Non-covalent inhibitors of thioredoxin glutathione reductase with schistosomicidal activity in vivo.

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.

GRIM19 is involved in WT1 expression and epithelial-to-mesenchymal transition in adenomyotic lesions.

The epithelial-to-mesenchymal transition may play a role in adenomyosis. GRIM19 expression is downregulated in adenomyotic lesions, and the effects of this downregulation in adenomyosis remain relatively unclear. In this study, we aimed to explore whether aberrant GRIM19 expression is associated with the epithelial-to-mesenchymal transition in adenomyosis and found that the expression of both GRIM19 and WT1 was low, and epithelial-to-mesenchymal transition, which included significant changes in CDH1, CDH2 and KRT8 expression, occurred in adenomyotic lesions, as confirmed by Western blotting and quantitative real-time PCR. We provided novel insights into WT1 expression in adenomyosis, revealing that WT1 expression was increased in the endometrial glands of adenomyotic lesions by immunohistochemistry. In vitro, knockdown of GRIM19 expression by small interfering RNA (siRNA) promoted the proliferation, migration and invasion of Ishikawa cells, as measured by Cell Counting Kit-8, wound healing assay and Transwell assays. Western blotting and quantitative real-time PCR confirmed that WT1 expression increased and epithelial-to-mesenchymal transition was induced, including the upregulation of CDH2 and downregulation of CDH1 and KRT8after transfecting the GRIM19 siRNA to Ishikawa cells. Furthermore, WT1 expression was upregulated and epithelial-to-mesenchymal transition was observed, including downregulation of CDH1 and KRT8in GRIM19 gene-knockdown mice. Upregulation of Wt1 expression in the endometrial glands of Grim19 knockdown mice was also verified by immunohistochemistry. Taken together, these results reveal that low expression of GRIM19 in adenomyosis may upregulate WT1 expression and induce epithelial-to-mesenchymal transition in the endometria, providing new insights into the pathogenesis of adenomyosis.

Enhanced antitumor effect of cisplatin in human oral squamous cell carcinoma cells by tumor suppressor GRIM­19.

Gene associated with retinoid­interferon­induced mortality 19 (GRIM­19) is a novel candidate tumor suppressor gene located on the human chromosome 19p13.1 region. Our previous study demonstrated that the upregulation of GRIM­19 in human oral squamous cell carcinoma (OSCC) cells significantly inhibited tumor cell growth in vitro and in vivo. In the present study, the combined effects of cationic liposome (LP)­mediated GRIM­19 gene (LP­pGRIM­19) and the low­dose chemotherapeutic drug, cisplatin (CDDP), on tumor cell growth in vitro and in vivo were examined, and the molecular mechanism of their mutual action was investigated by cell proliferation, colony formation, apoptosis, migration, invasion and western blotting assays in vitro, and a node nude tumor model. It was demonstrated that cationic LP­pGRIM­19 gene therapy sensitized the response of breast cancer cells to CDDP, and that LP­pGRIM­19 in combination with CDDP significantly induced apoptosis and inhibited proliferation, colony formation, migration and invasion of the cells, compared with CDDP treatment alone. In addition, systemic treatment with a combination of intravenous injection of LP­pGRIM­19 and intraperitoneal injection of low­dose CDDP into subcutaneous HSC3 human OSCC xenograft mice resulted in a significant inhibition of tumor growth (P<0.05). Further investigations indicated that the enhancement of CPPP­mediated antitumor effects by GRIM­19 may be associated with the upregulation of phosphorylated p53 and the downregulation of B cell lymphoma­2, cyclin D1, vascular endothelial growth factor, matrix metalloproteinase (MMP)­2 and MMP­9, the proteins of which are involved in the activation of signal transducer and activator of transcription 3. The results of the present study suggested that the combination of GRIM­19 gene therapy with low­dose CPPP­based chemotherapy may be a potent therapeutic strategy for the treatment of OSCC.

Identification of a soluble NADPH oxidoreductase (BmNOX) with antiviral activities in the gut juice of Bombyx mori.

