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Sample records for reductase prevents experimental

  1. Targeting 5α-reductase for prostate cancer prevention and treatment.

    PubMed

    Nacusi, Lucas P; Tindall, Donald J

    2011-07-01

    Testosterone is the most abundant circulating androgen, and can be converted to dihydrotestosterone (DHT), a more potent androgen, by the 5α-reductase enzymes in target tissues. Current treatments for prostate cancer consist of reducing androgen levels by chemical or surgical castration or pure antiandrogen therapy that directly targets the androgen receptor (AR). Although these therapies reduce tumor burden and AR activity, the cancer inevitably recurs within 18-30 months. An approach targeting the androgen-AR axis at different levels could, therefore, improve the efficacy of prostate cancer therapy. Inhibition of 5α-reductase is one such approach; however, the two largest trials to investigate the use of the 5α-reductase inhibitors (5ARIs) finasteride and dutasteride in patients with prostate cancer have shown that, although the incidence of cancer was reduced by 5ARI treatment, those cancers that were detected were more aggressive than in patients treated with placebo. Thus, the best practice for using these drugs to prevent and treat prostate cancer remains unclear. PMID:21629218

  2. Targeting 5α-reductase for prostate cancer prevention and treatment

    PubMed Central

    Nacusi, Lucas P.; Tindall, Donald J.

    2014-01-01

    Testosterone is the most abundant circulating androgen, and can be converted to dihydrotestosterone (DHT), a more potent androgen, by the 5α-reductase enzymes in target tissues. Current treatments for prostate cancer consist of reducing androgen levels by chemical or surgical castration or pure antiandrogen therapy that directly targets the androgen receptor (AR). Although these therapies reduce tumor burden and AR activity, the cancer inevitably recurs within 18–30 months. An approach targeting the androgen–AR axis at different levels could, therefore, improve the efficacy of prostate cancer therapy. Inhibition of 5α-reductase is one such approach; however, the two largest trials to investigate the use of the 5α-reductase inhibitors (5ARIs) finasteride and dutasteride in patients with prostate cancer have shown that, although the incidence of cancer was reduced by 5ARI treatment, those cancers that were detected were more aggressive than in patients treated with placebo. Thus, the best practice for using these drugs to prevent and treat prostate cancer remains unclear. PMID:21629218

  3. GP-1447, an inhibitor of aldose reductase, prevents the progression of diabetic cataract in rats.

    PubMed

    Kawakubo, Ken; Mori, Asami; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2012-01-01

    We examined the effects of GP-1447 (3-[(4,5,7-trifluorobenzothiazol-2-yl)methyl]-5-methylphenyl acetic acid) on existing cataracts and sorbitol content in the lens in rats with streptozotocin-induced diabetes. GP-1447 is an inhibitor of aldose reductase, which is the first enzyme in the polyol pathway. Cataracts in the central region of the lens were observed in 7 of 14 eyes (50%) by the fifth week after induction of diabetes, and development of mature cataracts was observed in most lenses by the ninth week. In diabetic rats that received GP-1447 treatment beginning in the fifth week after induction of diabetes, progression of cataracts was observed for 1 week after initiation of treatment. Thereafter, the severity of cataracts did not change substantially. Sorbitol levels in the lens peaked during the first week of diabetes, and this increase was maintained during the 9-week observation period. Elevated sorbitol levels in the lenses of diabetic rats gradually declined after GP-1447 treatment was started on the fifth week after induction of diabetes. Cataracts and sorbitol elevation were not observed in the lenses of controls or diabetic rats treated with GP-1447 immediately after induction of diabetes. These results suggest that the polyol pathway plays an important role in both the appearance and progression of cataracts in diabetic rats. Inhibition of aldose reductase could significantly prevent progression of existing cataracts. PMID:22687477

  4. Role of 5α-reductase inhibitors in prostate cancer prevention and treatment.

    PubMed

    Azzouni, Faris; Mohler, James

    2012-06-01

    Although testosterone is the most abundant serum androgen, dihydrotestosterone is the main prostatic androgen. Testosterone is converted to dihydrotestosterone by the enzyme 5α-reductase (5α-R). Dihydrotestosterone plays an important role in several human diseases, including benign prostate enlargement and prostate cancer. The observation that males born with 5α-R 2 deficiency have never been reported to develop prostate cancer stimulated interest in development of 5α-R inhibitors. Thus far, 2 5α-R inhibitors are approved for clinical use. Several trials evaluated the use of 5α-R inhibitors in prostate cancer prevention and treatment and will be reviewed in this article. PMID:22446342

  5. Bioactive fraction of Saraca indica prevents diabetes induced cataractogenesis: An aldose reductase inhibitory activity

    PubMed Central

    Somani, Gauresh; Sathaye, Sadhana

    2015-01-01

    Background: The present study was designed to investigate the effect of Saraca indica (SI) flowers extract and different bioactive fraction on in vitro aldose reductase (AR) inhibitory activity, high glucose-induced cataract in goat lens and in vivo streptozotocin (STZ; 45 mg/kg, i.p) induced cataract in rats. Methods: Extract of flowers of SI tested for inhibition against rat lens AR. Furthermore, bioactive fraction was investigated against high glucose-induced opacification of the lens in vitro lens culture and STZ induced diabetic cataract in rats. Identification of the bioactive component was attempted through high-performance thin-layer chromatography, high-performance liquid chromatography and liquid chromatography-mass spectrometry analysis. Results: Ethyl acetate fraction of S. indica (EASI) produced maximum inhibition that may be due to high phenolic content. Goat lenses in media containing glucose developed a distinctly opaque ring in 72 h and treatment with EASI fraction lowered lens opacity in 72 h. Prolonged treatment with EASI to STZ-induced diabetic rats inhibited the AR activity and delayed cataract progression in a dose dependent manner. Conclusion: Ethyl acetate fraction of S. indica fraction has potential to inhibit rat lens AR enzyme and prevent cataractogenesis not only in goat lens model (in vitro), but also in STZ induced diabetic rats (in vivo). This study is suggestive of the anticataract activity of EASI fraction that could be attributed to the phytoconstituents present in the same. PMID:25709218

  6. 5-α reductase inhibitors and prostate cancer prevention: where do we turn now?

    PubMed

    Hamilton, Robert J; Freedland, Stephen J

    2011-01-01

    With the lifetime risk of being diagnosed with prostate cancer so great, an effective chemopreventive agent could have a profound impact on the lives of men. Despite decades of searching for such an agent, physicians still do not have an approved drug to offer their patients. In this article, we outline current strategies for preventing prostate cancer in general, with a focus on the 5-α-reductase inhibitors (5-ARIs) finasteride and dutasteride. We discuss the two landmark randomized, controlled trials of finasteride and dutasteride, highlighting the controversies stemming from the results, and address the issue of 5-ARI use, including reasons why providers may be hesitant to use these agents for chemoprevention. We further discuss the recent US Food and Drug Administration ruling against the proposed new indication for dutasteride and the change to the labeling of finasteride, both of which were intended to permit physicians to use the drugs for chemoprevention. Finally, we discuss future directions for 5-ARI research. PMID:21920036

  7. The use of 5-alpha reductase inhibitors for the prevention of prostate cancer.

    PubMed

    Yu, Eun-mi; El-Ayass, Walid; Aragon-Ching, Jeanny B

    2010-07-01

    The use of 5-alpha-reductase inhibitors has been studied not only in benign prostatic hyperplasia, but as a chemopreventive strategy in prostate cancer. Both finasteride and dutasteride, 5 alpha-reductase inhibitors (5ARI), have been shown to decrease the risk of prostate cancer. The results of the REDUCE trial using the dual alpha-reductase isoenzyme inhibitor dutasteride, has recently been published by Andriole et al. in the New England Journal of Medicine. Certain considerations regarding its use and applicability to men with high risk of developing prostate cancer are herein discussed. PMID:20574153

  8. Molecular modeling study of dihydrofolate reductase inhibitors. Molecular dynamics simulations, quantum mechanical calculations, and experimental corroboration.

    PubMed

    Tosso, Rodrigo D; Andujar, Sebastian A; Gutierrez, Lucas; Angelina, Emilio; Rodríguez, Ricaurte; Nogueras, Manuel; Baldoni, Héctor; Suvire, Fernando D; Cobo, Justo; Enriz, Ricardo D

    2013-08-26

    A molecular modeling study on dihydrofolate reductase (DHFR) inhibitors was carried out. By combining molecular dynamics simulations with semiempirical (PM6), ab initio, and density functional theory (DFT) calculations, a simple and generally applicable procedure to evaluate the binding energies of DHFR inhibitors interacting with the human enzyme is reported here, providing a clear picture of the binding interactions of these ligands from both structural and energetic viewpoints. A reduced model for the binding pocket was used. This approach allows us to perform more accurate quantum mechanical calculations as well as to obtain a detailed electronic analysis using the quantum theory of atoms in molecules (QTAIM) technique. Thus, molecular aspects of the binding interactions between inhibitors and the DHFR are discussed in detail. A significant correlation between binding energies obtained from DFT calculations and experimental IC₅₀ values was obtained, predicting with an acceptable qualitative accuracy the potential inhibitor effect of nonsynthesized compounds. Such correlation was experimentally corroborated synthesizing and testing two new inhibitors reported in this paper. PMID:23834278

  9. Thioredoxin reductase inhibitor auranofin prevents membrane transport of diphtheria toxin into the cytosol and protects human cells from intoxication.

    PubMed

    Schnell, Leonie; Dmochewitz-Kück, Lydia; Feigl, Peter; Montecucco, Cesare; Barth, Holger

    2016-06-15

    During cellular uptake, diphtheria toxin delivers its catalytic domain DTA from acidified endosomes into the cytosol, which requires reduction of the disulfide linking DTA to the transport domain. In vitro, thioredoxin reduces this disulfide and thioredoxin reductase (TrxR) is part of a cytosolic complex facilitating DTA-translocation. We found that the TrxR-specific inhibitor auranofin prevented DTA delivery into the cytosol and intoxication of HeLa cells with diphtheria toxin, offering perspectives for novel pharmacological strategies against diphtheria. PMID:25911959

  10. S-nitrosoglutathione prevents experimental cerebral malaria

    PubMed Central

    Zanini, Graziela M.; Martins, Yuri C.; Cabrales, Pedro; Frangos, John A.; Carvalho, Leonardo J. M.

    2012-01-01

    Administration of the exogenous nitric oxide (NO) donor dipropylenetriamine-NONOate (DPTA-NO) to mice during Plasmodium berghei ANKA (PbA) infection largely prevents development of experimental cerebral malaria (ECM). However, a high dose (1mg/mouse twice a day) is necessary and causes potent side effects such as marked hypotension. In the present study we evaluated whether an alternative, physiologically relevant NO donor, S-nitrosoglutathione (GSNO), was able to prevent ECM at lower doses with minimal side effects. Prophylactic treatment with high (3.5mg), intermediate (0.35mg) or low (0.035mg) doses of GSNO decreased incidence of ECM in PbA-infected mice, decreasing also edema, leukocyte accumulation and hemorrhage incidence in the brain. The high dose inhibited parasite growth and also induced transient hypotension. Low and intermediate doses had no or only mild effects on parasitemia, blood pressure, and heart rate compared to saline-treated mice. PbA infection decreased brain total and reduced (GSH) glutathione levels. Brain levels of oxidized (GSSG) glutathione and the GSH/GSSG ratio were positively correlated with temperature and motor behavior. Low and intermediate doses of GSNO failed to restore the depleted brain total glutathione and GSH levels, suggesting that ECM prevention by GSNO was probably related to other effects such as inhibition of inflammation and vascular protection. These results indicate that ECM is associated with depletion of the brain glutathione pool and that GSNO is able to prevent ECM development in a wide range of doses, decreasing brain inflammation and inducing milder cardiovascular side effects. PMID:22391863

  11. Arabidopsis Peptide Methionine Sulfoxide Reductase2 Prevents Cellular Oxidative Damage in Long NightsW⃞

    PubMed Central

    Bechtold, Ulrike; Murphy, Denis J.; Mullineaux, Philip M.

    2004-01-01

    Peptide methionine sulfoxide reductase (PMSR) is a ubiquitous enzyme that repairs oxidatively damaged proteins. In Arabidopsis (Arabidopsis thaliana), a null mutation in PMSR2 (pmsr2-1), encoding a cytosolic isoform of the enzyme, exhibited reduced growth in short-day conditions. In wild-type plants, a diurnally regulated peak of total PMSR activity occurred at the end of the 16-h dark period that was absent in pmsr2-1 plants. This PMSR activity peak in the wild-type plant coincided with increased oxidative stress late in the dark period in the mutant. In pmsr2-1, the inability to repair proteins resulted in higher levels of their turnover, which in turn placed an increased burden on cellular metabolism. This caused increased respiration rates, leading to the observed higher levels of oxidative stress. In wild-type plants, the repair of damaged proteins by PMSR2 at the end of the night in a short-day diurnal cycle alleviates this potential burden on metabolism. Although PMSR2 is not absolutely required for viability of plants, the observation of increased damage to proteins in these long nights suggests the timing of expression of PMSR2 is an important adaptation for conservation of their resources. PMID:15031406

  12. Phenylcoumaran Benzylic Ether Reductase Prevents Accumulation of Compounds Formed under Oxidative Conditions in Poplar Xylem[W

    PubMed Central

    Niculaes, Claudiu; Morreel, Kris; Kim, Hoon; Lu, Fachuang; McKee, Lauren S.; Ivens, Bart; Haustraete, Jurgen; Vanholme, Bartel; Rycke, Riet De; Hertzberg, Magnus; Fromm, Jorg; Bulone, Vincent; Polle, Andrea; Ralph, John; Boerjan, Wout

    2014-01-01

    Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8–5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta. PMID:25238751

  13. Experimental Contact Lens to Prevent Glaucoma-Induced Blindness

    MedlinePlus

    ... Articles | Inside Life Science Home Page An Experimental Contact Lens to Prevent Glaucoma-Induced Blindness By Sharon Reynolds Posted January 23, 2014 An experimental contact lens design releases a glaucoma medicine at a ...

  14. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... gene provides instructions for making an enzyme called steroid 5-alpha reductase 2. This enzyme is involved ... external genitalia. Mutations in the SRD5A2 gene prevent steroid 5-alpha reductase 2 from effectively converting testosterone ...

  15. Preventive effect of long-term aldose reductase inhibition (ponalrestat) on nerve conduction and sural nerve structure in the spontaneously diabetic Bio-Breeding rat.

    PubMed Central

    Sima, A A; Prashar, A; Zhang, W X; Chakrabarti, S; Greene, D A

    1990-01-01

    To test the hypothesis that aldose reductase inhibition may prevent or delay the development of functional and structural neuropathy in the insulin-deficient diabetic Bio-Breeding rat (BB-rat), hyperglycemic rats were begun on the aldose reductase inhibitor (ARI) ponalrestat 25 mg/kg body wt soon after the onset of diabetes and followed for 4 or 6 mo. Ponalrestat treatment completely prevented the characteristic nerve conduction slowing and structural abnormalities of the node of Ranvier for 4 mo despite only partial preservation of axonal integrity. Ponalrestat treatment for 6 mo achieved a partial but significant prevention of nerve conduction slowing, axoglial dysjunction, and axonal degenerative changes. This incomplete but significant prevention of neuropathy by ponalrestat suggests that additional mechanisms besides polyol-pathway activation may be of importance in the pathogenesis of diabetic neuropathy. Alternatively, the dosage used in the present study may not have been sufficient to achieve a complete prevention. Despite the only partial protective effect of ARI treatment on degenerative peripheral nerve changes in hyperglycemic BB-rats, 6 mo of treatment resulted in a more than threefold increase in regenerating nerve fibers. These data suggest that prophylactic ARI treatment may be efficacious in delaying the development of diabetic neuropathy. Images PMID:2110189

  16. Effect of Genistein and L-Carnitine and Their Combination on Gene Expression of Hepatocyte HMG-COA Reductase and LDL Receptor in Experimental Nephrotic Syndrome

    PubMed Central

    YOUSEFINEJAD, Abbas; SIASSI, Fereydoon; MIRSHAFIEY, Abbas; ESHRAGHIAN, Mohammad-Reza; KOOHDANI, Fariba; JAVANBAKHT, Mohammad Hassan; SEDAGHAT, Reza; RAMEZANI, Atena; ZAREI, Mahnaz; DJALALI, Mahmoud

    2015-01-01

    Background: Nephrotic syndrome is a disorder that leads to hyperlipidemia. L-carnitine and genistein can effect on lipid metabolism and the syndrome. In the present study, we have delved into the separate and the twin-effects of L-carnitine and genistein on the gene expressions of HMG-COA reductase and LDL receptor in experimental nephrotic syndrome. Methods: In this controlled experimental study, 50 male Sprague–Dawley rats were randomly divided into five groups: NC (normal-control), PC (patient-control), LC (L-carnitine), G (genistein), LCG (L-carnitine-genistein). Adriamycin was used for inducing nephrotic syndrome and the spot urine samples and urine protein-to-creatinine ratio were measured. Hepatocytic RNA was extracted and real-time PCR was used for HMG-COA Reductase and LDL receptor gene Expression measurement. Results: The final weight of the patients groups were lower than the NC group (P=0.001), and weight gain of the NC group was higher than the other groups (P<0.001). The proteinuria and urine protein-to-creatinine ratio showed significant differences between PC group and LC, G and LCG groups at week 7 (P<0.001). The expression of HMGCOA Reductase mRNA down regulated in LC, G and LCG groups in comparison with PC group (P<0.001). ΔCT of LDLr mRNA showed significant differences between the PC group and the other patient groups (P<0.001). Conclusion: This study shows a significant decreasing (P<0.001) and non-significant increasing trend in HMG-COA Reductase and LDLr gene expression, respectively, and synergistic effect of L-carnitine and genistein on these genes in experimental nephrotic syndrome. PMID:26576346

  17. Sexual Violence Prevention through Bystander Education: An Experimental Evaluation

    ERIC Educational Resources Information Center

    Banyard, Victoria L.; Moynihan, Mary M.; Plante, Elizabethe G.

    2007-01-01

    The current study used an experimental design to evaluate a sexual violence prevention program based on a community of responsibility model that teaches women and men how to intervene safely and effectively in cases of sexual violence before, during, and after incidents with strangers, acquaintances, or friends. It approaches both women and men as…

  18. Prevention of experimental stroke by hypercapnic-hypoxic preconditioning.

    PubMed

    Yakushev, N N; Bespalov, A G; Kulikov, V P

    2008-09-01

    The effectiveness of hypercapnic hypoxic training in the prevention of acute disturbances in cerebral circulation was studied under experimental conditions. Hypercapnic hypoxic training was followed by a significant decrease in the severity of neurological deficit and locomotor and coordination disorders after cerebral ischemic injury. PMID:19240841

  19. Prevention of chronic experimental heart insufficiency by inosine.

    PubMed

    Belenkii, E E; Sokolov, I K; Kleimenova, N N; Suzdalnitskii, R S; Tunitskay, T A

    1975-01-01

    The administration of inosine dosed 25 mg/kg daily during 10 1/2 months prevented the development of excentric cardiac hypertrophy on the background of experimental aortic stenosis in rats, reduced the lowering of the working capacity of the animals, and partially inhibited the full development of functional and morphological myocardial changes, detected by electrocardiographic, vectorcardiographic, microscopic, and electronmicroscopic examinations. Differences were found in the relative weights of the heart, thymus, liver and other organs; these findings attested to an anabolic activity of inosine. The results obtained justify the application of inosine for the prevention of excentric hypertrophy and decompensation of the heart in cardiac failures varying in etiology; any efficient preventive measure is of importance, because the degree of excentric hypertrophy is a factor influencing the survival of the patients. PMID:125181

  20. Inhibition of squalene synthase but not squalene cyclase prevents mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis at a posttranscriptional level.

    PubMed

    Peffley, D M; Gayen, A K

    1997-01-15

    Previously, we found that mevalonate-derived products together with an oxysterol regulated reductase synthesis at a posttranscriptional level. To determine which products were responsible for this regulation, either the squalene synthase inhibitor zaragozic acid A or the squalene cyclase inhibitor 4,4,10-beta-trimethyl-trans-decal-3beta-ol (TMD) was added to lovastatin-treated Syrian hamster cells in conjunction with mevalonate. Mevalonate alone decreased reductase synthesis 50% compared with lovastatin-treated cells. In contrast, when both zaragozic acid A and mevalonate were added to lovastatin-treated cells, there was no change in reductase synthesis. With either treatment, reductase mRNA levels did not change compared with lovastatin-treated cells. When both 25-hydroxycholesterol and mevalonate were added to lovastatin-treated cells, reductase synthesis and mRNA levels were decreased 95 and 50%, respectively. The 10-fold difference between changes in reductase synthesis and mRNA levels under these conditions reflects a specific effect of mevalonate-derived isoprenoids on reductase synthesis at the translational level. In contrast, coincubation of cells with mevalonate plus 25-hydroxycholesterol in the presence of zaragozic acid decreased reductase synthesis and mRNA levels 60 and 50%, respectively, compared with lovastatin-treated cells. Moreover, degradation of reductase was increased approximately 7-fold in cells treated with mevalonate alone but only 3-fold in cells treated with mevalonate and zaragozic acid A. These results indicate that isoprenoid products between mevalonate and squalene affect reductase at a posttranslational level by increasing degradation but do not regulate reductase synthesis at a posttranscriptional level. In contrast, when both TMD and mevalonate were added to lovastatin-treated cells, reductase synthesis was decreased approximately 50% with no corresponding decrease in reductase mRNA levels, similar to mevalonate only. Reductase

  1. Thioredoxin reductase.

    PubMed

    Mustacich, D; Powis, G

    2000-02-15

    The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases. PMID:10657232

  2. Ribonucleotide reductase inhibition by metal complexes of Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone): A combined experimental and theoretical study

    PubMed Central

    Popović-Bijelić, Ana; Kowol, Christian R.; Lind, Maria E.S.; Luo, Jinghui; Himo, Fahmi; Enyedy, Éva A.; Arion, Vladimir B.; Gräslund, Astrid

    2012-01-01

    Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is currently the most promising chemotherapeutic compound among the class of α-N-heterocyclic thiosemicarbazones. Here we report further insights into the mechanism(s) of anticancer drug activity and inhibition of mouse ribonucleotide reductase (RNR) by Triapine. In addition to the metal-free ligand, its iron(III), gallium(III), zinc(II) and copper (II) complexes were studied, aiming to correlate their cytotoxic activities with their effects on the diferric/tyrosyl radical center of the RNR enzyme in vitro. In this study we propose for the first time a potential specific binding pocket for Triapine on the surface of the mouse R2 RNR protein. In our mechanistic model, interaction with Triapine results in the labilization of the diferric center in the R2 protein. Subsequently the Triapine molecules act as iron chelators. In the absence of external reductants, and in presence of the mouse R2 RNR protein, catalytic amounts of the iron(III)–Triapine are reduced to the iron(II)–Triapine complex. In the presence of an external reductant (dithiothreitol), stoichiometric amounts of the potently reactive iron (II)–Triapine complex are formed. Formation of the iron(II)–Triapine complex, as the essential part of the reaction outcome, promotes further reactions with molecular oxygen, which give rise to reactive oxygen species (ROS) and thereby damage the RNR enzyme. Triapine affects the diferric center of the mouse R2 protein and, unlike hydroxyurea, is not a potent reductant, not likely to act directly on the tyrosyl radical. PMID:21955844

  3. Muscle wasting and aging: Experimental models, fatty infiltrations, and prevention.

    PubMed

    Brioche, Thomas; Pagano, Allan F; Py, Guillaume; Chopard, Angèle

    2016-08-01

    Identification of cost-effective interventions to maintain muscle mass, muscle strength, and physical performance during muscle wasting and aging is an important public health challenge. It requires understanding of the cellular and molecular mechanisms involved. Muscle-deconditioning processes have been deciphered by means of several experimental models, bringing together the opportunities to devise comprehensive analysis of muscle wasting. Studies have increasingly recognized the importance of fatty infiltrations or intermuscular adipose tissue for the age-mediated loss of skeletal-muscle function and emphasized that this new important factor is closely linked to inactivity. The present review aims to address three main points. We first mainly focus on available experimental models involving cell, animal, or human experiments on muscle wasting. We next point out the role of intermuscular adipose tissue in muscle wasting and aging and try to highlight new findings concerning aging and muscle-resident mesenchymal stem cells called fibro/adipogenic progenitors by linking some cellular players implicated in both FAP fate modulation and advancing age. In the last part, we review the main data on the efficiency and molecular and cellular mechanisms by which exercise, replacement hormone therapies, and β-hydroxy-β-methylbutyrate prevent muscle wasting and sarcopenia. Finally, we will discuss a potential therapeutic target of sarcopenia: glucose 6-phosphate dehydrogenase. PMID:27106402

  4. Paraoxonase-1 overexpression prevents experimental abdominal aortic aneurysm progression.

    PubMed

    Burillo, Elena; Tarin, Carlos; Torres-Fonseca, Monica-Maria; Fernandez-García, Carlos-Ernesto; Martinez-Pinna, Roxana; Martinez-Lopez, Diego; Llamas-Granda, Patricia; Camafeita, Emilio; Lopez, Juan Antonio; Vega de Ceniga, Melina; Aviram, Michael; Egido, Jesus; Blanco-Colio, Luis-Miguel; Martín-Ventura, Jose-Luis

    2016-06-01

    Abdominal aortic aneurysm (AAA) is a permanent dilation of the aorta due to excessive proteolytic, oxidative and inflammatory injury of the aortic wall. We aimed to identify novel mediators involved in AAA pathophysiology, which could lead to novel therapeutic approaches. For that purpose, plasma from four AAA patients and four controls were analysed by a label-free proteomic approach. Among identified proteins, paraoxonase-1 (PON1) was decreased in plasma of AAA patients compared with controls, which was further validated in a bigger cohort of samples by ELISA. The phenylesterase enzymatic activity of PON1 was also decreased in serum of AAA patients compared with controls. To address the potential role of PON1 as a mediator of AAA, experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and human transgenic PON1 (HuTgPON1) mice. Similar to humans, PON1 activity was also decreased in serum of elastase-induced AAA mice compared with healthy mice. Interestingly, overexpression of PON1 was accompanied by smaller aortic dilation and higher elastin and vascular smooth muscle cell (VSMC) content in the AAA of HuTgPON1 compared with WT mice. Moreover, HuTgPON1 mice display decreased oxidative stress and apoptosis, as well as macrophage infiltration and monocyte chemoattractant protein-1 (MCP1) expression, in elastase-induced AAA. In conclusion, decreased circulating PON1 activity is associated with human and experimental AAA. PON1 overexpression in mice protects against AAA progression by reducing oxidative stress, apoptosis and inflammation, suggesting that strategies aimed at increasing PON1 activity could prevent AAA. PMID:26993251

  5. Preventive and Therapeutic Euphol Treatment Attenuates Experimental Colitis in Mice

    PubMed Central

    Bento, Allisson F.; Marcon, Rodrigo; Schmidt, Éder C.; Bouzon, Zenilda L.; Pianowski, Luiz F.; Calixto, João B.

    2011-01-01

    Background The tetracyclic triterpene euphol is the main constituent found in the sap of Euphorbia tirucalli. This plant is widely known in Brazilian traditional medicine for its use in the treatment of several kinds of cancer, including leukaemia, prostate and breast cancers. Here, we investigated the effect of euphol on experimental models of colitis and the underlying mechanisms involved in its action. Methodology/Principal Findings Colitis was induced in mice either with dextran sulfate sodium (DSS) or with 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the effect of euphol (3, 10 and 30 mg/kg) on colonic injury was assessed. Pro-inflammatory mediators and cytokines were measured by immunohistochemistry, enzyme-Linked immunoabsorbent assay (ELISA), real time-polymerase chain reaction (RT-PCR) and flow cytometry. Preventive and therapeutic oral administration of euphol attenuated both DSS- and TNBS-induced acute colitis as observed by a significant reduction of the disease activity index (DAI), histological/microscopic damage score and myeloperoxidase (MPO) activity in colonic tissue. Likewise, euphol treatment also inhibited colon tissue levels and expression of IL-1β, CXCL1/KC, MCP-1, MIP-2, TNF-α and IL-6, while reducing NOS2, VEGF and Ki67 expression in colonic tissue. This action seems to be likely associated with inhibition of activation of nuclear factor-κB (NF-κB). In addition, euphol decreased LPS-induced MCP-1, TNF-α, IL-6 and IFN-γ, but increased IL-10 secretion from bone marrow-derived macrophages in vitro. Of note, euphol, at the same schedule of treatment, markedly inhibited both selectin (P- and E-selectin) and integrin (ICAM-1, VCAM-1 and LFA-1) expression in colonic tissue. Conclusions/Significance Together, these results clearly demonstrated that orally-administered euphol, both preventive or therapeutic treatment were effective in reducing the severity of colitis in two models of chemically-induced mouse colitis and suggest this plant

  6. Quantifying immunohistochemical staining of phospho-eIF2alpha, heme oxygenase-2 and NADPH cytochrome P450 reductase in oligodendrocytes during experimental autoimmune encephalomyelitis.

