Reduced LINE-1 methylation is associated with arsenic-induced genotoxic stress in children

Early life exposure to arsenic has profound effect towards development of arsenic induced toxic outcomes. Some districts in the state of West Bengal, India are highly affected by arsenic, mainly through ground water. In children, not much of the toxic outcomes like dermatological lesions are observed but it is thought that the exposure leads to transient alteration in their biological processes that leads to various deleterious health effects later on. We evaluated the global methylation status by analyzing the LINE-1 methylation profile in children from arsenic exposed region between the age group 5–15 years along with the cytogenetic stress induced by arsenic as measured by lymphocyte micronucleus (MN) frequency. A total of 52 arsenic exposed and 32 unexposed children were analyzed. Whole blood DNA was used to measure the LINE-1 methylation by qRT-MSP. We found a significant association of MN-frequency in exposed individuals with highly depleted LINE-1 methylation compared to the exposed individuals with near baseline (which was comparable to unexposed control) methylation index as well as with those with the hypermethylated LINE-1 promoters. From our results, we interpret that LINE-1 methylation index may serve as a potent global epigenetic mark to detect the degree of arsenic genotoxicity at a very early age. We propose that this may be utilized to determine the extent of toxic influence exerted by arsenic, from a very early age.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H(2)O(2) with concurrent evolution of O(2). This pseudocatalatic degradation of H(2)O(2) to O(2) is solely dependent on ascorbate and is blocked by a spin trap, alpha-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H(2)O with subsequent regeneration of ascorbate by the reduction of MDA with H(2)O(2) evolving O(2) through the intermediate formation of O(2)(-). Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN(-) in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN(-). Further kinetic studies indicate that SCN(-) itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN(-) blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H(2)O(2) to O(2). Binding studies by optical difference spectroscopy indicate that SCN(-) binds LmAPX (Kd = 100 +/- 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN(-) acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Mutational analysis of the tetrahydrobiopterin-binding site in inducible nitric-oxide synthase.

Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.     

Bishydrazide glycoconjugates for lectin recognition and capture of bacterial pathogens

Bishydrazides are versatile linkers for attaching glycans to substrates for lectin binding and pathogen detection schemes. The α,ω-bishydrazides of carboxymethylated hexa(ethylene glycol) (4) can be conjugated at one end to unprotected oligosaccharides, then attached onto carrier proteins, tethered onto activated carboxyl-terminated surfaces, or functionalized with a photoactive cross-linking agent for lithographic patterning. Glycoconjugates of bishydrazide 4 can also be converted into dithiocarbamates (DTCs) by treatment with CS(2) under mild conditions, for attachment onto gold substrates. The immobilized glycans serve as recognition elements for cell-surface lectins and enable the detection and capture of bacterial pathogens such as Pseudomonas aeruginosa by their adsorption onto micropatterned substrates. A detection limit of 103 cfu/mL is demonstrated, using a recently introduced method based on optical pattern recognition.     

Overexpression of Mitochondrial Leishmania major Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage

Ascorbate peroxidase from Leishmania major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H₂O₂ or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca²⁺ attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H₂O₂-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H₂O₂- or CPT-induced cytochrome c and endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H₂O₂ or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.In multicellular organisms, mitochondria are the major physiological source of reactive oxygen species (ROS) within cells and also are important checkpoints for the control of programmed cell death (27). There are increasing numbers of reports that describe apoptosis- or programmed cell death-like processes in unicellular organisms also, such as trypanosomatids (4, 60), bacteria (20, 25), yeasts (34), and Plasmodium (3). Among the kinetoplastid parasites, Trypanosoma and Leishmania are the most carefully studied genera where apoptotic features are well established (49). Several reports have shown that mitochondrial dysfunction or an imbalance of antioxidant homeostasis causes an increase in mitochondrion-generated ROS, which include H₂O₂, superoxide radical anions, singlet oxygen, and hydroxyl radicals. These species have all been implicated in apoptosis (16, 26, 28, 41). Increasing evidence has been presented to support that ROS homeostasis regulates two major types of important physiological processes and exerts diverse functions within cells. One type of function includes damage or oxidation of cellular macromolecules (DNA, proteins, and lipids), which can lead to necrotic cell death or protein modification (7). The second type of function includes the activation of cellular signaling cascades that regulate proliferation, detoxification, DNA repair, or apoptosis (11). The detoxification of toxic mitochondrial ROS in cells occurs through a variety of cellular antioxidant enzymes, such as superoxide dismutase, which detoxifies cells from superoxide released into the mitochondrial matrix, and several other antioxidant proteins, such as catalase, glutathione (GSH) peroxidase, and peroxiredoxins, which are known to catalyze further degradation of H₂O₂ (44). During its life cycle, the Leishmania sp. encounters a pool of ROS that is generated either by its own physiological processes or as a result of host immune reaction and drug metabolism. However, unlike most eukaryotes, Leishmania lacks catalase- and selenium-containing GSH peroxidases, enzymes that play a front-line role in detoxifying ROS. Hence, the mechanism by which it resists the toxic effects of H₂O₂ remains poorly understood.Recently, we cloned, expressed and characterized the unusual heme-containing ascorbate peroxidase from Leishmania major (LmAPX) and observed that the expression of LmAPX is increased when Leishmania cells are treated with exogenous H₂O₂ (1, 18). This enzyme is a functional hybrid between cytochrome c peroxidase and APX, owing to its ability to use both ascorbate and cytochrome c as reducing electron donors (58). Colocalization studies by confocal microscopy, submitochondrial fractionation analysis of the isolated mitochondria, and subsequent Western blot analysis with anti-LmAPX antibody have confirmed that the mature enzyme is present in intermembrane space side of the inner membrane. It has also been shown that overexpression of LmAPX causes a decrease in the mitochondrial ROS burden, an increase in tolerance to H₂O₂, and protection against cardiolipin oxidation under oxidative stress (18). Although previous studies have shown that Leishmania species use superoxide dismutase (23), peroxiredoxins (8), intracellular thiols (14), lipophosphoglycan (13), trypanothione (5), HSP 70 (a heat shock protein) (36), tryparedoxin peroxidase (29), and APX (18) for detoxification of ROS, it is still unclear how the antioxidants protect against oxidative stress-induced apoptotic events in the unicellular organism Leishmania.Since the LmAPX protein is localized in the mitochondria, we hypothesized that it would be a key protein for the maintenance of mitochondrial functions due to its antioxidant properties via its ROS-scavenging function (18). To test this hypothesis, we overexpressed LmAPX in Leishmania major cells and investigated whether overexpression of LmAPX can confer resistance to oxidant-mediated mitochondrial damage as well as oxidative stress-induced cell death. In this study, we provide evidence that the overexpression of LmAPX in Leishmania cells can indeed protect against camptothecin (CPT) or H₂O₂-mediated mitochondrial damage as measured by various parameters, including disruption of mitochondrial membrane potential (Δψ_m), decrease of ATP production, and cytochrome c and endonuclease G release from mitochondria. Cells overexpressing LmAPX were also protected against oxidative stress-induced protein carbonylation, DNA fragmentation, and apoptosis. To the best of our knowledge, this is the first report of a mitochondrial hemeperoxidase that controls the ROS-induced mitochondrial death pathway.     

Endoplasmic reticulum stress-induced apoptosis in Leishmania through Ca2+-dependent and caspase-independent mechanism

Numerous reports have shown that mitochondrial dysfunctions play a major role in apoptosis of Leishmania parasites, but the endoplasmic reticulum (ER) stress-induced apoptosis in Leishmania remains largely unknown. In this study, we investigate ER stress-induced apoptotic pathways in Leishmania major using tunicamycin as an ER stress inducer. ER stress activates the expression of ER-localized chaperone protein BIP/GRP78 (binding protein/identical to the 78-kDa glucose-regulated protein) with concomitant generation of intracellular reactive oxygen species. Upon exposure to ER stress, the elevation of cytosolic Ca(2+) level is observed due to release of Ca(2+) from internal stores. Increase in cytosolic Ca(2+) causes mitochondrial membrane potential depolarization and ATP loss as ablation of Ca(2+) by blocking voltage-gated cation channels with verapamil preserves mitochondrial membrane potential and cellular ATP content. Furthermore, ER stress-induced reactive oxygen species (ROS)-dependent release of cytochrome c and endonuclease G from mitochondria to cytosol and subsequent translocation of endonuclease G to nucleus are observed. Inhibition of caspase-like proteases with the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or metacaspase inhibitor antipain does not prevent nuclear DNA fragmentation and phosphatidylserine exposure. Conversely, significant protection in tunicamycin-induced DNA degradation and phosphatidylserine exposure was achieved by either pretreatment of antioxidants (N-acetyl-L-cysteine, GSH, and L-cysteine), chemical chaperone (4-phenylbutyric acid), or addition of Ca(2+) chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester). Taken together, these data strongly demonstrate that ER stress-induced apoptosis in L. major is dependent on ROS and Ca(2+)-induced mitochondrial toxicity but independent of caspase-like proteases.     

