HNOXPred: a web tool for the prediction of gas-sensing H-NOX proteins from amino acid sequence.

SUMMARY: HNOXPred is a webserver for the prediction of gas-sensing heme-nitric oxide/oxygen (H-NOX) proteins from amino acid sequence. H-NOX proteins are gas-sensing hemoproteins found in diverse organisms ranging from bacteria to eukaryotes. Recently, gas-sensing complex multi-functional proteins containing only the conserved amino acids at the heme centers of H-NOX proteins, have been identified through a motif-based approach. Based on experimental data and H-NOX candidates reported in the literature, HNOXPred is created to automate and facilitate the identification of similar H-NOX centers across systems. The server features HNOXSCORES scaled from 0 to 1 that consider in its calculation, the physicochemical properties of amino acids constituting the heme center in H-NOX in addition to the conserved amino acids within the center. From user input amino acid sequence, the server returns positive hits and their calculated HNOXSCORES ordered from high to low confidence which are accompanied by interpretation guides and recommendations. The utility of this server is demonstrated using the human proteome as an example. AVAILABILITY AND IMPLEMENTATION: The HNOXPred server is available at https://www.hnoxpred.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Navigating the Unnatural Reaction Space: Directed Evolution of Heme Proteins for Selective Carbene and Nitrene Transfer.

Despite the astonishing diversity of naturally occurring biocatalytic processes, enzymes do not catalyze many of the transformations favored by synthetic chemists. Either nature does not care about the specific products, or if she does, she has adopted a different synthetic strategy. In many cases, the appropriate reagents used by synthetic chemists are not readily accessible to biological systems. Here, we discuss our efforts to expand the catalytic repertoire of enzymes to encompass powerful reactions previously known only in small-molecule catalysis: formation and transfer of reactive carbene and nitrene intermediates leading to a broad range of products, including products with bonds not known in biology. In light of the structural similarity of iron carbene (Fe═C(R1)(R2)) and iron nitrene (Fe═NR) to the iron oxo (Fe═O) intermediate involved in cytochrome P450-catalyzed oxidation, we have used synthetic carbene and nitrene precursors that biological systems have not encountered and repurposed P450s to catalyze reactions that are not known in the natural world. The resulting protein catalysts are fully genetically encoded and function in intact microbial cells or cell-free lysates, where their performance can be improved and optimized by directed evolution. By leveraging the catalytic promiscuity of P450 enzymes, we evolved a range of carbene and nitrene transferases exhibiting excellent activity toward these new-to-nature reactions. Since our initial report in 2012, a number of other heme proteins including myoglobins, protoglobins, and cytochromes c have also been found and engineered to promote unnatural carbene and nitrene transfer. Due to the altered active-site environments, these heme proteins often displayed complementary activities and selectivities to P450s.Using wild-type and engineered heme proteins, we and others have described a range of selective carbene transfer reactions, including cyclopropanation, cyclopropenation, Si-H insertion, B-H insertion, and C-H insertion. Similarly, a variety of asymmetric nitrene transfer processes including aziridination, sulfide imidation, C-H amidation, and, most recently, C-H amination have been demonstrated. The scopes of these biocatalytic carbene and nitrene transfer reactions are often complementary to the state-of-the-art processes based on small-molecule transition-metal catalysts, making engineered biocatalysts a valuable addition to the synthetic chemist's toolbox. Moreover, enabled by the exquisite regio- and stereocontrol imposed by the enzyme catalyst, this biocatalytic platform provides an exciting opportunity to address challenging problems in modern synthetic chemistry and selective catalysis, including ones that have eluded synthetic chemists for decades.

Quantitative imaging of intraerythrocytic hemozoin by transient absorption microscopy.