    PubMed

    Chakrabarty, Anuradha; Fleming, Kandace K; Marquis, Janet G; LeVine, Steven M

    2005-06-15

    As a consequence of inflammation associated with multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), stress responses are induced in many cells within the CNS, however, those that occur within the primary pathological target, the oligodendrocyte, are not fully established. Recently, we found that phosphorylated eukaryotic initiation factor-2alpha (eIF2alpha), an inhibitor of protein translation associated with the stress response, is expressed in a greater number of oligodendrocytes in EAE animals compared to controls. However, since numerous oligodendrocytes in control animals also expressed phospho-eIF2alpha, a method was developed to detect expression levels within oligodendrocytes that did not rely on the number of oligodendrocytes that were stained. This method utilized a high dilution of the primary antibody so that the staining density was kept below a maximum plateau which could eliminate expression differences. Furthermore, the staining density within oligodendrocytes, as determined by image analysis, was corrected by the background density or that within neurons. In either case, the density of staining was greater in oligodendrocytes from EAE animals versus controls. The expression of heme oxygenase-2 and NADPH cytochrome P450 reductase also were examined, but unlike phospho-eIF2alpha, neither was increased in oligodendrocytes from EAE animals compared to controls. In summary, a protocol involving a high dilution of primary antibody and image analysis revealed that the expression of phospho-eIF2alpha within oligodendrocytes was increased in EAE animals compared to control animals. PMID:15910982

  7. Estrogen treatment prevents gray matter atrophy in experimental autoimmune encephalomyelitis.

    PubMed

    MacKenzie-Graham, Allan J; Rinek, Gilda A; Avedisian, Andrea; Morales, Laurie B; Umeda, Elizabeth; Boulat, Benoit; Jacobs, Russell E; Toga, Arthur W; Voskuhl, Rhonda R

    2012-07-01

    Gray matter atrophy is an important correlate to clinical disability in multiple sclerosis (MS), and many treatment trials include atrophy as an outcome measure. Atrophy has been shown to occur in experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The clinical severity of EAE is reduced in estrogen-reated mice, but it remains unknown whether estrogen treatment can reduce gray matter atrophy in EAE. In this study, mice with EAE were treated with either estrogen receptor (ER)-α ligand or ER-β ligand, and diffusion tensor images (DTI) were collected and neuropathology was performed. DTI showed atrophy in the cerebellar gray matter of vehicle-treated EAE mice compared with healthy controls but not in ER-α or ER-β ligand-treated EAE mice. Neuropathology demonstrated that Purkinje cell numbers were decreased in vehicle-treated EAE mice, whereas neither ER ligand-treated EAE groups showed a decrease. This is the first report of a neuroprotective therapy in EAE that unambiguously prevents gray matter atrophy while sparing a major neuronal cell type. Fractional anisotropy (FA) in the cerebellar white matter was decreased in vehicle- and ER-β ligand-treated but not in ER-α ligand-treated EAE mice. Inflammatory cell infiltration was increased in vehicle- and ER-β ligand-treated but not in ER-α ligand-treated EAE mice. Myelin staining was decreased in vehicle-treated EAE mice and was spared in both ER ligand-treated groups. This is consistent with decreased FA as a potential biomarker for inflammation rather than myelination or axonal damage in the cerebellum in EAE. PMID:22411609

  8. Development of novel pyrazolone derivatives as inhibitors of aldose reductase: an eco-friendly one-pot synthesis, experimental screening and in silico analysis.

    PubMed

    Kadam, Aparna; Dawane, Bhaskar; Pawar, Manisha; Shegokar, Harshala; Patil, Kapil; Meshram, Rohan; Gacche, Rajesh

    2014-04-01

    Aldose reductase is the key enzyme of polypol pathway leading to accumulation of sorbitol. Sorbitol does not diffuse across the cell membranes easily and therefore accumulates within the cell, causing osmotic damage which leads to retinopathy (cataractogenesis), neuropathy and other diabetic complications. Currently, aldose reductase inhibitors like epalrestat, ranirestat and fidarestat are used for the amelioration of diabetic complications. However, such drugs are effective in patients having good glycemic control and less severe diabetic complications. In present study we have designed novel pyrazolone derivative and performed eco-friendly synthesis approach and tested the synthesized compounds as potential inhibitors of aldose reductase activity. Additional in silico analysis in current study indicates presence of highly conserved chemical environment in active site of goat lens aldose reductase. The reported data is expected to be useful for developing novel pyrazolone derivatives as lead compounds in the management of diabetic complications. PMID:24607578

  9. Inhibition of aldose reductase prevents growth factor-induced G1-S phase transition through the AKT/phosphoinositide 3-kinase/E2F-1 pathway in human colon cancer cells.

    PubMed

    Ramana, Kota V; Tammali, Ravinder; Srivastava, Satish K

    2010-04-01

    Colon cancer is the leading cause of cancer death in both men and women worldwide. The deregulated cell cycle control or decreased apoptosis of normal epithelial cells leading to uncontrolled proliferation is one of the major features of tumor progression. We have previously shown that aldose reductase (AR), a NADPH-dependent aldo-keto reductase, has been shown to be involved in growth factor-induced proliferation of colon cancer cells. Herein, we report that inhibition of AR prevents epidermal growth factor (EGF)- and basic fibroblast growth factor (bFGF)-induced HT29 cell proliferation by accumulating cells at G(1) phase of cell cycle. Similar results were observed in SW480 and HCT-116 colon cancer cells. Treatment of HT29 cells with AR inhibitor, sorbinil or zopolrestat, prevented the EGF- and bFGF-induced DNA binding activity of E2F-1 and phosphorylation of retinoblastoma protein. Inhibition of AR also prevented EGF- and bFGF-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G(1)-S transition regulatory proteins such as cyclin D1, cdk4, proliferating cell nuclear antigen, cyclin E, and c-myc. More importantly, inhibition of AR prevented the EGF- and bFGF-induced activation of phosphoinositide 3-kinase/AKT and reactive oxygen species generation in colon cancer cells. Further, inhibition of AR also prevented the tumor growth of human colon cancer cells in nude mouse xenografts. Collectively, these results show that AR mediates EGF- and bFGF-induced colon cancer cell proliferation by activating or expressing G(1)-S phase proteins such as E2F-1, cdks, and cyclins through the reactive oxygen species/phosphoinositide 3-kinase/AKT pathway, indicating the use of AR inhibitors in the prevention of colon carcinogenesis. Mol Cancer Ther; 9(4); 813-24. (c)2010 AACR. PMID:20354121

  10. Preventive effect of taurine on experimental type II diabetic nephropathy

    PubMed Central

    2010-01-01

    Background It has been verified that taurine has some preventive effects on diabetes and its complications when used alone or together with other drugs, but there are few reports about taurine on the prevention of diabetic nephropathy, the mechanisms of which are still unknown. Methods Taurine was administered to type Ⅱ diabetic rats induced by high fat high sugar diet combined with STZ injection. The preventive effect of taurine on diabetic nephropathy was investigated by detecting blood glucose, lipid metabolism, kidney function and glomerular basement membrane metabolism. Results Taurine could lower blood glucose, TG, TC, BUN, Scr, NAG, U-PRO, the expression of laminin B1( LBN1) mRNA, and increase HDL-C of diabetic rats. Conclusions The results indicated that taurine could prevent the occurrence and development of diabetic nephropathy by decreasing blood glucose, improving lipid metabolism, glomerular basement membrane metabolism, and kidney function. PMID:20804623

  11. [Experimental studies of the prevention of postoperative adhesions].

    PubMed

    Schwarz, J; Donat, H

    1989-01-01

    Female wistar rats were used for the testing of different substances to prevent postoperative intraabdominal adhesions. It could be demonstrated that the best results were obtained by dextran 70 with a concentration of 10%. Good effects were seen also by contrykal (aprotinin) and hylase (hyaluronidase). By the combination of dextran 70 30% with contrykal the spread of adhesions was very low but the effect was not significant better than with dextran 70 10% alone. For the prevention of intraabdominal adhesions operative techniques with minimal lesions are important too of the peritoneal epithelium. PMID:2466384

  12. Prevention praised, cure preferred: results of between-subjects experimental studies comparing (monetary) appreciation for preventive and curative interventions

    PubMed Central

    2013-01-01

    Background 'An ounce of prevention is worth a pound of cure’ is a common saying, and indeed, most health economic studies conclude that people are more willing to pay for preventive measures than for treatment activities. This may be because most health economic studies ask respondents to compare preventive measures with treatment, and thus prompt respondents to consider other uses of resources. However, psychological theorizing suggests that, when methods do not challenge subjects to consider other uses of resources, curative treatment is favored over prevention. Could it be that while prevention is praised, cure is preferred? Methods In two experimental studies, we investigated, from a psychological perspective and using a between-subjects design, whether prevention or treatment is preferred and why. In both studies, participants first read a lung cancer prevention or treatment intervention scenario that varied on the prevention-treatment dimension, but that were the same on factors like 'costs per saved life’ and kind of disease. Then participants completed a survey measuring appreciation (general and monetary) as well as a number of potential mediating variables. Results Both studies clearly demonstrated that, when the design was between-subjects, participants had greater (general and monetary) appreciation for treatment interventions than for preventive interventions with perceived urgency of the intervention quite consistently mediating this effect. Differences in appreciation of treatment over preventive treatment were shown to be .59 (Study 1) and .45 (Study 2) on a 5-point scale. Furthermore, participants thought that health insurance should compensate more for the treatment than for preventive measures, differences of 16% (Study 1), and 22% (Study 2). When participants were asked to directly compare both interventions on the basis of a short description, they preferred the preventive intervention. Conclusion It appears that people claim to prefer

  13. Experimental gram-negative bacterial sepsis: prevention of mortality not preventable by antibiotics alone.

    PubMed Central

    Greisman, S E; DuBuy, J B; Woodward, C L

    1979-01-01

    Outbred Swiss mice were inoculated intraperitoneally or intravenously with one 90 to 100% lethal dose of Escherichia coli O:18, Proteus mirabilis, or Klebsiella pneumoniae. After carefully timed intervals, aminoglycoside antibiotics were begun at dosages nnd intervals predetermined to constitute optimal therapy. With progressive increases in delay of antibiotic therapy, mortality rates increased progressively from 0% to 90 to 100%. Standardized models of infection were developed by selecting delay periods before initiating antibiotic therapy such that 50 to 70% mortalities resulted. Utilizing these models, agents with reputed anti-endotoxin activity were administered concomitantly with the delayed antibiotic therapy to determine if any could prevent gram-negative septic mortality no longer preventable by the antibiotics alone. The following were observed: (i) adrenal corticosteroids prevented mortality that was no longer preventable by optimal aminoglycoside antibiotics alone. The following were preventable by optimal aminoglycoside antibiotic therapy alone; (ii) specific antisera also did so, provided anaphylaxis was circumvented; (iii) in one model (P. mirabilis), such protection by adrenal corticosteroids and specific antiserum could be additive; (iv) adrenal corticosteroids and specific antiserum acted synergistically with the aminoglycoside antibiotics--no protection was achieved by delayed administration of the steroids or antiserum alone; (v) timing was crucial--the synergistic protective activity of adrenal corticosteroids and of specific antiserum with aminoglycosides declined rapidly as infection progressed; (vi) cyclophosphamide pretreatment markedly impaired the synergistic protective activity of specific antiserum and of adrenal corticosteroids with aminoglycosides; (vii) no reputed anti-endotoxin agents other than adrenal corticosteroids and specific antiserum proved capable of preventing mortality not preventable by aminoglycoside antibiotics alone

  14. Preventing MIC through experimental, on-line fouling monitoring

    SciTech Connect

    Bloch, K.P.; DiFranco, P.

    1995-10-01

    Classic water conditioning of large industrial cooling circuits requires a practical chemical approach to control corrosion, scaling, and fouling. One or more of these areas may be destabilized when a system experiences a change in its applied cooling water chemical treatment technology. When determining the consequences of alternative chemical water treatment programs, special attention to biofilm formation must be given, since this type of fouling represents the potential for subsequent microbiologically induced corrosion (MIC). During the evaluation stage, a fouling monitor can be used to assess the formation of a biofilm, to determine its effect on corrosion rates, and to evaluate treatment efficacy for the control of sessile microorganisms. Such a model was used to develop a water treatment recommendation for a cooling system which will be required to convert to an alternative water treatment technology. This approach to MIC prevention can be applied to related systems which may be subject to underdeposit corrosion.

  15. [Prevention from secondary nerve root adhesion: an experimental study].

    PubMed

    Yao, M; Sun, Y; Yan, J

    1996-06-01

    In the study, 27 dogs were divided into three groups: A, B and C. Then all of the dogs had their lumbar intervertebral disks removed. Into the wounded cavity of group A, 1 ml of dimethicone was dropped and gelatin sponge was applied on the surface of the nerve root of group B. Group C was served as the control. The dogs were killed and the operation area was removed respectively 2 weeks, 4 weeks, and 12 weeks after the operation for macroscopical observation, nerve root motility measurement and histological examination. The result of the experiment proved that dimethicone was fairly effective in the prevention from secondary nerve root adhesion. While gelatin sponge in the process of its absorption induced the formation of quite a few scar tissues, thus aggrevating nerve root adhesion. PMID:9594172

  16. [Preventive effect of changzhankang in experimental intestinal adhesions in rats].

    PubMed

    Wang, Y Q; Wei, J Q; Dai, D Z

    1991-08-01

    Intestinal adhesions were induced in rats by stabbing the terminal part of the ileum. Adhesion prevention by ibuprofen and changzhankang (CZK), which was composed by traditional Chinese medicines, was evaluated with a grading system. All of the 13 rats in the non-treated group created severe adhesions. The severity was significantly modified by orally administered CZK of 20 g/kg (in crude drugs) once or twice daily for five days (P less than 0.01 and P less than 0.05 compared with the non-treated). Intramuscular injection of ibuprofen (35 mg/kg, 3 times daily) also alleviated the severity of adhesions. There was no significant difference between the ibuprofen-treated and CZK-treated groups though some of the rats were virtually free from adhesion formation in the latter. It is plausible to expect CZK to become a promising drug used in treating intestinal adhesions, for the natural drug has greater security and less side effects than synthesized drugs. PMID:1954667

  17. Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis.

    PubMed Central

    Yolken, R H; Peterson, J A; Vonderfecht, S L; Fouts, E T; Midthun, K; Newburg, D S

    1992-01-01

    Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens. Images PMID:1331178

  18. 5α-Reductase Inhibition Prevents the Luteal Phase Increase in Plasma Allopregnanolone Levels and Mitigates Symptoms in Women with Premenstrual Dysphoric Disorder.

    PubMed

    Martinez, Pedro E; Rubinow, David R; Nieman, Lynnette K; Koziol, Deloris E; Morrow, A Leslie; Schiller, Crystal E; Cintron, Dahima; Thompson, Karla D; Khine, Khursheed K; Schmidt, Peter J

    2016-03-01

    Changes in neurosteroid levels during the luteal phase of the menstrual cycle may precipitate affective symptoms. To test this hypothesis, we stabilized neurosteroid levels by administering the 5α-reductase inhibitor dutasteride to block conversion of progesterone to its neurosteroid metabolite allopregnanolone in women with premenstrual dysphoric disorder (PMDD) and in asymptomatic control women. Sixteen women with prospectively confirmed PMDD and 16 control women participated in one of two separate randomized, double-blind, placebo-controlled, cross-over trials, each lasting three menstrual cycles. After one menstrual cycle of single-blind placebo, participants were randomized to receive, for the next two menstrual cycles, either double-blind placebo or dutasteride (low-dose 0.5 mg/day in the first eight PMDD and eight control women or high-dose 2.5 mg/day in the second group of women). All women completed the daily rating form (DRF) and were evaluated in clinic during the follicular and luteal phases of each menstrual cycle. Main outcome measures were the DRF symptoms of irritability, sadness, and anxiety. Analyses were performed with SAS PROC MIXED. In the low-dose group, no significant effect of dutasteride on PMDD symptoms was observed compared with placebo (ie, symptom cyclicity maintained), and plasma allopregnanolone levels increased in women with PMDD from follicular to the luteal phases, suggesting the absence of effect of the low-dose dutasteride on 5α-reductase. In contrast, the high-dose group experienced a statistically significant reduction in several core PMDD symptoms (ie, irritability, sadness, anxiety, food cravings, and bloating) on dutasteride compared with placebo. Dutasteride had no effect on mood in controls. Stabilization of allopregnanolone levels from the follicular to the luteal phase of the menstrual cycle by blocking the conversion of progesterone to its 5α-reduced neurosteroid metabolite mitigates symptoms in PMDD. These data

  19. The role of experimental research in the study of the prevention of malignant tumours

    PubMed Central

    Shabad, L. M.

    1962-01-01

    The author discusses the role of experimental oncological research in the prevention of malignant neoplasms, with special reference to the conclusions drawn from such research in the USSR. He points out that experimental research can contribute to cancer prevention in two ways: (a) by supplying information on the occurrence of carcinogenic substances in the human environment—in the atmosphere, in industry and in foodstuffs—and thus providing a rational basis for the introduction of measures to prevent cancer from arising; and (b) by throwing light on the series of tissue changes that may precede the development of the malignant tumour and hence making it possible, through the timely treatment and cure of known precancerous conditions, to prevent cancer from developing. PMID:13911073

  20. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

  1. Tomato Lycopene and Lung Cancer Prevention: From Experimental to Human Studies

    PubMed Central

    Palozza, Paola; Simone, Rossella E.; Catalano, Assunta; Mele, Maria Cristina

    2011-01-01

    Increasing evidence suggests that tomato lycopene may be preventive against the formation and the development of lung cancer. Experimental studies demonstrated that lycopene may inhibit the growth of several cultured lung cancer cells and prevent lung tumorigenesis in animal models through various mechanisms, including a modulation of redox status, cell cycle arrest and/or apoptosis induction, a regulation of growth factor signaling, changes in cell growth-related enzymes, an enhancement of gap junction communication and a prevention of smoke-induced inflammation. In addition, lycopene also inhibited cell invasion, angiogenesis, and metastasis. Several lycopene metabolites have been identified, raising the question as to whether the preventive effects of lycopene on cancer risk is, at least in part, due to its metabolites. Despite these promising reports, it is difficult at the moment to directly relate available experimental data to human pathophysiology. More well controlled clinical intervention trials are needed to further clarify the exact role of lycopene in the prevention of lung cancer cell growth. Such studies should take into consideration subject selection, specific markers of analysis, the levels of carotenoids being tested, metabolism and isomerization of lycopene, interaction with other bioactive food components. This article reviews data on the cancer preventive activities of lycopene, possible mechanisms involved, and the relationship between lycopene consumption and human cancer risk. PMID:24212813

  2. Prevention of hepatocellular carcinoma: potential targets, experimental models, and clinical challenges

    PubMed Central

    Hoshida, Yujin; Fuchs, Bryan C.; Tanabe, Kenneth K.

    2013-01-01

    Chronic fibrotic liver diseases such as viral hepatitis eventually develop liver cirrhosis, which causes occurrence of hepatocellular carcinoma (HCC). Given the limited therapeutic efficacy in advanced HCC, prevention of HCC development could be an effective strategy for improving patient prognosis. However, there is still no established therapy to meet the goal. Studies have elucidated a wide variety of molecular mechanisms and signaling pathways involved in HCC development. Genetically-engineered or chemically-treated experimental models of cirrhosis and HCC have been developed and shown their potential value in investigating molecular therapeutic targets and diagnostic biomarkers for HCC prevention. In this review, we overview potential targets of prevention and currently available experimental models, and discuss strategies to translate the findings into clinical practice. PMID:22873223

  3. Significance of 5,10-methylenetetrahydrofolate reductase gene variants in acute lymphoblastic leukemia in Indian population: an experimental, computational and meta-analysis.

    PubMed

    Bellampalli, Ravishankara; Phani, Nagaraja M; Bhat, Kamalakshi G; Prasad, Krishna; Bhaskaranand, Nalini; Guruprasad, Kanive P; Rai, Padmalatha S; Satyamoorthy, Kapaettu

    2015-05-01

    Acute lymphoblastic leukemia (ALL) arises due to several genetic alterations in progenitor cells, and methotrexate is frequently used as part of the treatment regimen. Although there is evidence for an effect of 5,10-methylenetetrahydrofolate reductase gene (MTHFR) C677T and A1298C variations on drug response in ALL, its risk association for ALL is still unresolved. In a case-control study of 203 patients with ALL and 246 controls and meta-analysis in the Indian population, we showed an insignificant association of MTHFR C677T and A1298C genotypes with childhood and adult ALL. Comprehensive in silico characterization of non-synonymous single nucleotide polymorphisms (nsSNPs) and SNPs of the 3' untranslated region (UTR) revealed nine nsSNPs as deleterious, and three SNPs in the 3'UTR could possibly alter the binding of miRNAs. The study revealed that several overlooked SNPs may contribute to the risk of ALL susceptibility and further studies of these SNPs with functional characterization in a large sample size are required to understand the significant role of MTHFR in ALL development. PMID:25115513

  4. Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase.

    PubMed Central

    Kern, Renée; Malki, Abderrahim; Holmgren, Arne; Richarme, Gilbert

    2003-01-01

    Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase. We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress. Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines. Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine. Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions. It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function. PMID:12549977

  5. An electrogenic nitric oxide reductase.

    PubMed

    Al-Attar, Sinan; de Vries, Simon

    2015-07-22

    Nitric oxide reductases (Nors) are members of the heme-copper oxidase superfamily that reduce nitric oxide (NO) to nitrous oxide (N₂O). In contrast to the proton-pumping cytochrome oxidases, Nors studied so far have neither been implicated in proton pumping nor have they been experimentally established as electrogenic. The copper-A-dependent Nor from Bacillus azotoformans uses cytochrome c₅₅₁ as electron donor but lacks menaquinol activity, in contrast to our earlier report (Suharti et al., 2001). Employing reduced phenazine ethosulfate (PESH) as electron donor, the main NO reduction pathway catalyzed by Cu(A)Nor reconstituted in liposomes involves transmembrane cycling of the PES radical. We show that Cu(A)Nor reconstituted in liposomes generates a proton electrochemical gradient across the membrane similar in magnitude to cytochrome aa₃, highlighting that bacilli using Cu(A)Nor can exploit NO reduction for increased cellular ATP production compared to organisms using cNor. PMID:26149211

  6. Preventative and Therapeutic Probiotic Use in Allergic Skin Conditions: Experimental and Clinical Findings

    PubMed Central

    Özdemir, Öner; Göksu Erol, Azize Yasemin

    2013-01-01

    Probiotics are ingested live microbes that can modify intestinal microbial populations in a way that benefits the host. The interest in probiotic preventative/therapeutic potential in allergic diseases stemmed from the fact that probiotics have been shown to improve intestinal dysbiosis and permeability and to reduce inflammatory cytokines in human and murine experimental models. Enhanced presence of probiotic bacteria in the intestinal microbiota is found to correlate with protection against allergy. Therefore, many studies have been recently designed to examine the efficacy of probiotics, but the literature on the allergic skin disorders is still very scarce. Here, our objective is to summarize and evaluate the available knowledge from randomized or nonrandomized controlled trials of probiotic use in allergic skin conditions. Clinical improvement especially in IgE-sensitized eczema and experimental models such as atopic dermatitis-like lesions (trinitrochlorobenzene and picryl chloride sensitizations) and allergic contact dermatitis (dinitrofluorobenzene sensitization) has been reported. Although there is a very promising evidence to recommend the addition of probiotics into foods, probiotics do not have a proven role in the prevention or the therapy of allergic skin disorders. Thus, being aware of possible measures, such as probiotics use, to prevent/heal atopic diseases is essential for the practicing allergy specialist. PMID:24078929

  7. Daptomycin (LY146032) for prevention and treatment of experimental aortic valve endocarditis in rabbits.

    PubMed Central

    Kennedy, S; Chambers, H F

    1989-01-01

    The efficacy of daptomycin (LY146032), a vancomycinlike lipopeptide antibiotic, was compared with that of antibiotics commonly in use for prevention and treatment of experimental aortic valve endocarditis in rabbits. Strains of Staphylococcus aureus. S. epidermidis, Streptococcus sanguis, and Enterococcus faecalis were used to establish endocarditis. A single 10-mg/kg dose of daptomycin and a single 25-mg/kg dose of vancomycin were both effective in prevention of endocarditis produced by strains of S. aureus and S. sanguis. Daptomycin was more effective than vancomycin for prevention of endocarditis caused by the strain of S. epidermidis. A single dose of daptomycin also was more effective in prevention of staphylococcal and enterococcal endocarditis than were single-dose regimens of cefazolin (100 mg/kg) and the combination of ampicillin (30 mg/kg) plus gentamicin (3 mg/kg), respectively. For treatment of endocarditis, daptomycin (10 mg/kg) as a single daily dose was as effective as regimens of either vancomycin or beta-lactam antibiotics for staphylococcal and enterococcal endocarditis. Daptomycin, however, was not as effective as a single daily dose of 600,000 U of procaine penicillin for endocarditis caused by the strain of S. sanguis. PMID:2554799

  8. Silicon prevents post laminectomy epidural root adhesions. An experimental study in rats.

    PubMed

    Hadani, M; Ram, Z; Horowitz, A; Shacked, I

    1993-01-01

    An experimental study was designed to test the efficacy of a silicon barrier around a nerve root in order to prevent post operative epidural root scar adhesions. In 32 Sprague-Dawley rats a lumbar nerve root was microsurgically exposed bilaterally. In 16 animals a silicon tube, prepared with a longitudinal cut along half of its wall, was placed around one nerve root, while the contralateral side served as control. In another group of 12 animals, an autologous subcutaneous fat graft was placed on the exposed root. A group of 4 animals served as the control group which had not been operated upon. No post operative neurological deficit was observed in any of the animals. All animals were sacrificed 60 days after the operation and a block of tissue including the nerve roots were resected bilaterally without removal of the silicon or fat graft. Longitudinal and transverse cuts of the roots were stained with Haematoxilin and Eosin and with Masson's trichrome collagen stain. The roots in the "unoperated" control group were clean of any scar tissue. In 13 out of 16 animals, silicon prevented scar formation around the root as opposed to scar adhesions around control root on the contralateral side and as compared to unoperated roots. In the silicon group, adhesions penetrated only through the longitudinal narrow cut edge of the tube. Fat did not prevent adhesions in 11 out of 12 animals. We conclude that a silicon barrier is an effective method preventing post operative epidural root scarring in rats. PMID:8237494

  9. Prevention

    MedlinePlus

    ... our e-newsletter! Aging & Health A to Z Prevention Basic Facts & Information Some factors that affect your ... control of the things that you can change. Preventive Recommendations for Adults Aged 65 and Older The ...

  10. Nanofiber nets in prevention of cicatrization in spinal procedures. Experimental study.

    PubMed

    Andrychowski, Jarosław; Frontczak-Baniewicz, Małgorzata; Sulejczak, Dorota; Kowalczyk, Tomasz; Chmielewski, Tomasz; Czernicki, Zbigniew; Kowalewski, Tomasz Aleksander

    2013-01-01

    Excessive cicatrisation or epidural fibrosis in the operative field is an inappropriate event occasionally occurring after neurosurgical procedures (i.e., spine procedures and craniotomies). This excessive process may disturb the postoperative course and render reoperations more difficult and risky. The literature describes this phenomenon as accompanying up to 20% of neurosurgical procedures. The scar tissue that forms postoperatively adheres to the dura mater, penetrates into the spinal canal and can cause narrowing symptoms, neurological deficits and pain. The incidence and spread of this excessive scar or epidural fibrosis can be prevented through the modification of the surgical technique by incorporating endoscopic or microscopic access to minimize the operative field and the use of isolating substances (autogenous or heterogeneous) administered intraoperatively. The aim of this experimental study was to morphologically assess the cicatrization process, adhesion and to prevent excessive scar formation with the local use of membranes manufactured by an electrospinning process (nanotechnology). We also investigated whether the biodegradable nanofibrous net triggers or modifies the immunological response or the local inflammatory process. Micro-nanofibrous membranes were produced by the electrospinning process. A biodegradable, medically certified copolymer poly(L-lactide-co-caprolactone) (PLCL) was used as the electrospun material. An experimental rat model was used in this study. Experimental and control groups were formed with specified follow-up times of 4, 14 and 30 days. During the operation, a two-level laminectomy in the thoracic segment was performed. The operative field was divided into two regions. Isolating material was used on the dura mater and surface of the spinal cord in the area where the laminectomy was performed. The material was analysed with the use of light and electron microscopy. Local cicatrisation can be modified using nanomaterials

  11. Experimental study on asphaltene adsorption onto formation rock: An approach to asphaltene formation damage prevention

    SciTech Connect

    Piro, G.; Barberis Canonica, L.; Galbariggi, G.; Bertero, L.; Carniani, C.