Silkworms show high variability in silk quality and disease resistance. Attempts are on to combine the disease tolerance of multivoltine races and the silk quality of bivoltine races to generate new races with desirable phenotypic traits. We report the identification of a 26.5-kDa protein that is overexpressed in the gut juice of disease-resistant multivoltine races and that has anti-BmNPV activity. We have characterized this protein as a soluble NADH-oxidoreductase-like protein (BmNOX). Treatment of live BmNPV particles with BmNOX inhibited the capability of the viral particles to infect BmN cells in vitro.

Systems-based design of bi-ligand inhibitors of oxidoreductases: filling the chemical proteomic toolbox.

Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features in their cofactor binding sites. Then we identify a ligand for this site and use NMR-based binding site mapping (NMR SOLVE) to determine where to extend a combinatorial library, such that diversity elements are directed into the adjacent substrate site. The cofactor mimic is reused in the library in a manner that parallels the reuse of cofactor domains in the oxidoreductase gene family. A library designed in this manner yielded specific inhibitors for multiple oxidoreductases.

Comparative study of the xenobiotic metabolising system in the digestive gland of the bivalve molluscs in different aquatic ecosystems and in aquaria experiments.

A comparative study has been performed on populations of Unionidae from the Lake Suszek and Brda river situated in the centre of Tucholski Landscape Park, around which there are no factories and the Pilica river--affected by the influence of the nearby town agglomeration. Mussels collected from Suszek were also treated (72 h) with various concentrations of dichlorophenol (DCP; 0.1, 0.15, 0.2 ppm) and paraquat (PQ; 1, 5, 10 ppm) in laboratory conditions (aquarium). The activities of glutathione S-transferase (GST) and cytochrome P450 monooxygenase system (NAD(P)H ferricyanide reductase, NAD(P)H cytochrome c reductase), cytochrome P450 content and b(5) in microsomal and cytosolic fractions of digestive gland were investigated. The differences in enzyme activities between groups of mussels, which were exposed to various concentrations of chemical pollutants, as well as the dependence on geographical distribution in Poland, were observed. In experiments with DCP the dose-dependent increase in GST activity was found, but no changes after PQ treatment were observed. Results, in experiments with DCP and PQ, have varied from no change to increase or decrease in the measured monooxygenase activities and cytochrome P450 content. Increases have been recorded in two cases (NADPH ferricyanide reductase and cytochrome P450) after exposure to DCP and in the case of NADH ferricyanide reductase following the exposure to PQ. NAD(P)H cytochrome c reductase activity and content of P450 decreased considerably in 5 and 10 ppm PQ-treated mussels. Thus, the treatment with DCP and PQ in water changed the properties of the mussels digestive gland cytochrome P450 monooxygenase system. These changes may be used as a bioindicator, at the molecular level, of exposure to those xenobiotics not only in controlled experiments (aquaria) but also in the natural environment.

Vascular superoxide production by NAD(P)H oxidase: association with endothelial dysfunction and clinical risk factors.

Superoxide anion plays important roles in vascular disease states. Increased superoxide production contributes to reduced nitric oxide (NO) bioactivity and endothelial dysfunction in experimental models of vascular disease. We measured superoxide production by NAD(P)H oxidase in human blood vessels and examined the relationships between NAD(P)H oxidase activity, NO-mediated endothelial function, and clinical risk factors for atherosclerosis. Endothelium-dependent vasorelaxations and direct measurements of vascular superoxide production were determined in human saphenous veins obtained from 133 patients with coronary artery disease and identified risk factors. The predominant source of vascular superoxide production was an NAD(P)H-dependent oxidase. Increased vascular NAD(P)H oxidase activity was associated with reduced NO-mediated vasorelaxation. Furthermore, reduced endothelial vasorelaxations and increased vascular NAD(P)H oxidase activity were both associated with increased clinical risk factors for atherosclerosis. Diabetes and hypercholesterolemia were independently associated with increased NADH-dependent superoxide production. The association of increased vascular NAD(P)H oxidase activity with endothelial dysfunction and with clinical risk factors suggests an important role for NAD(P)H oxidase-mediated superoxide production in human atherosclerosis. The full text of this article is available at http://www.circresaha.org.

Monofunctional adenine N-3 adducts of melphalan: occurrence at a mutational hotspot sequence and resistance to removal by AlkA protein.