Hemozoin, the heme detoxification end product in malaria parasites during their growth in the red blood cells (RBCs), serves as an important marker for diagnosis and treatment target of malaria disease. However, the current method for hemozoin-targeted drug screening mainly relies on in vitro ß-hematin inhibition assays, which may lead to false-positive events due to under-representation of the real hemozoin crystal. Quantitative in situ imaging of hemozoin is highly desired for high-throughput screening of antimalarial drugs and for elucidating the mechanisms of antimalarial drugs. We present transient absorption (TA) imaging as a high-speed single-cell analysis platform with chemical selectivity to hemozoin. We first demonstrated that TA microscopy is able to identify ß-hematin, the artificial form of hemozoin, from the RBCs. We further utilized time-resolved TA imaging to in situ discern hemozoin from malaria-infected RBCs with optimized imaging conditions. Finally, we quantitatively analyzed the hemozoin amount in RBCs at different infection stages by single-shot TA imaging. These results highlight the potential of TA imaging for efficient antimalarial drug screening and drug mechanism investigation.

Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins.

BACKGROUND: Most species of Shewanella harbor two ferrochelatase paralogues for the biosynthesis of c-type cytochromes, which are crucial for their respiratory versatility. In our previous study of the Shewanella loihica PV-4 strain, we found that the disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), but it is different in Shewanella oneidensis MR-1. Hence, the function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, are investigated in S. oneidensis MR-1. RESULT: In the present study, deletion of either hemH1 or hemH2 in S. oneidensis MR-1 did not lead to overproduction of extracellular protoporphyrin IX (PPIX) as previously described in the hemH1 mutants of S. loihica PV-4. Moreover, supplement of exogenous hemins made it possible to generate the hemH1 and hemH2 double mutant in MR-1, but not in PV-4. Under aerobic condition, exogenous hemins were required for the growth of MR-1ΔhemH1ΔhemH2, which also overproduced extracellular PPIX. These results suggest that heme is essential for aerobic growth of Shewanella species and MR-1 could also uptake hemin for biosynthesis of essential cytochrome(s) and respiration. Besides, the exogenous hemin mediated CymA cytochrome maturation and the cellular KatB catalase activity. Both hemH paralogues were transcribed in wild-type MR-1, and the hemH2 transcription was remarkably up-regulated in MR-1ΔhemH1 mutant to compensate for the loss of hemH1. The periplasmic glutathione peroxidase gene pgpD, located in the same operon with hemH2, and a large gene cluster coding for iron, heme (hemin) uptake systems are absent in the PV-4 genome. CONCLUSION: Our results indicate that the genetic divergence in gene content and gene expression between these Shewanella species, accounting for the phenotypic difference described here, might be due to their speciation and adaptation to the specific habitats (iron-rich deep-sea vent versus iron-poor freshwater) in which they evolved and the generated mutants could potentially be utilized for commercial production of PPIX.

Magnesium Lithospermate B Derived from Salvia miltiorrhiza Ameliorates Right Ventricle Remodeling in Pulmonary Hypertensive Rats via Inhibition of NOX/VPO1 Pathway.

Right ventricle (RV) remodeling is a major pathological feature in pulmonary arterial hypertension (PAH). Magnesium lithospermate B (MLB) is a compound isolated from the roots of Salvia miltiorrhiza and it possesses multiple pharmacological activities such as anti-inflammation and antioxidation. This study aims to investigate whether MLB is able to prevent RV remodeling in PAH and the underlying mechanisms. In vivo, SD rats were exposed to 10% O2 for 21 d to induce RV remodeling, which showed hypertrophic features (increases in the ratio of RV weight to tibia length, cellular size, and hypertrophic marker expression), accompanied by upregulation in expression of NADPH oxidases (NOX2 and NOX4) and vascular peroxidase 1 (VPO1), increases in hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production and elevation in phosphorylation levels of ERK; these changes were attenuated by treating rats with MLB. In vitro, the cultured H9c2 cells were exposed to 3% O2 for 24 h to induce hypertrophy, which showed hypertrophic features (increases in cellular size and hypertrophic marker expression). Administration of MLB or VAS2870 (a positive control for NOX inhibitor) could prevent cardiomyocyte hypertrophy concomitant with decreases in NOX (NOX2 and NOX4) and VPO1 expression, H2O2 and HOCl production, and ERK phosphorylation. Based on these observations, we conclude that MLB is able to prevent RV remodeling in hypoxic PAH rats through a mechanism involving a suppression of NOX/VPO1 pathway as well as ERK signaling pathway. MLB may possess the potential clinical value for PAH therapy.