    1995-12-31

    In this paper, through a comparative study on Static vs Dynamic adsorption of asphaltene onto formation rock, it is reported how, for the particular asphaltene/formation rock system here considered, the Dynamic asphaltene adsorption onto formation rock is a continuous phenomenon by which the quantity of adsorbed asphaltene increases continuously. In the authors` opinion this rather remarkable adsorption behavior may contribute to asphaltene formation damage. In the hypothesis that prevention may represent a more economical approach than removal, in this work is also reported a possible prevention approach based on formation rock treatment by means of specific chemicals more apt than asphaltenes to be adsorbed onto rock. As preliminary demonstration, with the aim at assessing qualitatively the potential of their approach, the authors have pre-treated the rock by means of commercially available asphaltene dispersant and flocculation inhibitors. Albeit the chosen additives are not commercialized on the base of their specific adsorption feature, a prevention effect has been effectively found. Experimental set ups and procedures used as a base for a test able to rank chemicals with respect to their asphaltene adsorption inhibitive effects are also reported.

  12. Human brain aldehyde reductases: relationship to succinic semialdehyde reductase and aldose reductase.

    PubMed

    Hoffman, P L; Wermuth, B; von Wartburg, J P

    1980-08-01

    Human brain contains multiple forms of aldehyde-reducing enzymes. One major form (AR3), as previously shown, has properties that indicate its identity with NADPH-dependent aldehyde reductase isolated from brain and other organs of various species; i.e., low molecular weight, use of NADPH as the preferred cofactor, and sensitivity to inhibition by barbiturates. A second form of aldehyde reductase ("SSA reductase") specifically reduces succinic semialdehyde (SSA) to produce gamma-hydroxybutyrate. This enzyme form has a higher molecular weight than AR3, and uses NADH as well as NADPH as cofactor. SSA reductase was not inhibited by pyrazole, oxalate, or barbiturates, and the only effective inhibitor found was the flavonoid quercetine. Although AR3 can also reduce SSA, the relative specificity of SSA reductase may enhance its in vivo role. A third form of human brain aldehyde reductase, AR2, appears to be comparable to aldose reductases characterized in several species, on the basis of its activity pattern with various sugar aldehydes and its response to characteristic inhibitors and activators, as well as kinetic parameters. This enzyme is also the most active in reducing the aldehyde derivatives of biogenic amines. These studies suggest that the various forms of human brain aldehyde reductases may have specific physiological functions. PMID:6778961

  13. Antiinflammatory Effect of Phytosterols in Experimental Murine Colitis Model: Prevention, Induction, Remission Study

    PubMed Central

    Aldini, Rita; Micucci, Matteo; Cevenini, Monica; Fato, Romana; Bergamini, Christian; Nanni, Cristina; Cont, Massimiliano; Camborata, Cecilia; Spinozzi, Silvia; Montagnani, Marco; Roda, Giulia; D'Errico-Grigioni, Antonia; Rosini, Francesca; Roda, Aldo; Mazzella, Giuseppe; Chiarini, Alberto; Budriesi, Roberta

    2014-01-01

    Phytosterols, besides hypocholesterolemic effect, present anti-inflammatory properties. Little information is available about their efficacy in Inflammatory Bowel Disease (IBD). Therefore, we have evaluated the effect of a mixture of phytosterols on prevention/induction/remission in a murine experimental model of colitis. Phytosterols were administered x os before, during and after colitis induction with Dextran Sodium Sulfate (DSS) in mice. Disease Activity Index (DAI), colon length, histopathology score, 18F-FDG microPET, oxidative stress in the intestinal tissue (ileum and colon) and gallbladder ileum and colon spontaneous and carbachol (CCh) induced motility, plasma lipids and plasma, liver and biliary bile acids (BA) were evaluated. A similar longitudinal study was performed in a DSS colitis control group. Mice treated with DSS developed severe colitis as shown by DAI, colon length, histopathology score, 18F-FDG microPET, oxidative stress. Both spontaneous and induced ileal and colonic motility were severely disturbed. The same was observed with gallbladder. DSS colitis resulted in an increase in plasma cholesterol, and a modification of the BA pattern. Phytosterols feeding did not prevent colitis onset but significantly reduced the severity of the disease and improved clinical and histological remission. It had strong antioxidant effects, almost restored colon, ileal and gallbladder motility. Plasmatic levels of cholesterol were also reduced. DSS induced a modification in the BA pattern consistent with an increase in the intestinal BA deconjugating bacteria, prevented by phytosterols. Phytosterols seem a potential nutraceutical tool for gastrointestinal inflammatory diseases, combining metabolic systematic and local anti-inflammatory effects. PMID:25268769

  14. Prevention

    MedlinePlus

    ... Prevention Treatment 2003 U.S. Outbreak African Rodent Importation Ban For Clinicians Clinical Recognition Specimen Collection Treatment Smallpox ... Examining Animals with Suspected Monkeypox African Rodent Importation Ban Resources Related Links Poxvirus Molluscum Contagiosum Orf Virus ( ...

  15. Prevention of chemically induced diabetes mellitus in experimental animals by virgin argan oil.

    PubMed

    Bellahcen, Said; Mekhfi, Hassane; Ziyyat, Abderrahim; Legssyer, Abdelkhaleq; Hakkou, Abdelkader; Aziz, Mohammed; Bnouham, Mohamed

    2012-02-01

    The argan tree plays an important socioeconomic and ecologic role in South Morocco. Moreover, there is much evidence for the beneficial effects of virgin argan oil (VAO) on human health. Thus, this study investigated whether administering VAO to rats can prevent the development of diabetes. VAO extracted by a traditional method from the almonds of Argania spinosa (2 mL/kg) was administered orally (for 7 consecutive days) to rats before and during intraperitoneal alloxan administration (75 mg/kg for 5 consecutive days). An alloxan diabetic-induced untreated group and treated by table oil were used as control groups. Body mass, blood glucose and hepatic glycogen were evaluated. In the present study, subchronic treatment with VAO at a dose of 2 mL/kg, before the experimental induction of diabetes, prevented the body mass loss, induced a significant reduction of blood glucose and a significant increase of hepatic glycogen level (p < 0.001) compared with the untreated diabetic group. In conclusion, the present study shows that argan oil should be further investigated in a human study to clarify its possible role in reducing weight loss in diabetics, and even in inhibiting the development or progression of diabetes. This antidiabetic effect could be due to the richness of VAO in tocopherols, phenolic compounds and unsaturated fatty acids. PMID:21584872

  16. Experimental concepts for toxicity prevention and tissue restoration after central nervous system irradiation

    PubMed Central

    Nieder, Carsten; Andratschke, Nicolaus; Astner, Sabrina T

    2007-01-01

    Several experimental strategies of radiation-induced central nervous system toxicity prevention have recently resulted in encouraging data. The present review summarizes the background for this research and the treatment results. It extends to the perspectives of tissue regeneration strategies, based for example on stem and progenitor cells. Preliminary data suggest a scenario with individually tailored strategies where patients with certain types of comorbidity, resulting in impaired regeneration reserve capacity, might be considered for toxicity prevention, while others might be "salvaged" by delayed interventions that circumvent the problem of normal tissue specificity. Given the complexity of radiation-induced changes, single target interventions might not suffice. Future interventions might vary with patient age, elapsed time from radiotherapy and toxicity type. Potential components include several drugs that interact with neurodegeneration, cell transplantation (into the CNS itself, the blood stream, or both) and creation of reparative signals and a permissive microenvironment, e.g., for cell homing. Without manipulation of the stem cell niche either by cell transfection or addition of appropriate chemokines and growth factors and by providing normal perfusion of the affected region, durable success of such cell-based approaches is hard to imagine. PMID:17603905

  17. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  18. An experimental evaluation of the All Stars prevention curriculum in a community after school setting.

    PubMed

    Gottfredson, Denise C; Cross, Amanda; Wilson, Denise; Rorie, Melissa; Connell, Nadine

    2010-06-01

    This study tested the effectiveness of a prevention curriculum, All Stars, as implemented in a year-long school-based after school program and provides an independent replication of the effects of All Stars on targeted mediators and problem behaviors using an experimental methodology. Middle school students (N = 447) who registered for the after school program were randomly assigned to the experimental or control condition. The sample included approximately equal proportions of males and females, was 70% African American, and 59% of the students received subsidized meals at school. All Stars was delivered with reasonable integrity to the program design, although with lower quality than reported in earlier efficacy trials. However, actual student exposure to the program was lower than expected due to low levels of attendance in the after school program. Students who ever attended received an average of 16 h of All Stars instruction. Results showed no differences between the treatment and control students at post-test on any of the outcomes or mediators. Further, no positive effects were found for youths receiving higher dosage, higher quality program delivery, or both. Insufficient time to achieve high quality implementation in the after school context and potential deviancy training are suggested as reasons for the failure to replicate positive program effects. PMID:19859806

  19. Understanding the molecular mechanisms of cancer prevention by dietary phytochemicals: From experimental models to clinical trials

    PubMed Central

    Maru, Girish B; Hudlikar, Rasika R; Kumar, Gaurav; Gandhi, Khushboo; Mahimkar, Manoj B

    2016-01-01

    Chemoprevention is one of the cancer prevention approaches wherein natural/synthetic agent(s) are prescribed with the aim to delay or disrupt multiple pathways and processes involved at multiple steps, i.e., initiation, promotion, and progression of cancer. Amongst environmental chemopreventive compounds, diet/beverage-derived components are under evaluation, because of their long history of exposure to humans, high tolerability, low toxicity, and reported biological activities. This compilation briefly covers and compares the available evidence on chemopreventive efficacy and probable mechanism of chemoprevention by selected dietary phytochemicals (capsaicin, curcumin, diallyl sulphide, genistein, green/black tea polyphenols, indoles, lycopene, phenethyl isocyanate, resveratrol, retinoids and tocopherols) in experimental systems and clinical trials. All the dietary phytochemicals covered in this review have demonstrated chemopreventive efficacy against spontaneous or carcinogen-induced experimental tumors and/or associated biomarkers and processes in rodents at several organ sites. The observed anti-initiating, anti-promoting and anti-progression activity of dietary phytochemicals in carcinogen-induced experimental models involve phytochemical-mediated redox changes, modulation of enzymes and signaling kinases resulting to effects on multiple genes and cell signaling pathways. Results from clinical trials using these compounds have not shown them to be chemopreventive. This may be due to our: (1) inability to reproduce the exposure conditions, i.e., levels, complexity, other host and lifestyle factors; and (2) lack of understanding about the mechanisms of action and agent-mediated toxicity in several organs and physiological processes in the host. Current research efforts in addressing the issues of exposure conditions, bioavailability, toxicity and the mode of action of dietary phytochemicals may help address the reason for observed mismatch that may ultimately

  20. Understanding the molecular mechanisms of cancer prevention by dietary phytochemicals: From experimental models to clinical trials.

    PubMed

    Maru, Girish B; Hudlikar, Rasika R; Kumar, Gaurav; Gandhi, Khushboo; Mahimkar, Manoj B

    2016-02-26

    Chemoprevention is one of the cancer prevention approaches wherein natural/synthetic agent(s) are prescribed with the aim to delay or disrupt multiple pathways and processes involved at multiple steps, i.e., initiation, promotion, and progression of cancer. Amongst environmental chemopreventive compounds, diet/beverage-derived components are under evaluation, because of their long history of exposure to humans, high tolerability, low toxicity, and reported biological activities. This compilation briefly covers and compares the available evidence on chemopreventive efficacy and probable mechanism of chemoprevention by selected dietary phytochemicals (capsaicin, curcumin, diallyl sulphide, genistein, green/black tea polyphenols, indoles, lycopene, phenethyl isocyanate, resveratrol, retinoids and tocopherols) in experimental systems and clinical trials. All the dietary phytochemicals covered in this review have demonstrated chemopreventive efficacy against spontaneous or carcinogen-induced experimental tumors and/or associated biomarkers and processes in rodents at several organ sites. The observed anti-initiating, anti-promoting and anti-progression activity of dietary phytochemicals in carcinogen-induced experimental models involve phytochemical-mediated redox changes, modulation of enzymes and signaling kinases resulting to effects on multiple genes and cell signaling pathways. Results from clinical trials using these compounds have not shown them to be chemopreventive. This may be due to our: (1) inability to reproduce the exposure conditions, i.e., levels, complexity, other host and lifestyle factors; and (2) lack of understanding about the mechanisms of action and agent-mediated toxicity in several organs and physiological processes in the host. Current research efforts in addressing the issues of exposure conditions, bioavailability, toxicity and the mode of action of dietary phytochemicals may help address the reason for observed mismatch that may ultimately

  1. Thioredoxin Reductase and its Inhibitors

    PubMed Central

    Saccoccia, Fulvio; Angelucci, Francesco; Boumis, Giovanna; Carotti, Daniela; Desiato, Gianni; Miele, Adriana E; Bellelli, Andrea

    2014-01-01

    Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis. PMID:24875642

  2. Experimental study of the efficiency of oxidized dextran for prevention of influenza A/H5N1.

    PubMed

    Shkurupy, V A; Potapova, O V; Sharkova, T V; Troitskii, A V; Gulyaeva, E P; Bystrova, T N; Shestopalov, A M

    2014-11-01

    Oxidized dextran is suggested for prevention of infection induced by influenza A/H5N1 viruses, methods of its use and doses are determined. Two intravenous injections of dextran 3 and 1 days before experimental infection of outbred mice by influenza A/H5N1 A/goose/Krasnoozerskoye/627/05 virus resulted in a high preventive dose-dependent effect: the mean lifespan was 25% prolonged, the mortality decreased 3-fold. PMID:25403410

  3. Prevention of passively transferred experimental autoimmune myasthenia gravis by a phage library-derived cyclic peptide

    PubMed Central

    Venkatesh, Natarajan; Im, Sin-Heyog; Balass, Moshe; Fuchs, Sara; Katchalski-Katzir, Ephraim

    2000-01-01

    Many pathogenic antibodies in myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are directed against the main immunogenic region (MIR) of the acetylcholine receptor (AcChoR). These antibodies are highly conformation dependent; hence, linear peptides derived from native receptor sequences are poor candidates for their immunoneutralization. We employed a phage-epitope library to identify peptide-mimotopes capable of preventing the pathogenicity of the anti-MIR mAb 198. We identified a 15-mer peptide (PMTLPENYFSERPYH) that binds specifically to mAb 198 and inhibits its binding to AcChoR. A 10-fold increase in the affinity of this peptide was achieved by incorporating flanking amino acid residues from the coat protein as present in the original phage library. This extended peptide (AEPMTLPENYFSERPYHPPPP) was constrained by the addition of cysteine residues on both ends of the peptide, thus generating a cyclic peptide that inhibited the binding of mAb 198 to AcChoR with a potency that is three orders of magnitude higher when compared with the parent library peptide. This cyclic peptide inhibited the in vitro binding of mAb 198 to AcChoR and prevented the antigenic modulation of AcChoR caused by mAb 198 in human muscle cell cultures. The cyclic peptide also reacted with several other anti-MIR mAbs and the sera of EAMG rats. In addition, this peptide blocked the ability of mAb 198 to passively transfer EAMG in rats. Further derivatization of the cyclic peptide may aid in the design of suitable synthetic mimotopes for modulation of MG. PMID:10639153

  4. Prevention or Modification of Epileptogenesis after Brain Insults: Experimental Approaches and Translational Research

    PubMed Central

    Brandt, Claudia

    2010-01-01

    Diverse brain insults, including traumatic brain injury, stroke, infections, tumors, neurodegenerative diseases, and prolonged acute symptomatic seizures, such as complex febrile seizures or status epilepticus (SE), can induce “epileptogenesis,” a process by which normal brain tissue is transformed into tissue capable of generating spontaneous recurrent seizures. Furthermore, epileptogenesis operates in cryptogenic causes of epilepsy. In view of the accumulating information about cellular and molecular mechanisms of epileptogenesis, it should be possible to intervene in this process before the onset of seizures and thereby either prevent the development of epilepsy in patients at risk or increase the potential for better long-term outcome, which constitutes a major clinical need. For identifying pharmacological interventions that prevent, interrupt or reverse the epileptogenic process in people at risk, two groups of animal models, kindling and SE-induced recurrent seizures, have been recommended as potentially useful tools. Furthermore, genetic rodent models of epileptogenesis are increasingly used in assessing antiepileptogenic treatments. Two approaches have been used in these different model categories: screening of clinically established antiepileptic drugs (AEDs) for antiepileptogenic or disease-modifying potential, and targeting the key causal mechanisms that underlie epileptogenesis. The first approach indicated that among various AEDs, topiramate, levetiracetam, carisbamate, and valproate may be the most promising. On the basis of these experimental findings, two ongoing clinical trials will address the antiepileptogenic potential of topiramate and levetiracetam in patients with traumatic brain injury, hopefully translating laboratory discoveries into successful therapies. The second approach has highlighted neurodegeneration, inflammation and up-regulation of immune responses, and neuronal hyperexcitability as potential targets for antiepileptogenesis

  5. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease.

    PubMed

    Flores, Alyssa M; Casey, Scott D; Felix, Christian M; Phuan, Puay W; Verkman, A S; Levin, Marc H

    2016-05-01

    Dry eye disorders, including Sjögren's syndrome, constitute a common problem in the aging population, with limited effective therapeutic options available. The cAMP-activated Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is a major prosecretory channel at the ocular surface. We investigated whether compounds that target CFTR can correct the abnormal tear film in dry eye. Small-molecule activators of human wild-type CFTR identified by high-throughput screening were evaluated in cell culture and in vivo assays, to select compounds that stimulate Cl(-)-driven fluid secretion across the ocular surface in mice. An aminophenyl-1,3,5-triazine, CFTRact-K089, fully activated CFTR in cell cultures with EC50 ∼250 nM and produced an ∼8.5 mV hyperpolarization in ocular surface potential difference. When delivered topically, CFTRact-K089 doubled basal tear volume for 4 h and had no effect in CF mice. CFTRact-K089 showed sustained tear film bioavailability without detectable systemic absorption. In a mouse model of aqueous-deficient dry eye produced by lacrimal ablation, topical administration of 0.1 nmol CFTRact-K089 3 times daily restored tear volume to basal levels, preventing corneal epithelial disruption when initiated at the time of surgery and reversing it when started after development of dry eye. Our results support the potential utility of CFTR-targeted activators as a novel prosecretory treatment for dry eye.-Flores, A. M., Casey, S. D., Felix, C. M., Phuan, P. W., Verkman, A. S., Levin, M. H. Small-molecule CFTR activators increase tear secretion and prevent experimental dry eye disease. PMID:26842854

  6. Lippia sidoides and Myracrodruon urundeuva gel prevents alveolar bone resorption in experimental periodontitis in rats.

    PubMed

    Botelho, M A; Rao, V S; Carvalho, C B M; Bezerra-Filho, J G; Fonseca, S G C; Vale, M L; Montenegro, D; Cunha, F; Ribeiro, R A; Brito, G A

    2007-09-25

    In Brazilian folk medicine, Lippia sidoides (Ls) and Myracrodruon urundeuva (Mu) have gained popularity and reputation as effective antimicrobial and anti-inflammatory agents. This work aimed to evaluate the effect of topical herbal gel from Ls 0.5% (v/w) and Mu 5% (w/w) in experimental periodontal disease (EPD) in rats. Wistar rats were subjected to ligature placement around the second upper left molars. Animals were treated topically with Ls and/or Mu-based gel, immediately after EPD induction and three times/day for 11 days until the rats were sacrificed (11th day). Saline-based gel was utilized as control for all experiments and doxycycline based gel 10% (w/w) was utilized as reference substance. Animals were weighed daily. Alveolar bone loss was measured as the difference (in millimeters) between the cusp tip and the alveolar bone. The periodontum and the surrounding gingivae were examined at histopathology, as well as the neutrophil influx into the gingivae was assayed using myeloperoxidase activity and cytokine production mainly tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) levels by ELISA method. The local bacterial flora was assessed through culture of the gingival tissue in standard aerobic and anaerobic media. Alveolar bone loss was significantly inhibited by Ls and Mu combined treatment compared to the saline control group. Ls and Mu combined treatment reduced tissue lesion at histopathology, with partial preservation of the periodontum, coupled to decreased myeloperoxidase activity as well as significantly inhibited TNF-alpha and IL-1beta production in gingival tissue compared to the saline control group. Ls and Mu combined treatment also prevented the growth of oral microorganisms and the weight loss. Ls and Mu combined based gel treatment preserved alveolar bone resorption and demonstrated anti-inflammatory and antibacterial activities in experimental periodontitis. PMID:17714897

  7. Endovascular Coil Embolization of Segmental Arteries Prevents Paraplegia After Subsequent TAAA Repair – An Experimental Model

    PubMed Central

    Geisbüsch, S; Stefanovic, A; Koruth, JS; Lin, HM; Morgello, S; Weisz, DJ; Griepp, RB; Di Luozzo, G

    2013-01-01

    Objective To test a strategy for minimizing ischemic spinal cord injury (SCI) following extensive thoracoabdominal aneurysm (TAAA) repair, we occluded a small number of segmental arteries (SAs) endovascularly one week before simulated aneurysm repair in an experimental model. Methods 30 juvenile Yorkshire pigs (25.2±1.7kg) were randomized into three groups. All SAs—intercostal and lumbar—were sacrificed by a combination of surgical ligation of the lumbar SAs and occlusion of intercostal SAs with thoracic endovascular stent grafting (TEVAR). 7–10 days before this simulated TAAA replacement, SAs in the lower thoracic/upper lumbar region were occluded using embolization coils: 1.5±0.5 SAs in Group 1 (T13/L1), and 4.5±0.5 in Group 2 (T11-L3). No SAs were coiled in the controls. Hind limb function was evaluated blindly from daily videotapes using a modified Tarlov score: 0=paraplegia; 9=full recovery. After sacrifice, each segment of spinal cord was graded histologically using the 9-point Kleinman score: 0=normal, 8=complete necrosis. Results Hind limb function remained normal after coil embolization. After simulated TAAA repair, paraplegia occurred in 6/10 control pigs, but only 2/10 pigs in Group 1: no pigs in Group 2 had SCI. Tarlov scores were significantly better in Group 2 (Control vs 1 p=0.06; Control vs 2 p= 0.0002; 1 vs 2 p=0.05). A dramatic reduction in histologic damage—most prominently in the coiled region—was seen when SAs were embolized before simulated TAAA repair. Conclusions Endovascular coiling of 2–4 SAs prevents paraplegia in an experimental model of extensive hybrid TAAA repair, and helps protect the spinal cord from ischemic histopathological injury. A clinical trial in a selected patient population at high risk for postoperative SCI may be appropriate. PMID:24220154

  8. Preventive effect of Zea mays L. (purple waxy corn) on experimental diabetic cataract.

    PubMed

    Thiraphatthanavong, Paphaphat; Wattanathorn, Jintanaporn; Muchimapura, Supaporn; Wipawee, Thukham-mee; Wannanon, Panakaporn; Terdthai, Tong-un; Suriharn, Bhalang; Lertrat, Kamol

    2014-01-01

    Recently, substances possessing antioxidant can prevent cataractogenesis of diabetic cataract. Therefore, this study was carried out to determine the anticataract effect of Zea mays L. (purple waxy corn), a flavonoids rich plant, in experimental diabetic cataract. Enucleated rat lenses were incubated in artificial aqueous humor containing 55 mM glucose with various concentrations of Zea mays L. (purple waxy corn) ranging between 2, 10, and 50 mg/mL at room temperature for 72 h. At the end of the incubation period, the evaluation of lens opacification, MDA level, and the activities of SOD, CAT, GPx, and AR in lens were performed. The results showed that both medium and high doses of extract decreased lens opacity together with the decreased MDA level. In addition, medium dose of extract increased GPx activity while the high dose decreased AR activity. No other significant changes were observed. The purple waxy corn seeds extract is the potential candidate to protect against diabetic cataract. The mechanism of action may occur via the decreased oxidative stress and the suppression of AR. However, further research in vivo is still essential. PMID:24527449

  9. Toxoplasma gondii Upregulates Interleukin-12 To Prevent Plasmodium berghei-Induced Experimental Cerebral Malaria

    PubMed Central

    Settles, Erik W.; Moser, Lindsey A.; Harris, Tajie H.

    2014-01-01

    A chronic infection with the parasite Toxoplasma gondii has previously been shown to protect mice against subsequent viral, bacterial, or protozoal infections. Here we have shown that a chronic T. gondii infection can prevent Plasmodium berghei ANKA-induced experimental cerebral malaria (ECM) in C57BL/6 mice. Treatment with soluble T. gondii antigens (STAg) reduced parasite sequestration and T cell infiltration in the brains of P. berghei-infected mice. Administration of STAg also preserved blood-brain barrier function, reduced ECM symptoms, and significantly decreased mortality. STAg treatment 24 h post-P. berghei infection led to a rapid increase in serum levels of interleukin 12 (IL-12) and gamma interferon (IFN-γ). By 5 days after P. berghei infection, STAg-treated mice had reduced IFN-γ levels compared to those of mock-treated mice, suggesting that reductions in IFN-γ at the time of ECM onset protected against lethality. Using IL-10- and IL-12βR-deficient mice, we found that STAg-induced protection from ECM is IL-10 independent but IL-12 dependent. Treatment of P. berghei-infected mice with recombinant IL-12 significantly decreased parasitemia and mortality. These data suggest that IL-12, either induced by STAg or injected as a recombinant protein, mediates protection from ECM-associated pathology potentially through early induction of IFN-γ and reduction in parasitemia. These results highlight the importance of early IL-12 induction in protection against ECM. PMID:24396042

  10. Prevention of experimental autoimmune encephalomyelitis by antibodies against α4βl integrin

    NASA Astrophysics Data System (ADS)

    Yednock, Ted A.; Cannon, Catherine; Fritz, Lawrence C.; Sanchez-Madrid, Francisco; Steinman, Lawrence; Karin, Nathan

    1992-03-01

    EXPERIMENTAL autoimmune encephalomyelitis (EAE) is an inflammatory condition of the central nervous system with similarities to multiple sclerosis1,2. In both diseases, circulating leukocytes penetrate the blood-brain barrier and damage myelin, resulting in impaired nerve conduction and paralysis3-5. We sought to identify the adhesion receptors that mediate the attachment of circulating leukocytes to inflamed brain endothelium in EAE, because this interaction is the first step in leukocyte entry into the central nervous system. Using an in vitro adhesion assay on tissue sections, we found that lymphocytes and monocytes bound selectively to inflamed EAE brain vessels. Binding was inhibited by antibodies against the integrin molecule α4βl, but not by antibodies against numerous other adhesion receptors. When tested in vivo, anti-α4 integrin effectively prevented the accumulation of leukocytes in the central nervous system and the development of EAE. Thus, therapies designed to interfere with α4βl integrin may be useful in treating inflammatory diseases of the central nervous system, such as multiple sclerosis.

  11. Equine 5α-reductase activity and expression in epididymis.

    PubMed

    Corbin, C J; Legacki, E L; Ball, B A; Scoggin, K E; Stanley, S D; Conley, A J

    2016-10-01

    The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies. PMID:27466384

  12. The prevention and treatment of cognitive decline and dementia: An overview of recent research on experimental treatments.

