Copper-zinc Superoxide Dismutase Research Articles

Low protein diet influences mineral absorption and utilization in medium-growing yellow-feathered broilers from 1 to 30 days of age

Reduced-protein diet can save protein ingredients and reduce nitrogen (N) losses. However, the effect of low protein diet on the mineral uptake and utilization in broilers needs to be explored. The aim of this study was to investigate the effect of low-protein diet on the growth performance, N deposition, mineral contents in serum, tissues and excreta, and the activities and gene expression of related enzymes in tissues of medium-growing yellow-feathered broilers, so as to elucidate the relationship between dietary protein level and the absorption and utilization of minerals in broilers. A total of 72 1-d-old Spotted-Brown male broilers were randomly allotted to 1 of 2 treatments with 6 replicate cages of 6 birds per cage for each treatment. The dietary crude protein (CP) levels for the two treatments were 21 % (the control treatment) and 19 % (low protein treatment), respectively. The experimental period was 30 d. The results showed that no differences (P > 0.05) were detected in average daily gain, average daily feed intake and feed: gain ratio of broilers during 1 to 30 d between the two treatments. However, low protein intake increased (P < 0.05) N retention rate, serum P, Cu and Mn, and excreta Cu, Mn and Zn, and decreased (P < 0.05) liver P and excreta P. In addition, birds fed low protein diet had higher (P < 0.05) manganese superoxide dismutase, and total superoxide dismutase activities in liver, and total antioxidant capacity and malondialdehyde content in heart, and lower (P < 0.05) copper-zinc superoxide dismutase (CuZnSOD) and succinate dehydrogenase activities in liver and CuZnSOD mRNA level in heart. In conclusion, the reduction of dietary CP content from 21 % to 19 % improved N retention, the absorption of P, Cu and Mn, as well as the antioxidant ability of liver and heart, and influenced metabolic utilization of P, Cu, Zn, Fe and Mn in medium-growing yellow-feathered broilers from 1 to 30 d of age.

Amyloid fibril structures and ferroptosis activation induced by ALS-causing SOD1 mutations.

Over 200 genetic mutations in copper-zinc superoxide dismutase (SOD1) have been linked to amyotrophic lateral sclerosis (ALS). Among these, two ALS-causing mutants, histidine-46→arginine (H46R) and glycine-85→arginine (G85R), exhibit a decreased capacity to bind metal ions. Here, we report two cryo-electron microscopy structures of amyloid fibrils formed by H46R and G85R. These mutations lead to the formation of amyloid fibrils with unique structures distinct from those of the native fibril. The core of these fibrils features a serpentine arrangement with seven or eight β strands, secured by a hydrophobic cavity and a salt bridge between arginine-85 and aspartic acid-101 in the G85R fibril. We demonstrate that these mutant fibrils are notably more toxic and capable of promoting the aggregation of wild-type SOD1 more effectively, causing mitochondrial impairment and activating ferroptosis in cell cultures, compared to wild-type SOD1 fibrils. Our study provides insights into the structural mechanisms by which SOD1 mutants aggregate and induce cytotoxicity in ALS.

Protective effect of betaine supplementation in low-met diet against Aeromonas hydrophila-induced intestinal injury in grass carp (Ctenopharyngodon idella)

This study evaluated the effect of supplemental betaine in low-methionine (Met) diet concerning the regulatory role of bacteria-induced intestinal injury in fish. 540 grass carp (Ctenopharyngodon Idella) (210 ± 0.68 g) were selected and randomly divided into 6 groups, which contained a reference group fed with the diet supplemented with DL-Met (3.1 g kg−1 diet) and 5 groups fed on the diets added with increasing levels of betaine (0–6.4 g kg−1 diet) for 60 days. Then, a 14-day Aeromonas hydrophila challenge was conducted. Compared with BET0 group, results indicated that appropriate betaine supplementation (1) enhanced antioxidant enzymes activities (except glutathione S-transferase pi-1 (GSTP1) and copper zinc superoxide dismutase (CuZnSOD) and glutathione (GSH) contents, and NF-E2-related-factor 2 (Nrf2) protein nuclear translocation, while reduced reactive oxygen species (ROS), and representative oxidative damage markers; (2) up-regulated the transcription abundance of anti-apoptosis-related molecules while reduced DNA-ladder occurrence and the transcription levels of pro-apoptosis-related genes; (3) down-regulated the transcription of tight junction-related proteins (such as zonula occludens-1 (ZO-1) etc.) in fish intestines. Furthermore, correlation analysis revealed that feeding betaine ameliorating the oxidative damage, apoptosis and tight junction breakdown was possibly related to intestinal Nrf2, p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinases (JNK), and myosin light chain kinase (MLCK) signaling, respectively. According to the regression analysis for intestinal ROS abundance, claudin-b and caspase-2 transcription, the appropriate betaine supplementations were estimated to be 3.82, 3.98, and 3.62 g kg−1 diet, respectively. Notably, betaine has the potential to spare part Met and the efficacy of betaine relative to Met was 206.67 % based on intestinal ROS contents in grass carp (210–776 g).

