbal epithelium, although they were also present in the central epithelium to a minor extent. Among the proteins primarily expressed in the limbal fraction we further identified cytokeratin (CK) 15, CK19 and alpha enolase, which have been reported previously to be related to the limbal basal epithelium. The basal limbal epithelium consists of clusters of slow cycling limbal stem cells and rapid cycling transient amplifying cells. Ideally, proteins exclusively expressed in the limbal part of the epithelium may serve as markers for the basal limbal cells. SOD2 and CK15 identify clusters of limbal basal cells and therefore they may serve as markers for limbal stem cells in conjunction with the earliest transient amplifying cells. |
| 2008 | Troglitazone-induced hepatic mitochondrial proteome expression dynamics in heterozygous Sod2(+/-) mice: two-stage oxidative injury. | Toxicol Appl Pharmacol | The determinants of susceptibility to troglitazone-induced idiosyncratic liver injury have not yet been determined; however, troglitazone has been shown to target mitochondria and induce mitochondria-mediated hepatocellular injury in vitro. The aim of this study was to use a systems approach to analyze the dynamics of mitochondrial changes at the proteome level and more clearly define the mechanisms and time course of troglitazone hepatotoxicity by using a previously characterized mouse model that is highly sensitized to troglitazone hepatotoxicity. Mice heterozygous in mitochondrial superoxide dismutase-2 (Sod2(+/-)) were injected intraperitoneally with troglitazone (30 mg/kg/day) or vehicle daily for 2 or 4 weeks. Hepatic mitochondria were isolated, purified, and subjected to two-dimensional difference gel electrophoresis (2D-DIGE). We found that among the ~1500 resolved hepatic mitochondrial proteins, 70 exhibited significantly altered abundance after troglitazone treatment. MALDI-TOF/TOF MS/MS analysis revealed that early changes (2 weeks) included increased levels of heat shock protein family members (mortalin, HSP7C), Lon protease, and catalase, indicating induction of a mitochondrial stress response. In contrast, after 4 weeks, a number of critical proteins including ATP synthase beta-subunit, aconitase-2, and catalase exhibited decreased abundance, and total protein carbonyls were significantly increased, suggesting uncompensated oxidative damage. Aconitase-2 (ACO2) was decreased at both time points, making this protein a potential sensitive and early biomarker for mitochondrial oxidant stress. These results show that, in this murine model of underlying clinically silent mitochondrial stress, superimposed troglitazone induces a two-stage response: an initial adaptive response, followed by a toxic response involving oxidant injury to mitochondrial proteins. |
| Proteomic based identification of manganese superoxide dismutase 2 (SOD2) as a metastasis marker for oral squamous cell carcinoma. | Cancer Genomics Proteomics | Metastasis is a critical event in oral squamous cell carcinoma (OSCC) progression. To identify proteomic biomarkers for OSCC metastasis, 3 paired OSCC cell lines (UM1/UM2, 1386Tu/1386Ln, 686Tu/686Ln) with different metastatic potential were examined. Among those 3 cell lines, UM1, 1386Ln and 686Ln exhibited a higher degree of metastatic potential than their paired cell lines UM2, 1386Tu and 686Tu, respectively, as measured using an in vitro cell invasion assay. A total of 40 differentially expressed proteins were identified using 2D-PAGE/MS proteomic approach. Selected protein candidates (superoxide dismutase 2 and heat shock protein 27) were further investigated by immuno-histochemistry (IHC) method using independent OSCC patient tissue samples. The statistically significantly increases in IHC staining for manganese superoxide dismutase 2 (SOD2) were observed in lymph node metastatic disease when compared with the paired primary OSCC. Our results thus indicated that elevated SOD2 levels is associated with lymph node metastasis in OSCC and may provide predictive values for diagnosis of metastasis. |