    PubMed

    Andrade, Chittaranjan; Radhakrishnan, Rajiv

    2009-01-01

    The prevention and treatment of cognitive impairment in the elderly has assumed increasing importance in an aging population. This article presents a qualitative review of recent research on experimental interventions for the prevention and treatment of mild cognitive impairment and Alzheimer's disease in elderly subjects. Interventions addressed range from lifestyle measures to pharmacological treatments. Epidemiological studies suggest that dietary measures, physical exercise, and mental activity may reduce the risk of cognitive impairment and Alzheimer's disease in elderly subjects. Statins may protect against incident dementia, and lithium may convey similar benefits to bipolar patients. Ginkgo appears ineffective as a primary preventive measure. Donepezil but not Vitamin E may benefit persons with mild cognitive impairment. Experimental treatments potentially useful for Alzheimer's disease include dimebon, PBT2 and etanercept; the safety and efficacy of the Alzheimer's vaccine remains to be proven, and growth hormone secretagogue and tarenflurbil are likely ineffective. Herbal treatments merit study in elderly subjects with cognitive syndromes. PMID:19742190

  13. Profound prevention of experimental brain metastases of breast cancer by temozolomide in an MGMT-dependent manner

    PubMed Central

    Palmieri, Diane; Duchnowska, Renata; Woditschka, Stephan; Hua, Emily; Qian, Yongzhen; Biernat, Wojciech; Sosińska-Mielcarek, Katarzyna; Gril, Brunilde; Stark, Andreas; Hewitt, Stephen; Liewehr, David J; Steinberg, Seth M; Jassem, Jacek; Steeg, Patricia S

    2014-01-01

    Purpose Brain metastases of breast cancer cause neurocognitive damage and are incurable. We evaluated a role for temozolomide in the prevention of brain metastases of breast cancer in experimental brain metastasis models. Experimental Design Temozolomide was administered in mice following earlier injection of brain-tropic human epidermal growth factor receptor 2 (HER2)-positive Jimt1-BR3 and triple negative 231-BR-EGFP sublines, the latter with and without expression of 06-methylguanine-DNA methyltransferase (MGMT). Additionally, the percentage of MGMT-positive tumor cells in 62 patient-matched sets of breast cancer primary tumors and resected brain metastases was determined immunohistochemically. Results Temozolomide, when dosed at 50, 25, 10 or 5 mg/kg, 5 days/week, beginning 3 days after inoculation, completely prevented the formation of experimental brain metastases from MGMT-negative 231-BR-EGFP cells. At a 1 mg/kg dose, temozolomide prevented 68% of large brain metastases, and was ineffective at a dose of 0.5 mg/kg. When the 50 mg/kg dose was administered beginning on days 18 or 24, temozolomide efficacy was reduced or absent. Temozolomide was ineffective at preventing brain metastases in MGMT-transduced 231-BR-EGFP and MGMT-expressing Jimt-1-BR3 sublines. In 62 patient-matched sets of primary breast tumors and resected brain metastases, 43.5% of the specimens had concordant low MGMT expression, while in another 14.5% of sets high MGMT staining in the primary tumor corresponded with low staining in the brain metastasis. Conclusions Temozolomide profoundly prevented the outgrowth of experimental brain metastases of breast cancer in an MGMT-dependent manner. These data provide compelling rationale for investigating the preventive efficacy of temozolomide in a clinical setting. PMID:24634373

  14. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  15. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  16. Preventive effects of cranberry products on experimental colitis induced by dextran sulphate sodium in mice.

    PubMed

    Xiao, Xiao; Kim, Jonggun; Sun, Quancai; Kim, Daeyoung; Park, Cheon-Seok; Lu, Tzong-Shi; Park, Yeonhwa

    2015-01-15

    With the prevalence of inflammatory bowel disease (IBD) and its associated risk for development of colorectal cancer, it is of great importance to prevent and treat IBD. However, due to the complexity of etiology and potentially serious adverse effects, treatment options for IBD are relatively limited. Thus, the purpose of this study was to identify a safe food-based approach for the prevention and treatment of IBD. In this study, we tested the effects of cranberry products on preventing dextran sulphate sodium-induced murine colitis. Our results suggest that both cranberry extract and dried cranberries-fed groups had a significantly reduced disease activity index, where dried cranberries were more effective in preventing colitis than cranberry extract. Shortening of colon length, colonic myeloperoxidase activity and production of pro-inflammatory cytokines were attenuated in animals fed dried cranberries compared to the controls. The current report suggests that cranberries can be applied to prevent and reduce the symptoms of IBD. PMID:25149009

  17. Steroidal pyrazolines evaluated as aromatase and quinone reductase-2 inhibitors for chemoprevention of cancer.

    PubMed

    Abdalla, Mohamed M; Al-Omar, Mohamed A; Bhat, Mashooq A; Amr, Abdel-Galil E; Al-Mohizea, Abdullah M

    2012-05-01

    The aromatase and quinone reductase-2 inhibition of synthesized heterocyclic pyrazole derivatives fused with steroidal structure for chemoprevention of cancer is reported herein. All compounds were interestingly less toxic than the reference drug (Cyproterone(®)). The aromatase inhibitory activities of these compounds were much more potent than the lead compound resveratrol, which has an IC(50) of 80 μM. In addition, all the compounds displayed potent quinone reductase-2 inhibition. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). The aromatase and quinone reductase-2 inhibitors resulting from this study have potential value in the treatment and prevention of cancer. PMID:22361454

  18. Magnesium Supplementation Prevents and Reverses Experimentally Induced Movement Disturbances in Rats: Biochemical and Behavioral Parameters.

    PubMed

    Kronbauer, Maikel; Segat, Hecson J; De David Antoniazzi, Caren Tatiane; Roversi, Karine; Roversi, Katiane; Pase, Camila S; Barcelos, Raquel C S; Burger, Marilise E

    2015-08-01

    Reserpine administration results in a predictable animal model of orofacial dyskinesia (OD) that has been largely used to access movement disturbances related to extrapyramidal oxidative damage. Here, OD was acutely induced by reserpine (two doses of 0.7 mg/kg subcutaneous (s.c.)), every other day for 3 days), which was administered after (experiment 1) and before (experiment 2) magnesium (Mg) supplementation (40 mg/kg/mL, peroral (p.o.)). In experiment 1, Mg was administered for 28 days before reserpine treatment, while in experiment 2, it was initiated 24 h after the last reserpine administration and was maintained for 10 consecutive days. Experiment 1 (prevention) showed that Mg supplementation was able to prevent reserpine-induced OD and catalepsy development. Mg was also able to prevent reactive species (RS) generation, thus preventing increase of protein carbonyl (PC) levels in both cortex and substantia nigra, but not in striatum. Experiment 2 (reversion) showed that Mg was able to decrease OD and catalepsy at all times assessed. In addition, Mg was able to decrease RS generation, with lower levels of PC in both cortex and striatum, but not in substantia nigra. These outcomes indicate that Mg is an important metal that should be present in the diet, since its intake is able to prevent and minimize the development of movement disorders closely related to oxidative damage in the extrapyramidal brain areas, such as OD. PMID:25686766

  19. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases. PMID:12626517

  20. Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases.

    PubMed

    Min, Tongpil; Kasahara, Hiroyuki; Bedgar, Diana L; Youn, Buhyun; Lawrence, Paulraj K; Gang, David R; Halls, Steven C; Park, HaJeung; Hilsenbeck, Jacqueline L; Davin, Laurence B; Lewis, Norman G; Kang, ChulHee

    2003-12-12

    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed. PMID:13129921

  1. Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases

    NASA Technical Reports Server (NTRS)

    Min, Tongpil; Kasahara, Hiroyuki; Bedgar, Diana L.; Youn, Buhyun; Lawrence, Paulraj K.; Gang, David R.; Halls, Steven C.; Park, HaJeung; Hilsenbeck, Jacqueline L.; Davin, Laurence B.; Lewis, Norman G.; Kang, ChulHee

    2003-01-01

    Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

  2. Comparison of montelukast and cabergoline for prevention of ovarian hyperstimulation syndrome: in an experimental rat model.

    PubMed

    Akman, Levent; Sahin, Gulnaz; Erbas, Oytun; Aktug, Huseyin; Akdogan, Aysin; Goker, Ege Nazan Tavmergen; Taskiran, Dilek; Tavmergen, Erol

    2015-05-01

    Ovarian hyperstimulation syndrome (OHSS) is a serious iatrogenic complication that can occur during assisted reproductive techniques. The aim of this study is to investigate the effects of the leukotriene receptor antagonist (montelukast) treatment in prevention of OHSS and compare to cabergoline treatment. Twenty-four immature female Wistar rats were assigned to four groups. Group 1 was the control group. In the remaining three groups, OHSS was induced through ovarian stimulation with gonadotropins. No treatment was given to Group 2. Group 3 was administered a low-dose 100 mg/kg cabergoline treatment and Group 4 was received 20 mg/kg montelukast. Body weight, ovarian weight, vasculary permability (VP), peritoneal fluid vascular endothelial growth factor (VEGF) values and VEGF immune-expression were compared between the groups. Both cabergoline and montelukast prevented progression of OHSS compared to the OHSS group. Body weight, ovarian weight, VP, peritoneal fluid VEGF values and VEGF expression were significantly lower in both cabergoline- and montelukast-treated rats than in those not treated OHSS group. In conclusion, montelukast is an effective option for prevention of OHSS, as well as cabergoline. Montelukast may be a new treatment option to prevent and control the OHSS. PMID:25599748

  3. Dietary Agents and Phytochemicals in the Prevention and Treatment of Experimental Ulcerative Colitis

    PubMed Central

    Saxena, Arpit; Kaur, Kamaljeet; Hegde, Shweta; Kalekhan, Faizan M; Baliga, Manjeshwar Shrinath; Fayad, Raja

    2014-01-01

    Inflammatory bowel diseases (IBDs), consisting mainly of ulcerative colitis (UC) and Crohn's disease (CD), are important immune-mediated diseases of the gastrointestinal tract. The etiology of the disease includes environmental and genetic factors. Its management presents a constant challenge for gastroenterologists and conventional surgeon. 5-Amninosalicylates, antibiotics, steroids, and immune modulators have been used to reduce the symptoms and for maintenance of remission. Unfortunately, long-term usage of these agents has been found to lead to severe toxicities, which are deterrent to the users. Pre-clinical studies carried out in the recent past have shown that certain dietary agents, spices, oils, and dietary phytochemicals that are consumed regularly possess beneficial effects in preventing/ameliorating UC. For the first time, this review addresses the use of these dietary agents and spices in the treatment and prevention of IBD and also emphasizes on the mechanisms responsible for their effects. PMID:25379461

  4. Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase

    SciTech Connect

    Hallenbeck, P.C. ); Clark, M.A.; Barrett, E.L. )

    1989-06-01

    Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions. Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (<0.33%). It was also prevented by the presence of 0.1 mM nitrate. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (10{sup {minus}7} M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10{sup {minus}7}M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADHP and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.

  5. Intranasal vaccination with proinsulin DNA induces regulatory CD4+ T cells that prevent experimental autoimmune diabetes.

    PubMed

    Every, Alison L; Kramer, David R; Mannering, Stuart I; Lew, Andrew M; Harrison, Leonard C

    2006-04-15

    Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (T(reg)) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4+ T(reg) that suppressed diabetes development, both after adoptive cotransfer with "diabetogenic" spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4+ CD25+ T(reg), CD4+ T(reg) induced by proinsulin DNA were both CD25+ and CD25- and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of T(reg) and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective T(reg), but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity. PMID:16585551

  6. Preventing Depression in Later Life: Translation From Concept to Experimental Design and Implementation

    PubMed Central

    Sriwattanakomen, Roy; Ford, Angela F.; Thomas, Stephen B.; Miller, Mark D.; Stack, Jacqueline A.; Morse, Jennifer Q.; Kasckow, John; Brown, Charlotte; Reynolds, Charles F.

    2009-01-01

    Objective The authors detail the public health need for depression prevention research and the decisions made in designing an experiment testing problem solving therapy as “indicated” preventive intervention for high-risk older adults with subsyndromal depression. Special attention is given to the recruitment of African Americans because of well-documented inequalities in mental health services and depression treatment outcomes between races. Methods A total of 306 subjects (half white, half African American) with scores of 16 or higher on the Center for Epidemiological Studies of Depression Scale, but with no history of major depressive disorder in the past 12 months, are being recruited and randomly assigned to either problem solving therapy-primary care or to a dietary education control condition. Time to, and rate of, incident episodes of major depressive disorder are to be modeled using survival analysis. Level of depressive symptoms will be analyzed via a mixed models approach. Results Twenty-two subjects have been recruited into the study, and to date eight have completed the randomly assigned intervention and postintervention assessment. Four of 22 have exited after developing major depressive episodes. None have complained about study procedures or demands. Implementation in a variety of community settings is going well. Conclusion The data collected to date support the feasibility of translating from epidemiology to RCT design and implementation of empirical depression prevention research in later life. PMID:18515690

  7. Superiority of preventive antibiotic treatment compared with standard treatment of poststroke pneumonia in experimental stroke: a bed to bench approach

    PubMed Central

    Hetze, Susann; Engel, Odilo; Römer, Christine; Mueller, Susanne; Dirnagl, Ulrich; Meisel, Christian; Meisel, Andreas

    2013-01-01

    Stroke patients are prone to life-threatening bacterial pneumonia. Previous experimental stroke studies have demonstrated that preventive antibiotic treatment (PAT) improves outcome compared with placebo treatment, which however does not model the clinical setting properly. Here we investigate whether PAT is superior to the current clinical ‘gold standard' for treating poststroke infections. Therefore, we modeled stroke care according to the current stroke guidelines recommending early antibiotic treatment after diagnosing infections. To reliably diagnose pneumonia in living mice, we established a general health score and a magnetic resonance imaging protocol for radiologic confirmation. Compared with standard treatment after diagnosis by these methods, PAT not only abolished pneumonia successfully but also improved general medical outcome. Both, preventive and standard antibiotic treatment using enrofloxacin improved survival in a similar way compared with placebo treatment. However, in contrast to standard treatment, only PAT improved functional outcome assessed by gait analysis. In conclusion, standard and preventive treatment approach reduced poststroke mortality, however at the cost of a worse neurologic outcome compared with preventive approach. These data support the concept of PAT for treating patients at risk for poststroke infections and warrant phase III trials to prove this concept in clinical setting. PMID:23361393

  8. Preventive effects of lignan extract from flax hulls on experimentally induced benign prostate hyperplasia.

    PubMed

    Bisson, Jean-François; Hidalgo, Sophie; Simons, Rudy; Verbruggen, Marian

    2014-06-01

    Consumption of diet rich in lignans may decrease the risk of some chronic hormonal conditions such as benign prostatic hyperplasia (BPH). This study investigated whether a lignan-rich extract from flaxseed hulls, LinumLife EXTRA (LLE), could prevent BPH using the testosterone propionate (TP)-induced BPH rat model. Male Wistar-Unilever rats were randomly divided into four groups of 12 rats each: a negative control group fed with control diet and receiving daily subcutaneous injections of corn oil without TP, and three groups fed with control diet (positive control), diet containing 0.5% LLE (LLE 0.5) or 1.0% LLE (LLE 1.0) and receiving daily subcutaneous injections of TP in corn oil. Treatments with diets started 2 weeks before the induction of BPH and were carried out for 5 consecutive weeks. The influence of TP and LLE on body weight (BW), food and water consumptions, and enterolactone (ENL) levels in serum and urine of rats was examined at the end of the 5-week treatment period. TP significantly diminished the mean body weight gain (MBWG) of positive control rats and their food and water consumptions while LLE reduced significantly this MBWG reduction in a dose-dependent manner. The lignan-rich extract significantly inhibited TP-induced prostate size ratio (prostate weight/rat BW) increase in comparison with positive controls (P<.001). This effect was dose dependent. Higher serum and urine levels of ENL correlated well with the dose of extract provided to rats. It was concluded that the lignan-rich flaxseed hull extract prevented the TP-induced BPH indicating it might be beneficial in the prevention of BPH. PMID:24460407

  9. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

  10. Aldose reductase, oxidative stress, and diabetic mellitus.

    PubMed

    Tang, Wai Ho; Martin, Kathleen A; Hwa, John

    2012-01-01

    Diabetes mellitus (DM) is a complex metabolic disorder arising from lack of insulin production or insulin resistance (Diagnosis and classification of diabetes mellitus, 2007). DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR; ALR2; EC 1.1.1.21), a key enzyme in the polyol pathway, catalyzes nicotinamide adenosine dinucleotide phosphate-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues of DM including the heart, vasculature, neurons, eyes, and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis) and myocardium (heart failure) leading to severe morbidity and mortality (reviewed in Heather and Clarke, 2011). In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis, and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications. PMID:22582044

  11. Aldose reductase inhibitory compounds from Xanthium strumarium.

    PubMed

    Yoon, Ha Na; Lee, Min Young; Kim, Jin-Kyu; Suh, Hong-Won; Lim, Soon Sung

    2013-09-01

    As part of our ongoing search for natural sources of therapeutic and preventive agents for diabetic complications, we evaluated the inhibitory effects of components of the fruit of Xanthium strumarium (X. strumarium) on aldose reductase (AR) and galactitol formation in rat lenses with high levels of glucose. To identify the bioactive components of X. strumarium, 7 caffeoylquinic acids and 3 phenolic compounds were isolated and their chemical structures were elucidated on the basis of spectroscopic evidence and comparison with published data. The abilities of 10 X. strumarium-derived components to counteract diabetic complications were investigated by means of inhibitory assays with rat lens AR (rAR) and recombinant human AR (rhAR). From the 10 isolated compounds, methyl-3,5-di-O-caffeoylquinate showed the most potent inhibition, with IC₅₀ values of 0.30 and 0.67 μM for rAR and rhAR, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate, methyl-3,5-di-O-caffeoylquinate showed competitive inhibition of rhAR. Furthermore, methyl-3,5-di-O-caffeoylquinate inhibited galactitol formation in the rat lens and in erythrocytes incubated with a high concentration of glucose, indicating that this compound may be effective in preventing diabetic complications. PMID:23604720

  12. Prevention of the formation of arterial thrombi using different antiplatelet drugs: Experimental study in dogs

    SciTech Connect

    Escudero-Vela, M.C.; Alvarez, L.; Rodriguez, V.; del Moral, J.H.; Millan, I.; Castillo-Olivares, J.L.

    1989-05-01

    We have induced the formation of arterial thrombosis in dogs by means of an intima lesion produced by continuous current. The platelets were labeled with 111-In-oxine. Groups of 7 mongrel dogs received treatment for 7 days prior to the trial: Group I, control; Group II, 5 mg/kg body weight/day acetylsalicylic acid; Group III, 20 mg/kg body wt/day acetylsalicylic acid; Group IV, 15 mg/kg body wt/day triflusal + 5 mg/kg body wt/day dipyridamole; Group V, 15 mg/kg body wt/day triflusal; and Group VI, 5 mg/kg body wt/day acetylsalicylic acid + 5 mg/kg body wt/day dipyridamole. The only effective treatment for arterial thrombosis prevention was that employed in Group II (p less than 0.05).

  13. Chinese Herbal Compounds for the Prevention and Treatment of Atherosclerosis: Experimental Evidence and Mechanisms

    PubMed Central

    Li, Jianping; Hartstone-Rose, Adam; Wang, Jing; Li, Jiqiang; Janicki, Joseph S.

    2015-01-01

    Atherosclerosis is a leading cause of disability and death worldwide. Research into the disease has led to many compelling hypotheses regarding the pathophysiology of atherosclerotic lesion formation and the resulting complications such as myocardial infarction and stroke. Herbal medicine has been widely used in China as well as other Asian countries for the treatment of cardiovascular diseases for hundreds of years; however, the mechanisms of action of Chinese herbal medicine in the prevention and treatment of atherosclerosis have not been well studied. In this review, we briefly describe the mechanisms of atherogenesis and then summarize the research that has been performed in recent years regarding the effectiveness and mechanisms of antiatherogenic Chinese herbal compounds in an attempt to build a bridge between traditional Chinese medicine and cellular and molecular cardiovascular medicine. PMID:26089946

  14. Methylene blue prevents retinal damage in an experimental model of ischemic proliferative retinopathy.

    PubMed

    Rey-Funes, Manuel; Larrayoz, Ignacio M; Fernández, Juan C; Contartese, Daniela S; Rolón, Federico; Inserra, Pablo I F; Martínez-Murillo, Ricardo; López-Costa, Juan J; Dorfman, Verónica B; Martínez, Alfredo; Loidl, César F

    2016-06-01

    Perinatal asphyxia induces retinal lesions, generating ischemic proliferative retinopathy, which may result in blindness. Previously, we showed that the nitrergic system was involved in the physiopathology of perinatal asphyxia. Here we analyze the application of methylene blue, a well-known soluble guanylate cyclase inhibitor, as a therapeutic strategy to prevent retinopathy. Male rats (n = 28 per group) were treated in different ways: 1) control group comprised born-to-term animals; 2) methylene blue group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30 and 5 min before delivery; 3) perinatal asphyxia (PA) group comprised rats exposed to perinatal asphyxia (20 min at 37°C); and 4) methylene blue-PA group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30 and 5 min before delivery, and then the pups were subjected to PA as above. For molecular studies, mRNA was obtained at different times after asphyxia, and tissue was collected at 30 days for morphological and biochemical analysis. Perinatal asphyxia produced significant gliosis, angiogenesis, and thickening of the inner retina. Methylene blue treatment reduced these parameters. Perinatal asphyxia resulted in a significant elevation of the nitrergic system as shown by NO synthase (NOS) activity assays, Western blotting, and (immuno)histochemistry for the neuronal isoform of NOS and NADPH-diaphorase activity. All these parameters were also normalized by the treatment. In addition, methylene blue induced the upregulation of the anti-angiogenic peptide, pigment epithelium-derived factor. Application of methylene blue reduced morphological and biochemical parameters of retinopathy. This finding suggests the use of methylene blue as a new treatment to prevent or decrease retinal damage in the context of ischemic proliferative retinopathy. PMID:26984891

  15. Preventive activity of banana peel polyphenols on CCl4-induced experimental hepatic injury in Kunming mice

    PubMed Central

    WANG, RUI; FENG, XIA; ZHU, KAI; ZHAO, XIN; SUO, HUAYI

    2016-01-01

    The aim of the present study was to evaluate the preventive effects of banana peel polyphenols (BPPs) against hepatic injury. Mice were divide into normal, control, 100 mg/kg and 200 mg/kg banana peel polyphenol and silymarin groups. All the mice except normal mice were induced with hepatic damage using CCl4. The serum and tissue levels of mice were determined by a kit and the tissues were further examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. BPPs reduced the serum levels of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase in a CCl4-induced mouse model of hepatic injury. Furthermore, BPPs reduced the levels of malondialdehyde and triglyceride, while increasing glutathione levels in the serum and liver tissues of mice. In addition, the effects of 200 mg/kg treatment were more evident, and these effects were comparable to those of the drug silymarin. Serum levels of the cytokines, interleukin (IL)-6, IL-12, tumor necrosis factor (TNF)-α and interferon-γ, were reduced in the mice treated with BPPs compared with injury control group mice, and these levels were comparable to those of the normal and silymarin-treated groups. Histopathological examination indicated that BPPs were able to reduce the extent of CCl4-induced liver tissue injury and protect the liver cells. Furthermore, the mRNA and protein expression levels of the inflammation-associated factors cyclooxygenase-2, nitric oxide synthase, TNF-α and IL-1β were reduced in mice treated with BPPs compared with the control group mice. Mice that received 200 mg/kg BPP exhibited reduced expression levels of these factors compared with mice that received 100 mg/kg BPP. In conclusion, the results of the present study suggested that BPPs exert a good preventive effect against hepatic injury. PMID:27168833

  16. Quercetin prevents experimental glucocorticoid-induced osteoporosis: a comparative study with alendronate.

    PubMed

    Derakhshanian, Hoda; Djalali, Mahmoud; Djazayery, Abolghassem; Nourijelyani, Keramat; Ghadbeigi, Sajad; Pishva, Hamideh; Saedisomeolia, Ahmad; Bahremand, Arash; Dehpour, Ahmad Reza

    2013-05-01

    Glucocorticoid-induced osteoporosis (GIO) is the most common type of secondary osteoporosis. The aim of this study was to compare the efficacy of quercetin, a plant-derived flavonoid, with alendronate in the prevention of GIO. Fifty-six Sprague-Dawley rats were randomly distributed among 7 groups (8 rats per group) and treated for 6 weeks with one of the following: (i) normal saline; (ii) 40 mg methylprednisolone sodium succinate (MP)/kg body mass; (iii) MP + 40 μg alendronate/kg; (iv) MP + 50 mg quercetin/kg; (v) MP + 40 μg alendronate/kg + 50 mg quercetin/kg; (vi) MP + 150 mg quercetin/kg; and (vii) MP + 40 μg alendronate/kg + 150 mg quercetin/kg. MP and alendronate were injected subcutaneously and quercetin was administered by oral gavage 3 days a week. At the end of the study, femur breaking strength was significantly decreased as a consequence of MP injection. This decrease was completely compensated for in groups receiving 50 mg quercetin/kg plus alendronate, and 150 mg quercetin/kg with or without alendronate. Quercetin noticeably elevated osteocalcin as a bone formation marker, while alendronate did not show such an effect. In addition, administration of 150 mg quercetin/kg increased femoral trabecular and cortical thickness by 36% and 22%, respectively, compared with the MP-treated group. These data suggest that 150 mg quercetin/kg, alone or in combination with alendronate, can completely prevent GIO through its bone formation stimulatory effect. PMID:23656499

  17. Oral administration of an immunodominant T-cell epitope downregulates Th1/Th2 cytokines and prevents experimental myasthenia gravis

    PubMed Central

    Baggi, Fulvio; Andreetta, Francesca; Caspani, Elisabetta; Milani, Monica; Longhi, Renato; Mantegazza, Renato; Cornelio, Ferdinando; Antozzi, Carlo

    1999-01-01

    The mucosal administration of the native antigen or peptide fragments corresponding to immunodominant regions is effective in preventing or treating several T cell–dependent models of autoimmune disease. No data are yet available on oral tolerance with immunodominant T-cell peptides in experimental autoimmune myasthenia gravis (EAMG), an animal model of B cell–dependent disease. We report that oral administration of the T-cell epitope α146-162 of the Torpedo californica acetylcholine receptor (TAChR) α-subunit suppressed T-cell responses to AChR and ameliorated the disease in C57Bl/6 (B6) mice. Protection from EAMG was associated with reduced serum Ab’s to mouse AChR and reduced AChR loss in muscle. The effect of Tα146-162 feeding was specific; treatment with a control peptide did not affect EAMG manifestations. The protective effect induced by peptide Tα146-162 was mediated by reduced production of IFN-γ, IL-2, and IL-10 by TAChR-reactive cells, suggesting T-cell anergy. TGF-β–secreting Th3 cells did not seem to be involved in tolerance induction. We therefore demonstrate that feeding a single immunodominant epitope can prevent an Ab-mediated experimental model of autoimmune disease. PMID:10545527

  18. High density lipoproteins and prevention of experimental atherosclerosis with special reference to tree shrews.

    PubMed

    She, M P; Xia, R Y; Ran, B F; Wong, Z L

    1990-01-01

    According to data obtained from epidemiological and experimental survey, serum HDL level is known to be correlated conversely with the incidence of atherosclerosis. Experimental data collected in this article explained part of its mechanism, which is described in four parts as follows: 1. The result of 3 successive experiments on experimental atherosclerosis in tree shrews (total of 96 animals available including 40 as the controls) showed that the serum HDL level had been kept persistantly to 69-88% of the total serum lipoproteins even after a high cholesterol intake for 32 weeks. The incidence of atheromatous lesions developed was only 0-9%, but the incidence of gall stone was very high, 48-84% by gross examination by the end of these experiments. 2. HDL are also capable of (1) promotion of monocyte migration activity; (2) enhancement of cholesterol clearance rate of aortic smooth muscle cells originally isolated from either rabbits or tree shrews; (3) inhibition of 20% of LDL degradation but with no inhibitory effect obtained on Ac-LDL degradation in the endothelial cells; (4) presence of specific binding sites for apo E free HDL on the surface of aortic smooth muscle cells from either rabbits or tree shrews which recognizes apo A1 as a ligand. 3. Data from 2 successive experiments in rabbits showed that HDL lipoproteins (mainly apo A1) possess an inhibitory effect on the development of atheromatous plaques, but not a very strong one. 4. The colesterol clearance effect of smooth muscle cells was markedly enhanced by apo A1/phospholipid liposomes (the apo A1 used was isolated from either rabbit's or tree shrew's serum) in vitro. PMID:2123379

  19. Experimental investigation of contamination prevention techniques to cryogenic surfaces on board orbiting spacecraft

    NASA Technical Reports Server (NTRS)

    Hetrick, M. A.; Rantanen, R. O.; Ress, E. B.; Froechtenigt, J. F.

    1978-01-01

    Within the simulation limitations of on-orbit conditions, it was demonstrated that a helium purge system could be an effective method for reducing the incoming flux of contaminant species. Although a generalized purge system was employed in conjunction with basic telescope components, the simulation provided data that could be used for further modeling and design of a specific helium injection system. Experimental telescope pressures required for 90% attenuation appeared to be slightly higher (factor of 2 to 5). Cooling the helium purge gas and telescope components from 300 to 140 K had no measurable effect on stopping efficiency of a given mass flow of helium from the diffuse injector.

