MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis

In the noninfectious soil saprophyte Mycobacterium smegmatis, intracellular levels of the stress alarmones guanosine tetraphosphate and guanosine pentaphosphate, together termed (p)ppGpp, are regulated by the enzyme Rel(Msm). This enzyme consists of a single, bifunctional polypeptide chain that is capable of both synthesizing and hydrolyzing (p)ppGpp. The rel(Msm), knockout strain of M. smegmatis (Delta rel(Msm)) is expected to show a (p)ppGpp null (p)ppGpp(0)] phenotype. Contrary to this expectation, the strain is capable of synthesizing (p)ppGpp in vivo. In this study, we identify and functionally characterize the open reading frame (ORF), MSMEG_5849, that encodes a second functional (p)ppGpp synthetase in M. smegmatis. In addition to (p)ppGpp synthesis, the 567-amino-acid-long protein encoded by this gene is capable of hydrolyzing RNA(.)DNA hybrids and bears similarity to the conventional RNase HII enzymes. We have classified this protein as actRel(Msm) in accordance with the recent nomenclature proposed and have named it MS_RHII-RSD, indicating the two enzymatic activities present RHII, RNase HII domain, originally identified as (d) under bar omain of (u) under bar nknown (f) under bar unction 429 (DUF429), and RSD, RelA_SpoT nucleotidyl transferase domain, the SYNTH domain responsible for (p)ppGpp synthesis activity]. MS_RHII-RSD is expressed and is constitutively active in vivo and behaves like a monofunctional (p)ppGpp synthetase in vitro. The occurrence of the RNase HII and (p)ppGpp synthetase domains together on the same polypeptide chain is suggestive of an in vivo role for this novel protein as a link connecting the essential life processes of DNA replication, repair, and transcription to the highly conserved stress survival pathway, the stringent response.

Spirodiazaselenuranes: synthesis, structure and antioxidant activity

In this paper, the synthesis, characterization and glutathione peroxidase and peroxynitrite scavenging activities of a series of stable spirodiazaselenuranes are described. The spiro compounds were synthesized in good yields by oxidative cyclization of diaryl selenides bearing amide moieties. All the selenides and spiro derivatives were characterized by H-1, C-13 and Se-77 NMR spectroscopy, mass spectral techniques and the structures of some of the spirodiazaselenuranes were confirmed by single crystal X-ray crystallography. The structures reveal that the selenium atom occupies the center of a distorted trigonal bipyramid core with two nitrogen atoms occupying the apical positions and two carbon atoms and the selenium lone pair occupying the equatorial positions. Mechanistic investigations indicate that the spirocyclization occurs via the formation of selenoxide intermediates. The new compounds were evaluated for their glutathione peroxidase (GPx) mimetic activity by using H2O2 as a substrate and glutathione (GSH) as a co-substrate. It was found that the substituents attached to the nitrogen atom of the selenazole ring have a significant effect on the GPx activity. While the introduction of electron withdrawing groups such as -Cl, -Br etc. to the phenyl ring decreases the activity, the introduction of electron donating groups such as -OH, -OMe significantly enhances the GPx activity of both diaryl selenides and spirodiazaselenuranes. In addition to GPx activity, the selenides and spiro derivatives were studied for their ability to inhibit peroxynitrite (PN)-mediated nitration of bovine serum albumin (BSA) and oxidation of dihydrorhodamine 123. These studies indicate that the diarylselenides effectively inhibit the PN-mediated nitration and oxidation reactions by reacting with PN to produce the corresponding spirodiazaselenuranes.

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/ mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2(-/-) mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnf alpha and Ifn gamma in sera are not significantly affected, Nos2(-/-) mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.

Antioxidant and oxidative stress parameters in brain of Heteropneustes fossilis under air exposure condition; role of mitochondrial electron transport chain