| 2007 | Impaired cytoprotective mechanisms in eyes with pseudoexfoliation syndrome/glaucoma. | Invest Ophthalmol Vis Sci | Evidence suggests that chronically increased stress conditions in the anterior eye segment constitute major mechanisms involved in the pathobiology of pseudoexfoliation (PEX) syndrome. The expression of stress-related genes in eyes from patients with and without PEX syndrome/glaucoma was investigated to determine whether PEX syndrome is associated with an altered cellular stress response.cDNA array hybridization, quantitative real-time PCR, Western blot analysis, and immunohistochemistry were applied to analyze the mRNA and protein expression of stress-related genes in anterior segment tissues of PEX eyes, with and without glaucoma, and to compare them with normal and glaucomatous control eyes.Hybridization of cDNA arrays identified a set of stress-related candidate genes for differential expression in PEX syndrome/glaucoma, of which 10 were confirmed by real-time PCR in ciliary processes and iris tissue. The expression of MAPKp38, heat shock proteins (HSP40, HSP60), and superoxide dismutase (SOD2) was increased up to threefold in PEX specimens. In contrast, a large set of cytoprotective gene products, including antioxidant defense enzymes (glutathione S-transferases mGST1 and GSTT1), ubiquitin-conjugating enzymes (UBE2A, UBE2B), the DNA repair protein MLH1, and the stress-inducible transcription factor GADD153, were found to be consistently downregulated up to threefold in PEX specimens on both the mRNA and protein levels.The present findings provide evidence of alterations in cytoprotective mechanisms including antioxidant defense, proteasome function, endoplasmic reticulum-related stress response, and DNA repair in anterior segment tissues of PEX eyes. The resultant enhanced sensitivity and vulnerability to cellular stress conditions may therefore be one contributing factor in the pathobiology of PEX syndrome. |
| 2008 | Proteomic analysis of liver cancer cells treated with suberonylanilide hydroxamic acid. | Cancer Chemother Pharmacol | Suberonylanilide hydroxamic acid (SAHA) is an orally administered histone deacetylase inhibitor (HDACI) that has shown significant antitumor activity in a variety of tumor cells. To evaluate if SAHA has an activity against liver cancer, and with an aim to identify the altered cellular factors upon SAHA treatment, human HepG2 cancer cell line was used as a model, and proteomic approach was utilized to elucidate the molecular mechanisms underlying SAHA's antitumor activity.Cell growth inhibition was measured by MTT method, and apoptosis was detected by means of flow cytometry analysis and TUNEL assay. Protein expression profiles were analyzed by 2-DE coupled with MALDI-Q-TOF MS/MS analysis.A total of 55 differentially expressed proteins were visualized by 2-DE and Coomassie Brilliant Blue (CBB) staining. Of these, 34 proteins were identified via MS/MS analysis. Among the identified proteins, six proteins also displayed significant expression changes at earlier time points upon SAHA treatment, and such alterations were further confirmed by semi-quantitative RT-PCR. Together, at both the mRNA and protein levels, SAHA suppressed the expression of reticulocalbin 1 precursor (RCN1), annexin A3 (ANXA3) and heat shock 27 kDa protein 1 (HSP27), while increasing the expression of aldose reductase (AR), triosephosphate isomerase 1 (TPI) and manganese superoxide dismutase (SOD2).SAHA remarkably inhibited proliferation of HepG2 cancer cells, and induced apoptosis in vitro. Using proteomics approaches, a variety of differentially expressed proteins were identified in HepG2 cancer cells before and after treatment with SAHA. This study will enable a better understanding of the molecular mechanisms underlying SAHA-mediated antitumor effects at the protein level. |
| 2007 | Ste50 adaptor protein influences Ras/cAMP-driven stress-response and cell survival in Saccharomyces cerevisiae. | Curr Genet | The Ste50 adaptor protein is involved in a variety of cellular pathways that yeast cells use to adapt rapidly to environmental changes. A highly activated Ras-cyclic AMP (cAMP) pathway by deletion of the high-affinity cAMP-dependent phosphodiesterase 2 (PDE2) leads to repression of a stress mediated response and cell survival. Here we show that inactivation of STE50 confers a synthetic genetic interaction with pde2Delta. A hyperosmotic stress growth defect of ste50Delta pde2Delta cells is exacerbated by extracellular cAMP or by galactose as the sole carbon source in the medium. The inactivation of the serine/threonine protein-kinase Akt homologue Sch9 increase stress resistance and extend chronological life span. By pde2Delta-dependent increase of the Ras-cAMP pathway activity, inactivation of STE50 results in an extreme shortening of life span and oxidative stress sensitivity of sch9Delta mutants. Furthermore, sch9Delta can promote transcription of the small heat shock protein HSP26 in a PDE2-dependent manner; however, sch9Delta can promote transcription of the mitochondrial superoxide dismutase SOD2 in a PDE2- and STE50-dependent manner. These data indicate that inactivation of STE50 influences stress tolerance in mutants of the Ras-cAMP pathway, which is a major determinant of intrinsic stress tolerance and cell survival of the Saccharomyces cerevisiae. |