  20. Icariin Prevents Cartilage and Bone Degradation in Experimental Models of Arthritis

    PubMed Central

    Chao Wei, Chen; Qi Ping, Dai; Tian You, Fan; Yong Qiang, Chen; Tao, Che

    2016-01-01

    Background. Icariin (ICA) is an active compound extracted from Epimedium brevicornum Maxim. Previous reports have shown that icariin has a clinically significant therapeutic effect on rheumatoid arthritis. However, little is known about the mechanism by which icariin inhibits cartilage and bone degradation. Methods. New Zealand rabbits were immunized with antigen-induced arthritis (AIA) and treated with icariin. Joint tissues from rabbits were studied by histological analysis, transmission electron microscopy (TEM), and micro-CT. The expression levels of receptor activator of nuclear factor-B ligand (RANKL) and osteoprotegerin (OPG) in joint tissues were determined using immunohistochemistry and real-time PCR analysis. Results. Histological analysis and TEM sections of cartilage in the ICA treated group showed a low level of chondrocyte destruction. Micro-CT analysis showed that the bone mineral density value and bone structural level in ICA treated rabbits were significantly higher compared with those in the AIA group. Immunohistochemistry and real-time PCR analysis showed that icariin treatment reduced RANKL expression and enhanced OPG expression levels, as compared to the AIA group. Conclusion. These data indicate that ICA suppresses articular bone loss and prevents joint destruction. This study also determined that ICA regulated articular bone loss in part by regulating RANKL and OPG expression. PMID:27199510

  1. Icariin Prevents Cartilage and Bone Degradation in Experimental Models of Arthritis.

    PubMed

    Chao Wei, Chen; Qi Ping, Dai; Tian You, Fan; Yong Qiang, Chen; Tao, Che

    2016-01-01

    Background. Icariin (ICA) is an active compound extracted from Epimedium brevicornum Maxim. Previous reports have shown that icariin has a clinically significant therapeutic effect on rheumatoid arthritis. However, little is known about the mechanism by which icariin inhibits cartilage and bone degradation. Methods. New Zealand rabbits were immunized with antigen-induced arthritis (AIA) and treated with icariin. Joint tissues from rabbits were studied by histological analysis, transmission electron microscopy (TEM), and micro-CT. The expression levels of receptor activator of nuclear factor-B ligand (RANKL) and osteoprotegerin (OPG) in joint tissues were determined using immunohistochemistry and real-time PCR analysis. Results. Histological analysis and TEM sections of cartilage in the ICA treated group showed a low level of chondrocyte destruction. Micro-CT analysis showed that the bone mineral density value and bone structural level in ICA treated rabbits were significantly higher compared with those in the AIA group. Immunohistochemistry and real-time PCR analysis showed that icariin treatment reduced RANKL expression and enhanced OPG expression levels, as compared to the AIA group. Conclusion. These data indicate that ICA suppresses articular bone loss and prevents joint destruction. This study also determined that ICA regulated articular bone loss in part by regulating RANKL and OPG expression. PMID:27199510

  2. Exercise prevents the effects of experimental arthritis on the metabolism and function of immune cells.

    PubMed

    Navarro, Francisco; Bacurau, Aline V N; Almeida, Sandro S; Barros, Carlos C; Moraes, Milton R; Pesquero, Jorge L; Ribeiro, Sandra M L; Araújo, Ronaldo C; Costa Rosa, Luis F B P; Bacurau, Reury F P

    2010-06-01

    Active lymphocytes (LY) and macrophages (MPhi) are involved in the pathophysiology of rheumatoid arthritis (RA). Due to its anti-inflammatory effect, physical exercise may be beneficial in RA by acting on the immune system (IS). Thus, female Wistar rats with type II collagen-induced arthritis (CIA) were submitted to swimming training (6 weeks, 5 days/week, 60 min/day) and some biochemical and immune parameters, such as the metabolism of glucose and glutamine and function of LY and MPhi, were evaluated. In addition, plasma levels of some hormones and of interleukin-2 (IL-2) were also determined. Results demonstrate that CIA increased lymphocyte proliferation (1.9- and 1.7-fold, respectively, in response to concanavalin A (ConA) and lipopolysaccharide (LPS)), as well as macrophage H(2)O(2) production (1.6-fold), in comparison to control. Exercise training prevented the activation of immune cells, induced by CIA, and established a pattern of substrate utilization similar to that described as normal for these cells. Exercise also promoted an elevation of plasma levels of corticosterone (22.2%), progesterone (1.7-fold) and IL-2 (2.6-fold). Our data suggest that chronic exercise is able to counterbalance the effects of CIA on cells of the IS, reinforcing the proposal that the benefits of exercise may not be restricted to aerobic capacity and/or strength improvement. PMID:20517889

  3. Spirulina maxima prevents fatty liver formation in CD-1 male and female mice with experimental diabetes.

    PubMed

    Rodríguez-Hernández, A; Blé-Castillo, J L; Juárez-Oropeza, M A; Díaz-Zagoya, J C

    2001-07-20

    The dietary administration of 5% Spirulina maxima (SM) during four weeks to diabetic mice, starting one week after a single dose of alloxan, 250 mg/Kg body weight, prevented fatty liver production in male and female animals. The main action of SM was on triacylglycerol levels in serum and liver. There was also a moderate hypoglycemia in male mice. The thiobarbituric acid reactive substances also decreased in serum and liver after SM administration. There was also a decrease in the percentage of HDL in diabetic mice that was reverted by the SM administration. The sum of LDL + VLDL percentages was also partially normalized in diabetic animals by the SM administration. An additional observation was the lower incidence of adherences between the liver and the intestine loops in the diabetic mice treated with SM compared with diabetic mice without SM. Male and female mice showed differences to diabetes susceptibility and response to SM, the female being more resistant to diabetes induction by alloxan and more responsive to the beneficial effects of SM. It is worth future work of SM on humans looking for better quality of life and longer survival of diabetic patients. PMID:11508645

  4. Heating stents with radio frequency energy to prevent tumor ingrowth: modeling and experimental results

    NASA Astrophysics Data System (ADS)

    Ryan, Thomas P.; Lawes, Kate; Goldberg, S. Nahum

    1998-04-01

    Stents are often inserted into internal orifices to treat blockage due to tumor ingrowth. Stents are favored due to their minimally invasive nature, possible avoidance of a surgical procedure, and their ability to palliate surgically non-resectable disease. Because of rapid tumor growth however, a treatment means to prevent overgrowth through the stent and resultant blockage is required. To further this goal, experiments were performed in which a stent was placed in tissue and heated with radiofrequency (RF) energy to coagulate a cylinder of tissue, thereby eradicating viable tissue in the proximity of the stent. Temperatures were measured at the central stent surface and edges over time during a 5 - 10 minute heating in phantom and in fresh tissue. In addition, a finite element model was used to simulate the electric field and temperature distribution. Blood flow was also introduced in the model by evaluating RF application to stents to determine effectiveness of the energy applications. Changing perfusion and tissue electrical conductivity as a function of temperature was applied as the tissue was heated to 100 degree(s)C. Results from the electric field model will be shown as well as the thermal distribution over time from the simulations. Lastly, results from the damage integral will be discussed.

  5. The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

    PubMed Central

    Profant, Deborah A.; Roberts, Christopher J.; Koning, Ann J.; Wright, Robin L.

    1999-01-01

    In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain. PMID:10512876

  6. In vitro expression of rat lens aldose reductase in Escherichia coli.

    PubMed Central

    Old, S E; Sato, S; Kador, P F; Carper, D A

    1990-01-01

    Aldose reductase (alditol:NADP+ oxidoreductase, EC 1.1.1.21), an enzyme that converts glucose to sorbitol, the first step of the polyol pathway, has been implicated in secondary complications of diabetes, such as cataracts, retinopathy, neuropathy, and nephropathy. Aldose reductase inhibitors have been observed to prevent or delay the onset of these complications; however, more potent and specific inhibitors are needed. Development of new inhibitors necessitates a better understanding of the molecular structure of this protein. To elucidate the structure-function relationships of aldose reductase and to develop methods of regulating this enzyme, large and homogeneous quantities of rat lens aldose reductase have been expressed in bacterial cells. A construction of the complete coding sequence and 3' untranslated region for rat lens aldose reductase was assembled in the expression vector pKK233-2 (Pharmacia). This construction expresses an active enzyme that has been purified and demonstrates kinetic, immunological, and inhibitory properties similar to rat lens aldose reductase. Images PMID:2114645

  7. Myricetin Prevents Alveolar Bone Loss in an Experimental Ovariectomized Mouse Model of Periodontitis

    PubMed Central

    Huang, Jialiang; Wu, Chuanlong; Tian, Bo; Zhou, Xiao; Ma, Nian; Qian, Yufen

    2016-01-01

    Periodontitis is a common chronic inflammatory disease, which leads to alveolar bone resorption. Healthy and functional alveolar bone, which can support the teeth and enable their movement, is very important for orthodontic treatment. Myricetin inhibited osteoclastogenesis by suppressing the expression of some genes, signaling pathways, and cytokines. This study aimed to investigate the effects of myricetin on alveolar bone loss in an ovariectomized (OVX) mouse model of periodontitis as well as in vitro osteoclast formation and bone resorption. Twenty-four healthy eight-week-old C57BL/J6 female mice were assigned randomly to four groups: phosphate-buffered saline (PBS) control (sham) OVX + ligature + PBS (vehicle), and OVX + ligature + low or high (2 or 5 mg∙kg−1∙day−1, respectively) doses of myricetin. Myricetin or PBS was injected intraperitoneally (i.p.) every other day for 30 days. The maxillae were collected and subjected to further examination, including micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, and tartrate-resistant acid phosphatase (TRAP) staining; a resorption pit assay was also performed in vitro to evaluate the effects of myricetin on receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis. Myricetin, at both high and low doses, prevented alveolar bone resorption and increased alveolar crest height in the mouse model and inhibited osteoclast formation and bone resorption in vitro. However, myricetin was more effective at high dose than at low dose. Our study demonstrated that myricetin had a positive effect on alveolar bone resorption in an OVX mouse model of periodontitis and, therefore, may be a potential agent for the treatment of periodontitis and osteoporosis. PMID:27011174

  8. Dihydropteridine reductase from Escherichia coli.

    PubMed Central

    Vasudevan, S G; Shaw, D C; Armarego, W L

    1988-01-01

    A dihydropteridine reductase from Escherichia coli was purified to apparent homogeneity. It is a dimeric enzyme with identical subunits (Mr 27000) and a free N-terminal group. It can use NADH (Vmax./Km 3.36 s-1) and NADPH (Vmax./Km 1.07 s-1) when 6-methyldihydro-(6H)-pterin is the second substrate, as well as quinonoid dihydro-(6H)-biopterin (Vmax./Km 0.69 s-1), dihydro-(6H)-neopterin (Vmax./Km 0.58 s-1), dihydro-(6H)-monapterin 0.66 s-1), 6-methyldihydro-(6H)-pterin and cis-6,7-dimethyldihydro-(6H)-pterin (Vmax./Km 0.66 s-1) when NADH is the second substrate. The pure reductase has a yellow colour and contains bound FAD. The enzyme also has pterin-independent NADH and NADPH oxidoreductase activities when potassium ferricyanide is the electron acceptor. Images Fig. 2. PMID:3060113

  9. Aldose reductase inhibitory activity of compounds from Zea mays L.

    PubMed

    Kim, Tae Hyeon; Kim, Jin Kyu; Kang, Young-Hee; Lee, Jae-Yong; Kang, Il Jun; Lim, Soon Sung

    2013-01-01

    Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1-7) and 5 anthocyanins (compound 8-12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC(50), 4.78 μ M). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. PMID:23586057

  10. Prevention of Axonal Injury using Calpain Inhibitor in Chronic Progressive Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Hassen, Getaw Worku; Feliberti, Jason; Kesner, Leo; Stracher, Alfred; Mokhtarian, Foroozan

    2011-01-01

    Axonal injury is the major correlate of permanent disability in neurodegenerative diseases such as multiple sclerosis (MS), especially in secondary-progressive MS following relapsing-remitting disease course. Proteolytic enzyme, calpain, is a potential candidate for causing axonal injury. Most current treatment options only target the inflammatory component of MS. Previous work using calpain inhibitor CYLA in our laboratory showed significant reduction in clinical sign, demyelination and tissue calpain content in acute experimental autoimmune encephalomyelitis (EAE). Here we evaluated markers of axonal injury (amyloid precursor protein, Nav1.6 channels), neuronal calpain content and the effect of CYLA on axonal protection using histological methods in chronic EAE [myelin oligodendrocyte glycoprotein (MOG) – induced disease model of MS]. Intraperitoneal application of CYLA (2mg/mouse/day) significantly reduced the clinical signs, tissue calpain content, demyelination and inflammatory infiltration of EAE. Similarly, markers for axonal injury were barely detectable in the treated mice. Thus, this novel drug, which markedly suppresses the disease course, axonal injury and its progression, is a candidate for the treatment of a neurodegenerative disease such as multiple sclerosis. PMID:18725211

  11. Limitations in Bonding to Dentin and Experimental Strategies to Prevent Bond Degradation

    PubMed Central

    Liu, Y.; Tjäderhane, L.; Breschi, L.; Mazzoni, A.; Li, N.; Mao, J.; Pashley, D.H.; Tay, F.R.

    2011-01-01

    The limited durability of resin-dentin bonds severely compromises the lifetime of tooth-colored restorations. Bond degradation occurs via hydrolysis of suboptimally polymerized hydrophilic resin components and degradation of water-rich, resin-sparse collagen matrices by matrix metalloproteinases (MMPs) and cysteine cathepsins. This review examined data generated over the past three years on five experimental strategies developed by different research groups for extending the longevity of resin-dentin bonds. They include: (1) increasing the degree of conversion and esterase resistance of hydrophilic adhesives; (2) the use of broad-spectrum inhibitors of collagenolytic enzymes, including novel inhibitor functional groups grafted to methacrylate resins monomers to produce anti-MMP adhesives; (3) the use of cross-linking agents for silencing the activities of MMP and cathepsins that irreversibly alter the 3-D structures of their catalytic/allosteric domains; (4) ethanol wet-bonding with hydrophobic resins to completely replace water from the extrafibrillar and intrafibrillar collagen compartments and immobilize the collagenolytic enzymes; and (5) biomimetic remineralization of the water-filled collagen matrix using analogs of matrix proteins to progressively replace water with intrafibrillar and extrafibrillar apatites to exclude exogenous collagenolytic enzymes and fossilize endogenous collagenolytic enzymes. A combination of several of these strategies should result in overcoming the critical barriers to progress currently encountered in dentin bonding. PMID:21220360

  12. Complement C5 in Experimental Autoimmune Encephalomyelitis (EAE) Facilitates Remyelination and Prevents Gliosis

    PubMed Central

    Weerth, Susanna H.; Rus, Horea; Shin, Moon L.; Raine, Cedric S.

    2003-01-01

    Activation of the classical complement system is known to play a central role in autoimmune demyelination. We have analyzed the role of complement component C5 in experimental autoimmune encephalomyelitis (EAE) using C5-deficient (C5-d) and C5-sufficient (C5-s) mice. Both groups of mice displayed early onset EAE, a short recovery phase, and similar stable chronic courses. However, in contrast to the clinical similarities, marked differences were apparent by histopathology. During acute EAE in C5-d, a delay in inflammatory cell infiltration and tissue damage was observed along with restricted lesion areas, which in C5-s mice were more extensive and diffuse. More striking were the differences in chronic lesions. In C5-d mice, inflammatory demyelination and Wallerian degeneration were followed by axonal depletion and severe gliosis, while in C5-s, the same initial signs were followed by axonal sparing and extensive remyelination. In C5-d, immunohistochemistry and Western blotting showed an increase in glial fibrillary acidic protein and a decrease in neurofilament protein, proteolipid protein, and several pro-inflammatory markers. These results in the EAE model indicate that absence of C5 resulted in fiber loss and extensive scarring, whereas presence of C5-favored axonal survival and more efficient remyelination. PMID:12937147

  13. Euphol prevents experimental autoimmune encephalomyelitis in mice: evidence for the underlying mechanisms.

    PubMed

    Dutra, Rafael Cypriano; de Souza, Paula Roberta de Cezaro; Bento, Allisson Freire; Marcon, Rodrigo; Bicca, Maíra Assunção; Pianowski, Luiz Francisco; Calixto, João B

    2012-02-15

    Multiple sclerosis (MS) is a severe chronic T cell-mediated autoimmune inflammatory disease of the central nervous system (CNS), the existing therapy of which is only partially effective and is associated with undesirable side effects. Euphol, an alcohol tetracyclic triterpene, has a wide range of pharmacological properties and is considered to have anti-inflammatory action. However there are no reports about the effects and mechanisms of euphol in experimental autoimmune encephalomyelitis (EAE), an established model of MS. Here we report the effects and the underlying mechanisms of action of euphol in EAE. Euphol (1-10mg/kg) was administered orally at different time-points of EAE. Immunological and inflammatory responses were evaluated by real-time PCR, Western blot and flow cytometry assays. We provide evidence that euphol significantly attenuates neurological signs of EAE. These beneficial effects of euphol seem to be associated with the down-regulation of mRNA and protein expression of some pro-inflammatory mediators such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the CNS. Furthermore, in vitro, euphol consistently inhibited the T cell-mediated immune response including the production of T(H)1 and T(H)17 cytokines in spleen cells of untreated EAE animals. Likewise, oral euphol treatment inhibited the infiltration of T(H)17 myelin-specific cells into the CNS through the adhesion molecule, lymphocyte function-associated antigen 1 (LFA-1). Our findings reveal that oral administration of euphol consistently reduces and limits the severity and development of EAE. Therefore, euphol might represent a potential molecule of interest for the treatment of MS and other T(H)17 cell-mediated inflammatory diseases. PMID:22155310

  14. Blockage of mitochondrial calcium uniporter prevents iron accumulation in a model of experimental subarachnoid hemorrhage

    SciTech Connect

    Yan, Huiying; Hao, Shuangying; Sun, Xiaoyan; Zhang, Dingding; Gao, Xin; Yu, Zhuang; Li, Kuanyu; Hang, Chun-Hua

    2015-01-24

    Highlights: • Iron accumulation was involved in the acute phase following SAH. • Blockage of MCU could attenuate cellular iron accumulation following SAH. • Blockage of MCU could decrease ROS generation and improve cell energy supply following SAH. • Blockage of MCU could alleviate apoptosis and brain injury following SAH. - Abstract: Previous studies have shown that iron accumulation is involved in the pathogenesis of brain injury following subarachnoid hemorrhage (SAH) and chelation of iron reduced mortality and oxidative DNA damage. We previously reported that blockage of mitochondrial calcium uniporter (MCU) provided benefit in the early brain injury after experimental SAH. This study was undertaken to identify whether blockage of MCU could ameliorate iron accumulation-associated brain injury following SAH. Therefore, we used two reagents ruthenium red (RR) and spermine (Sper) to inhibit MCU. Sprague–Dawley (SD) rats were randomly divided into four groups including sham, SAH, SAH + RR, and SAH + Sper. Biochemical analysis and histological assays were performed. The results confirmed the iron accumulation in temporal lobe after SAH. Interestingly, blockage of MCU dramatically reduced the iron accumulation in this area. The mechanism was revealed that inhibition of MCU reversed the down-regulation of iron regulatory protein (IRP) 1/2 and increase of ferritin. Iron–sulfur cluster dependent-aconitase activity was partially conserved when MCU was blocked. In consistence with this and previous report, ROS levels were notably reduced and ATP supply was rescued; levels of cleaved caspase-3 dropped; and integrity of neurons in temporal lobe was protected. Taken together, our results indicated that blockage of MCU could alleviate iron accumulation and the associated injury following SAH. These findings suggest that the alteration of calcium and iron homeostasis be coupled and MCU be considered to be a therapeutic target for patients suffering from SAH.

  15. Prevention of Vasa Vasorum Neovascularization Attenuates Early Neointima Formation in Experimental Hypercholesterolemia

    PubMed Central

    Gössl, Mario; Herrmann, Jörg; Tang, Hui; Versari, Daniele; Galili, Offer; Mannheim, Dallit; Rajkumar, S Vincent; Lerman, Lilach O; Lerman, Amir

    2010-01-01

    Objective Vasa vasorum (VV) neovascularization is a key feature of early atherosclerosis and adds substantial endothelial exchange-surface to the coronary vessel wall. Thus, it is conceivable that VV neovascularization favors the entry of pro-inflammatory and pro-atherosclerotic blood components into the coronary vessel wall. We sought to investigate the effects of Thalidomide (Th) a potent anti-angiogenic drug on vasa vasorum (VV) neovascularization, vessel wall inflammation and neointima formation in early experimental atherosclerosis. Methods and Results Female domestic swine, 3-months old, were fed normal (N, n=12) or high-cholesterol diet (HC, n=12) for 3 months. In each group six pigs were randomized to 200 mg Thalidomide daily for the diet period (N+Th, HC+Th). LADs were scanned with micro-CT (20μm cubic voxel size) to determine VV spatial density (#/mm²). Fresh-frozen coronary tissue was used for Western Blotting (VEGF, TNF-α , LOX-1, IkBα and Gro-α ) and electrophoretic mobility shift assay (EMSA, NFkB). Treatment with Thalidomide preserved VV spatial density (2.7±0.3 (N), 6.4±0.7 (HC), 3.5±0.8 (HC+Th); p=ns HC+Th vs. N) and inhibited the expression of VEGF, TNF-alpha and LOX-1 but not NFkB activity in the coronary vessel wall. Immunofluorescense analyses revealed co-localization of vWF but not SMA and NFkB, TNF-α as well as VEGF in HC and HC+Th coronaries. Intima-media thickness was significantly inhibited in HC+Th compared to HC. Serum levels of hs-CRP and TNF-α did not differ among the groups. Conclusions Our study supports a role of VV neovascularization in the development of and a therapeutic potential for anti-angiogenic intervention in early atherosclerosis. PMID:19458984

  16. The Mood-Stabilizer Lithium Prevents Hippocampal Apoptosis and Improves Spatial Memory in Experimental Meningitis

    PubMed Central

    Liechti, Fabian D.; Stüdle, Nicolas; Theurillat, Regula; Grandgirard, Denis; Thormann, Wolfgang; Leib, Stephen L.

    2014-01-01

    Pneumococcal meningitis is associated with high morbidity and mortality rates. Brain damage caused by this disease is characterized by apoptosis in the hippocampal dentate gyrus, a morphological correlate of learning deficits in experimental paradigms. The mood stabilizer lithium has previously been found to attenuate brain damage in ischemic and inflammatory diseases of the brain. An infant rat model of pneumococcal meningitis was used to investigate the neuroprotective and neuroregenerative potential of lithium. To assess an effect on the acute disease, LiCl was administered starting five days prior to intracisternal infection with live Streptococcus pneumoniae. Clinical parameters were recorded, cerebrospinal fluid (CSF) was sampled, and the animals were sacrificed 42 hours after infection to harvest the brain and serum. Cryosections of the brains were stained for Nissl substance to quantify brain injury. Hippocampal gene expression of Bcl-2, Bax, p53, and BDNF was analyzed. Lithium concentrations were measured in serum and CSF. The effect of chronic lithium treatment on spatial memory function and cell survival in the dentate gyrus was evaluated in a Morris water maze and by quantification of BrdU incorporation after LiCl treatment during 3 weeks following infection. In the hippocampus, LiCl significantly reduced apoptosis and gene expression of Bax and p53 while it increased expression of Bcl-2. IL-10, MCP-1, and TNF were significantly increased in animals treated with LiCl compared to NaCl. Chronic LiCl treatment improved spatial memory in infected animals. The mood stabilizer lithium may thus be a therapeutic alternative to attenuate neurofunctional deficits as a result of pneumococcal meningitis. PMID:25409333

  17. Autogenic-Feedback Training (AFT) as a preventive method for space motion sickness: Background and experimental design

    NASA Technical Reports Server (NTRS)

    Cowings, Patricia S.; Toscano, William B.

    1993-01-01

    Finding an effective treatment for the motion sickness-like symptoms that occur in space has become a high priority for NASA. The background research is reviewed and the experimental design of a formal life sciences shuttle flight experiment designed to prevent space motion sickness in shuttle crew members is presented. This experiment utilizes a behavioral medicine approach to solving this problem. This method, Autogenic-Feedback Training (AFT), involves training subjects to voluntarily control several of their own physiological responses to environmental stressors. AFT has been used reliably to increase tolerance to motion sickness during ground-based tests in over 200 men and women under a variety of conditions that induce motion sickness, and preliminary evidence from space suggests that AFT may be an effective treatment for space motion sickness as well. Proposed changes to this experiment for future manifests are included.

  18. Aldose reductase mediates retinal microglia activation.

    PubMed

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy. PMID:27033597

  19. Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells.

    PubMed

    Rezende, Rafael M; Oliveira, Rafael P; Medeiros, Samara R; Gomes-Santos, Ana C; Alves, Andrea C; Loli, Flávia G; Guimarães, Mauro A F; Amaral, Sylvia S; da Cunha, André P; Weiner, Howard L; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M C

    2013-02-01

    Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. PMID:22939403

  20. Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

    PubMed Central

    Rezende, Rafael M.; Oliveira, Rafael P.; Medeiros, Samara R.; Gomes-Santos, Ana C.; Alves, Andrea C.; Loli, Flávia G.; Guimarães, Mauro A.F.; Amaral, Sylvia S.; da Cunha, André P.; Weiner, Howard L.; Azevedo, Vasco; Miyoshi, Anderson; Faria, Ana M.C.

    2013-01-01

    Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice. PMID:22939403

  1. Effects of chronic normobaric hypoxic and hypercapnic exposure in rats: Prevention of experimental chronic mountain sickness by hypercapnia

    NASA Astrophysics Data System (ADS)

    Lincoln, B.; Bonkovsky, H. L.; Ou, Lo-Chang

    1987-09-01

    A syndrome of experimental chronic mountain sickness can be produced in the Hilltop strain of Sprague-Dawley rats by chronic hypobaric hypoxic exposure. This syndrome is characterized by polycythemia, plasma hemoglobinemia, pulmonary hypertension and right ventricular hypertrophy with eventual failure and death. It has generally been assumed that these changes are caused by chronic hypoxemia, not by hypobaric exposure per se. We have now confirmed this directly by showing that chronic normobaric hypoxic exposure (10.5% O2) produces similar hematologic and hemodynamic changes. Further, the addition of hypercapnic exposure to the hypoxic exposure blunted or prevented the effects of the hypoxic exposure probably by stimulating respiration, thus increasing the rate of oxygen delivery to the cells. Changes in the rate-controlling enzymes of hepatic heme metabolism, 5-aminolevulinate synthase and heme oxygenase, and in cytochrome(s) P-450, the major hepatic hemoprotein(s), were also measured in hypoxic and hypercapnic rats. Hypoxia decreased 5-aminolevulinate synthase and increased cytochrome(s) P-450, probably by increasing the size of a “regulatory” heme pool within hepatocytes. These changes were also prevented by the addition of hypercapnic to hypoxic exposure.

  2. Medicine Sellers for Prevention and Control of Sexually Transmitted Infections: Effect of a Quasi-Experimental Training Intervention in Bangladesh

    PubMed Central

    Alam, Nazmul; Alam, Anadil; Fournier, Pierre

    2015-01-01

    This study used a quasi-experimental pre-post design to test whether short training can improve medicine sellers' (MSs) practices and skills for prevention and control of sexually transmitted infections (STIs) in Bangladesh. The training included lectures, printed materials, and identification of referral sites. Difference-in-differences estimation was used to determine the effects of intervention on key primary and secondary outcomes. Advice given by the MSs in intervention group for partner treatment and condoms use increased significantly by 11% and 9%, respectively, after adjusting for baseline differences in education, religion, age, duration of training, and study site. Referral of clients to qualified service providers increased by 5% in the intervention group compared to the comparison group, but this change was not found to be statistically significant. Significantly higher proportion of MSs in the intervention group recognized the recommended medications as per the national syndromic management guidelines in Bangladesh for treatment of urethral discharge and genital ulcer symptoms. Short training intervention was found to be effective in improving MSs' practice of promoting condom use and partner treatment to the clients. We anticipate the need for broad based training programs of MSs to improve their skills for the prevention and control of STI/HIV in Bangladesh. PMID:26491678

  3. Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme

    SciTech Connect

    Hinojosa-Leon, M.; Dubourdieu, M.; Sanchez-Crispin, J.A.; Chippaux, M.

    1986-04-29

    Use of radioactive molybdenum demonstrates that the tetrathionate reductase of Salmonella typhimurium is a molydenum containing enzyme. It is proposed that this enzyme shares with other molybdo-proteins, such as nitrate reductase, a common molybdenum containing cofactor the defect of which leads to the loss of the tetrathionate reductase and nitrate reductase activities.