Many fishes are exposed to air in their natural habitat or during their commercial handling. In natural habitat or during commercial handling, the cat fish Heteropneustes fossilis is exposed to air for > 24 h. Data on its oxidative metabolism in the above condition are not available. Oxidative stress (OS) indices (lipid and protein oxidation), toxic reactive oxygen species (ROS: H2O2) generation, antioxidative status (levels of superoxide dismutase, catalase, glutathione peroxidase and reductase, ascorbic acid and nonprotein sulfhydryl) and activities of electron transport chain (ETC) enzymes (complex I-IV) were investigated in brain tissue of H. fossilis under air exposure condition (0, 3, 6, 12 and 18 h at 25 degrees C). Decreased activities of antioxidant (except catalase) and ETC enzymes (except complex II) with increased H2O2 and OS levels were observed in the tissue under water deprivation condition. Positive correlation was observed for complex II activity and non-protein thiol groups with time period of air exposure. The critical time period to induce OS and to reduce most of the studied antioxidant level in brain was found to be 3-6 h air exposure. The data can be useful to minimize the stress generated during commercial handling of the live fishes those exposed to air in general and H. fossilis in particular. (C) 2013 Elsevier Inc. All rights reserved.

The Modified Selenenyl Amide, M-hydroxy Ebselen, Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-beta (TGF-beta) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-beta-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation.

Antitumor and antioxidant status of Terminalia catappa against Ehrlich ascites carcinoma in Swiss albino mice

Objective: The present study was undertaken to evaluate the antitumor and antioxidant status of ethanol extract of Terminalia catappa leaves against Ehrlich ascites carcinoma (EAC) in Swiss albino mice. Materials and Methods: The leaves powder was extracted with Soxhlet apparatus and subjected to hot continuous percolation using ethanol (95% v/v). Tumor bearing animals was treated with 50 and 200 mg/kg of ethanol extract. EAC induced in mice by intraperitoneal injection of EAC cells 1 x 10(6) cells/mice. The study was assed using life span of EAC-bearing hosts, hematological parameters, volume of solid tumor mass and status of antioxidant enzymes such as lipid peroxidation (LPO), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities. Total phenolics and flavonoids contents from the leaves extract were also determined. Results: Total phenolics and flavonoids contents from the leaves extract were found 354.02 and 51.67 mg/g extract. Oral administration of ethanol extract of T. catappa (50 and 200 mg/kg) increased the life span (27.82% and 60.59%), increased peritoneal cell count (8.85 +/- 0.20 and 10.37 +/- 0.26) and significantly decreased solid tumor mass (1.16 +/- 0.14 cm(2)) at 200 mg/kg as compared with EAC-tumor bearing mice (P < 0.01). Hematological profile including red blood cell count, white blood cell count, hemoglobin (11.91 +/- 0.47 % g) and protein estimation were found to be nearly normal levels in extract-treated mice compared with tumor bearing control mice. Treatment with T. catappa significantly decreased levels of LPO and GSH, and increased levels of SOD and CAT activity (P < 0.01). Conclusion: T. catappa exhibited antitumor effect by modulating LPO and augmenting antioxidant defense systems in EAC bearing mice. The phenolic and flavonoid components in this extract may be responsible for antitumor activity.

Inhibition of Lactoperoxidase-Catalyzed Oxidation by Imidazole-Based Thiones and Selones: A Mechanistic Study

Herein, we describe the synthesis and biomimetic activity of a series of N,N-disubstituted thiones and selones that contain an imidazole pharmacophore. The N,N-disubstituted thiones do not show any inhibitory activity towards LPO-catalyzed oxidation reactions, but their corresponding N,N-disubstituted selones exhibit inhibitory activity towards LPO-catalyzed oxidation reactions. Substituents on the N atom of the imidazole ring appear to have a significant effect on the inhibition of LPO-catalyzed oxidation and iodination reactions. Selones 16, 17, and 19, which contain methyl, ethyl, and benzyl substituents, exhibit similar inhibition activities towards LPO-catalyzed oxidation reactions with IC50 values of 24.4, 22.5, and 22.5M, respectively. However, their activities are almost three-fold lower than that of the commonly used anti-thyroid drug methimazole (MMI). In contrast, selone 21, which contains a NCH2CH2OH substituent, exhibits high inhibitory activity, with an IC50 value of 7.2M, which is similar to that of MMI. The inhibitory activity of these selones towards LPO-catalyzed oxidation/iodination reactions is due to their ability to decrease the concentrations of the co-substrates (H2O2 and I2), either by catalytically reducing H2O2 (anti-oxidant activity) or by forming stable charge-transfer complexes with oxidized iodide species. The inhibition of LPO-catalyzed oxidation/iodination reactions by N,N-disubstituted selones can be reversed by increasing the concentration of H2O2. Interestingly, all of the N,N-disubstituted selones exhibit high anti-oxidant activities and their glutathione peroxidase (GPx)-like activity is 4-12-fold higher than that of the well-known GPx-mimic ebselen. These experimental and theoretical studies suggest that the selones exist as zwitterions, in which the imidazole ring contains a positive charge and the selenium atom carries a large negative charge. Therefore, the selenium moieties of these selones possess highly nucleophilic character. The 77SeNMR chemical shifts for the selones show large upfield shift, thus confirming the zwitterionic structure in solution.