| 2007 | Comparative proteomic analysis with postmortem prefrontal cortex tissues of suicide victims versus controls. | J Psychiatr Res | The origin of suicidal behaviour is multifactorial including genetic, neurobiological and psychosocial correlates. Although there is no doubt that serotonin has a central role, the overall genetic findings with candidate genes of the serotonergic pathway are relatively inconsistent and suggests that other, yet unidentified, genes and gene products are also contributing to the vulnerability of suicidality. Proteomics is a powerful method to investigate modifications in protein expression.We performed comparative proteomic analysis with prefrontal cortex tissues of 17 suicide victims and 9 controls.Applying two dimensional gel electrophoresis and image analysis we detected five protein spots to differ significantly in intensities between both groups. Three of them appeared only in suicide victims and could be identified by means of MALDI-TOF-MS analysis and protein database search as alpha crystallin chain B (CRYAB), glial fibrillary acidic protein (GFAP) and manganese superoxide dismutase (SOD2). CRYAB belongs to the low molecular heat shock proteins and GFAP is known as a marker of astrocytic activation in gliosis. SOD2 is a major antioxidant enzyme protecting cells against oxidative injury. Two further spots revealed higher intensities in the control group but had no unambiguous protein to match.Our findings suggest that proteins, being involved in glial function, neurodegeneration and oxidative stress neuronal injury, might also have an impact upon the neurobiological cascade leading to suicidality. As animal data provide evidence for an up-regulation of GFAP synthesis in astrocytes due to alterations in 5-HT levels, similar mechanisms of interaction might also be relevant in humans. |
| 2006 | Dihydroxyacetone detoxification in Saccharomyces cerevisiae involves formaldehyde dissimilation. | Mol Microbiol | To investigate Saccharomyces cerevisiae physiology during growth on the conditionally toxic triose dihydroxyacetone (DHA), protein expression was studied in strains overexpressing either of the two dihydroxyacetone kinase isogenes, DAK1 or DAK2, that grow well utilizing DHA as a carbon and energy source. DHA metabolism was found mostly similar to ethanol utilization, involving a strong component of glucose derepression, but also involved DHA-specific regulatory changes. A specific and strong (10- to 30-fold induction of formaldehyde dehydrogenase, Fdhlp, indicated activation of the formaldehyde dissimilation pathway in DHA medium. The importance of this pathway was further supported by impaired adaptation to DHA growth and DHA survival in a glutathione-dependent formaldehyde dehydrogenase (SFA1) deletion mutant. Glutathione synthase (GSH1) deletion led to decreased DHA survival in agreement with the glutathione cofactor requirement for the SFA1-encoded activity. DHA toxicity did, however, not solely appear related to formaldehyde accumulation, because SFA1 overexpression only enhanced formaldehyde but not DHA tolerance. In further agreement with a low DHA-to-formaldehyde flux, GSH supplements in the low microM range also fully suppressed the DHA sensitivity of a gsh1Delta strain. Under growth reduction on high (100 mM) DHA medium we report increased levels of advanced glycation end-product (AGE) formation on total protein. Under these high-DHA conditions expression of several stress-related proteins, e.g. a heat-shock protein (Hsp104p) and the oxidative stress indicator, alkyl hydroperoxide reductase (Ahp1p) was also found induced. However, hallmark determinants of oxidative stress tolerance (e.g. YAP1, SKN7, HYR1/GPX3 and SOD2) were redundant for DHA tolerance, thus indicating mechanisms of DHA toxicity largely independent of central oxidative stress defence mechanisms. We conclude that mechanisms for DHA growth and detoxification appear complex and that the evolutionary strive to minimize detrimental effects of this intracellular metabolite links to both formaldehyde and glutathione metabolism. |
| 2005 | Response of fission yeast to toxic cations involves cooperative action of the stress-activated protein kinase Spc1/Sty1 and the Hal4 protein kinase. | Mol Cell Biol | Stress-activated protein kinases (SAPKs), members of a mitogen-activated protein kinase (MAPK) subfamily, are highly conserved among eukaryotes. Studies of yeasts demonstrated that SAPKs play pivotal roles in survival responses to high osmolarity, oxidative stress, and heat shock. Here we report a novel physiological role of the fission yeast Spc1 SAPK in cellular resistance to certain cations, such as Na(+), Li(+), and Ca(2+). Strains lacking Spc1 or its activator, Wis1 MAPK kinase, are hypersensitive to these cations. Spc1 positively regulates expression of sod2(+) encoding