Evaluation of the Dietary Arginine Supplementation on Yellow Catfish: From a Low-Temperature Farming Perspective

The yellow catfish is an economically significant freshwater fish with increasing importance in aquaculture. However, the low temperature environments prevalent in certain regions pose challenges to its growth, development, and overall health. This study aimed to explore the impact of dietary arginine (Arg) addition on the growth, digestive capacity, and intestinal antioxidant response in fish under low temperature acclimation (18 °C). Total 720 fish were randomly distributed into six groups, each containing 120 fish. Over the course of eight weeks, each group was fed with diets about varying Arg concentrations (1.79–3.26 g/kg). The results indicated that Arg supplementation resulted in an increase in specific growth rate (SGR), feed intake (FI), feed efficiency (FE), as well as pancreatic enzyme activities in both pancreas and intestine. Conversely, malondialdehyde (MDA) and protein carbonyl (PC) contents initially decreased but increased with higher Arg concentrations. Glutathione peroxidase 1a (GPX1a) showed a positive correlation with nuclear factor-erythroid 2-related factor-2 (Nrf2), showing its role in antioxidative capacity. Furthermore, this study revealed that Arg significantly enhanced the activities of anti-superoxide anion, anti-hydroxyl radical, and anti-oxidative enzymes, along with the relative mRNA abundance of Copper-Zinc superoxide dismutase (CuZnSOD), catalase, GPX1a, glutamate-cysteine ligase catalytic subunit (GCLC), and Nrf2 in the intestine. It was determined that yellow catfish weighing between 61.0 g and 89.0 g require an intake of 26.8 g of Arg per kilogram of diet based on polynomial regression analysis of specific growth rate (SGR), which is equivalent to 37.0 g of dietary protein, under sub-low temperature conditions of 18 °C.

Evolutionary Conserved and Divergent Responses to Copper Zinc Superoxide Dismutase Inhibition in Plants.

After an initial evolution in a reducing environment, life got successively challenged by reactive oxygen species (ROS), especially during the great oxidation event (GOE) that followed the development of photosynthesis. Therefore, ROS are deeply intertwined into the physiological, morphological and transcriptional responses of most present-day organisms. Copper-zinc superoxide dismutases (CuZnSODs) evolved during the GOE and are present in charophytes and extant land plants, but nearly absent from chlorophytes. The chemical inhibitor of CuZnSOD, lung cancer screen 1 (LCS-1), could greatly facilitate the study of SODs in diverse plants. Here, we determined the impact of chemical inhibition of plant CuZnSOD activity, on plant growth, transcription and metabolism. We followed a comparative approach by using different plant species, including Marchantia Polymorpha and Physcomitrium patens, representing bryophytes, the sister lineage to vascular plants, and Arabidopsis thaliana. We show that LCS-1 causes oxidative stress in plants and that the inhibition of CuZnSODs provoked a similar core response that mainly impacted glutathione homoeostasis in all plant species analysed. That said, Physcomitrium and Arabidopsis, which contain multiple CuZnSOD isoforms showed a more complex and exacerbated response. In addition, an untargeted metabolomics approach revealed a specific metabolic signature for each plant species. Our comparative analysis exposes a conserved core response at the physiological and transcriptional level towards LCS-1, while the metabolic response largely varies. These differences correlate with the number and localization of the CuZnSOD isoforms present in each species.

Effect of increasing levels of Hermetia illucens in a fishmeal-free diet at sea bream (Sparus aurata, L.) gastrointestinal level

The impact of a fishmeal-free, high plant-protein-based diet, in which vegetable proteins (VPs) were partially substituted with the proteins derived from Hermetia illucens pupae meal (Hi) was examined in the gilthead sea bream (Sparus aurata). The physiological response at the gastrointestinal level was investigated by considering the gene expression responses and histology at the liver, stomach, pyloric caeca, foregut, midgut, and hindgut. To this end, three iso-proteic (crude protein, Nx6.25, 45 %) and iso-lipidic (crude fat, 20 %) extruded diets were formulated. A control diet (CV), comprising a substantial proportion of vegetable plant-protein (VPs) derivatives, was formulated alongside two test diets. These were created by replacing graded levels of the VPs (20 and 40 %, respectively) with protein derived from Hi (Hi20 and Hi40, respectively). In all dietary treatments, the foregut, midgut, and hindgut exhibited general structural integrity, displaying intact intestinal folds, enterocytes, and no signs of inflammation. However, occasional mild lymphocytic infiltrations of the epithelium and lamina propria were observed in the pyloric caeca of the CV and Hi20 diets. The histological examination of the stomach revealed that the CV diet had induced moderately-severe lymphocytic gastritis, accompanied by a down-regulation of heat shock protein 70 (hsp70). Moderate lymphocytic infiltrations were observed in liver of fish fed with the CV diet, which correlated to a down-regulation of copper-zinc superoxide dismutase (Sod1). In the hindgut, an increase in the expression of Sod1, manganese superoxide dismutase (Sod2), and intestinal alkaline phosphatase (iap) and a decrease in the expression of hsp70 were observed in insect meal-containing diet relative to the CV. Overall, the present study showed that in sea bream a plant-protein based diet lacking in fishmeal negatively affects stomach mucosa, but HI included in a plant-protein-based diet helps in ameliorate histological distortion of tissue through specific correlated gene activations.