In vitro antiplasmodial activity and prophylactic potentials of extract and fractions of Trema orientalis (Linn.) stem bark.

BACKGROUND: Trema orientalis (T. orientalis Linn) has been used in the management of malaria in the western part of Nigeria and despite its application in ethnomedicine, there is dearth of scientific evidence to justify the acclaimed prophylactic antimalarial usage of the plant. The aim of this study is to assess the in vitro antiplasmodial cell-free assay and chemopreventive efficacy of the methanol extract of the stem bark of T. orientalis and its fractions as a prophylactic regimen for malaria prevention. Also, the antimicrobial activities of the extract and the fractions were investigated. METHOD: Vacuum liquid chromatography was used to obtain dichloromethane, ethylacetate and methanol fractions from the methanol extract of T. orientalis. The fractions were tested for their prophylactic and cell-free antimalarial activity using murine models and ß-hematin formation assay respectively. Disc diffusion method was used to determine the antibacterial activity of the extract and its fractions against both Gram-positive and Gram-negative bacteria. RESULTS: In the prophylactic experiment, dichloromethane (DCMF), methanol fraction (MF) and extract (ME) (in this order) showed significant chemopreventive effects against P. berghei invasion of the red blood cells when compared with both Sulfadoxine-Pyrimethamine (SP) and untreated controls. Results of the in vitro study showed that the DCMF had the highest effect in preventing the formation of ß-hematin when compared with other fractions. The DCMF also had the highest percentage inhibition of ß-hematin formation when compared with chloroquine. The extract and fractions showed a concentration dependent antibacterial activity. Methanol extract had a pronounced inhibitory effect on Enterobacter cloaca ATCC 13047 and Enterococcus faecalis ATCC 29212. Serratia mercescens ATCC 9986 and Pseudomonas aeruginosa ATCC 19582 were the most susceptible bacteria. CONCLUSION: The results obtained showed that both extract and fractions of T. orientalis possessed antiplasmodial and antimicrobial activity.

Synthesis, ß-hematin inhibition studies and antimalarial evaluation of dehydroxy isotebuquine derivatives against Plasmodium berghei.

Diverse dehydroxy-isotebuquine derivatives were prepared by using a five step synthetic sequence in good yields. All these new 4-aminoquinolines were evaluated as inhibitors of haemozoin formation, where most of them showed a significant inhibition value (% IHF >97). The best inhibitors were tested in vivo as potential antimalarials in mice infected with Plasmodium berghei ANKA chloroquine susceptible strain, three of them (11b, 11d and 11h) displayed an antimalarial activity comparable to that of chloroquine.

Evaluation of in vitro antimalarial activity of different extracts of Artemisia aucheri Boiss. and A. armeniaca Lam. and fractions of the most potent extracts.

Ten extracts with different polarity from two Iranian Artemisia species, A. armeniaca Lam. and A. aucheri Boiss, were screened for their antimalarial properties by in vitro ß -hematin formation assay. Dichloromethane (DCM) extracts of both plants showed significant antimalarial activities with IC50 values of 1.36±0.01 and 1.83±0.03 mg/mL and IC90 values of 2.12±0.04 and 2.62±0.09 mg/mL for A. armeniaca and A. aucheri, respectively. Bioactivity-guided fractionation of DCM extracts of both plants by vacuum liquid chromatography (VLC) over silica gel with solvent mixtures of increasing polarities afforded seven fractions. Two fractions from DCM extract of A. armeniaca and four fractions from DCM extract of A. aucheri showed potent antimalarial activity with reducing IC50 and IC90 values compared to extracts. The most potent fraction belonged to DCM extract of A. armeniaca with IC50 and IC90 values of 0.47±0.006 and 0.71±0.006 mg/mL, respectively.