a Na(+)/H(+) antiporter through Atf1 and other transcription factors. In addition, we have identified a novel Spc1-interacting protein, Hal4, which is highly homologous to the budding yeast Sat4/Hal4 protein kinase. Like its budding yeast counterpart, the fission yeast Hal4 kinase is essential for cellular resistance to Na(+), Li(+), and Ca(2+). The hal4-null phenotype is complemented by overexpression of the Trk1 potassium transporter or increased K(+) in the growth medium, suggesting that Hal4 promotes K(+) uptake, which consequently increases cellular resistance to other cations. Interestingly, the Spc1-Hal4 interaction appears to be required for cellular resistance to Ca(2+) but not Na(+) and Li(+). We propose that Spc1 SAPK and Hal4 kinase cooperatively function to protect cells from the toxic cations. |
| 2005 | Selective neuronal vulnerability and inadequate stress response in superoxide dismutase mutant mice. | Free Radic Biol Med | To understand the role of oxidative stress and mitochondrial defects in the development of neurodegeneration, we examined the age-related pathological changes and corresponding gene expression profiles in homozygous mutant mice deficient in the mitochondrial form of superoxide dismutase (MnSOD, SOD2). These Sod2-/- mice, generated on a B6D2F1 background, developed ataxia at Postnatal Day (P) 11 and progressively deteriorated with frequent seizures by P14. Histopathological examination revealed neurodegenerative changes consistent with the neurological signs. Vacuolar degeneration was observed in neurons and neuropil throughout the brainstem and rostral cortex. The motor trigeminal nucleus in brainstem and the deeper layers of the motor cortex were the earliest regions to degenerate, with the thalamus and hippocampus affected at later stages. Oligonucleotide microarrays were used to compare gene expression profiles in the brainstem and thalamus of Sod2+/+ and -/- mice from birth to P18. Notably, a large set of heat-shock protein genes was transcriptionally down regulated, and this was most likely due to a reduction in the heat-shock transcription factor 1 (HSF1). Other major classes of differentially expressed genes include lipid biosynthesis and ROS metabolism. |
| 2002 | Heat-shock protein 25 (Hspb1) regulates manganese superoxide dismutase through activation of Nfkb (NF-kappaB). | Radiat Res | We previously demonstrated that overexpression of HSP25 (now known as Hspb1) conferred increased resistance to ionizing radiation (Radiat Res. 154, 421-428, 2000). In the present study, L929 cells overexpressing Hspb1 were shown to have increased expression of the manganese superoxide dismutase gene (now known as SOD2) and its enzyme activity. To elucidate Hspb1-induced pathways leading to activation of these antioxidant enzymes, the production of the tumor necrosis factor alpha (Tnf) and interleukin 1 beta (Il1b) genes was examined. Increased expression of Tnf and Il1b resulting from Hspb1 overexpression was detected by RT-PCR. Increased activation of Nfkb (degradation of Ikb, a member of the Nfkb family) was also found in Hspb1-overexpressing cells. When treated with Tnf, Nfkb activation and SOD2 gene expression were increased more by Hspb1 overexpression. Moreover, transfection with the Hspb1 antisense gene abrogated all of the Hspb1-mediated phenomena. To further elucidate the exact relationship between induction of SOD2 and Nfkb activation, a dominant negative I-kBalpha (now known as Nfkb1a) construct was transfected into Hspb1-overexpressing cells. The dominant negative Nfkb1a inhibited Hspb1-mediated SOD2 gene expression. In addition, Hspb1-mediated radioresistance was blocked by dominant negative Nfkb1a transfection. When the SOD2 gene was transfected into L929 cells, a somewhat increased radioresistance was detected by a clonogenic survival assay compared to control cells. Hspb1 produced Tnf and Il1b and facilitated SOD2 gene expression through Nfkb activation, possibly resulting in Hspb1-mediated radioresistance. |
| 2002 | Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically. | Toxicol Sci | Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir. |