Previous work showed that a CTAAA sequence in the supF gene of the shuttle plasmid pZ189 was a hotspot for mutagenesis by the aromatic nitrogen mustards melphalan and chlorambucil, and indirect evidence suggested adenine N-3 adducts as premutagenic lesions. In order to characterize the adducts formed at this sequence more directly, a substrate was prepared in which the three adjacent adenines in the CTAAA sequence were 3H-labeled. Following treatment of this substrate with [14C]melphalan, thermolabile adducts were depurinated and analyzed by HPLC. Only a single peak bearing both 3H and 14C label was detected and it coeluted with the single major adduct formed by the reaction of melphalan with free adenine base. Various spectrometric analyses of this species were all consistent with its identification as a monofunctional adenine N-3 adduct of melphalan. There was no evidence for any bifunctional adducts involving the labeled adenines. There was little if any release of the adenine N-3 adduct of melphalan by Escherichia coli AlkA protein, under conditions where 3-methyladenine was quantitatively released. The results support the proposal that monofunctional adenine N-3 adducts are intermediates in the generation of A.T-->T.A and A.T-->C.G transversions by aromatic nitrogen mustards.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism symptoms in man, has been examined as a substrate of recombinant human cytochrome P450 2D6. When cumene hydroperoxide is used as an oxygen and electron donor, a single product is formed, identified as 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) for formation of this product (130 microM) is in agreement with the dissociation constants for MPTP binding to the enzyme determined by optical and nuclear magnetic resonance (NMR) spectroscopy. When the reaction is carried out with nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and recombinant human NADPH-cytochrome P450 reductase, a second product, identified as 1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine, is formed in addition to 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) values for formation of these two products are 19 microM and 120 microM, respectively. Paramagnetic relaxation experiments have been used to measure distances between the protons of bound MPTP and the heme iron, and these have been used to construct models for the position and orientation of MPTP in the active site. For the cytochrome alone, a single mode of binding was observed, with the N-methyl close to the heme iron in a position appropriate for the observed N-demethylation reaction. In the presence of the reductase, the data were not consistent with a single mode of binding but could be explained by the existence of two alternative orientations of MPTP in the active site. One of these, characterized by a dissociation constant of 150 microM, is essentially identical to that observed in the absence of the reductase. In the second, which has a K(d) of 25 microM, the MPTP is oriented so that the aromatic ring is close to the heme iron, in a position appropriate for p-hydroxylation leading to the formation of the product seen only in the presence of the reductase. In the case of codeine, another substrate for cytochrome P450 2D6, the addition of reductase had no effect on the nature of the product formed, the dissociation constant, or the orientation in the binding site. These observations show that NADPH-cytochrome P450 reductase has an allosteric effect on the active site of cytochrome P450 2D6 that affects the binding of some substrates but not others.

Preventive effect of isoflurane on destruction of cytochrome P450 during reductive dehalogenation of carbon tetrachloride in guinea-pig liver microsomes.

Effects of isoflurane on cytochrome P450 loss during anaerobic dechlorination of carbon tetrachloride in guinea pig liver microsomes were examined. Under anaerobic conditions, chloroform was produced from carbon tetrachloride by the microsomes in the presence of NADPH, and production of chloroform was increased 1.80 times by the addition of isoflurane. The concentration of microsomal cytochrome P450 decreased to 71.6% after 7 minutes incubation with carbon tetrachloride and NADPH. With the addition of isoflurane in the same incubation system, the decrease in cytochrome P450 was to 84.9%. The essential components for the loss of cytochrome P450 during the dechlorination of carbon tetrachloride were microsomes, NADPH and carbon tetrachloride. Addition of carbon monoxide reduced the cytochrome P450 loss to negligible. These findings indicate that isoflurane interacts with cytochrome P450 to prevent the cytochrome P450 destruction during the anaerobic dechlorination of carbon tetrachloride in guinea-pig liver microsomes. These results also suggest that the destruction of cytochrome P450 during anaerobic dechlorination of carbon tetrachloride in microsomes was caused by direct attack by the trichloromethyl radical, rather than by carbon tetrachloride-induced lipid peroxidation.

In vitro metabolism of imipramine by brain microsomes: effects of inhibitors and exogenous cytochrome P450 reductase.

