Catalase activity is stimulated by H2O2 in rich culture medium and is required for H2O2 resistance and adaptation in yeast

AbstractCatalases are efficient scavengers of H2O2 and protect cells against H2O2 stress. Examination of the H2O2 stimulon in Saccharomyces cerevisiae revealed that the cytosolic catalase T (Ctt1) protein level increases 15-fold on H2O2 challenge in synthetic complete media although previous work revealed that deletion of the CCT1 or CTA1 genes (encoding peroxisomal/mitochondrial catalase A) does not increase the H2O2 sensitivity of yeast challenged in phosphate buffer (pH 7.4). This we attributed to our observation that catalase activity is depressed when yeast are challenged with H2O2 in nutrient-poor media. Hence, we performed a systematic comparison of catalase activity and cell viability of wild-type yeast and of the single catalase knockouts, ctt1∆ and cta1∆, following H2O2 challenge in nutrient-rich medium (YPD) and in phosphate buffer (pH 7.4). Ctt1 but not Cta1 activity is strongly induced by H2O2 when cells are challenged in YPD but suppressed when cells are challenged in buffer. Consistent with the activity results, exponentially growing ctt1∆ cells in YPD are more sensitive to H2O2 than wild-type or cta1∆ cells, whereas in buffer all three strains exhibit comparable H2O2 hypersensitivity. Furthermore, catalase activity is increased during adaptation to sublethal H2O2 concentrations in YPD but not in buffer. We conclude that induction of cytosolic Ctt1 activity is vital in protecting yeast against exogenous H2O2 but this activity is inhibited by H2O2 when cells are challenged in nutrient-free media

Effect of copper on antioxidant enzyme activities and mineral nutrition of white lupin plants grown in nutrient solution

We analyzed the effect of different copper (Cu) concentrations (0.10, 0.15, 0.20 and 0.35 mM) and time (1 day to 9 days) on several growth and biochemical parameters of roots and shoots of white lupin plants (Lupinus albus cv Estoril) grown in nutrient solution. A significant decrease in leaf fresh weight and leaf area was detected. Copper accumulated in the roots, and an impairment of nutrient translocation was only observed after six days at the highest Cu concentrations applied. A transient increase in the activity of polyphenoloxidase (EC 1.10.3.1) enforces a role for lignification as a defense strategy under enhanced Cu levels. The activities of several antioxidative enzymes were enhanced after Cu application. Our results indicate that Lupinus albus cv ‘Estoril’ is a rather resistant plant that can cope with moderate concentrations of copper, mostly by controlling up to a certain point, the uptake of excessive amounts of this meta

Ctt1 catalase activity potentiates antifungal azoles in the emerging opportunistic pathogen Saccharomyces cerevisiae

Fungi respond to antifungal drugs by increasing their antioxidant stress response. How this impacts antifungal efficacy remains controversial and not well understood. Here we examine the role of catalase activity in the resistance of Saccharomyces cerevisiae to the common antifungals, fluconazole and miconazole, for which we report minimum inhibitory concentrations (MICs) of 104 and 19 μM, respectively. At sub-MIC concentrations, fluconazole and miconazole stimulate catalase activity 2-3-fold but, unexpectedly, deletion of cytosolic catalase (ctt1) makes cells more resistant to these azoles and to clotrimazole, itraconazole and posaconazole. On the other hand, upregulating Ctt1 activity by preconditioning with 0.2 mM H2O2 potentiates miconazole 32-fold and fluconazole 4-fold. Since H2O2 preconditioning does not alter the resistance of ctt1Δ cells, which possess negligible catalase activity, we link azole potentiation with Ctt1 upregulation. In contrast, sod2Δ cells deleted for mitochondrial superoxide dismutase are 4–8-fold more azole sensitive than wild-type cells, revealing that Sod2 activity protects cells against azole toxicity. In fact, the ctt1Δ mutant has double the Sod2 activity of wild-type cells so ctt1 deletion increases azole resistance in part by Sod2 upregulation. Notably, deletion of peroxisomal/mitochondrial cta1 or cytosolic sod1 does not alter fluconazole or miconazole potency

The Impact of Feet Callosities, Arm Posture, and Usage of Electrolyte Wipes on Body Composition by Bioelectrical Impedance Analysis in Morbidly Obese Adults

Objective: This study evaluated the impact of feet callosities, arm posture, and use of electrolyte wipes on body composition measurements by bioelectrical impedance analysis (BIA) in morbidly obese adults. Methods: 36 morbidly obese patients (13 males, aged 28-70 years, BMI 41.6 ± 4.3 kg/m2) with moderate/severe feet callosities participated in this study. Body composition (percent body fat (%BF)) was measured while fasting using multi-frequency BIA (InBody 720®), before and after removal of callosities, with and without InBody® electrolyte wipes and custom-built auxiliary pads (to assess arm posture impact). Results from BIA were compared to air displacement plethysmography (ADP, BodPod®). Results: Median %BF was significantly higher with auxiliary pads than without (50.1 (interquartile range 8.2) vs. 49.3 (interquartile range 9.1); p < 0.001), while no differences were found with callosity removal (49.3 (interquartile range 9.1) vs. 50.0 (interquartile range 7.9); NS) or use of wipes (49.6 (interquartile range 8.5) vs. 49.3 (interquartile range 9.1); NS). No differences in %BF were found between BIA and ADP (49.1 (IQR: 8.9) vs. 49.3 (IQR: 9.1); NS). Conclusion: Arm posture has a significant impact on %BF assessed by BIA, contrary to the presence of feet callosities and use of electrolyte wipes. Arm posture standardization during BIA for body composition assessment is, therefore, recommended.This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only