  4. Prevention and therapy of experimental autoimmune neuritis by an antibody against T cell receptors-alpha/beta.

    PubMed

    Jung, S; Krämer, S; Schluesener, H J; Hünig, T; Toyka, K; Hartung, H P

    1992-06-15

    The mAb R73 directed to the TCR-alpha/beta of rat lymphocytes was tested for its therapeutic potential during the effector phase of experimental autoimmune neuritis (EAN) in Lewis rats. EAN can be actively induced by immunization with bovine peripheral nerve myelin, bovine P2 protein, or a peptide containing its neuritogenic epitope and serves as a model of the human Guilain-Barré syndrome. Adoptive transfer of activated P2-specific T lymphocytes also produces the monophasic disease (AT-EAN) characterized by inflammation and demyelination of peripheral nerves and highlights the central role of T lymphocytes in the pathogenesis of EAN. A single administration of the mAb R73 immediately after injection of activated P2-specific T line cells completely prevented the development of clinical and electrophysiologic signs of EAN in most animals and greatly alleviated the disease in the others. In further experiments mAb R73 was applied after the appearance of first clinical signs of EAN actively induced by immunization with a neuritogenic peptide or bovine peripheral nerve myelin. In both cases the anti-TCR-alpha/beta mAb reversed clinical signs of EAN and prevented the development of peripheral nerve dysfunction. In vivo and in vitro data suggest that impairment of Ag recognition and T cell function by occupancy of the TCR and R73-induced TCR-modulation rather than depletion of TCR-alpha/beta-bearing lymphocytes is the decisive mechanism underlying suppression of EAN that is apparent already within 48 h of the first R73 injection. PMID:1376340

  5. Genetics Home Reference: sepiapterin reductase deficiency

    MedlinePlus

    ... reductase enzyme. This enzyme is involved in the production of a molecule called tetrahydrobiopterin (also known as ... is responsible for the last step in the production of tetrahydrobiopterin. Tetrahydrobiopterin helps process several building blocks ...

  6. A Quasi-experimental Study to Assess an Interactive Educational Intervention on Nurses' Knowledge of Pressure Ulcer Prevention in Nigeria.

    PubMed

    Ekama Ilesanmi, Rose; Morohunfoluwa Oluwatosin, Odunayo

    2016-04-01

    Educational intervention programs are an important element to improve nurses' knowledge of pressure ulcer (PU) prevention. Various teaching methods have been used with diverse results but none have been analyzed in Nigeria. A quasi- experimental study using a pretest/post test design was conducted among 193 registered nurses with >6 months experience who worked in purposefully selected wards (neuroscience, orthopedics, renal, and cardiac) in 3 teaching hospitals to examine the level of knowledge retention after interactive instruction. Participants were randomized to intervention (IG, n = 127 from 2 hospitals) and control (CG, n = 66 from 1 hospital) groups; the IG was provided a 5-day, face-to-face interactive lecture, and the CG engaged in a 1-day, 4-hour discussion of PU prevention practices. The Pressure Ulcer Knowledge Tool, a 47-item questionnaire in which a correct answer = 1 point and an incorrect/"I don't know" answer = 0 (maximum score 47), was used to assess and compare knowledge retention at 3 time points: baseline (T1), immediately after instruction (T2), and after 3 months (T3). Three trained research assistants assisted with registration of participants and distribution and collection of the questionnaires. All questionnaires were retrieved at T1 before the intervention be- gan. Respondents were encouraged to respond to all questions. Data were analyzed using t-test and ANOVA (P = 0.05). At T1, knowledge scores were comparable between the IG and CG (32.5 ± 4.2 and 30.8 ± 5.0 for IG and CG, respectively). At T2, knowledge scores increased significantly only in the IG to 40.7 ± 3.4 (d = 1.94, P less than 0.05). The mean difference between T1 and T2 was -8.2 ± 5.4, t = -17.0, P = 0.000. Similarly, mean scores increased significantly from T2 to T3 in the IG (mean= -2.0 ± 5.5, t = -4.1, P = 0.000); scores in the CG were -6.2 ± 7.2, t = -6.3 (P = 0.000). A face-to-face interactive lecture was shown to be an effective method of program delivery for

  7. Preventive and therapeutic anti-inflammatory properties of the sesquiterpene α-humulene in experimental airways allergic inflammation

    PubMed Central

    Rogerio, Alexandre P; Andrade, Edinéia L; Leite, Daniela FP; Figueiredo, Cláudia P; Calixto, João B

    2009-01-01

    Background and purpose: α-Humulene and trans-caryophyllene are plant sesquiterpenes with pronounced anti-inflammatory properties. Here, we evaluated the effects of these compounds in an experimental model of airways allergic inflammation. Experimental approach: Female BALB/c mice, sensitized to and challenged with ovalbumin received daily α-humulene or trans-caryophyllene (50 mg·kg−1, orally) or α-humulene (1 mg·mL−1, by aerosol) as either a preventive (for 22 days) or therapeutic (from the 18th to the 22nd day) treatment. Dexamethasone or budesonide was used as a positive control drug. Inflammation was determined on day 22 post-immunization by leukocyte recruitment, interleukin-5 (IL-5), CCL11, interferon-γ (IFN-γ) and leukotriene (LT)B4 levels in bronchoalveolar lavage fluid (BALF). In addition, transcription factors [nuclear factor κB (NF-κB), activator protein 1 (AP-1)] and P-selectin in lung tissue were measured by immunohistochemistry and mucus secretion by histochemistry. Key results: Preventive or therapeutic treatments with α-humulene, but not with trans-caryophyllene, significantly reduced the eosinophil recruitment to the BALF. In addition, α-humulene recovery INF-γ and reduced the IL-5, CCL11 and LTB4 levels in BALF, as well as the IL-5 production in mediastinal lymph nodes (in vitro assay). Furthermore, α-humulene decreased the NF-kB and the AP-1 activation, the expression of P-selectin and the increased mucus secretion in the lung. Conclusions and implications: α-Humulene, given either orally or by aerosol, exhibited marked anti-inflammatory properties in a murine model of airways allergic inflammation, an effect that seemed to be mediated via reduction of inflammatory mediators, adhesion molecule expression and transcription factors activation. This article is part of a themed issue on Mediators and Receptors in the Resolution of Inflammation. To view this issue visit http://www3.interscience.wiley.com/journal/121548564/issueyear

  8. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

    Grant, M. A.; Hochstein, L. I.

    1984-01-01

    Paracoccus halodenitrificans produced a membrane-associated nitrite reductase. Spectrophotometric analysis showed it to be associated with a cd-cytochrome and located on the inner side of the cytoplasmic membrane. When supplied with nitrite, membrane preparations produced nitrous oxide and nitric oxide in different ratios depending on the electron donor employed. The nitrite reductase was maximally active at relatively low concentrations of sodium chloride and remained attached to the membranes at 100 mM sodium chloride.

  9. Evaluation of bacteriophages for prevention and treatment of diarrhea due to experimental enterotoxigenic Escherichia coli O149 infection of pigs.

    PubMed

    Jamalludeen, Nidham; Johnson, Roger P; Shewen, Patricia E; Gyles, Carlton L

    2009-04-14

    The objective of this study was to determine the efficacy of selected phages individually and in combination in prevention and treatment of diarrhea due to experimental O149:H10:F4 enterotoxigenic Escherichia coli (ETEC) in weaned pigs. For prophylaxis, the phages were administered orally shortly after challenge, and for therapeutic use, were given 24h after challenge, following the onset of diarrhea. The parameters used to assess outcomes were weight change, duration of diarrhea, severity of diarrhea, composite diarrhea score, and extent of shedding of the challenge ETEC over 6 days. Six phages that were tested individually in a prophylactic mode were effective as determined by a significant change in each of the parameters, although the phages were not present at titres greater than 10(3)PFU/g of feces. A modified protocol involving pre-treatment of the pigs with florfenicol and oral administration of sodium bicarbonate prior to the ETEC challenge and phage administration resulted in high levels of phages in the feces. Using this protocol, a combination of three phages that was tested in the prophylactic mode significantly reduced the severity of diarrhea and the composite diarrhea score. A mixture of two phages given therapeutically significantly improved each of the outcome parameters, without perturbation of the total fecal E. coli flora. Enumeration of phages in feces after treatment indicated that the phages were replicating to high titres in the intestinal tract of ETEC infected pigs within 1-2 days before declining progressively. These findings indicate that the selected phages were effective in moderating the course of experimental O149:H10:F4 ETEC diarrhea in weaned pigs when given prophylactically or therapeutically. PMID:19058927

  10. Induction of autophagy by cystatin C: a potential mechanism for prevention of cerebral vasospasm after experimental subarachnoid hemorrhage

    PubMed Central

    2013-01-01

    Background Studies have demonstrated that autophagy pathways are activated in the brain after experimental subarachnoid hemorrhage (SAH) and this may play a protective role in early brain injury. However, the contribution of autophagy in the pathogenesis of cerebral vasospasm (CVS) following SAH, and whether up-regulated autophagy may contribute to aggravate or release CVS, remain unknown. Cystatin C (CysC) is a cysteine protease inhibitor that induces autophagy under conditions of neuronal challenge. This study investigated the expression of autophagy proteins in the walls of basilar arteries (BA), and the effects of CysC on CVS and autophagy pathways following experimental SAH in rats. Methods All SAH animals were subjected to injection of 0.3 mL fresh arterial, non-heparinized blood into the cisterna magna. Fifty rats were assigned randomly to five groups: control group (n = 10), SAH group (n = 10), SAH + vehicle group (n = 10), SAH + low dose of CysC group (n = 10), and SAH + high dose of CysC group (n = 10). We measured proteins by western blot analysis, CVS by H&E staining method, morphological changes by electron microscopy, and recorded neuro-behavior scores. Results Microtubule-associated protein light chain-3, an autophagosome biomarker, and beclin-1, a Bcl-2-interacting protein required for autophagy, were significantly increased in the BA wall 48 h after SAH. In the CysC-handled group, the degree of CVS, measured as the inner BA perimeter and BA wall thickness, was significantly ameliorated in comparison with vehicle-treated SAH rats. This effect paralleled the intensity of autophagy in the BA wall induced by CysC. Conclusions These results suggest that the autophagy pathway is activated in the BA wall after SAH and CysC-induced autophagy may play a beneficial role in preventing SAH-induced CVS. PMID:23816364

  11. KIDNEY STONE INCIDENCE AND METABOLIC URINARY CHANGES AFTER MODERN BARIATRIC SURGERY: REVIEW OF CLINICAL STUDIES, EXPERIMENTAL MODELS, AND PREVENTION STRATEGIES

    PubMed Central

    Canales, Benjamin K.; Hatch, Marguerite

    2014-01-01

    Bariatric surgery has been associated with increased metabolic kidney stone risk and post-operative stone formation. A MEDLINE search, performed for articles published between January 2005 and November 2013, identified 24 pertinent studies containing 683 bariatric patients with 24-hour urine profiles, 6,777 bariatric patients with kidney stone incidence, and 7,089 non-stone forming controls. Of all procedures reviewed, only Roux-en-Y gastric bypass (RYGB) was linked to post-operative kidney stone development, increasing stone incidence two-fold in non-stone formers (8.5%) and four-fold in patients with previous stone history (16.7%). High quality evidence from 7 studies (n=277 patients) before and after RYGB identified the following post-RYGB urinary lithogenic risk factors: 30% reduction in urine volume (the main driver of urinary crystal saturation), 40% reduction in urinary citrate (a potent stone inhibitor), and 50% increase in urinary oxalate (a stone promotor). Based on this, a summary of strategies to reduce calcium oxalate stone risk following RYGB is provided. Furthermore, recent experimental RYGB studies are assessed for insights into the pathophysiology of oxalate handling, and the literature in gut anion (oxalate) transport is reviewed. Finally, as a potential probiotic therapy for hyperoxaluria, primary data from our laboratory is presented, demonstrating a 70% reduction in urinary oxalate levels in four experimental RYGB animals after colonization with Oxalobacter formigines, a non-pathogenic gut commensal that uses oxalate as an energy source. Overall, urine profiles and kidney stone risk following bariatric surgery appear modifiable by dietary adjustments, appropriate supplementation, and lifestyle changes. For hyperoxaluria resistant to dietary oxalate restriction and calcium binding, well-designed human investigations are needed to identify additional means of lowering urinary oxalate, such as Oxalobacter colonization or empiric pyridoxine therapy

  12. Multiple aldehyde reductases of human brain.

    PubMed

    Hoffman, P L; Wermuth, B; von Wartburg, J P

    1980-01-01

    Human brain contains four forms of aldehyde reducing enzymes. One major activity, designated AR3, has properties indicating its identity with the NADPH-dependent aldehyde reductase, EC 1.1.1.2. The other major form of human brain enzyme, AR1, which is also NADPH-dependent, reduces both aldehyde and ketone-containing substrates, including vitamin K3 (menadione) and daunorubicin, a cancer chemotherapeutic agent. This enzyme is very sensitive to inhibition by the flavonoids quercitrin and quercetine, and may be analogous to a daunorubicin reductase previously described in liver of other species. One minor form of human brain aldehyde reductase, AR2, demonstrates substrate specificity and inhibitor sensitivity which suggest its similarity to aldose reductases found in lens and other tissues of many species. This enzyme, which can also use NADH as cofactor to some extent, is the most active in reducing the aldehyde derivatives of the biogenic amines. The fourth human brain enzyme ("SSA reductase") differs from the other forms in its ability to use NADH as well as or better than NADPH as cofactor, and in its molecular weight, which is nearly twice that of the other forms. It is quite specific for succinic semialdehyde (SSA) as substrate, and was found to be significantly inhibited only by quercetine and quercitrin. AR3 can also reduce SSA, and both enzymes may contribute to the production of gamma-hydroxybutyric acid in vivo. These results indicate that the human brain aldehyde reductases can play relatively specific physiologic roles. PMID:7424738

  13. Preventive and therapeutic effects of adenanthin on experimental autoimmune encephalomyelitis by inhibiting NF-κB signaling.

    PubMed

    Yin, Qian-Qian; Liu, Chuan-Xu; Wu, Ying-Li; Wu, Shao-Fang; Wang, Yan; Zhang, Xia; Hu, Xiao-Juan; Pu, Jian-Xin; Lu, Ying; Zhou, Hu-Chen; Wang, Hong-Lin; Nie, Hong; Sun, Han-Dong; Chen, Guo-Qiang

    2013-09-01

    Adenanthin, a diterpenoid isolated from the leaves of Isodon adenanthus, has been reported to possess antileukemic activity through targeting peroxiredoxin I/II. However, its other potential activities remain to be explored. Using myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we report in this study that adenanthin exerts efficaciously preventive and therapeutic effects on EAE accompanied by significant restriction of infiltration of inflammatory cells and demyelination in CNS. Adenanthin-presented immunomodulatory effects on EAE are correlated with suppressed proliferation of MOG35-55-reactive T cells, decreased Th1 and Th17 cells, increased regulatory T cell populations, decreased production of serum proinflammatory cytokines, and reduced stimulatory capacity of APCs, which might be mediated by its inhibitory action on NF-κB signaling pathway. Our results propose that, as a novel NF-κB inhibitor, adenanthin has potent immunomodulatory activity for the treatment of multiple sclerosis and possibly other autoimmune disorders. PMID:23964105

  14. Characterization of thyroidal glutathione reductase

    SciTech Connect

    Raasch, R.J.

    1989-01-01

    Glutathione levels were determined in bovine and rat thyroid tissue by enzymatic conjugation with 1-chloro-2,4-dinitrobenzene using glutathione S-transferase. Bovine thyroid tissue contained 1.31 {+-} 0.04 mM reduced glutathione (GSH) and 0.14 {+-} 0.02 mM oxidized glutathione (GSSG). In the rat, the concentration of GSH was 2.50 {+-} 0.05 mM while GSSG was 0.21 {+-} 0.03 mM. Glutathione reductase (GR) was purified from bovine thyroid to electrophoretic homogeneity by ion exchange, affinity and molecular exclusion chromatography. A molecular weight range of 102-109 kDa and subunit size of 55 kDa were determined for GR. Thyroidal GR was shown to be a favoprotein with one FAD per subunit. The Michaelis constants of bovine thyroidal GR were determined to be 21.8 {mu}M for NADPH and 58.8 {mu}M for GSSG. The effect of thyroid stimulating hormone (TSH) and thyroxine (T{sub 4}) on in vivo levels of GR and glucose 6-phosphate dehydrogenase were determined in rat thyroid homogenates. Both enzymes were stimulated by TSH treatment and markedly reduced following T{sub 4} treatment. Lysosomal hydrolysis of ({sup 125}I)-labeled and unlabeled thyroglobulin was examined using size exclusion HPLC.

  15. Distinguishing two groups of flavin reductases by analyzing the protonation state of an active site carboxylic acid.

    PubMed

    Dumit, Verónica I; Cortez, Néstor; Matthias Ullmann, G

    2011-07-01

    Flavin-containing reductases are involved in a wide variety of physiological reactions such as photosynthesis, nitric oxide synthesis, and detoxification of foreign compounds, including therapeutic drugs. Ferredoxin-NADP(H)-reductase (FNR) is the prototypical enzyme of this family. The fold of this protein is highly conserved and occurs as one domain of several multidomain enzymes such as the members of the diflavin reductase family. The enzymes of this family have emerged as fusion of a FNR and a flavodoxin. Although the active sites of these enzymes are very similar, different enzymes function in opposite directions, that is, some reduce oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) and some oxidize reduced nicotinamide adenine dinucleotide phosphate (NADPH). In this work, we analyze the protonation behavior of titratable residues of these enzymes through electrostatic calculations. We find that a highly conserved carboxylic acid in the active site shows a different titration behavior in different flavin reductases. This residue is deprotonated in flavin reductases present in plastids, but protonated in bacterial counterparts and in diflavin reductases. The protonation state of the carboxylic acid may also influence substrate binding. The physiological substrate for plastidic enzymes is NADP(+), but it is NADPH for the other mentioned reductases. In this article, we discuss the relevance of the environment of this residue for its protonation and its importance in catalysis. Our results allow to reinterpret and explain experimental data. PMID:21538544

  16. The Role of Friends’ Disruptive Behavior in the Development of Children’s Tobacco Experimentation: Results from a Preventive Intervention Study

    PubMed Central

    Huizink, Anja; Vuijk, Patricia

    2010-01-01

    Having friends who engage in disruptive behavior in childhood may be a risk factor for childhood tobacco experimentation. This study tested the role of friends’ disruptive behavior as a mediator of the effects of a classroom based intervention on children’s tobacco experimentation. 433 Children (52% males) were randomly assigned to the Good Behavior Game (GBG) intervention, a universal preventive intervention targeting disruptive behavior, and facilitating positive prosocial peer interactions. Friends’ disruptive behavior was assessed from age 7–10 years. Participants’ experimentation with tobacco was assessed annually from age 10–13. Reduced rates in tobacco experimentation and friends’ disruptive behavior were found among GBG children, as compared to controls. Support for friends’ disruptive behavior as a mediator in the link between intervention status and tobacco experimentation was found. These results remained after controlling for friends’ and parental smoking status, and child ADHD symptoms. The results support the role of friends’ disruptive behavior in preadolescents’ tobacco experimentation. PMID:20694577

  17. High- and Low-Level Dissonance-Based Eating Disorder Prevention Programs with Young Women with Body Image Concerns: An Experimental Trial

    ERIC Educational Resources Information Center

    McMillan, Whitney; Stice, Eric; Rohde, Paul

    2011-01-01

    Objective: As cognitive dissonance is theorized to contribute to the effects of dissonance-based eating disorder prevention programs, we evaluated a high-dissonance version of this program against a low-dissonance version and a wait-list control condition to provide an experimental test of the mechanism of intervention effects. Method: Female…

  18. A Quasi-Experimental Evaluation of a Community-Based HIV Prevention Intervention for Mexican American Female Adolescents: The SHERO's Program

    ERIC Educational Resources Information Center

    Harper, Gary W.; Bangi, Audrey K.; Sanchez, Bernadette; Doll, Mimi; Pedraza, Ana

    2009-01-01

    This article describes a quasi-experimental evaluation of a community-based, culturally and ecologically tailored HIV prevention intervention for Mexican American female adolescents grounded in the AIDS risk reduction model. A total of 378 Mexican American female adolescents (mean age = 15.2) participated in either the nine-session SHERO's (a…

  19. Can empirical hypertonic saline or sodium bicarbonate treatment prevent the development of cardiotoxicity during serious amitriptyline poisoning? Experimental research

    PubMed Central

    Sukru Paksu, Muhammet; Zengin, Halit; Uzun, Adem; Ilkaya, Fatih; Guzel, Hasan; Paksu, Sule; Ucar, Durmus; Alacam, Hasan; Duran, Latif; Murat, Naci; Guzel, Ahmet

    2015-01-01

    Summary Objective The aim of this experimental study was to investigate whether hypertonic saline or sodium bicarbonate administration prevented the development of cardiotoxicity in rats that received toxic doses of amitriptyline. Method Thirty-six Sprague Dawley rats were used in the study. The animals were divided into six groups. Group 1 received toxic doses of i.p. amitriptyline. Groups 2 and 3 toxic doses of i.p. amitriptyline, plus i.v. sodium bicarbonate and i.v. hypertonic saline, respectively. Group 4 received only i.v. sodium bicarbonate, group 5 received only i.v. hypertonic saline, and group 6 was the control. Electrocardiography was recorded in all rats for a maximum of 60 minutes. Blood samples were obtained to measure the serum levels of sodium and ionised calcium. Results The survival time was shorter in group 1. In this group, the animals’ heart rates also decreased over time, and their QRS and QTc intervals were significantly prolonged. Groups 2 and 3 showed less severe changes in their ECGs and the rats survived for a longer period. The effects of sodium bicarbonate or hypertonic saline treatments on reducing the development of cardiotoxicity were similar. The serum sodium levels decreased in all the amitriptyline-applied groups. Reduction of serum sodium level was most pronounced in group 1. Conclusion Empirical treatment with sodium bicarbonate or hypertonic saline can reduce the development of cardiotoxicity during amitriptyline intoxication. As hypertonic saline has no adverse effects on drug elimination, it should be considered as an alternative to sodium bicarbonate therapy. PMID:25939777

  20. Inhibition of human anthracycline reductases by emodin - A possible remedy for anthracycline resistance.

    PubMed

    Hintzpeter, Jan; Seliger, Jan Moritz; Hofman, Jakub; Martin, Hans-Joerg; Wsol, Vladimir; Maser, Edmund

    2016-02-15

    The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC50- and Ki-values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. PMID:26773812

  1. Structural and mechanistic insights on nitrate reductases.

    PubMed

    Coelho, Catarina; Romão, Maria João

    2015-12-01

    Nitrate reductases (NR) belong to the DMSO reductase family of Mo-containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane-bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data. PMID:26362109

  2. Respiratory arsenate reductase as a bidirectional enzyme

    USGS Publications Warehouse

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  3. Respiratory arsenate reductase as a bidirectional enzyme

    SciTech Connect

    Richey, Christine; Chovanec, Peter; Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282 ; Hoeft, Shelley E.; Oremland, Ronald S.; Basu, Partha; Stolz, John F.

    2009-05-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe-S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  4. Phylogenomics of Mycobacterium Nitrate Reductase Operon.

    PubMed

    Huang, Qinqin; Abdalla, Abualgasim Elgaili; Xie, Jianping

    2015-07-01

    NarGHJI operon encodes a nitrate reductase that can reduce nitrate to nitrite. This process enhances bacterial survival by nitrate respiration under anaerobic conditions. NarGHJI operon exists in many bacteria, especially saprophytic bacteria living in soil which play a key role in the nitrogen cycle. Most actinomycetes, including Mycobacterium tuberculosis, possess NarGHJI operons. M. tuberculosis is a facultative intracellular pathogen that expands in macrophages and has the ability to persist in a non-replicative form in granuloma lifelong. Nitrogen and nitrogen compounds play crucial roles in the struggle between M. tuberculosis and host. M. tuberculosis can use nitrate as a final electron acceptor under anaerobic conditions to enhance its survival. In this article, we reviewed the mechanisms regulating nitrate reductase expression and affecting its activity. Potential genes involved in regulating the nitrate reductase expression in M. tuberculosis were identified. The conserved NarG might be an alternative mycobacterium taxonomic marker. PMID:25980349

  5. Interspecific variation for thermal dependence of glutathione reductase in sainfoin.

    PubMed

    Kidambi, S P; Mahan, J R; Matches, A G

    1990-05-01

    Understanding the biochemical and physiological consequences of species variation would expedite improvement in agronomically useful genotypes of sainfoin (Onobrychis spp.) Information on variation among sainfoin species is lacking on thermal dependence of glutathione reductase (B.C. 1.6.4.2.), which plays an important role in the protection of plants from both high and low temperature stresses by preventing harmful oxidation of enzymes and membranes. Our objective was to investigate the interspecific variation for thermal dependency of glutathione reductase in sainfoin. Large variation among species was found for: (i) the minimum apparent Km (0.4-2.5 μM NADPH), (ii) the temperature at which the minimum apparent Km was observed (15°-5°C), and (iii) the thermal kinetic windows (2°-30°C width) over a 15°-45°C temperature gradient. In general, tetraploid species had narrower (≤17°C) thermal kinetic windows than did diploid species (∼30°C), with one exception among the diploids. Within the tetraploid species, the cultivars of O. viciifolia had a broader thermal kinetic window (≥7°C) than the plant introduction (PI 212241, >2 °C) itself. PMID:24226572

  6. Properties of seleno-methionine substituted assimilatory nitrate reductase

    SciTech Connect

    Solomonson, L.P.; Barber, M.J. )

    1991-03-11

    Assimilatory NADH:nitrate reductase contains FAD, heme and Mo-pterin arranged in an NADH{yields}FAD{yields}heme{yields}Mo-Pterin{yields}NO{sub 3} electron transfer sequence. A functional Mo-pterin center is essential for all nitrate-reducing activities. To assess the possible functional role of Met, a Se-Met substituted NR was obtained by addition of Se-Met to ammonia-grown Chlorella cells prior to induction of NR activity. Increase in NADH:dehydrogenase partial activities and nitrate reductase protein proceeded normally following induction but little or no nitrate-reducing activity was expressed. This effect was observed with as little as 10{sup {minus}5} Se-Met and was prevented by a 10-fold excess of Met. A less pronounced effect was observed with 10{sup {minus}4}M Se-Cys. The purified Se-Met substituted enzyme exhibited the same apparent physical size, spectral properties and NADH dehydrogenase activities as control NR but was devoid of nitrate-reducing activities. These results suggest that one or more Met residues are essential for the catalytic function of the molybdo-pterin center of assimilatory NR.

  7. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase

    PubMed Central

    Schumacher, Marc M; Elsabrouty, Rania; Seemann, Joachim; Jo, Youngah; DeBose-Boyd, Russell A

    2015-01-01

    Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD. DOI: http://dx.doi.org/10.7554/eLife.05560.001 PMID:25742604

  8. The Cellular Thioredoxin-1/Thioredoxin Reductase-1 Driven Oxidoreduction Represents a Chemotherapeutic Target for HIV-1 Entry Inhibition

    PubMed Central

    Curbo, Sophie; Pannecouque, Christophe; Noppen, Sam; Liekens, Sandra; Engman, Lars; Lundberg, Mathias; Balzarini, Jan; Karlsson, Anna

    2016-01-01

    Background The entry of HIV into its host cell is an interesting target for chemotherapeutic intervention in the life-cycle of the virus. During entry, reduction of disulfide bridges in the viral envelope glycoprotein gp120 by cellular oxidoreductases is crucial. The cellular thioredoxin reductase-1 plays an important role in this oxidoreduction process by recycling electrons to thioredoxin-1. Therefore, thioredoxin reductase-1 inhibitors may inhibit gp120 reduction during HIV-1 entry. In this present study, tellurium-based thioredoxin reductase-1 inhibitors were investigated as potential inhibitors of HIV entry. Results The organotellurium compounds inhibited HIV-1 and HIV-2 replication in cell culture at low micromolar concentrations by targeting an early event in the viral infection cycle. Time-of-drug-addition studies pointed to virus entry as the drug target, more specifically: the organotellurium compound TE-2 showed a profile similar or close to that of the fusion inhibitor enfuvirtide (T-20). Surface plasmon resonance-based interaction studies revealed that the compounds do not directly interact with the HIV envelope glycoproteins gp120 and gp41, nor with soluble CD4, but instead, dose-dependently bind to thioredoxin reductase-1. By inhibiting the thioredoxin-1/thioredoxin reductase-1-directed oxidoreduction of gp120, the organotellurium compounds prevent conformational changes in the viral glycoprotein which are necessary during viral entry. Conclusion Our findings revealed that thioredoxin-1/thioredoxin reductase-1 acts as a cellular target for the inhibition of HIV entry. PMID:26816344

  9. IN VITRO INHIBITION OF GLUTATHIONE REDUCTASE BY ARSENOTRI-GLUTATHIONE

    EPA Science Inventory

    Arsenotriglutathione, a product of the reduction of arsenate and the complexation of arsenite by glutathione, is a mixed type inhibitor of the reduction of glutathione disulfide by purified yeast glutathione reductase or the glutathione reductase activity in rabbit erythrocyte ly...