Bioinformatics analysis reveals SOD1 is a prognostic factor in lung adenocarcinoma.

Lung cancer is a major cause of cancer-related deaths worldwide. Unfortunately, non-small cell lung cancer (NSCLC) often lacks clear clinical symptoms and molecular markers for early diagnosis, which can hinder the initiation of timely treatments. In this study, we conducted an extensive bioinformatics analysis of copper-zinc superoxide dismutase (SOD1), a molecule linked to lung adenocarcinoma (LUAD) to enhance early detection and treatment approaches for this condition. A bioinformatics analysis was conducted using a dataset from The Cancer Genome Atlas (TCGA) database. Several analytical methods, such as a differential expression analysis, a Kaplan-Meier survival analysis, a clinicopathological analysis, an enrichment analysis, protein-protein interaction (PPI) network construction using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and an immunoreactivity analysis of SOD1 expression in LUAD using TIMER were employed. We further validated the expression of SOD1 in LUAD through in vitro experiments using quantitative polymerase chain reaction (qPCR) and Western blot. Our findings indicate that LUAD tissues exhibited significantly higher expression levels of SOD1 than healthy tissues. The univariate Cox analysis showed that the elevated level was linked to unfavorable overall survival (OS) rates. Further, the Cox regression analysis of multiple variables suggested that elevated SOD1 expression levels acted as an autonomous prognosticator for unfavorable OS. We also conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, and a gene set enrichment analysis (GSEA) and observed differential pathway enrichment among patients with high SOD1 expression. In addition, a correlation between SOD1 and immune cell infiltration was found. The in vitro experiments confirmed that SOD1 expression was upregulated in LUAD. SOD1 could serve as a reliable prognostic indicator in individuals diagnosed with LUAD. Our findings may prove valuable in the development of therapeutic and prognostic markers for LUAD. The potential clinical utility of SOD1 in LUAD requires further investigation.

In-silico analysis of ethanol peel extract of Citrus sinensis on mercury chloride-induced prefrontal cortex damage in Wistar rat

This study evaluated the effect of ethanolic extract of Citrus sinensis peel on mercury chloride-induced changes in the prefrontal cortex of adult Wistar rats. Twenty-five Wistar rats were purchased and divided into five groups containing five animals in each group. Group 1 was administered with distilled water, Group 2, Group 3, Group 4, and Group 5 were administered with 5 mg/kg of mercury chloride only, 5 mg/kg of mercury chloride and 140 mg/kg Silymarin, 5 mg/kg mercury chloride and 1000 mg/kg of ethanoic extract of orange peel, and 5 mg/kg mercury chloride and 1500 mg/kg of ethanoic extract of orange peel. All administration was done orally and the administration lasted for 14 days. The animals were sacrificed humanely using 75 mg/kg ketamine intraperitoneally and prefrontal was dissected out. Part was homogenized in 0.1M phosphate buffered saline for the estimation of superoxide dismutase (SOD), and catalase (CAT) activities, and Malondialdehyde (MDA) level and the other part was fixed by immersion in 10 neutral buffered formal-saline for histological evaluation. The chemical structure of sixteen compounds from Citrus sinensis peel obtained from GC-MS analysis reported in the literature were retrieved from the PubChem compound database and the crystal structure of human peroxisome proliferator- activated receptor gamma (PPARγ; PDB:ID: 2P4Y) reported to increase the expression of catalase gene, human extracellular copper-zinc superoxide dismutase enzyme (PDB ID: 2JLP) and mammalian 15-lipoxygenase - 1 enzyme (PDB ID: 2P0M) responsible for the production of lipid peroxidation were retrieved from the PDB database. Ligands and protein preparation, docking and MM-GBSA relative binding free energy calculation were done in Maestro 2023.1. Data obtained was analyzed using the IBM SPSS (version 25) using one-way ANOVA and p-value &lt; 0.05 was considered statistically significant. The results indicate that the MDA level in control Group(13.29±1.183 μmol) is statistically significantly lower compared to the Group 2 (21.88±1.96 μmol), group 3 (27.44±3.52 μmol), Group 4 (22.20±1.16 μmol) and Group 5 (25.40±3.13 μmol). No statistically significant difference was observed in SOD activity. The CAT activities in control group (53.51±6.83 nmol/ml) was statistically significantly lower compared to the group 2 (26.21±3.70 nmol/ml), Group 3 (27.17±5.32 nmol/ml), Group 4 (25.30±4.24 nmol/ml) and Group5 (26.59±4.22 nmol/ml).The present study suggests, ethanolic extract of Citrus sinensis peel have protective effects on mercury-induced changes in prelimbic part of the prefrontal cortex of adult Wistar rats.

Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) Ameliorate Heart Failure through Reductions in Oxidative Stress: A Systematic Review and Meta-Analysis.

The objectives of this study were to explore the role that eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) plays in heart failure (HF), highlighting the potential connection to oxidative stress pathways. Following PRISMA guidelines, we conducted electronic searches of the literature in MEDLINE and EMBASE focusing on serum EPA and/or DHA and EPA and/or DHA supplementation in adult patients with heart failure or who had heart failure as an outcome of this study. We screened 254 studies, encompassing RCTs, observational studies, and cohort studies that examined HF outcomes in relation to either serum concentrations or dietary supplementation of EPA and/or DHA. The exclusion criteria were pediatric patients, non-HF studies, abstracts, editorials, case reports, and reviews. Eleven studies met our criteria. In meta-analyses, high serum concentrations of DHA were associated with a lower rate of heart failure with a hazard ratio of 0.74 (CI = 0.59-0.94). High serum concentrations of EPA also were associated with an overall reduction in major adverse cardiovascular events with a hazard ratio of 0.60 (CI = 0.46-0.77). EPA and DHA, or n3-PUFA administration, were associated with an increased LVEF with a mean difference of 1.55 (CI = 0.07-3.03)%. A potential explanation for these findings is the ability of EPA and DHA to inhibit pathways by which oxidative stress damages the heart or impairs cardiac systolic or diastolic function producing heart failure. Specifically, EPA may lower oxidative stress within the heart by reducing the concentration of reactive oxygen species (ROS) within cardiac tissue by (i) upregulating nuclear factor erythroid 2-related factor 2 (Nrf2), which increases the expression of antioxidant enzyme activity, including heme oxygenase-1, thioredoxin reductase 1, ferritin light chain, ferritin heavy chain, and manganese superoxide dismutase (SOD), (ii) increasing the expression of copper-zinc superoxide dismutase (MnSOD) and glutathione peroxidase, (iii) targeting Free Fatty Acid Receptor 4 (Ffar4), (iv) upregulating expression of heme-oxygenase-1, (v) lowering arachidonic acid levels, and (vi) inhibiting the RhoA/ROCK signaling pathway. DHA may lower oxidative stress within the heart by (i) reducing levels of mitochondrial-fission-related protein DRP-1(ser-63), (ii) promoting the incorporation of cardiolipin within the mitochondrial membrane, (iii) reducing myocardial fibrosis, which leads to diastolic heart failure, (iv) reducing the expression of genes such as Appa, Myh7, and Agtr1α, and (v) reducing inflammatory cytokines such as IL-6, TNF-α. In conclusion, EPA and/or DHA have the potential to improve heart failure, perhaps mediated by their ability to modulate oxidative stress.

Flaxseed Oil, Casitose, and Its Combination Diets Increase Antioxidant Activity and Upregulates Aging-related Genes in Drosophila melanogaster

Abstract Background: Nutrigenomics refers to the study of how diet influences the genomic expression and function of an organism. Diet is a significant factor that impacts metabolism, aging, and lifespan. Long-term dietary patterns can influence the risk of age-related diseases, including diabetes, cardiovascular diseases, hypertension, and cancer. While research in Drosophila melanogaster has focused on areas such as calorie restriction, high-sugar and high-fat diets, micronutrients, and disease-specific nutritional interventions, the effects of different combinations of macronutrients have not been adequately studied. This study aimed to analyze the impact of individual macronutrients, specifically flaxseed oil (a source of fat) and casitose (hydrolyzed milk protein), as well as their combinations, on longevity, antioxidant activity, and the expression of aging-related genes in D. melanogaster. Methods: The study examined the in vivo effects of different concentrations of flaxseed oil (120 μl, 240 μl, 480 μl, and 960 μl), casitose (125 mg, 250 mg, 500 mg, and 1000 mg), and their combinations with a base diet on longevity, antioxidant activity, and the expression of aging-related genes, namely manganese superoxide dismutase (Mn-SOD or SOD1) and copper–zinc SOD (Cu-Zn SOD or SOD2) in D. melanogaster. Results: The study found that flaxseed oil, casitose, and their combinations increased lipid peroxidation (LPO) activity, leading to the production of reactive oxygen species. However, these substances also enhanced the activity of the antioxidant enzymes SOD and catalase, which help combat-free radicals. Furthermore, the gene expression of SOD1 and SOD2 was upregulated, suggesting a potential mechanism for the observed improvements in lifespan. Interestingly, high doses of flaxseed oil (960 μl) were associated with decreased lifespan in the flies. Conclusion: The findings indicate that the optimal levels of flaxseed oil, casitose, or their combination can increase lifespan in Drosophila by upregulating the expression of the antioxidant enzymes SOD1 and SOD2. This provides insights into the potential of targeted nutrient combinations as a dietary approach to combat aging-related diseases.