Single-Center Experience With Liver Transplant Using Donors With Very High Transaminase Levels

OBJECTIVES: Elevation of transaminases has been used as a marker of hepatic ischemic injury and as a crucial parameter for liver graft assessment. However, analysis of serum transaminases has limitations regarding the quantitative assessment of liver necrosis and is not a reliable predictor of outcomes. MATERIALS AND METHODS: We retrospectively reviewed the medical records of all liver transplants (N = 238) performed at the UMass Memorial Medical Center from 2009 to 2013. RESULTS: Fourteen liver grafts showed high peak aminotransferases alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at \u3e 1000 U/L. This high aminotransferase group was compared with 224 donors with low transaminase levels (ALT/AST \u3c 1000 U/L). The high transaminase donors had a median peak AST level of 3216 U/L (range, 1823-13?030 U/L) and ALT level of 2677 U/L (range, 812-7080 U/L). The high transaminase donors showed higher levels of lactate dehydrogenase, international normalized ratio, total bilirubin, and gamma-glutamyltransferase compared with low transaminase donors; however, only lactate dehydrogenase results reached statistical significance. None of the grafts from the high transaminase donors showed primary nonfunction. Three-year graft and patient survival rates were similar in both groups (75% vs 80% [P = .48] and 72% vs 82% [P = .33], respectively). In an analysis of the discard rate of livers over a 10-year period in the United States using the Scientific Registry of Transplant Recipients database, the discard rate of livers with high aminotransferase levels was 69.14% compared with 22.23% for livers with low transaminase levels. CONCLUSIONS: Liver grafts from donors with high transaminase levels can lead to clinical results that are similar to liver grafts from donors who had lower peak transaminase levels

Cross-Presentation of a Spread-Defective MCMV Is Sufficient to Prime the Majority of Virus-Specific CD8+ T Cells

CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection

Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1)

The response of tobacco plants genetically engineered with the AtTPS1 gene to stress induced by excess Cu and Cd was evaluated in hydroponic solution (100 and 400 lM Cu and 50 and 200 lM Cd) after a 48 h exposure. Two transgenic lines, transformed with the AtTPS1 (trehalose-6-phosphate synthase) gene from Arabidopsis, with different levels of trehalose-6-phosphate synthase expression (B5H, higher and B1F, lower), and a wild type (WT) were investigated. Protein content, antioxidative enzymes (CAT, POD, SOD, and APX), glucose, fructose, lipid peroxidation, hydrogen peroxide and Cd and Cu contents were determined in leaves. The two transgenic lines were differently influenced by Cd and Cu exposure as they induced a different antioxidant enzymatic defense response. B1F and B5H plants showed a better acclimation to Cd and excess Cu compared to WT. Furthermore B1F was more tolerant than B5H to Cd and excess Cu. B1F accumulated less Cd and Cu in leaves, probably due to a more efficient exclusion mechanism. Catalase was shown to be the most important enzyme in the antioxidative system of these plants

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification

A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery

CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the constitutive pathogen response 5 (CPR5) gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targeted deep sequencing strategies. We were able to identify different mutation patterns within insertions and deletions (InDels) between + 5 nt and –30 bp and mutation frequency ranging from 4.2 to 18.1% in the GmCPR5 locus. Our results showed that DNA-free delivery of Cas9 complexes to protoplasts is a useful approach to perform early-stage genetic screens and anticipated analysis of Cas9 activity in soybeans.publishedVersio

Potential of sorghum polyphenols to prevent and treat Alzheimer\u27s disease: A review article

Alzheimer’s disease (AD) is characterized by the excessive deposition of extracellular amyloid-beta peptide (Aβ) and the build-up of intracellular neurofibrillary tangles containing hyperphosphorylated tau proteins. This leads to neuronal damage, cell death and consequently results in memory and learning impairments leading to dementia. Although the exact cause of AD is not yet clear, numerous studies indicate that oxidative stress, inflammation, and mitochondrial dysfunction significantly contribute to its onset and progression. There is no effective therapeutic approach to stop the progression of AD and its associated symptoms. Thus, early intervention, preferably, pre-clinically when the brain is not significantly affected, is a better option for effective treatment. Natural polyphenols (PP) target multiple AD-related pathways such as protecting the brain from Aβ and tau neurotoxicity, ameliorating oxidative damage and mitochondrial dysfunction. Among natural products, the cereal crop sorghum has some unique features. It is one of the major global grain crops but in the developed world, it is primarily used as feed for farm animals. A broad range of PP, including phenolic acids, flavonoids, and condensed tannins are present in sorghum grain including some classes such as proanthocyanidins that are rarely found in others plants. Pigmented varieties of sorghum have the highest polyphenolic content and antioxidant activity which potentially makes their consumption beneficial for human health through different pathways such as oxidative stress reduction and thus the prevention and treatment of neurodegenerative diseases. This review summarizes the potential of sorghum PP to beneficially affect the neuropathology of AD