| 2001 | Acquisition of tolerance against oxidative damage in Saccharomyces cerevisiae. | BMC Microbiol | Living cells constantly sense and adapt to redox shifts by the induction of genes whose products act to maintain the cellular redox environment. In the eukaryote Saccharomyces cerevisiae, while stationary cells possess a degree of constitutive resistance towards oxidants, treatment of exponential phase cultures with sub-lethal stresses can lead to the transient induction of protection against subsequent lethal oxidant conditions. The sensors of oxidative stress and the corresponding transcription factors that activate gene expression under these conditions have not yet been completely identified.We report the role of SOD1, SOD2 and TPS1 genes (which encode the cytoplasmic Cu/Zn-superoxide dismutase, the mitochondrial Mn-isoform and trehalose-6-phosphate synthase, respectively) in the development of resistance to oxidative stress. In all experimental conditions, the cultures were divided into two parts, one was immediately submitted to severe stress (namely: exposure to H2O2, heat shock or ethanol stress) while the other was initially adapted to 40 degrees C for 60 min. The deficiency in trehalose synthesis did not impair the acquisition of tolerance to H2O2, but this disaccharide played an essential role in tolerance against heat and ethanol stresses. We also verified that the presence of only one Sodp isoform was sufficient to improve cellular resistance to 5 mM H2O2. On the other hand, while the lack of Sod2p caused high cell sensitivity to ethanol and heat shock, the absence of Sod1p seemed to be beneficial to the process of acquisition of tolerance to these adverse conditions. The increase in oxidation-dependent fluorescence of crude extracts of sod1 mutant cells upon incubation at 40 degrees C was approximately 2-fold higher than in sod2 and control strain extracts. Furthermore, in Western blots, we observed that sod mutants showed a different pattern of Hsp104p and Hsp26p expression also different from that in their control strain.Trehalose seemed not to be essential in the acquisition of tolerance to H2O2 stress, but its absence was strongly felt under water stress conditions such as heat and alcoholic stresses. On the other hand, Sod1p could be involved in the control of ROS production; these reactive molecules could signal the induction of genes implicated within cell tolerance to heat and ethanol. The effects of this deletion needs further investigation. |
| 2000 | Role of glutathione in heat-shock-induced cell death of Saccharomyces cerevisiae. | Biochem J | Previously we reported that expression of GSH1 (gamma-glutamylcysteine synthetase) and GSH2 (glutathione synthetase) of the yeast Saccharomyces cerevisiae was increased by heat-shock stress in a Yap1p-dependent fashion and consequently intracellular glutathione content was increased [Sugiyama, Izawa and Inoue (2000) J. Biol. Chem. 275, 15535-15540]. In the present study, we discuss the physiological role of glutathione in the heat-shock stress response in this yeast. Both gsh1 and gsh2 mutants could acquire thermotolerance by mild heat-shock stress and induction of Hsp104p in both mutants was normal; however, mutant cells died faster by heat shock than their parental wild-type strain. After pretreatment at a sublethal temperature, the number of respiration-deficient mutants increased in a gsh1 mutant strain in the early stages of exposure to a lethal temperature, although this increase was partially suppressed by the addition of glutathione. These results lead us to suspect that an increase of glutathione synthesis during heat-shock stress is to protect mitochondrial DNA from oxidative damage. To investigate the correlation between mitochondrial DNA damage and glutathione, mitochondrial Mn-superoxide dismutase (the SOD2 gene product) was disrupted. As a result, the rate of generation of respiration-deficient mutants of a sod2 delta strain was higher than that of the isogenic wild-type strain and treatment of the sod2 delta mutant with buthionine sulphoximine, an inhibitor of glutathione synthesis, inhibited cell growth. These results suggest that glutathione synthesis is induced by heat shock to protect the mitochondrial DNA from oxidative damage that may lead to cell death. |
| 1995 | Characterization of Na+/H(+)-antiporter gene closely related to the salt-tolerance of yeast Zygosaccharomyces rouxii. | Yeast | In order to clarify the relationship between salt-tolerance of Zygosaccharomyces rouxii and the function of Na+/H(+)-antiporter, a gene was isolated from Z. rouxii which exhibited homology to the Na+/H(+)-antiporter gene (sod2) from Schizosaccharomyces pombe. This newly isolated gene (Z-SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by its Sz. pombe homologue. The predicted amino-acid sequence of Z-Sod2p was highly homologous to that of the Sz. pombe protein, but included an extra-hydrophilic stretch in the C-terminal region. The expression of Z-SOD2 was constitutive and independent of NaCl-shock. Z-SOD2-disruptants of Z. rouxii did not grow in media supplemented with 3 M-NaCl, but grew well in the presence of 50% sorbitol, indicating that the function of Z-SOD2 was closely related to the salt-tolerance of Z. rouxii. Several genes are also compared and discussed in relation to the salt-tolerance of Z. rouxii. |