Preparation of nitrophorin 7(Δ1-3) from Rhodnius prolixus without start-methionine using recombinant expression in Escherichia coli.

The heterologous recombinant expression of proteins in Escherichia coli without start-methionine is a common problem. The nitrophorin 7 heme properties and function strongly depend on the accurate N-terminal amino acid sequence. Leading protein expression into the periplasm by fusion with the leader peptide pelB yields functional protein; however, the folded protein sticks to the cell debris. Therefore, the periplasmic fraction was dissolved in guanidinium chloride and folded by a drop-in method. Separation from impurities including residual pelB-nitrophorin 7 required establishing an unconventional chromatographic technique using calcium-loaded Chelating Sepharose as cation exchanger and elution by a linear CaCl2 gradient.

Rutaecarpine inhibits angiotensin II-induced proliferation in rat vascular smooth muscle cells.

OBJECTIVE: To evaluate the effects and possible mechanisms of rutaecarpine on angiotensin II (Ang II)-induced proliferation in cultured rat vascular smooth muscle cells (VSMCs). METHODS: VSMCs were isolated from Male Sprague-Dawley rat aorta, and cultured by enzymic dispersion method. Experiments were performed with cells from passages 3-8. The cultured VSMCs were randomly divided into control, model (Ang II 0.1 µmol/L), and rutaecarpine (0.3-3.0 µmol/L) groups. VMSC proliferation was induced by Ang II, and was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and cell counting. To examine the mechanisms involved in anti-proliferative effects of rutaecarpine, nitric oxide (NO) levels and NO synthetase (NOS) activity were determined. Expressions of VSMC proliferation-related genes including endothelial nitric oxide synthase (eNOS), and c-myc hypertension related gene-1 (HRG-1) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Rutaecarpine (0.3-3.0 µmol/L) inhibited Ang II-induced VSMC proliferation and the best effects were achieved at 3.0 µmol/L. The Ang II-induced decreases in cellular NO contents and NOS activities were antagonized by rutaecarpine (P <0.05). Ang II administration suppressed the expressions of eNOS and HRG-1, while increased c-myc expression (P <0.05). All these effects were attenuated by 3.0 µmol/L rutaecarpine (P <0.05). CONCLUSION: Rutaecarpine is effective against Ang II-induced rat VSMC proliferation, and this effect is due, at least in part, to NO production and the modulation of VMSC proliferation-related gene expressions.

Hemozoin and antimalarial drug discovery.

Recent initiatives to develop more effective and affordable drugs, controlling mosquitoes and development of a preventative vaccine have been launched with the goal of completely eradicating malaria. To this end, Novartis (Surrey, UK) and GlaxoSmithKline (Middlesex, UK) screened their chemical libraries of approximately two million small molecules for antimalarial properties, which resulted in a set of over 20,000 'highly druggable' initial hits. Efforts in academia are centered on specific pathway targets. One such high-throughput screening effort has been focused on hemozoin formation, a unique heme detoxification pathway found in the malaria parasite. This review discusses the current approaches and limitations of high-throughput screening discovery of hemozoin inhibitors. In the future, new methods must be developed to validate the mechanism of action of these hit compounds within the parasite.

The heme-binding protein Dap1 links iron homeostasis to azole resistance via the P450 protein Erg11 in Candida glabrata.