Pro-inflammatory cytokines and antioxidative enzymes as salivary biomarkers of dentofacial infections in children.

Dentofacial infection resulting from untreated dental caries or periodontal disease is a serious disease that can spread to deeper tissues of the face and neck. The present study aimed to analyze the salivary cytokine profile and oxidative stress parameters as potential biomarkers of acute odontogenic infections in children. The prospective study group (DI) consisted of 28 children aged 3-17 years with acute dentofacial infections, and the control group (CG) comprised 52 children aged 4-17 years with uncomplicated dental caries. The cytokine profile was analyzed using the Bio-Plex Pro™ Human Cytokine 27-plex kit. In addition, oxidative stress parameters, such as catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), manganese SOD (Mn-SOD), copper-zinc SOD (CuZn-SOD), total antioxidant capacity (TAC), total oxidant status (TOS), and malondialdehyde (MDA), in the saliva of children in both groups were compared. The levels of interleukin 6 (IL-6), macrophage inflammatory protein 1 alpha (MIP-1α) and tumor necrosis factor alpha (TNF-α) were significantly increased in children with dentofacial infections as compared to CG. In contrast, the levels of other pro-inflammatory cytokines, such as IL-1β, IL-1 receptor agonist (IL-Ra), IL-8, monocyte chemoattractant protein 1 (MCP-1), and MIP-1β, did not show statistically significant differences between the 2 groups. Among the measured oxidative stress and antioxidative parameters, only CAT and GR were elevated in children with dentofacial infections as compared to controls. IL-6, MIP-1α, TNF-α, CAT, and GR can serve as selective biomarkers of oral cavity inflammation in children. These biomarkers can be useful in identifying and monitoring the progress and treatment of bacterial infections resulting in dentofacial inflammation.

Elevated Renal Oxidative Stress and Na+ Transporters are Associated with Hypertension in Postpartum Preeclamptic Rats

Background: Approximately 5-10% of US pregnancies result in preeclampsia (PE). PE is characterized by new onset hypertension (HTN) during pregnancy and is usually accompanied by end-organ damage, especially in the kidneys. Postpartum (PP) women and dams that had PE have an increased risk of developing HTN and chronic kidney disease (CKD) later in life. However, mechanisms linking PE to the long-term development of HTN and CKD are unknown. One aspect that may contribute to renal injury in PP PE women and dams is oxidative stress. Elevated concentrations of oxidative stress have been shown to augment the abundance and activity of renal transporters to increase sodium (Na+) reabsorption and blood volume. These alterations in renal transporters can consequently increase Na+ and water to facilitate HTN. Thus, we hypothesize that at 6 weeks PP (~3 human years), PE dams will display increased oxidative stress, renal Na+ transporter abundance, and elevated blood pressure (BP). Methods: Pregnant Sprague Dawley rats were randomly assigned to two groups: normal (CON) and PE dams. On gestational day 14, the reduced uterine perfusion pressure surgery was performed to mimic placental ischemia and generate a model of PE. Dams gave birth naturally and weaned for 3 weeks. After 6 weeks PP, BP was measured via carotid catheterization, and kidneys were removed and sectioned. Western blots were used to quantify renal Na+ transporters: Na+ K+ 2Cl− transporter (NKCC2) in the kidney medulla (KM) and epithelial Na+ channel (ENaC) in both the kidney cortex (KC) and KM. Oxidative stress was evaluated by heat shock protein 1 (HSP-1), copper zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) via Western blots. Hydrogen peroxide (H2O2) and antioxidant capacity concentrations were assessed via colorimetric assays. Results: PP PE dams had increased BP (126.3±6.18vs105.7±3.74 mmHg, p&lt;0.05) at 6 weeks after birth. KC HSP-1, H2O2, MnSOD, and antioxidant capacity were unchanged between groups. However, KC CuZnSOD protein abundance was decreased in PP PE dams (69.51±11.64vs100±5.73 IU/Protein/Control%, p&lt;0.05). In the KM, HSP-1 abundance (113.7±3.3vs100±5.07 IU/Protein/Control%, p=0.06) and H2O2 concentrations (1.97±0.11vs1.31± 0.38 nM H2O2/mg Protein, p=0.08) were elevated in PP PE dams. MnSOD, CuZnSOD, and antioxidant capacity were unchanged between groups in the KM. No changes occurred in KC and KM ENaC protein abundance. However, NKCC2 protein abundance was elevated by ~50% in PP PE dams (151.71±22.17vs100±5.59 IU/Protein/Control%, p=0.06). Conclusion: In summary, BP, oxidative stress, and NKCC2 were elevated in PP PE dams at 6 weeks. Perhaps, the presence of oxidative stress in the KM may lead to increased NKCC2 abundance. However, more studies are warranted to make this conclusion. NKCC2 elevation may result in increased Na+ and water reabsorption, leading to an increase in BP. Future studies will assess the role of renal oxidative stress to regulate Na+ transporters in PP PE dams and determine the timeline after birth (PP) in which changes in oxidative stress, Na+ transporters, and BP occur. In summary, this study is clinically relevant because it indicates that both oxidative stress and NKCC2 in the KM, separately or together, may have a formative role in the pathogenesis of HTN and CKD in PP PE women later in life. This research was supported by the Neurobiology of Aging and Alzheimer’s Disease Training Program (funded by NIH training grant T32 AG020494). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Electrochemical biosensing platform based on AuNWs/rGO-CMC-PEDOT:PSS composite for the detection of superoxide anion released from living cells