The metabolism of imipramine in the brains of rats was analyzed to study the activity of cytochrome P450 in brain microsomes. Brain microsomes were capable of metabolizing imipramine to both hydroxylated and N-demethylated products. The use of selective inhibitors of different cytochromes P450 effected varying changes in the metabolic profiles of formed metabolites consistent with the involvement of several P450 forms in imipramine metabolism. Quinidine inhibited the hydroxylation of imipramine competitively by 60% and 98% at concentrations of 10 microM and 100 microM, respectively. Ketoconazole and 7,8-benzoflavone at a concentration of 100 microM inhibited N-demethylation of imipramine by 75% and 30%, respectively, with a lower effect on imipramine hydroxylation. Results from studies on the incorporation of cytochrome P450 reductase into the brain microsomal system reveal a reductase concentration-dependent increase in imipramine metabolism and suggest that the reductase level in brain is an important factor for the study of catalytic activities in brain microsomal systems.

[Control of buccal peroxidases by a bacterial NADH-hypothiocyanite oxidoreductase]. / Contrôle des peroxydases buccales par une NADH-hypothiocyanite-oxydoréductase bactérienne.

Oral peroxidases (myeloperoxidase, sialoperoxidase) catalyze thiocyanate peroxidation into hypothiocyanite which is bacteriostatic or bactericidal against numerous bacterial species. NADH-hypothiocyanite-oxidoreductase is thought to protect bacteria which can express it; up to now, this enzyme activity was never purified. The present study analyzes, on one hand, the susceptibility of periodontal bacteria against hypothiocyanite and, on the other hand, proposes a purification design for the NADH-hypothiocyanite-oxidoreductase from Streptococcus sanguis, a commensal micro-organism of dental surfaces. The data suggest the importance of the bacterial biofilm on dental surfaces for production of antiseptic oxidants and for the control of their toxicity.

Modification of neutrophil oxidant production with diphenyleneiodonium and its effect on bacterial killing.

Diphenyleneiodonium (DPI), an inhibitor of the NADPH oxidase, has been used to distinguish between oxidative and nonoxidative killing of Staphylococcus aureus and Escherichia coli by neutrophils. The rate of killing of S. aureus was inhibited by 77% in the presence of 10 microM DPI, compared to 81% measured under anaerobic conditions. DPI represents a convenient and accessible alternative to an anaerobic environment or using neutrophils from patients with chronic granulomatous disease, for eliminating oxidative killing. The killing of E. coli was also inhibited by DPI. The effect was more apparent at 30 min than at 10 min, suggesting that E. coli can be killed rapidly by nonoxidative mechanisms that become less efficient at later times. DPI was used at concentrations less than 10 microM to determine how this affected production of the three major neutrophil oxidants, superoxide, hydrogen peroxide, and hypochlorous acid, and to determine the effect of partial inhibition of oxidant production on the killing of S. aureus. Unexpectedly, lower concentrations of DPI (0.1-2 microM) inhibited hydrogen peroxide and hypochlorous acid production 10-30% more than they inhibited superoxide production. Correlation of hydrogen peroxide or hypochlorous acid production with the killing of S. aureus showed that up to 30% inhibition had no effect on the rate of killing, implying that agents that impair neutrophil oxidant production less than this will not compromise bacterial killing. Higher inhibition of oxidant production led to a linear decline in the rate of killing.

Pyrroloquinoline quinone acts with flavin reductase to reduce ferryl myoglobin in vitro and protects isolated heart from re-oxygenation injury.

Pyrroloquinoline quinone has been isolated from bacteria and recently has been detected in mammalian tissues and fluids. We report in vitro studies which show that pyrroloquinoline quinone serves as a high-affinity substrate for an erythrocyte "flavin reductase" and that the pyrroloquinoline quinol generated by this catalysis reacts rapidly with ferryl myoglobin radical. Western blot analysis of rat and rabbit heart homogenates detects a cross-reactive protein which has a molecular weight identical to the erythrocyte reductase from the same species. Low concentrations of pyrroloquinoline quinone protect isolated rabbit heart from re-oxygenation injury, serving as an effective tissue-protective agent in this model for cellular oxidative damage. We propose that this tissue protection is due to a pyrroloquinoline quinol-mediated reduction of reactive oxygen species.

Inhibitory effects of non-steroidal anti-inflammatory drugs on superoxide generation.