The pathogenic fungus Candida glabrata is relatively resistant to azole antifungals, which target lanosterol 14α-demethylase (Erg11p) in the ergosterol biosynthesis pathway. Our study revealed that C. glabrata exhibits increased azole susceptibility under low-iron conditions. To investigate the molecular basis of this phenomenon, we generated a strain lacking the heme (iron protoporphyrin IX)-binding protein Dap1 in C. glabrata. The Δdap1 mutant displayed growth defects under iron-limited conditions, decreased azole tolerance, decreased production of ergosterol, and increased accumulation of 14α-methylated sterols lanosterol and squalene. All the Δdap1 phenotypes were complemented by wild-type DAP1, but not by DAP1(D91G) , in which a heme-binding site is mutated. Furthermore, azole tolerance of the Δdap1 mutant was rescued by exogenous ergosterol but not by iron supplementation alone. These results suggest that heme binding by Dap1 is crucial for Erg11 activity and ergosterol biosynthesis, thereby being required for azole tolerance. A Dap1-GFP fusion protein predominantly localized to vacuolar membranes and endosomes, and the Δdap1 cells exhibited aberrant vacuole morphologies, suggesting that Dap1 is also involved in the regulation of vacuole structures that could be important for iron storage. Our study demonstrates that Dap1 mediates a functional link between iron homeostasis and azole resistance in C. glabrata.

Design, synthesis of 4-aminoquinoline-derived thiazolidines and their antimalarial activity and heme polymerization inhibition studies.

The present study describes the synthesis of a series of new 4-aminoquinoline-derived thiazolidines and evaluation of their antimalarial activity against a NF-54 strain of Plasmodium falciparum in vitro and N-67 strain of Plasmodium yoelii in vivo. Among the series, two compounds, 2-(4-chloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide hydrochloride (14) and 2-(2,6-dichloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide hydrochloride (22) exhibited significant suppression of parasitaemia in the in vivo assay. All the analogues were found to form strong complex with haematin and inhibited the ß-haematin formation in vitro. These results suggest that these compounds act on heme polymerization target.

[Use of vitamins for correction of the functional state of cytochrome P450 systems in experimental allergic encephalomyelitis].

It is known that inflammatory cytokines, which level is significantly increased in the pathogenesis of multiple sclerosis (MS), as well as interferon-beta, which is used to treat autoimmune diseases, can inhibit cytochrome P450-dependent processes of detoxification and biotransformation. The uncontrolled decrease of the activity of these processes may have a negative affect on the state of patients, so it is urgent to study the functional state of the cytochrome P450 system and to develop effective means for its regulation in these conditions. The effect of vitamin D3 and efficiency of its composition with vitamins B1, B2, B6, PP, E, alpha-lipoic, alpha-linolenoic acid and mineral substances (Mg, Zn, Se) in prevention of a functional state changes of cytochrome P450- and b5-dependent systems of the rat brain and liver endoplasmic reticulum at EAE are investigated. It has been shown that the essential decrease of the level of these cytochromes is observed both in the brain and liver. In addition the level of activity of NADH- and NADPH-oxidoreductases, which are part of microsomal electron transport chain components and coupled with monooxigenases, was reduced. These changes confirm the disturbances of a redox state and functional activity of detoxication and biotransformation systems in the studied animal tissues. Supplement of vitamin D3 as well as the composition of biologically active substances, which we developed earlier, effectively eliminated the decrease of the level of cytochromes and activities of NADH-oxidoreductase in immunised rat tissues. Normalization of these disturbances can be explained by antioxidant and membrane-stabilizing properties of applied substances, and also by the ability to reduce the activity of inflammatory reactions by regulation of the level of inflammatory cytokines in rat organism at EAE. Thus the studied vitamin-mineral composition appeared to be more effective to normalize the found disturbances and it can be useful for prevention of exacerbations and for improvement of a status of patients with multiple sclerosis and other diseases, which are accompanied with hyperactivation of immune system.

Study of streptococcal hemoprotein receptor (Shr) in iron acquisition and virulence of M1T1 group A streptococcus.