Detection of superoxide anion (O2·-) levels holds significant importance for the diagnosis and even clinical treatments of oxidative stress-related diseases. Herein, we prepared a composite electrode material to encapsulate copper-zinc superoxide dismutase (SOD1) for biosensing of O2·-. The sensing material consists of gold nanowires (AuNWs), reduced graphene oxide (rGO), carboxymethyl cellulose (CMC) and PEDOT:PSS. CMC provides abundant -COOH to bind SOD1, with a high adsorption coverage of 1.499 × 10−9 mol cm−2 on the sensor surface. rGO and PEDOT endow the composite with significant conductivity, whereas PSS has antifouling capability. Moreover, AuNWs exhibit excellent electrical conductivity and a high aspect ratio, which promotes electron transfer, and ultimately enhances the catalytic performance of the enzyme. Meanwhile, SOD1(Cu2+) catalyzes the dismutation of O2·- to O2 and H2O2, and H2O2 is then electrochemically oxidized to generate amperometric signals for determination of O2·-. The sensor demonstrates outstanding detection performance for O2·- with a low detection limit of 2.52 nM, and two dynamic ranges (14.30 nM–1.34 μM and 1.34 μM–42.97 μM) with corresponding sensitivity of 0.479 and 0.052 μA μM−1cm−2, respectively. Additionally, the calculated apparent Michaelis constant (Kmapp) of 1.804 μM for SOD1 demonstrates the outstanding catalytic activity and the surface-immobilized enzyme's substrate affinity. Furthermore, the sensor shows the capability to dynamically detect the level of O2·- released from living HepG2 cells. This study provides an inovative design to obtain a biocompatible electrochemical sensing platform with plenty of immobilization sites for biomolecules, large surface area, high conductivity and flexibility.

Semirational Design Based on Consensus Sequences to Balance the Enzyme Activity-Stability Trade-Off.

In this study, the phenomenon of the stability-activity trade-off, which is increasingly recognized in enzyme engineering, was explored. Typically, enhanced stability in enzymes correlates with diminished activity. Utilizing Rosa roxburghii copper-zinc superoxide dismutase (RrCuZnSOD) as a model, single-site mutations were introduced based on a semirational design derived from consensus sequences. The initial set of mutants was selected based on activity, followed by combinatorial mutation. This approach yielded two double-site mutants, D25/A115T (18,688 ± 206 U/mg) and A115T/S135P (18,095 ± 1556 U/mg), exhibiting superior enzymatic properties due to additive and synergistic effects. These mutants demonstrated increased half-lives (T1/2) at 80 °C by 1.2- and 1.6-fold, respectively, and their melting temperatures (Tm) rose by 3.4 and 2.5 °C, respectively, without any loss in activity relative to the wild type. Via an integration of structural analysis and molecular dynamics simulations, we elucidated the underlying mechanism facilitating the concurrent enhancement of both thermostability and enzymatic activity.