Modulation of redox regulatory molecules and electron transport chain activity in muscle of air breathing fish Heteropneustes fossilis under air exposure stress

Responses of redox regulatory system to long-term survival (> 18 h) of the catfish Heteropneustes fossilis in air are not yet understood. Lipid and protein oxidation level, oxidant (H2O2) generation, antioxidative status (levels of superoxide dismutase, catalase, glutathione peroxidase and reductase, ascorbic acid and non-protein sulfhydryl) and activities of respiratory complexes (I, II, III and IV) in mitochondria were investigated in muscle of H. fossilis under air exposure condition (0, 3, 6, 12 and 18 h at 25 A degrees C). The increased levels of both H2O2 and tissue oxidation were observed due to the decreased activities of antioxidant enzymes in muscle under water deprivation condition. However, ascorbic acid and non-protein thiol groups were the highest at 18 h air exposure time. A linear increase in complex II activity with air exposure time and an increase up to 12 h followed by a decrease in activity of complex I at 18 h were observed. Negative correlation was observed for complex III and V activity with exposure time. Critical time to modulate the above parameters was found to be 3 h air exposure. Dehydration induced oxidative stress due to modulation of electron transport chain and redox metabolizing enzymes in muscle of H. fossilis was clearly observed. Possible contribution of redox regulatory system in muscle tissue of the fish for long-term survival in air is elucidated. Results of the present study may be useful to understand the redox metabolism in muscle of fishes those are exposed to air in general and air breathing fishes in particular.

Temozolomide-modulated glioma proteome: Role of interleukin-1 receptor-associated kinase-4 (IRAK4) in chemosensitivity

The current treatment for glioblastoma includes temozolomide (TMZ) chemotherapy, yet the mechanism of action of TMZ is not thoroughly understood. Here, we investigated the TMZ-induced changes in the proteome of the glioma-derived cell line (U251) by 2D DIGE. We found 95 protein spots to be significantly altered in their expression after TMZ treatment. MS identified four upregulated spots: aspartyl tRNA synthetase glutathione synthetase, interleukin-1 receptor-associated kinase-4 (IRAK4), and breast carcinoma amplified sequence-1 and one downregulated spot: optineurin. TMZ-induced regulation of these five genes was validated by RT-qPCR andWestern blot analysis. RNAi-mediated knockdown of IRAK4, an important mediator of Toll-like receptors signaling and chemoresistance, rendered the glioma cells resistant to TMZ. High levels of IRAK4 induced upon TMZ treatment resulted in IRAK1 downregulation and inhibition of NFkB pathway. Endogenous IRAK4 protein, but not transcript levels in glioma cell lines, correlated with TMZ sensitivity. Thus, we have identified several TMZ-modulated proteins and discovered an important novel role for IRAK4 in determining TMZ sensitivity of glioma cells through its ability to inhibit Toll-like receptor signaling and NFkB pathway.

FOLD TYPE II PYRIDOXAL 5 `-PHOSPHATE DEPENDENT ENZYMES: STRUCTURE, SUBSTRATE RECOGNITION AND CATALYSIS

Enzymes utilizing pyridoxal 5'-phosphate dependent mechanism for catalysis are observed in all cellular forms of living organisms. PLP-dependent enzymes catalyze a wide variety of reactions involving amino acid substrates and their analogs. Structurally, these ubiquitous enzymes have been classified into four major fold types. We have carried out investigations on the structure and function of fold type I enzymes serine hydroxymethyl transferase and acetylornithine amino transferase, fold type n enzymes catabolic threonine deaminase, D-serine deaminase, D-cysteine desulfhydrase and diaminopropionate ammonia lyase. This review summarizes the major findings of investigations on fold type II enzymes in the context of similar studies on other PLP-dependent enzymes. Fold type II enzymes participate in pathways of both degradation and synthesis of amino acids. Polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity are briefly described. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance.