Streptococcus pyogenes (group A streptococcus, GAS) is a human bacterial pathogen of global significance, causing severe invasive diseases associated with serious morbidity and mortality. To survive within the host and establish an infection, GAS requires essential nutrients, including iron. The streptococcal hemoprotein receptor (Shr) is a surface-localized GAS protein that binds heme-containing proteins and extracellular matrix components. In this study, we employ targeted allelic exchange mutagenesis to investigate the role of Shr in the pathogenesis of the globally disseminated serotype M1T1 GAS. The shr mutant exhibited a growth defect in iron-restricted medium supplemented with ferric chloride, but no significant differences were observed in neutrophil survival, antimicrobial peptide resistance, cell surface charge, fibronectin-binding or adherence to human epithelial cells and keratinocytes, compared with wild-type. However, the shr mutant displayed a reduction in human blood proliferation, laminin-binding capacity and was attenuated for virulence in in vivo models of skin and systemic infection. We conclude that Shr augments GAS adherence to laminin, an important extracellular matrix attachment component. Furthermore, Shr-mediated iron uptake contributes to GAS growth in human blood, and is required for full virulence of serotype M1T1 GAS in mouse models of invasive disease.

An unconventional hexacoordinated flavohemoglobin from Mycobacterium tuberculosis.

Being an obligate aerobe, Mycobacterium tuberculosis faces a number of energetic challenges when it encounters hypoxia and environmental stress during intracellular infection. Consequently, it has evolved innovative strategies to cope with these unfavorable conditions. Here, we report a novel flavohemoglobin (MtbFHb) from M. tuberculosis that exhibits unique features within its heme and reductase domains distinct from conventional FHbs, including the absence of the characteristic hydrogen bonding interactions within the proximal heme pocket and mutations in the FAD and NADH binding regions of the reductase domain. In contrast to conventional FHbs, it has a hexacoordinate low-spin heme with a proximal histidine ligand lacking imidazolate character and a distal heme pocket with a relatively low electrostatic potential. Additionally, MtbFHb carries a new FAD binding site in its reductase domain similar to that of D-lactate dehydrogenase (D-LDH). When overexpressed in Escherichia coli or Mycobacterium smegmatis, MtbFHb remained associated with the cell membrane and exhibited D-lactate:phenazine methosulfate reductase activity and oxidized D-lactate into pyruvate by converting the heme iron from Fe(3+) to Fe(2+) in a FAD-dependent manner, indicating electron transfer from D-lactate to the heme via FAD cofactor. Under oxidative stress, MtbFHb-expressing cells exhibited growth advantage with reduced levels of lipid peroxidation. Given the fact that D-lactate is a byproduct of lipid peroxidation and that M. tuberculosis lacks the gene encoding D-LDH, we propose that the novel D-lactate metabolizing activity of MtbFHb uniquely equips M. tuberculosis to balance the stress level by protecting the cell membrane from oxidative damage via cycling between the Fe(3+)/Fe(2+) redox states.

Synthesis and antimalarial activities of a diverse set of triazole-containing furamidine analogues.

Four different series of triazole diamidines have been prepared by the Pinner method from the corresponding triazole dinitriles. Copper-catalyzed "click chemistry" was used for the synthesis of 1,4- and 4,5-substituted triazoles, aryl magnesium acetylide reagents for the 1,5-substituted triazoles, with a thermal dipolar addition reaction employed for the 2,4-substituted triazoles. In vitro antimalarial activity against two different PfCRT-modified parasite lines (Science 2002, 298, 210-213) of Plasmodium falciparum and inhibition of hemozoin formation were determined for each compound. Several diamidines with potent nanomolar antimalarial activities were identified, and selected molecules were resynthesized as their diamidoxime triazole prodrugs. One of these prodrugs, OB216, proved to be highly potent in vivo with an ED50 value of 5 mg kg(-1) (po) and an observed 100 % cure rate (CD100) of just 10 mg kg(-1) by oral (po) administration in mice infected with P. vinckei.