  10. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

    Streeter, J.G.; DeVine, P.J.

    1983-08-01

    Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase. 18 references

  11. Crystal Structure of Saccharomyces Cerevisiae 3'-Phosphoadenosine-5'-Phosphosulfate Reductase Complexed With Adenosine 3',5'-Bisphosphate

    SciTech Connect

    Yu, Z.; Lemongello, D.; Segel, I.H.; Fisher, A.J.

    2009-05-28

    Most assimilatory bacteria, fungi, and plants species reduce sulfate (in the activated form of APS or PAPS) to produce reduced sulfur. In yeast, PAPS reductase reduces PAPS to sulfite and PAP. Despite the difference in substrate specificity and catalytic cofactor, PAPS reductase is homologous to APS reductase in both sequence and structure, and they are suggested to share the same catalytic mechanism. Metazoans do not possess the sulfate reduction pathway, which makes APS/PAPS reductases potential drug targets for human pathogens. Here, we present the 2.05 A resolution crystal structure of the yeast PAPS reductase binary complex with product PAP bound. The N-terminal region mediates dimeric interactions resulting in a unique homodimer assembly not seen in previous APS/PAPS reductase structures. The 'pyrophosphate-binding' sequence (47)TTAFGLTG(54) defines the substrate 3'-phosphate binding pocket. In yeast, Gly54 replaces a conserved aspartate found in APS reductases vacating space and charge to accommodate the 3'-phosphate of PAPS, thus regulating substrate specificity. Also, for the first time, the complete C-terminal catalytic motif (244)ECGIH(248) is revealed in the active site. The catalytic residue Cys245 is ideally positioned for an in-line attack on the beta-sulfate of PAPS. In addition, the side chain of His248 is only 4.2 A from the Sgamma of Cys245 and may serve as a catalytic base to deprotonate the active site cysteine. A hydrophobic sequence (252)RFAQFL(257) at the end of the C-terminus may provide anchoring interactions preventing the tail from swinging away from the active site as seen in other APS/PAPS reductases.

  12. The 15kDa Selenoprotein and Thioredoxin Reductase 1 Promote Colon Cancer by Different Pathways

    PubMed Central

    Tsuji, Petra A.; Carlson, Bradley A.; Yoo, Min-Hyuk; Naranjo-Suarez, Salvador; Xu, Xue-Ming; He, Yiwen; Asaki, Esther; Seifried, Harold E.; Reinhold, William C.; Davis, Cindy D.; Gladyshev, Vadim N.; Hatfield, Dolph L.

    2015-01-01

    Selenoproteins mediate much of the cancer-preventive properties of the essential nutrient selenium, but some of these proteins have been shown to also have cancer-promoting effects. We examined the contributions of the 15kDa selenoprotein (Sep15) and thioredoxin reductase 1 (TR1) to cancer development. Targeted down-regulation of either gene inhibited anchorage-dependent and anchorage-independent growth and formation of experimental metastases of mouse colon carcinoma CT26 cells. Surprisingly, combined deficiency of Sep15 and TR1 reversed the anti-cancer effects observed with down-regulation of each single gene. We found that inflammation-related genes regulated by Stat-1, especially interferon-γ-regulated guanylate-binding proteins, were highly elevated in Sep15-deficient, but not in TR1-deficient cells. Interestingly, components of the Wnt/β-catenin signaling pathway were up-regulated in cells lacking both TR1 and Sep15. These results suggest that Sep15 and TR1 participate in interfering regulatory pathways in colon cancer cells. Considering the variable expression levels of Sep15 and TR1 found within the human population, our results provide insights into new roles of selenoproteins in cancer. PMID:25886253

  13. Comprehensive experimental study on prevention of land subsidence caused by dewatering in deep foundation pit with hanging waterproof curtain

    NASA Astrophysics Data System (ADS)

    Yang, T. L.; Yan, X. X.; Wang, H. M.; Huang, X. L.; Zhan, G. H.

    2015-11-01

    Land subsidence caused by dewatering of deep foundations pit has currently become the focus of prevention and control of land subsidence in Shanghai. Because of the reliance on deep foundation dewatering pit projects, two comprehensive test sites were established to help prevent land subsidence. Through geological environmental monitoring during dewatering of a deep foundation pit, the analysis of the relation between artesian water level and soil subsidence, some basic features of land subsidence caused by dewatering of deep foundation pits are elucidated. The results provide a scientific basis for prevention and control of land subsidence caused by dewatering in deep foundation pits.

  14. Curcumin is a tight-binding inhibitor of the most efficient human daunorubicin reductase--Carbonyl reductase 1.

    PubMed

    Hintzpeter, Jan; Hornung, Jan; Ebert, Bettina; Martin, Hans-Jörg; Maser, Edmund

    2015-06-01

    Curcumin is a major component of the plant Curcuma longa L. It is traditionally used as a spice and coloring in foods and is an important ingredient in curry. Curcuminoids have anti-oxidant and anti-inflammatory properties and gained increasing attention as potential neuroprotective and cancer preventive compounds. In the present study, we report that curcumin is a potent tight-binding inhibitor of human carbonyl reductase 1 (CBR1, Ki=223 nM). Curcumin acts as a non-competitive inhibitor with respect to the substrate 2,3-hexandione as revealed by plotting IC50-values against various substrate concentrations and most likely as a competitive inhibitor with respect to NADPH. Molecular modeling supports the finding that curcumin occupies the cofactor binding site of CBR1. Interestingly, CBR1 is one of the most effective human reductases in converting the anthracycline anti-tumor drug daunorubicin to daunorubicinol. The secondary alcohol metabolite daunorubicinol has significantly reduced anti-tumor activity and shows increased cardiotoxicity, thereby limiting the clinical use of daunorubicin. Thus, inhibition of CBR1 may increase the efficacy of daunorubicin in cancer tissue and simultaneously decrease its cardiotoxicity. Western-blots demonstrated basal expression of CBR1 in several cell lines. Significantly less daunorubicin reduction was detected after incubating A549 cell lysates with increasing concentrations of curcumin (up to 60% less with 50 μM curcumin), suggesting a beneficial effect in the co-treatment of anthracycline anti-tumor drugs together with curcumin. PMID:25541467

  15. An Evaluation of a Teat Dip with Dodecyl Benzene Sulfonic Acid in Preventing Bovine Mammary Gland Infection from Experimental Exposure to Streptococcus agalactiae and Staphylococcus aureus

    PubMed Central

    Barnum, D. A.; Johnson, R. E.; Brooks, B. W.

    1982-01-01

    The effectiveness of a teat dip with dodecyl benzene sulfonic acid (1.94%) for the prevention of intramammary infections was determined in cows experimentally challenged with Streptococcus agalactiae and Staphylococcus aureus. The infection rates with Streptococcus agalactiae and Staphylococcus aureus were 62.5% and 75% in undipped quarters, 12.5% and 21.5% in dipped quarters with a reduction rate of 80% and 71% respectively. The significance of some findings in relation to mastitis control are discussed. PMID:17422110

  16. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite, which is the first step in the nitrate assimilation pathway, but can also reduce nitrite to nitric oxide (NO), an important signaling molecule that is thought to mediate a wide array of of developmental and physiological processes...

  17. Promiscuity and diversity in 3-ketosteroid reductases.

    PubMed

    Penning, Trevor M; Chen, Mo; Jin, Yi

    2015-07-01

    Many steroid hormones contain a Δ(4)-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1-AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1-AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'. PMID:25500069

  18. Promiscuity and diversity in 3-ketosteroid reductases

    PubMed Central

    Penning, Trevor M.; Chen, Mo; Jin, Yi

    2014-01-01

    Many steroid hormones contain a Δ4-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1–AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1–AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled ‘Steroid/Sterol signaling’. PMID:25500069

  19. Ferrisiderophore reductase activity in Agrobacterium tumefaciens.

    PubMed Central

    Lodge, J S; Gaines, C G; Arceneaux, J E; Byers, B R

    1982-01-01

    Reduction of the iron in ferriagrobactin by the cytoplasmic fraction of Agrobacterium tumefaciens strictly required NaDH as the reductant. Addition of flavin mononucleotide and anaerobic conditions were necessary for the reaction; when added with flavin mononucleotide, magnesium was stimulatory. This ferrisiderophore reductase activity may be a part of the iron assimilation process in A. tumefaciens. PMID:7056702

  20. Material and Social Incentives to Participation in Behavioral Interventions: A Meta-Analysis of Gender Disparities in Enrollment and Retention in Experimental Human Immunodeficiency Virus Prevention Interventions

    PubMed Central

    Durantini, Marta R.; Albarracín, Dolores

    2013-01-01

    Objective A meta-analysis was conducted to test theoretical hypotheses about the predictors of enrollment and completion of condom-use-promotion interventions among men and women. Design A meta-analysis summarized research reports of the efficacy of experimental interventions on human immunodeficiency virus (HIV) prevention. Main Outcome Measures The outcome measure consisted of (a) a measure of participation, obtained by subtracting the actual number of participants from the number of the invited people, and (b) a measure of retention was obtained by subtracting the number of participants who completed the intervention from the number of commencers. Results Experimental interventions providing instrumental and financial resources (e.g., payments) increased initiation and retention more among predominantly male samples, whereas experimental interventions using group formats increased initiation and retention more among predominantly female samples. These patterns remained while controlling for past condom use, other HIV-risk behaviors, and demographics associated with gender composition. Conclusion People seek out HIV-prevention interventions to fulfill gender-specific needs, and these differences must be taken into account in the design of HIV-prevention interventions. PMID:19751090

  1. An electron-bifurcating caffeyl-CoA reductase.

    PubMed

    Bertsch, Johannes; Parthasarathy, Anutthaman; Buckel, Wolfgang; Müller, Volker

    2013-04-19

    A low potential electron carrier ferredoxin (E0' ≈ -500 mV) is used to fuel the only bioenergetic coupling site, a sodium-motive ferredoxin:NAD(+) oxidoreductase (Rnf) in the acetogenic bacterium Acetobacterium woodii. Because ferredoxin reduction with physiological electron donors is highly endergonic, it must be coupled to an exergonic reaction. One candidate is NADH-dependent caffeyl-CoA reduction. We have purified a complex from A. woodii that contains a caffeyl-CoA reductase and an electron transfer flavoprotein. The enzyme contains three subunits encoded by the carCDE genes and is predicted to have, in addition to FAD, two [4Fe-4S] clusters as cofactor, which is consistent with the experimental determination of 4 mol of FAD, 9 mol of iron, and 9 mol of acid-labile sulfur. The enzyme complex catalyzed caffeyl-CoA-dependent oxidation of reduced methyl viologen. With NADH as donor, it catalyzed caffeyl-CoA reduction, but this reaction was highly stimulated by the addition of ferredoxin. Spectroscopic analyses revealed that ferredoxin and caffeyl-CoA were reduced simultaneously, and a stoichiometry of 1.3:1 was determined. Apparently, the caffeyl-CoA reductase-Etf complex of A. woodii uses the novel mechanism of flavin-dependent electron bifurcation to drive the endergonic ferredoxin reduction with NADH as reductant by coupling it to the exergonic NADH-dependent reduction of caffeyl-CoA. PMID:23479729

  2. A defect in sodium-dependent amino acid uptake in diabetic rabbit peripheral nerve. Correction by an aldose reductase inhibitor or myo-inositol administration.

    PubMed Central

    Greene, D A; Lattimer, S A; Carroll, P B; Fernstrom, J D; Finegold, D N

    1990-01-01

    A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake. PMID:2185278

  3. Functional studies of aldo-keto reductases in Saccharomyces cerevisiae*

    PubMed Central

    Chang, Qing; Griest, Terry A.; Harter, Theresa M.; Petrash, J. Mark

    2007-01-01

    SUMMARY We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast. PMID:17140678

  4. The Role of Friends' Disruptive Behavior in the Development of Children's Tobacco Experimentation: Results from a Preventive Intervention Study

    ERIC Educational Resources Information Center

    van Lier, Pol A. C.; Huizink, Anja; Vuijk, Patricia

    2011-01-01

    Having friends who engage in disruptive behavior in childhood may be a risk factor for childhood tobacco experimentation. This study tested the role of friends' disruptive behavior as a mediator of the effects of a classroom based intervention on children's tobacco experimentation. 433 Children (52% males) were randomly assigned to the Good…

  5. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase

    PubMed Central

    Leavitt, William D.; Bradley, Alexander S.; Santos, André A.; Pereira, Inês A. C.; Johnston, David T.

    2015-01-01

    The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major (34S/32S) and minor (33S/32S, 36S/32S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in 34S/32S (hereafter, 34εDsrAB) to be 15.3 ± 2‰, 2σ. The accompanying minor isotope effect in 33S, described as 33λDsrAB, is calculated to be 0.5150 ± 0.0012, 2σ. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3–0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in 34εDsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of 34εDsrAB is similar to the median value of experimental observations compiled from all known published work, where 34εr−p = 16.1‰ (r–p indicates reactant vs. product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments (34εSO4−H2S =  17.3 ± 1.5‰, 2σ) and in modern marine sediments (34εSO4−H2S =  17.3 ± 3.8‰). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the biogeochemical and geobiological sulfur isotope records in

  6. Sulfur Isotope Effects of Dissimilatory Sulfite Reductase.

    PubMed

    Leavitt, William D; Bradley, Alexander S; Santos, André A; Pereira, Inês A C; Johnston, David T

    2015-01-01

    The precise interpretation of environmental sulfur isotope records requires a quantitative understanding of the biochemical controls on sulfur isotope fractionation by the principle isotope-fractionating process within the S cycle, microbial sulfate reduction (MSR). Here we provide the only direct observation of the major ((34)S/(32)S) and minor ((33)S/(32)S, (36)S/(32)S) sulfur isotope fractionations imparted by a central enzyme in the energy metabolism of sulfate reducers, dissimilatory sulfite reductase (DsrAB). Results from in vitro sulfite reduction experiments allow us to calculate the in vitro DsrAB isotope effect in (34)S/(32)S (hereafter, [Formula: see text]) to be 15.3 ± 2‰, 2σ. The accompanying minor isotope effect in (33)S, described as [Formula: see text], is calculated to be 0.5150 ± 0.0012, 2σ. These observations facilitate a rigorous evaluation of the isotopic fractionation associated with the dissimilatory MSR pathway, as well as of the environmental variables that govern the overall magnitude of fractionation by natural communities of sulfate reducers. The isotope effect induced by DsrAB upon sulfite reduction is a factor of 0.3-0.6 times prior indirect estimates, which have ranged from 25 to 53‰ in (34)εDsrAB. The minor isotope fractionation observed from DsrAB is consistent with a kinetic or equilibrium effect. Our in vitro constraints on the magnitude of [Formula: see text] is similar to the median value of experimental observations compiled from all known published work, where (34)ε r-p = 16.1‰ (r-p indicates reactant vs. product, n = 648). This value closely matches those of MSR operating at high sulfate reduction rates in both laboratory chemostat experiments ([Formula: see text] 17.3 ± 1.5‰, 2σ) and in modern marine sediments ([Formula: see text] 17.3 ± 3.8‰). Targeting the direct isotopic consequences of a specific enzymatic processes is a fundamental step toward a biochemical foundation for reinterpreting the

  7. Experimental lung injury promotes alterations in energy metabolism and respiratory mechanics in the lungs of rats: prevention by exercise.

    PubMed

    da Cunha, Maira J; da Cunha, Aline A; Scherer, Emilene B S; Machado, Fernanda Rossato; Loureiro, Samanta O; Jaenisch, Rodrigo B; Guma, Fátima; Lago, Pedro Dal; Wyse, Angela T S

    2014-04-01

    In the present study we investigated the effects of lung injury on energy metabolism (succinate dehydrogenase, complex II, cytochrome c oxidase, and ATP levels), respiratory mechanics (dynamic and static compliance, elastance and respiratory system resistance) in the lungs of rats, as well as on phospholipids in bronchoalveolar lavage fluid. The protective effect of physical exercise on the alterations caused by lung injury, including lung edema was also evaluated. Wistar rats were submitted to 2 months of physical exercise. After this period the lung injury was induced by intratracheal instillation of lipopolysaccharide. Adult Wistar rats were submitted to 2 months of physical exercise and after this period the lung injury was induced by intratracheal instillation of lipopolysaccharide in dose 100 μg/100 g body weight. The sham group received isotonic saline instillation. Twelve hours after the injury was performed the respiratory mechanical and after the rats were decapitated and samples were collected. The rats subjected to lung injury presented a decrease in activities of the enzymes of the electron transport chain and ATP levels in lung, as well as the formation of pulmonary edema. A decreased lung dynamic and static compliance, as well as an increase in respiratory system resistance, and a decrease in phospholipids content were observed. Physical exercise was able to totally prevent the decrease in succinate dehydrogenase and complex II activities and the formation of pulmonary edema. It also partially prevented the increase in respiratory system resistance, but did not prevent the decrease in dynamic and static compliance, as well as in phospholipids content. These findings suggest that the mitochondrial dysfunction may be one of the important contributors to lung damage and that physical exercise may be beneficial in this pathology, although it did not prevent all changes present in lung injury. PMID:24378995

  8. An Experimental Study to Determine the Role of Inferior Vena Cava Filter in Preventing Bone Cement Implantation Syndrome

    PubMed Central

    Guo, Wangang; Zheng, Qiangsun; Li, Bingling; Shi, Xiaoqin; Xiang, Dingcheng; Wang, Chen

    2015-01-01

    Background: Inferior vena cava filters (IVCF) are frequently used for preventing pulmonary embolism (PE) following deep venous thromboembolism. Objectives: The present study was designed to investigate whether IVCF could prevent or impede the occurrence of bone cement implantation syndrome (BCIS), since PE is considered as the central mechanism of BCIS. Materials and Methods: Fifteen sheep were divided into three groups: bone cement free (BCF) group, cement implantation (CI) group and IVCF group. In all the groups, an osteotomy proximal to the greater trochanter of left femur was carried out. In BCF group, the femoral canal was not reamed out or packed with any bone cement. In CI and IVCF groups, the left femoral canals were packed with bone cement, to simulate the cementing procedures carried out in hip replacement. An OptEase® filter was placed and released in inferior vena cava, prior to packing cement in the femoral canal in IVCF group, while the IVCF was not released in the CI group. The BCF group was considered as control. Results: Systolic blood pressure (SBP), saturation of oxygen (SaO2) and partial pressure of carbon dioxide (PaCO2) declined significantly 10 min after the bone cement packing, in CI group, compared to those in BCF group. This was accompanied by a rise in the arterial pH. However, IVCF prevented those changes in the CI group. On ultrasonography, there were dotted echoes in right atrium in the CI group, after bone cement packing, while such echoes were hardly seen in the IVCF group. Conclusion: This study demonstrates that IVCF could prevent BCIS effectively, and, as a corollary, suggests that PE represents the leading cause of the constellation of BCIS symptoms. PMID:26557267

  9. Mechanism of inhibition of ribonucleotide reductase with motexafin gadolinium (MGd)

    SciTech Connect

    Zahedi Avval, Farnaz; Berndt, Carsten; Pramanik, Aladdin; Holmgren, Arne

    2009-02-13

    Motexafin gadolinium (MGd) is an expanded porphyrin anticancer agent which selectively targets tumor cells and works as a radiation enhancer, with promising results in clinical trials. Its mechanism of action is oxidation of intracellular reducing molecules and acting as a direct inhibitor of mammalian ribonucleotide reductase (RNR). This paper focuses on the mechanism of inhibition of RNR by MGd. Our experimental data present at least two pathways for inhibition of RNR; one precluding subunits oligomerization and the other direct inhibition of the large catalytic subunit of the enzyme. Co-localization of MGd and RNR in the cytoplasm particularly in the S-phase may account for its inhibitory properties. These data can elucidate an important effect of MGd on the cancer cells with overproduction of RNR and its efficacy as an anticancer agent and not only as a general radiosensitizer.

  10. Covered Stent Membrane Design for Treatment of Atheroembolic Disease at Carotid Artery Bifurcation and Prevention of Thromboembolic Stroke: An In Vitro Experimental Study.

    PubMed

    Kabinejadian, Foad; Nezhadian, Mercedeh Kaabi; Cui, Fangsen; Ho, Pei; Leo, Hwa Liang

    2016-02-01

    In this study, a polymeric membrane has been designed and developed for carotid stents to prevent detachment of emboli from the arterial wall and subsequent stroke, while maintaining side-branch flow. Prototypes of different geometrical design parameters have been fabricated and their performance has been evaluated in vitro under physiological pulsatile flow condition in a life-size silicone anastomotic model of carotid artery bifurcation. These evaluations include both quantitative and qualitative experimental (in vitro) assessments of emboli prevention capability, side-branch flow preservation, and flow visualization. The covered stents with the novel membrane demonstrated significantly higher emboli prevention capability than the corresponding bare nitinol stent as well as some earlier related designs, while preserving more than 93% of the original flow of the external carotid artery (ECA). Flow in the ECA through these covered stents was uniform without evidence of undesirable flow recirculation or retrograde flow that might predispose the vessel wall to intimal thickening and atherosclerotic plaque formation. This study demonstrated the potential of these novel covered stent designs for the treatment of carotid atherosclerotic stenosis and prevention of late embolic stroke. However, further in vivo investigations of biological effects and mechanical performance of this covered stent design (e.g., its thrombogenicity potential and biocompatibility) are warranted. PMID:26147531

  11. Structure of aldose reductase from Giardia lamblia

    PubMed Central

    Ferrell, M.; Abendroth, J.; Zhang, Y.; Sankaran, B.; Edwards, T. E.; Staker, B. L.; Van Voorhis, W. C.; Stewart, L. J.; Myler, P. J.

    2011-01-01

    Giardia lamblia is an anaerobic aerotolerant eukaryotic parasite of the intestines. It is believed to have diverged early from eukarya during evolution and is thus lacking in many of the typical eukaryotic organelles and biochemical pathways. Most conspicuously, mitochondria and the associated machinery of oxidative phosphorylation are absent; instead, energy is derived from substrate-level phosphorylation. Here, the 1.75 Å resolution crystal structure of G. lamblia aldose reductase heterologously expressed in Escherichia coli is reported. As in other oxidoreductases, G. lamblia aldose reductase adopts a TIM-barrel conformation with the NADP+-binding site located within the eight β-strands of the interior. PMID:21904059

  12. Methylenetetrahydrofolate reductase deficiency: importance of early diagnosis.

    PubMed

    Fattal-Valevski, A; Bassan, H; Korman, S H; Lerman-Sagie, T; Gutman, A; Harel, S

    2000-08-01

    Methylenetetrahydrofolate reductase deficiency is the most common inborn error of folate metabolism and should be suspected when homocystinuria is combined with hypomethioninemia. The main clinical findings are neurologic signs such as severe developmental delay, marked hypotonia, seizures, microcephaly, apnea, and coma. Most patients present in early life. The infantile form is severe, with rapid deterioration leading to death usually within 1 year. Treatment with betaine has been shown to be efficient in lowering homocysteine concentrations and returning methionine to normal, but the clinical response is variable. We report two brothers with methylenetetrahydrofolate reductase deficiency: the first was undiagnosed and died at 8 months of age from neurologic deterioration and apnea, while his brother, who was treated with betaine from the age of 4 months, is now 3 years old and has developmental delay. PMID:10961793

  13. A Technology-Mediated Behavioral Weight Gain Prevention Intervention for College Students: Controlled, Quasi-Experimental Study

    PubMed Central

    Monroe, Courtney M; Turner-McGrievy, Gabrielle; Sundstrom, Beth; Larsen, Chelsea; Magradey, Karen; Wilcox, Sara; Brandt, Heather M

    2016-01-01

    Background Both men and women are vulnerable to weight gain during the college years, and this phenomenon is linked to an increased risk of several chronic diseases and mortality. Technology represents an attractive medium for the delivery of weight control interventions focused on college students, given its reach and appeal among this population. However, few technology-mediated weight gain prevention interventions have been evaluated for college students. Objective This study examined a new technology-based, social media-facilitated weight gain prevention intervention for college students. Methods Undergraduates (n =58) in two sections of a public university course were allocated to either a behavioral weight gain prevention intervention (Healthy Weight, HW; N=29) or a human papillomavirus (HPV) vaccination awareness intervention (control; N=29). All students were enrolled, regardless of initial body weight or expressed interest in weight management. The interventions delivered 8 lessons via electronic newsletters and Facebook postings over 9 weeks, which were designed to foster social support and introduce relevant educational content. The HW intervention targeted behavioral strategies to prevent weight gain and provided participants with a Wi-Fi-enabled scale and an electronic physical activity tracker to facilitate weight regulation. A repeated-measures analysis of variance was conducted to examine within- and between-group differences in measures of self-reported weight control practices and objectively measured weight. Use of each intervention medium and device was objectively tracked, and intervention satisfaction measures were obtained. Results Students remained weight stable (HW: −0.48+1.9 kg; control: −0.45+1.4 kg), with no significant difference between groups over 9 weeks (P =.94). However, HW students reported a significantly greater increase in the number of appropriate weight control strategies than did controls (2.1+4.5 vs −1

  14. Steroid 5 α-reductase inhibitors targeting BPH and prostate cancer.

    PubMed

    Schmidt, Lucy J; Tindall, Donald J

    2011-05-01

    Steroid 5 alpha-reductase inhibitors (5ARIs) have been approved for use clinically in treatment of benign prostate hyperplasia (BPH) and accompanying lower urinary tract symptoms (LUTS) and have also been evaluated in clinical trials for prevention and treatment of prostate cancer. There are currently two steroidal inhibitors in use, finasteride and dutasteride, both with distinct pharmacokinetic properties. This review will examine the evidence presented by various studies supporting the use of these steroidal inhibitors in the prevention and treatment of prostate disease. Article from the Special issue on Targeted Inhibitors. PMID:20883781

  15. Characterization of erythrose reductases from filamentous fungi.

    PubMed

    Jovanović, Birgit; Mach, Robert L; Mach-Aigner, Astrid R

    2013-01-01

    Proteins with putative erythrose reductase activity have been identified in the filamentous fungi Trichoderma reesei, Aspergillus niger, and Fusarium graminearum by in silico analysis. The proteins found in T. reesei and A. niger had earlier been characterized as glycerol dehydrogenase and aldehyde reductase, respectively. Corresponding genes from all three fungi were cloned, heterologously expressed in Escherichia coli, and purified. Subsequently, they were used to establish optimal enzyme assay conditions. All three enzymes strictly require NADPH as cofactor, whereas with NADH no activity could be observed. The enzymatic characterization of the three enzymes using ten substrates revealed high substrate specificity and activity with D-erythrose and D-threose. The enzymes from T. reesei and A. niger herein showed comparable activities, whereas the one from F. graminearum reached only about a tenth of it for all tested substrates. In order to proof in vivo the proposed enzyme function, we overexpressed the erythrose reductase-encoding gene in T. reesei. An increased production of erythritol by the recombinant strain compared to the parental strain could be detected. PMID:23924507

  16. Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

    PubMed Central

    Ma, Yan; Ludden, Paul W.

    2001-01-01

    Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding. PMID:11114923

  17. Molecular modeling toward selective inhibitors of dihydrofolate reductase from the biological warfare agent Bacillus anthracis.

    PubMed

    Giacoppo, Juliana O S; Mancini, Daiana T; Guimarães, Ana P; Gonçalves, Arlan S; da Cunha, Elaine F F; França, Tanos C C; Ramalho, Teodorico C

    2015-02-16

    In the present work, we applied docking and molecular dynamics techniques to study 11 compounds inside the enzymes dihydrofolate reductase (DHFR) from the biological warfare agent Bacillus anthracis (BaDHFR) and Homo sapiens sapiens (HssDHFR). Six of these compounds were selected for a study with the mutant BaF96IDHFR. Our results corroborated with experimental data and allowed the proposition of a new molecule with potential activity and better selectivity for BaDHFR. PMID:24985033

  18. Exercise training prevents increased intraocular pressure and sympathetic vascular modulation in an experimental model of metabolic syndrome

    PubMed Central

    Castro, E.F.S.; Mostarda, C.T.; Rodrigues, B.; Moraes-Silva, I.C.; Feriani, D.J.; De Angelis, K.; Irigoyen, M.C.