An antioxidant nanozyme that uncovers the cytoprotective potential of vanadia nanowires

Nanomaterials with enzyme-like properties has attracted significant interest, although limited information is available on their biological activities in cells. Here we show that V2O5 nanowires (Vn) functionally mimic the antioxidant enzyme glutathione peroxidase by using cellular glutathione. Although bulk V2O5 is known to be toxic to the cells, the property is altered when converted into a nanomaterial form. The Vn nanozymes readily internalize into mammalian cells of multiple origin (kidney, neuronal, prostate, cervical) and exhibit robust enzyme-like activity by scavenging the reactive oxygen species when challenged against intrinsic and extrinsic oxidative stress. The Vn nanozymes fully restore the redox balance without perturbing the cellular antioxidant defense, thus providing an important cytoprotection for biomolecules against harmful oxidative damage. Based on our findings, we envision that biocompatible Vn nanowires can provide future therapeutic potential to prevent ageing, cardiac disorders and several neurological conditions, including Parkinson's and Alzheimer's disease.

Lactoylglutathione lyase, a critical enzyme in methylglyoxal detoxification, contributes to survival of Salmonella in the nutrient rich environment

Glyoxalase I which is synonymously known as lactoylglutathione lyase is a critical enzyme in methylglyoxal (MG) detoxification. We assessed the STM3117 encoded lactoylglutathione lyase (Lgl) of Salmonella Typhimurium, which is known to function as a virulence factor, due in part to its ability to detoxify methylglyoxal. We found that STM3117 encoded Lgl isomerises the hemithioacetal adduct of MG and glutathione (GSH) into S-lactoylglutathione. Lgl was observed to be an outer membrane bound protein with maximum expression at the exponential growth phase. The deletion mutant of S. Typhimurium (lgl) exhibited a notable growth inhibition coupled with oxidative DNA damage and membrane disruptions, in accordance with the growth arrest phenomenon associated with typical glyoxalase I deletion. However, growth in glucose minimal medium did not result in any inhibition. Endogenous expression of recombinant Lgl in serovar Typhi led to an increased resistance and growth in presence of external MG. Being a metalloprotein, Lgl was found to get activated maximally by Co2+ ion followed by Ni2+, while Zn2+ did not activate the enzyme and this could be attributed to the geometry of the particular protein-metal complex attained in the catalytically active state. Our results offer an insight on the pivotal role of the virulence associated and horizontally acquired STM3117 gene in non-typhoidal serovars with direct correlation of its activity in lending survival advantage to Salmonella spp.

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 mu M) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading Delta psi m dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.

Epithelial atrophy in oral submucous fibrosis is mediated by copper (II) and arecoline of areca nut

Exposure of oral cavity to areca nut is associated with several pathological conditions including oral submucous fibrosis (OSF). Histopathologically OSF is characterized by epithelial atrophy, chronic inflammation, juxtaepithelial hyalinization, leading to fibrosis of submucosal tissue and affects 0.5% of the population in the Indian subcontinent. As the molecular mechanisms leading to atrophied epithelium and fibrosis are poorly understood, we studied areca nut actions on human keratinocyte and gingival fibroblast cells. Areca nut water extract (ANW) was cytotoxic to epithelial cells and had a pro-proliferative effect on fibroblasts. This opposite effect of ANW on epithelial and fibroblast cells was intriguing but reflects the OSF histopathology such as epithelial atrophy and proliferation of fibroblasts. We demonstrate that the pro-proliferative effects of ANW on fibroblasts are dependent on insulin-like growth factor signalling while the cytotoxic effects on keratinocytes are dependent on the generation of reactive oxygen species. Treatment of keratinocytes with arecoline which is a component of ANW along with copper resulted in enhanced cytotoxicity which becomes comparable to IC50 of ANW. Furthermore, studies using cyclic voltammetry, mass spectrometry and plasmid cleavage assay suggested that the presence of arecoline increases oxidation reduction potential of copper leading to enhanced cleavage of DNA which could generate an apoptotic response. Terminal deoxynucleotidyl transferase dUTP Nick End Labeling assay and Ki-67 index of OSF tissue sections suggested epithelial apoptosis, which could be responsible for the atrophy of OSF epithelium.

Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein, Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.