Correlation between tumor-associated proteins and response to neoadjuvant treatment in patients with advanced squamous-cell esophageal cancer.

BACKGROUND: Possible predictive markers of response to neoadjuvant radiochemotherapy (NRCT) of esophageal cancer have been identified. PATIENTS AND METHODS: Patient biopsies were obtained from both tumor and normal tissue before the NRCT of locally advanced esophageal squamous cell carcinoma. Protein solutions were separated and immunoblot analysis was performed with heat shock protein (Hsp)16.2, heme-binding protein 2 (SOUL), BCL2-associated X protein (Bax), B-cell-associated leukemia protein 2 (Bcl-2) and heat shock protein 90 (Hsp90) antibodies. Following NRCT, the patients were restaged according to the Response Evaluation Criteria In Solid Tumors (RECIST). Following resections the pathological down-staging was evaluated. RESULTS: Clinical restaging revealed a response rate of 65%. Pathological examination revealed down-staging in 30% and 25% of the cases for the T and N categories respectively. Compared to the normal esophageal mucosa, a decreased expression of Hsp16.2, Hsp90 and SOUL proteins and an increased Bax/Bcl-2 ratio was found in the responding tumors. CONCLUSION: Hsp16,2, Hsp90 and SOUL expression and Bax/ Bcl-2 ratio correlates to the efficacy of NRCT and predict outcome in patients with locally advanced squamous-cell esophageal cancer.

Effects of magnesium supplementation on the glutathione redox system in atopic asthmatic children.

OBJECTIVE: Our aim was to investigate the effects of 12-week oral magnesium (Mg) supplementation on the RBC redox system in stable, persistent, moderately asthmatic children (N = 40, 24 boys, 16 girls) aged 4-16 years in a randomized, double-blind, placebo-controlled study. DESIGN: Oxidized (GSSG) and reduced (GSH) glutathione, oxyhaemoglobin, methaemoglobin (metHb), hemichrome and bilirubin levels before and after treatment were determined, and GSH stability tests were performed. RESULT: The GSH concentration was significantly higher in the Mg-treated than in the placebo-treated patients after the treatment period. There was a positive correlation between the decreased plasma metHb and hemichrome levels and the decreased plasma haemoglobin concentrations in the Mg-treated patients at the end of the study. CONCLUSION: Mg in the given doses exerts antioxidant activity and influences the glutathione redox system.

Effect of zinc on hepatic drug metabolism under ethanol toxicity.

The effects of zinc on drug-metabolizing enzymes in the liver were examined in male Wistar rats following ethanol intoxication. Rats were orally fed 3 mL of 30% ethanol daily for either two, four, or eight weeks and were orally administered zinc sulfate (ZnSO4.7H2O) at a dose level of 227 mg/L. Levels of reduced glutathione (GSH) and the activities of cytochrome P-450, cytochrome b(5), NADPH cytochrome-C-reductase and glutathione-S-transferase (GST) were determined in liver after two, four, and eight weeks. Significant elevation was observed in the activities of the enzymes of the mixed function oxidase system in response to toxicity induced by ethanol at all the intervals. These effects of were ascribed to the enhanced activity of the microsomal ethanol oxidizing system and the associated increase in reactive oxygen species production. Zinc supplementation to these ethanol-intoxicated animals resulted in normalization of these elevated values significantly, but still they do not attain normal levels. Significant increase was observed in reduced glutathione content in animals after four and eight weeks of ethanol feeding, which appeared to be further elevated in combined zinc and ethanol treatment. Significant elevation in the activity of GST was illustrated on ethanol-fed animals at all the three treatment intervals. Furthermore, the activity of this enzyme was only moderately normalized following zinc treatment. This was accredited to the antioxidant potential of zinc, as well as its ability to induce metallothionein content, which provide protection against the toxic effects of ethanol. To conclude, zinc was able to normalize the effects of ethanol in the liver.