Current potential pathogenic mechanisms of copper-zinc superoxide dismutase 1 (SOD1) in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease which damages upper and lower motor neurons (UMN and LMN) innervating the muscles of the trunk, extremities, head, neck and face in cerebrum, brain stem and spinal cord, which results in the progressive weakness, atrophy and fasciculation of muscle innervated by the related UMN and LMN, accompanying with the pathological signs leaded by the cortical spinal lateral tract lesion. The pathogenesis about ALS is not fully understood, and no specific drugs are available to cure and prevent the progression of this disease at present. In this review, we reviewed the structure and associated functions of copper-zinc superoxide dismutase 1 (SOD1), discuss why SOD1 is crucial to the pathogenesis of ALS, and outline the pathogenic mechanisms of SOD1 in ALS that have been identified at recent years, including glutamate-related excitotoxicity, mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress, axonal transport disruption, prion-like propagation, and the non-cytologic toxicity of glial cells. This review will help us to deeply understand the current progression in this field of SOD1 pathogenic mechanisms in ALS.

Dual-Site Biomimetic Cu/Zn-MOF for Atopic Dermatitis Catalytic Therapy via Suppressing FcγR-Mediated Phagocytosis.

Atopic dermatitis (AD) is a prevalent chronic inflammatory skin disease that carries a significant global economic burden. Elevated levels of reactive oxygen species (ROS) have been recognized as contributing to AD exacerbation, making them a potential therapeutic target for AD treatment. Here, we introduce a dual-site biomimetic copper/zinc metal-organic framework (Cu/Zn-MOF) featuring four types of enzyme-like activities for AD treatment via suppressing the Fcγ receptor (FcγR)-mediated phagocytosis signal by mimicking the bimetallic sites of natural copper-zinc superoxide dismutase (CuZn-SOD). Interestingly, the neighboring Cu and Zn sites in both Cu/Zn-MOF and CuZn-SOD are at similar distances of ∼5.98 and ∼6.3 Å from each other, respectively, and additionally, both Cu and Zn sites are coordinated to nitrogen atoms in both structures, and the coordinating ligands to Cu and Zn are both imidazole rings. Cu/Zn-MOF exhibits remarkable SOD-like activity as well as its glutathione peroxidase (GPx)-, thiol peroxidase (TPx)-, and ascorbate peroxidase (APx)-like activities to continuously consume ROS and mitigate oxidative stress in keratinocytes. Animal experiments show that Cu/Zn-MOF outperforms halcinonide solution (a potent steroid medication) in terms of preventing mechanical injuries, reducing cutaneous water loss, and inhibiting inflammatory responses while presenting favorable biosafety. Mechanistically, Cu/Zn-MOF functions through an FcγR-mediated phagocytosis signal pathway, decreasing the continuous accumulation of ROS in AD and ultimately suppressing disease progression. These findings will provide an effective paradigm for AD therapy and contribute to the development of two-site bionics (TSB).

Olanzapine Effects on Parvalbumin/GAD67 Cell Numbers in Layers/Subregions of Dorsal Hippocampus of Chronically Socially Isolated Rats.

Depression is linked to changes in GABAergic inhibitory neurons, especially parvalbumin (PV) interneurons, which are susceptible to redox dysregulation. Olanzapine (Olz) is an atypical antipsychotic whose mode of action remains unclear. We determined the effect of Olz on PV-positive (+) and glutamate decarboxylase 67 (GAD67) + cell numbers in the layers of dorsal hippocampus (dHIPP) cornu ammonis (CA1-CA3) and dentate gyrus (DG) subregions in rats exposed to chronic social isolation (CSIS), which is an animal model of depression. Antioxidative enzymes and proinflammatory cytokine levels were also examined. CSIS decreased the PV+ cell numbers in the Stratum Oriens (SO) and Stratum Pyramidale (SP) of dCA1 and dDG. It increased interleukin-6 (IL-6), suppressor of cytokine signaling 3 (SOCS3), and copper-zinc superoxide dismutase (CuZnSOD) levels, and it decreased catalase (CAT) protein levels. Olz in CSIS increased the number of GAD67+ cells in the SO and SP layers of dCA1 with no effect on PV+ cells. It reduced the PV+ and GAD67+ cell numbers in the Stratum Radiatum of dCA3 in CSIS. Olz antagonizes the CSIS-induced increase in CuZnSOD, CAT and SOCS3 protein levels with no effect on IL-6. Data suggest that the protective Olz effects in CSIS may be mediated by altering the number of PV+ and GAD67+ cells in dHIPP subregional layers.

Direct relationship between dimeric form and activity in the acidic copper-zinc superoxide dismutase from lemon.

The copper-zinc superoxide dismutase (CuZnSOD) from lemon (SOD_CL) is active in an acidic environment and resists proteolytic degradation. The enzyme occurs as a dimer, which has an indirect effect on the enzyme activity as the monomer retains only ∼35% of the activity. Here, the crystal structure of SOD_CL at 1.86 Å resolution is reported that may explain this peculiarity. Thecrystal belonged to space group P21, with unit-cell parameters a = 61.11, b=74.55, c = 61.69 Å, β = 106.86°, and contained four molecules in the asymmetric unit. The overall structure of SOD_CL resembles that of CuZnSOD from plants. The structure of SOD_CL shows a unique arrangement of surface loop IV that connects the dimer interface and the active site, which is located away from the dimer-interface region. This arrangement allows direct interaction between the residues residing in the dimer interface and those in the active site. The arrangement also includes Leu62 and Gln164, which are conserved in cytoplasmic CuZnSOD. This supports the classification of SOD_CL as a cytoplasmic CuZnSOD despite sharing the highest amino-acid sequence homology with CuZnSODs from spinach and tomato, which are chloroplastic.