Modulation of physiological responses with TiO<Subscript>2</Subscript> nano-particle in <Emphasis Type="Italic">Azolla pinnata</Emphasis> R.Br. under 2,4-D toxicity

The present work is emphasised with the herbicidal tolerance of Azolla pinnata R.Br. and its modulation with TiO₂ nano-particle. Both carbohydrate and nitrogen metabolism were effected with 2,4-D as herbicide and in few cases TiO₂-NP had recovered few detrimental effects. From the nutrient status in Azolla it recorded the recovery of nitrogen as well as potassium by TiO₂-NP but not in case of phosphorus. However, a conversion of nitrate to ammonium was more induced by TiO₂-NP under herbicidal toxicity. Similar results were obtained for inter-conversion of amino acid–nitrate pool, but no changes with glutamine synthase activity with TiO₂-NP. Initially, the effects of 2,4-D was monitored with changes of chlorophyll content but had not been recovered with nanoparticle. Photosynthetic reserves expressed as both total and reducing sugar were insensitive to TiO₂-NP interference but activity of soluble and wall bound invertase was in reverse trend as compared to control. The 2,4-D mediated changes of redox and its oxidative stress was ameliorated in plants with over expressed ADH activity. As a whole the Azolla bio system with TiO₂ supplementation may be useful in sustenance against 2,4-D toxicity through recovery of nitrogen metabolism. Thus, Azolla-TiO₂-NP bio system would be realised to monitor the herbicidal toxicity in soil and its possible bioremediation.     

Regulation of the properties of the heme-NO complexes in nitric-oxide synthase by hydrogen bonding to the proximal cysteine

Nitric-oxide synthase (NOS) catalyzes the formation of NO and citrulline from l-arginine and oxygen. However, the NO so formed has been found to auto-inhibit the enzymatic activity significantly. We hypothesized that the NO reactivity is in part controlled by hydrogen bonding between the conserved tryptophan residue (position 409 in the neuronal isoform of NOS (nNOS)) and the cysteine residue that forms the proximal bond to the heme. By using resonance Raman spectroscopy and NO as a probe of the heme environment, we show that in the W409F and W409Y mutants of the oxygenase domain of the neuronal enzyme (nNOSox), the Fe-NO bond in the Fe3+NO complex is weaker than in the wild type enzyme, consistent with the loss of a hydrogen bond on the sulfur atom of the proximal cysteine residue. The weaker Fe-NO bond in the W409F and W409Y mutants might result in a faster rate of NO dissociation from the ferric heme in the Trp-409 mutants as compared with the wild type enzyme, which could contribute to the lower accumulation of the inhibitory NO-bound complexes observed during catalysis with the Trp-409 mutants (Adak, S., Crooks, C., Wang, Q., Crane, B. R., Tainer, J. A., Getzoff, E. D., and Stuehr, D. J. (1999) J. Biol. Chem. 274, 26907-26911). The optical and resonance Raman spectra of the Fe2+NO complexes of the Trp-409 mutants differ from those of the wild type enzyme and indicate that a significant population of a five-coordinate Fe2+NO complex is present. These data show that the hydrogen bond provided by the Trp-409 residue is necessary to maintain the thiolate coordination when NO binds to the ferrous heme. Taken together our results indicate that the heme environment on the proximal side of nNOS is critical for the formation of a stable iron-cysteine bond and for the control of the electronic properties of heme-NO complexes.