Effects of non-steroidal anti-inflammatory drugs (NSAID: amfenac sodium, diclofenac sodium, indomethacin and ketoprofen) on the generation of superoxide anion (O2-) by isolated rat polymorphonuclear leukocytes (PMN) were studied spectrophotometrically using cytochrome c. The effects of these drugs were also studied on O2- production by the xanthine-xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-NADPH oxidase systems. Amfenac sodium, at 0.1 mM, inhibited significantly O2- generation in rat PMN induced by opsonized zymosan. At 0.5 mM, diclofenac sodium and indomethacin inhibited the O2- generation in rat PMN. All of the above drugs slightly inhibited O2- production by the xanthine-xanthine oxidase system. On the other hand, O2- production by the NADPH-NADPH oxidase system was significantly inhibited by the addition of amfenac sodium, ketoprofen or indomethacin. These results suggest that non-steroidal anti-inflammatory drugs do not work as an O2- scavenger and block O2- production by the NADPH-NADPH oxidase system of rat PMN. It is concluded that amfenac sodium and the other drugs are able to inhibit granulocyte O2- production by blocking the activation of NADPH-oxidase.

Catalase and glutathione peroxidase activity in cells with trisomy 21.

CuZnSOD is produced in overdose in cells with trisomy 21. This has been considered to be a cause of increased oxidative stress. In the present work we have studied the catalase and glutathione peroxidase activity in fibroblasts from 6, and blood cells from 30, subjects affected by Down syndrome. In the fibroblasts, catalase and glutathione peroxidase activities did not differ significantly from control cells. In platelets, lymphocytes, polymorphs and erythrocytes, no significant increase of catalase activity was found while glutathione peroxidase activity appeared significantly increased in platelets, polymorphs and erythrocytes but not in lymphocytes. These data seem to indicate that the increase of CuZnSOD in trisomy 21 cells does not affect the production of catalase. An increase, instead, of glutathione peroxidase has been detected in all blood cells, except in lymphocytes; this is a sign of a greater need for protection against the risk of lipoperoxidation. The fact that the enhancement of glutathione peroxidase activity could be assessed only in some types of cells examined suggests that the observed increase in those cells is probably a result of an additive effect of the overproduction of CuZnSOD due to gene dosage and the ordinarily higher content of oxygen radicals and peroxides.

NADPH and "cocktails" containing polyarginine reactivate superoxide generation in leukocytes lysed by membrane-damaging agents.

Human blood leukocytes generated large amounts of superoxide (O2-) following stimulation by certain "cocktails" of soluble agents consisting of poly-L-arginine (PARG), phytohemagglutinin, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine and polyanethole sulfanote (liquoid). A variety of cytochalasins, which markedly boosted O2- generation by the soluble cocktails, markedly depressed luminol-dependent chemiluminescence (LDCL) which had been induced either by opsonized streptococci or by soluble agents. Glutathione, which totally reversed the inhibition of LDCL induced by cytochalasin A, failed to reverse the inhibition of LDCL induced by cytochalasin B. Generation of O2- by all the soluble agents employed, except PMA, was strongly inhibited either by the omission of extracellular calcium and magnesium or by treatment with the calcium blocker TMB-8. Generation of O2- was enhanced following stimulation of leukocytes with soluble agents if the cells had been exposed to slightly hypotonic buffers. Leukocytes, which had been preincubated for short periods (5 min) with PARG, saponin, digitonin, or lysolecithin (LL) and which lost their viability, and their O2- and LDCL-generating capacities following stimulation by soluble agents containing cytochalasin B, nevertheless regained these activities by the addition of NADPH. It is suggested that the lytic agents induced the leakage out of NADPH rather than acting as inactivators of the oxidase in the leukocyte membranes. Prolonged incubation of leukocytes with lytic agents failed to allow restoration, by NADPH, of the generation of SOD-inhibitable O2- generation. Since PARG acted both as a cytolytic agent and as a inducer of O2- generation, we postulate that lytic agents might also act as "primers" of the nascent membrane oxidase which could, however, be further potentiated and activated by soluble agents acting in "multiple hits," PARG could be totally replaced either by LL or by digitonin in the generation of O2- provided that both PHA and cytochalasin B were present in the reaction mixtures. We suggest that the various ingredients of the soluble "cocktails" may help to assemble components of the NADPH oxidase. Such an assembly and regulations are prerequisite for stimulation of the NADPH oxidase and the generation of oxygen radicals in leukocytes.