Extreme diversity of 12 cations in folding ALS-linked hSOD1 unveils novel hSOD1-dependent mechanisms for Fe2+/Cu2+-induced cytotoxicity

153-Residue copper-zinc superoxide dismutase 1 (hSOD1) is the first gene whose mutation was linked to FALS. To date, > 180 ALS-causing mutations have been identified within hSOD1, yet the underlying mechanism still remains mysterious. Mature hSOD1 is exceptionally stable constrained by a disulfide bridge to adopt a Greek-key β-barrel fold that accommodates copper/zinc cofactors. Conversely, nascent hSOD1 is unfolded and susceptible to aggregation and amyloid formation, requiring Zn2+ to initiate folding to a coexistence of folded and unfolded states. Recent studies demonstrate mutations that disrupt Zn2+-binding correlate with their ability to form toxic aggregates. Therefore, to decode the role of cations in hSOD1 folding provides not only mechanistic insights, but may bear therapeutic implications for hSOD1-linked ALS. Here by NMR, we visualized the effect of 12 cations: 8 essential for humans (Na+, K+, Ca2+, Zn2+, Mg2+, Mn2+, Cu2+, Fe2+), 3 mimicking zinc (Ni2+, Cd2+, Co2+), and environmentally abundant Al3+. Surprisingly, most cations, including Zn2+-mimics, showed negligible binding or induction for folding of nascent hSOD1. Cu2+ exhibited extensive binding to the unfolded state but led to severe aggregation. Unexpectedly, for the first time Fe2+ was deciphered to have Zn2+-like folding-inducing capacity. Zn2+ was unable to induce folding of H80S/D83S-hSOD1, while Fe2+ could. In contrast, Zn2+ could trigger folding of G93A-hSOD1, but Fe2+ failed. Notably, pre-existing Fe2+ disrupted the Zn2+-induced folding of G93A-hSOD1. Comparing with the ATP-induced folded state, our findings delineate that hSOD1 maturation requires: (1) intrinsic folding capacity encoded by the sequence; (2) specific Zn2+-coordination; (3) disulfide formation and Cu-load catalyzed by hCCS. This study unveils a previously-unknown interplay of cations in governing the initial folding of hSOD1, emphasizing the pivotal role of Zn2+ in hSOD1-related ALS and implying new hSOD1-dependent mechanisms for Cu2+/Fe2+-induced cytotoxicity, likely relevant to aging and other diseases.

Copper-Zinc Superoxide Dismutase (CuZn-SOD) Electrochemical Catalytic Amplification Sensing at Pt Ultramicroelectrodes

Abstract. Copper/zinc superoxide dismutase (CuZnSOD), a 32.5 kDa metalloprotein with a radius of ca. 2.1 nm, catalyses the superoxide to hydrogen peroxide and molecular oxygen. At the femtomolar concentration range, has been sensed through electrochemical catalytic amplification using a Pt ultramicroelectrode. During amperometric (i vs. t) analysis, cathodic and anodic current transitions peaks were seen, in agreement with the metalloprotein catalytic mechanism. The current amplitudes were analyzed and correspond to the CuZnSOD dimensions. Thermal treatment of metalloprotein samples at 80 °C showed larger current spikes suggesting aggregation without losing its catalytic capability. The size was confirmed by transmission electron microscopy. Resumen. Cuprozinc superóxido dismutasa (CuZnSOD), es una metaloproteina de 32.5 kDa con un radio de aproximadamente 2.1 nm. Esta enzima cataliza la reacción de superóxido a peróxido y oxígeno molecular. Por primera vez, esta proteína es detectada a concentraciones femtomolares haciendo uso de la técnica electroquímica conocida como amplificación catalítica y la tecnología de ultra-microelectrodos de Pt. Durante un análisis amperométrico (curvas i vs. t), se observaron picos transitorios de corriente catódica y anódica que concuerdan con el mecanismo catalítico de la enzima. Al analizar la amplitud de la corriente, la misma concuerda con las dimensiones de CuZnSOD. Luego de exponer la proteína a un tratamiento térmico de 80 °C, CuZnSOD mostró picos de corriente transitorias que sugieren aglomeración de la enzima sin perder su capacidad catalítica. El tamaño fue confirmado por microscopía electrónica de transmisión.