    2015-01-01

    The present study aimed to study the effects of exercise training (ET) performed by rats on a 10-week high-fructose diet on metabolic, hemodynamic, and autonomic changes, as well as intraocular pressure (IOP). Male Wistar rats receiving fructose overload in drinking water (100 g/L) were concomitantly trained on a treadmill for 10 weeks (FT group) or kept sedentary (F group), and a control group (C) was kept in normal laboratory conditions. The metabolic evaluation comprised the Lee index, glycemia, and insulin tolerance test (KITT). Arterial pressure (AP) was measured directly, and systolic AP variability was performed to determine peripheral autonomic modulation. ET attenuated impaired metabolic parameters, AP, IOP, and ocular perfusion pressure (OPP) induced by fructose overload (FT vs F). The increase in peripheral sympathetic modulation in F rats, demonstrated by systolic AP variance and low frequency (LF) band (F: 37±2, 6.6±0.3 vs C: 26±3, 3.6±0.5 mmHg2), was prevented by ET (FT: 29±3, 3.4±0.7 mmHg2). Positive correlations were found between the LF band and right IOP (r=0.57, P=0.01) and left IOP (r=0.64, P=0.003). Negative correlations were noted between KITT values and right IOP (r=-0.55, P=0.01) and left IOP (r=-0.62, P=0.005). ET in rats effectively prevented metabolic abnormalities and AP and IOP increases promoted by a high-fructose diet. In addition, ocular benefits triggered by exercise training were associated with peripheral autonomic improvement. PMID:25714884

  19. Inhibition of cytokine-induced microvascular arrest of tumor cells by recombinant endostatin prevents experimental hepatic melanoma metastasis.

    PubMed

    Mendoza, Lorea; Valcárcel, María; Carrascal, Teresa; Egilegor, Eider; Salado, Clarisa; Sim, B Kim Lee; Vidal-Vanaclocha, Fernando

    2004-01-01

    We investigated effects of endostatin (ES) in the prometastatic microenvironment of inflammation occurring during the microvascular phase of cancer cell infiltration in the liver. We used a model of intrasplenic injection of B16 melanoma (B16M) cells leading to hepatic metastasis through vascular cell adhesion molecule-(VCAM-1)-mediated capillary arrest of cancer cells via interleukin-18 (IL-18)-dependent mechanism. We show that administration of 50 mg/kg recombinant human (rh) ES 30 min before B16M, plus repetition of same dose for 3 additional days decreased metastasis number by 60%. A single dose of rhES before B16M injection reduced hepatic microvascular retention of luciferase-transfected B16M by 40% and inhibited hepatic production of tumor necrosis factor alpha (TNF-alpha) and IL-18 and VCAM-1 expression by hepatic sinusoidal endothelia (HSE). Consistent with these data, rhES inhibited VCAM-1-dependent B16M cell adhesion to primary cultured HSE receiving B16M conditioned medium, and it abolished the HSE cell production of TNF-alpha and IL-18 induced by tumor-derived vascular endothelial cell growth factor (VEGF). rhES abrogated recombinant murine VEGF-induced tyrosine phosphorylation of KDR/flk-1 receptor in HSE cells, preventing the proinflammatory action of tumor-derived VEGF on HSE. rhES also abolished hepatic production of TNF-alpha, microvascular retention of luciferase-transfected B16M, and adhesion of B16M cells to isolated HSE cells, all of them induced in mice given 5 micro g/kg recombinant murine VEGF for 18 h. This capillary inflammation-deactivating capability constitutes a nonantiangiogenic antitumoral action of endostatin that decreases cancer cell arrest within liver microvasculature and prevents metastases promoted by proinflammatory cytokines induced by VEGF. PMID:14729638

  20. Exercise training prevents increased intraocular pressure and sympathetic vascular modulation in an experimental model of metabolic syndrome.

    PubMed

    Castro, E F S; Mostarda, C T; Rodrigues, B; Moraes-Silva, I C; Feriani, D J; De Angelis, K; Irigoyen, M C

    2015-04-01

    The present study aimed to study the effects of exercise training (ET) performed by rats on a 10-week high-fructose diet on metabolic, hemodynamic, and autonomic changes, as well as intraocular pressure (IOP). Male Wistar rats receiving fructose overload in drinking water (100 g/L) were concomitantly trained on a treadmill for 10 weeks (FT group) or kept sedentary (F group), and a control group (C) was kept in normal laboratory conditions. The metabolic evaluation comprised the Lee index, glycemia, and insulin tolerance test (KITT). Arterial pressure (AP) was measured directly, and systolic AP variability was performed to determine peripheral autonomic modulation. ET attenuated impaired metabolic parameters, AP, IOP, and ocular perfusion pressure (OPP) induced by fructose overload (FT vs F). The increase in peripheral sympathetic modulation in F rats, demonstrated by systolic AP variance and low frequency (LF) band (F: 37±2, 6.6±0.3 vs C: 26±3, 3.6±0.5 mmHg2), was prevented by ET (FT: 29±3, 3.4±0.7 mmHg2). Positive correlations were found between the LF band and right IOP (r=0.57, P=0.01) and left IOP (r=0.64, P=0.003). Negative correlations were noted between KITT values and right IOP (r=-0.55, P=0.01) and left IOP (r=-0.62, P=0.005). ET in rats effectively prevented metabolic abnormalities and AP and IOP increases promoted by a high-fructose diet. In addition, ocular benefits triggered by exercise training were associated with peripheral autonomic improvement. PMID:25714884

  1. Efficacy and mechanism of tanshinone IIA liquid nanoparticles in preventing experimental postoperative peritoneal adhesions in vivo and in vitro

    PubMed Central

    Qin, Fei; Ma, Yun; Li, Xiao; Wang, Xian; Wei, Yuanyi; Hou, Chuqi; Lin, Si; Hou, Lianbing; Wang, Chengxi

    2015-01-01

    Up to 90% of patients develop adhesion following laparotomy. Upregulating fibrinolysis within the peritoneum reduces adhesions. Tanshinone IIA (Tan IIA) promotes fibrinolysis in hepatic fibrosis and the cardiovascular system and may play a role in preventing adhesions. We report preparation and characterization of liquid nanoparticles of Tan IIA for intravenous administration and investigate its feasibility in clinical practice. Tan IIA liquid nanoparticles (Tan IIA-NPs) were prepared using the emulsion/solvent evaporation method. Adhesions were induced in Sprague–Dawley rats by injuring the parietal peritoneum and cecum, followed by intravenous administration of various Tan IIA-NP dosages. The adhesion scores for each group were collected 7 days after the initial laparotomy. The activity of tissue-type plasminogen activator (tPA) was measured from the peritoneal lavage fluid. The messenger RNA and protein expression levels of plasminogen activator inhibitor-1 (PAI-1) were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. TGF-β1 and collagen I expressions were measured immunohistochemically in the ischemic tissues. The effects of Tan IIA-NPs and free-Tan IIA on tPA and PAI-1 were measured in vitro in TGF-β1-induced HMrSV5 cells. Tan IIA-NPs exhibited small particle size, high encapsulation efficiency, good stability for storage, and safety for intravenous administration. Tan IIA-NPs were effective in preventing adhesion. Tan IIA-NPs increased tPA activity in peritoneal lavage fluid, and tPA mRNA and protein expression, and decreased PAI-1 mRNA and protein expression in the ischemic tissues. Moreover, Tan IIA-NPs decreased TGF-β1 and collagen I expressions in the ischemic tissues. Tan IIA-NPs administered via tail veins upregulated fibrinolysis in the peritoneum. In vitro studies showed that these effects may be mediated by the TGF-β signal pathway. PMID:26056449

  2. Identification of imine reductase-specific sequence motifs.

    PubMed

    Fademrecht, Silvia; Scheller, Philipp N; Nestl, Bettina M; Hauer, Bernhard; Pleiss, Jürgen

    2016-05-01

    Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database (www.IRED.BioCatNet.de) was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED-Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx5 [ATS]x4 Gx4 [VIL]WNR[TS]x2 [KR] and the active site motif Gx[DE]x[GDA]x[APS]x3 {K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes. Proteins 2016; 84:600-610. © 2016 Wiley Periodicals, Inc. PMID:26857686

  3. Total Saponin from Anemone flaccida Fr. Schmidt Prevents Bone Destruction in Experimental Rheumatoid Arthritis via Inhibiting Osteoclastogenesis.

    PubMed

    Liu, Chunfang; Yang, Yue; Sun, Danni; Wang, Chao; Wang, Hui; Jia, Shiwei; Liu, Liang; Lin, Na

    2015-12-01

    Anemone flaccida Fr. Schmidt is used in the clinical compound prescription for the treatment of rheumatoid arthritis (RA) in China and has the traditional use of draining dampness, diminishing swelling, and relieving pain. Total saponins (TS) are the characteristic components and also the main active ingredients of A. flaccida. Previous reports indicated that TS possess anti-inflammatory and immunoregulatory properties; however, the effects of TS on bone destruction of RA have not been evaluated. In this study, our data first showed the therapeutic effects of TS on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) rats. Then, by microfocal computed tomography (CT) quantification, TS significantly increased bone mineral density, bone volume fraction, and trabecular thickness and decreased trabecular separation of inflamed joints both at peri-articular and extra-articular locations. TS also diminished the level of the bone resorption marker CTX-I and simultaneously increased the bone formation marker osteocalcin in sera of CIA rats. Interestingly, TS prevented bone destruction by reducing the number of osteoclasts in inflamed joints, reducing the expression of receptor activator of nuclear factor-κF (RANK) ligand (RANKL) and RANK, increasing the expression of osteoprotegerin (OPG), at both mRNA and protein levels, and decreasing the ratio of RANKL to OPG in inflamed joints and sera of CIA rats. This was further confirmed in the co-culture system of human fibroblast-like synovial and peripheral blood mononuclear cells. In addition, TS inhibited the levels of pro-inflammatory cytokines implicated in bone resorption, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), IL-6, IL-17, and IL-23 in sera and joints. These findings offer convincing evidence that TS attenuate RA partially by preventing both focal bone destruction and systemic bone loss. This anti-erosive effect results in part from inhibiting osteoclastogenesis

  4. The Role of Eugenol in the Prevention of Acute Pancreatitis-Induced Acute Kidney Injury: Experimental Study

    PubMed Central

    Markakis, Charalampos; Tsaroucha, Alexandra; Papalois, Apostolos E.; Lambropoulou, Maria; Spartalis, Eleftherios; Tsigalou, Christina; Romanidis, Konstantinos; Simopoulos, Constantinos

    2016-01-01

    Aim. Acute pancreatitis is an inflammatory intra-abdominal disease, which takes a severe form in 15–20% of patients and can result in high mortality especially when complicated by acute renal failure. The aim of this study is to assess the possible reduction in the extent of acute kidney injury after administration of eugenol in an experimental model of acute pancreatitis. Materials and Methods. 106 male Wistar rats weighing 220–350 g were divided into 3 groups: (1) Sham, with sham surgery; (2) Control, with induction of acute pancreatitis, through ligation of the biliopancreatic duct; and (3) Eugenol, with induction of acute pancreatitis and eugenol administration at a dose of 15 mg/kg. Serum urea and creatinine, histopathological changes, TNF-α, IL-6, and MPO activity in the kidneys were evaluated at predetermined time intervals. Results. The group that was administered eugenol showed milder histopathological changes than the Control group, TNF-α activity was milder in the Eugenol group, and there was no difference in activity for MPO and IL-6. Serum urea and creatinine levels were lower in the Eugenol group than in the Control group. Conclusions. Eugenol administration was protective for the kidneys in an experimental model of acute pancreatitis in rats. PMID:26884642

  5. Prevention and Mitigation of Experimental Autoimmune Encephalomyelitis by Murine β-Defensins via Induction of Regulatory T Cells.

    PubMed

    Bruhs, Anika; Schwarz, Thomas; Schwarz, Agatha

    2016-01-01

    The antimicrobial peptide murine β-defensin-14 (mBD14) was found to exert, in addition to its antimicrobial activity, the capacity to induce regulatory T cells as demonstrated in the model of contact hypersensitivity. Because it is induced by ultraviolet radiation, mBD14 may contribute to the antigen-specific immunosuppression by ultraviolet radiation. To prove whether this applies also for other immunologic models and because ultraviolet radiation appears to have beneficial effects on multiple sclerosis, we utilized the model of experimental autoimmune encephalomyelitis. Injection of mBD14 into mice before immunization with myelin oligodendrocyte glycoprotein caused amelioration of the disease with less central nervous system inflammation and decreased levels of proinflammatory cytokines and cytotoxic T cells. The beneficial effect was due to Foxp3(+) regulatory T cells because it was lost on in vivo depletion of regulatory T cells. mBD14, however, also acts in a therapeutic setting, because injection of mBD14 into mice with clinical features of experimental autoimmune encephalomyelitis reduced the clinical score significantly. Human β-defensin-3, the human orthologue of mBD14, induced in vitro regulatory T cell-specific markers in CD4(+)CD25(-) T cells, shifting these nonregulatory cells into a regulatory phenotype with suppressive features. Thus, defensins may represent candidates worth being further pursued for the therapy of multiple sclerosis. PMID:26763437

  6. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    SciTech Connect

    Iyanagi, Takashi . E-mail: iyanagi@spring8.or.jp

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.

  7. Indian food ingredients and cancer prevention - an experimental evaluation of anticarcinogenic effects of garlic in rat colon.

    PubMed

    Sengupta, Archana; Ghosh, Samit; Bhattacharjee, Shamee; Das, Sukta

    2004-01-01

    The major food items of Indian cuisine include rice, wheat, diary products, and abundant fruits and vegetables. Beside these, there are several kinds of herbs and spices as important ingredients, containing many phytochemicals with medicinal properties, adding taste to Indian cuisine. An impressive body of data exists in support of the concept that Indian food ingredients can be used in preventive strategies aimed at reducing the incidence and mortality of different types of cancers because of their antioxidative, antimutagenic and anticarcinogenic properties. Vital ingredients used in Indian cooking include turmeric, cloves, ginger, aniseed, mustard, saffron, cardamom and garlic Garlic is an indispensable ingredient of Indian food and this report concerns the chemopreventive efficacy of garlic in an azoxymethane induced rodent colon carcinogenesis model. The effect of garlic was evaluated in terms of aberrant crypt foci, putative preneoplastic lesions in the colon. In addition, cell proliferation and levels of apoptosis were determined and the expression of cyclooxygenase-2 protein was analyzed. Following treatment, significant inhibition of cell proliferation and induction of apoptosis, as well as suppression of cyclooxygenase-2 activity were observed, associated with significant reduction in the incidence of aberrant crypt foci. The study points to combined protective effects of garlic components on colon carcinogenesis. PMID:15244513

  8. Peptidoglycan Recognition Proteins Protect Mice from Experimental Colitis by Promoting Normal Gut Flora and Preventing Induction of Interferon-γ

    PubMed Central

    Saha, Sukumar; Jing, Xuefang; Park, Shin Yong; Wang, Shiyong; Li, Xinna; Gupta, Dipika; Dziarski, Roman

    2010-01-01

    SUMMARY There are multiple mechanisms of maintaining tolerance in the gut that protect the intestine from excessive inflammatory response to intestinal microorganisms. We report here that all four mammalian Peptidoglycan Recognition Proteins (PGRPs or Pglyrps) protect the host from colitis induced by dextran sulfate sodium (DSS). Pglyrp1−/−, Pglyrp2−/−, Pglyrp3−/−, and Pglyrp4−/− mice are all more sensitive than wild type (WT) mice to DSS-induced colitis due to changes to more inflammatory gut microflora, higher production of interferon-γ and interferon-inducible genes, and increase in NK cells in the colon upon initial exposure to DSS, which leads to severe hyperplasia of the lamina propria, loss of epithelial cells, and ulceration in the colon. Thus in WT mice PGRPs protect the colon from early inflammatory response and loss of the barrier function of intestinal epithelium by promoting normal bacterial flora and by preventing damaging production of interferon-γ by NK cells in response to injury. PMID:20709292

  9. Reduction of tetrathionate by mammalian thioredoxin reductase

    PubMed Central

    Narayan, Vivek; Kudva, Avinash K.; Prabhu, K. Sandeep

    2016-01-01

    Tetrathionate, a polythionate oxidation product of microbial hydrogen sulfide and reactive oxygen species from immune cells in the gut, serves as a terminal electron acceptor to confer growth advantage for Salmonella and other enterobacteria. Here we show that the rat liver selenoen-zyme thioredoxin reductase (Txnrd1; TR1) efficiently reduces tetrathionate in vitro. Furthermore, lysates of selenium-supplemented murine macrophages also displayed activity towards tetrathionate, while cells lacking TR1 were unable to reduce tetrathionate. These studies suggest that upregulation of TR1 expression, via selenium supplementation, may modulate the gut microbiome, particularly during inflammation, by regulating the levels of tetrathionate. PMID:26252619

  10. [Prevention of osteoporosis by foods and dietary supplements. Milk basic protein (MBP) induces alveolar bone formation in rat experimental periodontitis].

    PubMed

    Seto, Hiroyuki; Nagata, Toshihiko

    2006-10-01

    Periodontitis is a chronic inflammatory disease caused by infection of periodontopathic bacteriae, which induced alveolar bone resorpotion. Milk basic protein (MBP) has been reported to be useful as a supplement because of increasing bone formation in animal and human studies. We examined the effect of MBP for alveolar bone formation in rat experimental periodontitis. After alveolar bone resorption was induced by ligature technique, the diets containing low and high dose of MBP were given to rats for 90 days. Micro-focus computed tomography and histological observation revealed a recovery of alveolar bone in high-dose MBP group compared to the control group. Osteoid thickness of alveolar bone crest significantly increased in low and high-dose MBP groups. These findings indicate that MBP may be effective for the recovery of alveolar bone resorption in periodontitis. PMID:17012815

  11. HDAC6 Regulates the Chaperone-Mediated Autophagy to Prevent Oxidative Damage in Injured Neurons after Experimental Spinal Cord Injury

    PubMed Central

    Su, Min; Guan, Huaqing; Zhang, Fan; Gao, Yarong; Teng, Xiaomei; Yang, Weixin

    2016-01-01

    Hypoxia-ischemia- (HI-) induced oxidative stress plays a role in secondary pathocellular processes of acute spinal cord injury (SCI) due to HI from many kinds of mechanical trauma. Increasing evidence suggests that the histone deacetylase-6 (HDAC6) plays an important role in cell homeostasis in both physiological and abnormal, stressful, pathological conditions. This paper found that inhibition of HDAC6 accelerated reactive oxygen species (ROS) generation and cell apoptosis in response to the HI. Deficiency of HDAC6 hindered the chaperone-mediated autophagy (CMA) activity to resistance of HI-induced oxidative stress. Furthermore, this study provided the experimental evidence for the potential role of HDAC6 in the regulation of CMA by affecting HSP90 acetylation. Therefore, HDAC6 plays an important role in the function of CMA pathway under the HI stress induced by SCI and it may be a potential therapeutic target in acute SCI model. PMID:26649145

  12. Hygiene in the prevention of udder infections. V. Efficacy of teat dips under experimental exposure to mastitis pathogens.

    PubMed

    Philpot, W N; Pankley, J W

    1978-07-01

    Efficacy (reduction of new intramammary infections) was determined against Staphylococcus aureus in 14 trials and against Streptococcus agalactiae in 5 trials. Against Staphylococcus aureus seven 1% iodophor products had an average efficacy of 89.9% while three .5% iodophor products averaged 76.4%. A solution of sodium dichloro-s-triazenetrione reduced infections 79.0% and 2% dodecyl benzene sulfonic acid was 50.4% effective. Efficacies of two quaternary ammonium products at .18 and .5% were 84.8 and 60.9%. A placebo vehicle and .15 M saline as teat dips reduced infections 8.9 and 9.2%. Infection rates with Streptococcus agalactiae were low; efficacy ranged from 50.5 to 100.0%, average 65.6%. The model of experimental exposure provided a better estimate of effectiveness of a teat dip than would be possible under conditions of most commercial dairying. PMID:359605

  13. Does cholesterol lowering prevent stroke?

    PubMed

    Henry, R Y; Kendall, M J

    1998-10-01

    The importance of lowering plasma cholesterol to reduce the incidence of coronary events is well established. However, in the prevention of stroke disease, control of hypertension has been the main aim of treatment and lipid lowering therapy has not hitherto been considered to be desirable or necessary. In this review, the evidence from large multicentre trials, imaging studies and meta-analyses is presented. It shows convincingly that HMG-CoA reductase inhibitors (Statins) reduce stroke risk. PMID:9875681

  14. L-glutamine supplementation prevents the development of experimental diabetic cardiomyopathy in streptozotocin-nicotinamide induced diabetic rats.

    PubMed

    Badole, Sachin L; Jangam, Ganesh B; Chaudhari, Swapnil M; Ghule, Arvindkumar E; Zanwar, Anand A

    2014-01-01

    The objective of the present investigation was to evaluate the effect of L-glutamine on cardiac myopathy in streptozotocin-nicotinamide induced diabetic rats. Diabetes was induced in overnight fasted Sprague Dawely rats by using intraperitonial injection of streptozotocin (55 mg/kg). Nicotinamide (100 mg/kg, i.p.) was administered 20 min before administration of streptozotocin. Experimental rats were divided into Group I: non-diabetic control (distilled water; 10 ml/kg, p.o.), II: diabetic control (distilled water, 10 ml/kg, p.o.), III: L-glutamine (500 mg/kg, p.o.) and IV: L-glutamine (1000 mg/kg, p.o.). All groups were diabetic except group I. The plasma glucose level, body weight, electrocardiographic abnormalities, hemodynamic changes and left ventricular contractile function, biological markers of cardiotoxicity, antioxidant markers were determined after 4 months after STZ with nicotinamide injection. Histopathological changes of heart tissue were carried out by using H and E stain. L-glutamine treatment improved the electrocardiographic, hemodynamic changes; LV contractile function; biological markers; oxidative stress parameters and histological changes in STZ induced diabetic rats. Results from the present investigation demonstrated that L-glutamine has seemed a cardioprotective activity. PMID:24651718

  15. The preventive effect of Brassica napus L. oil on pathophysiological changes of respiratory system in experimental asthmatic rat

    PubMed Central

    Kabiri rad, Mehdi; Neamati, Ali; Boskabady, Mohammad Hossein; Mahdavi-Shahri, Naser; Mahmoudabady, Maryam

    2013-01-01

    Objective: Asthma is an airway complex disease defined by reversible airway narrowing and obstruction, chronic airway inflammation, airway hyperresponsiveness, and tissue remodeling. The purpose of this study was to determine the effect of Brassica napus L. (B. napus) on airway pathologic changes in a rat model of asthma. Materials and Methods: Twenty-four rats were divided into 4 groups: control, asthmatic, asthmatic treated with 0.5 mg/kg B. napus oil, and asthmatic treated with 0.75 mg/kg B. napus oil. To induce the experimental asthma, rats in groups 2, 3, and 4 received an i.p. injection of ovalbumin and aerosolized ovalbumin. Simultaneously, rats in groups 3 and 4 received B. napus oil daily by gavage. After 31 days, in all groups, thoracotomy was done and lung tissue samples were taken. For pathological evaluation, microscopic slides were prepared. The eosinophil numbers in the submucosal layer and thicknesses of smooth muscle layer of bronchioles were detected. Results: Eosinophil numbers in the submucosal layer, as well as smooth muscle layer thicknesses were significantly lower in the rat group treated with 0.75 mg/kg B. napus oil as compared with asthmatic group (p<0.01, p<0.05). Conclusion: B. napus could be useful as adjuvant therapy in rat model of asthma. This effect was probably related to its antioxidants components that reduce the levels of inflammatory mediators such as leukotrienes, IL-4, IL-5, and IL-13. PMID:25050259

  16. Suppression of electron transfer to dioxygen by charge transfer and electron transfer complexes in the FAD-dependent reductase component of toluene dioxygenase.

    PubMed

    Lin, Tzong-Yuan; Werther, Tobias; Jeoung, Jae-Hun; Dobbek, Holger

    2012-11-01

    The three-component toluene dioxygenase system consists of an FAD-containing reductase, a Rieske-type [2Fe-2S] ferredoxin, and a Rieske-type dioxygenase. The task of the FAD-containing reductase is to shuttle electrons from NADH to the ferredoxin, a reaction the enzyme has to catalyze in the presence of dioxygen. We investigated the kinetics of the reductase in the reductive and oxidative half-reaction and detected a stable charge transfer complex between the reduced reductase and NAD(+) at the end of the reductive half-reaction, which is substantially less reactive toward dioxygen than the reduced reductase in the absence of NAD(+). A plausible reason for the low reactivity toward dioxygen is revealed by the crystal structure of the complex between NAD(+) and reduced reductase, which shows that the nicotinamide ring and the protein matrix shield the reactive C4a position of the isoalloxazine ring and force the tricycle into an atypical planar conformation, both factors disfavoring the reaction of the reduced flavin with dioxygen. A rapid electron transfer from the charge transfer complex to electron acceptors further reduces the risk of unwanted side reactions, and the crystal structure of a complex between the reductase and its cognate ferredoxin shows a short distance between the electron-donating and -accepting cofactors. Attraction between the two proteins is likely mediated by opposite charges at one large patch of the complex interface. The stability, specificity, and reactivity of the observed charge transfer and electron transfer complexes are thought to prevent the reaction of reductase(TOL) with dioxygen and thus present a solution toward conflicting requirements. PMID:22992736

  17. Biliverdin reductase: a target for cancer therapy?

    PubMed Central

    Gibbs, Peter E. M.; Miralem, Tihomir; Maines, Mahin D.

    2015-01-01

    Biliverdin reductase (BVR) is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s) of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1, and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ, and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation. PMID:26089799

  18. Flavodiiron Oxygen Reductase from Entamoeba histolytica

    PubMed Central

    Gonçalves, Vera L.; Vicente, João B.; Pinto, Liliana; Romão, Célia V.; Frazão, Carlos; Sarti, Paolo; Giuffrè, Alessandro; Teixeira, Miguel

    2014-01-01

    Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr271 and Lys53 in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2, pointing to a role of the mutated residues in substrate selectivity. PMID:25151360

  19. Effectiveness of anonymised information sharing and use in health service, police, and local government partnership for preventing violence related injury: experimental study and time series analysis

    PubMed Central

    Florence, Curtis; Brennan, Iain; Simon, Thomas

    2011-01-01

    Objective To evaluate the effectiveness of anonymised information sharing to prevent injury related to violence. Design Experimental study and time series analysis of a prototype community partnership between the health service, police, and local government partners designed to prevent violence. Setting Cardiff, Wales, and 14 comparison cities designated “most similar” by the Home Office in England and Wales. Intervention After a 33 month development period, anonymised data relevant to violence prevention (precise violence location, time, days, and weapons) from patients attending emergency departments in Cardiff and reporting injury from violence were shared over 51 months with police and local authority partners and used to target resources for violence prevention. Main outcome measures Health service records of hospital admissions related to violence and police records of woundings and less serious assaults in Cardiff and other cities after adjustment for potential confounders. Results Information sharing and use were associated with a substantial and significant reduction in hospital admissions related to violence. In the intervention city (Cardiff) rates fell from seven to five a month per 100 000 population compared with an increase from five to eight in comparison cities (adjusted incidence rate ratio 0.58, 95% confidence interval 0.49 to 0.69). Average rate of woundings recorded by the police changed from 54 to 82 a month per 100 000 population in Cardiff compared with an increase from 54 to 114 in comparison cities (adjusted incidence rate ratio 0.68, 0.61 to 0.75). There was a significant increase in less serious assaults recorded by the police, from 15 to 20 a month per 100 000 population in Cardiff compared with a decrease from 42 to 33 in comparison cities (adjusted incidence rate ratio 1.38, 1.13 to 1.70). Conclusion An information sharing partnership between health services, police, and local government in Cardiff, Wales, altered policing

  20. 5alpha-reductase inhibitors in benign prostatic hyperplasia and prostate cancer risk reduction.

    PubMed

    Rittmaster, Roger S

    2008-04-01

    Androgens play an essential role in prostatic development and function, but are also involved in prostate disease pathogenesis. The primary prostatic androgen, dihydrotestosterone (DHT), is synthesized from testosterone by 5alpha-reductase types 1 and 2. Inhibition of the 5alpha-reductase isoenzymes therefore has potential therapeutic benefit in prostate disease. The two currently approved 5alpha-reductase inhibitors (5ARIs), finasteride and dutasteride, have demonstrated long-term efficacy and safety in the treatment of benign prostatic hyperplasia. Finasteride, a type-2 5ARI, has also been studied for its ability to reduce the incidence of biopsy-detectable prostate cancer in the Prostate Cancer Prevention Trial. Treatment with dutasteride, a dual 5ARI, has been shown to result in a greater degree and consistency of DHT suppression compared with finasteride. Two large-scale studies of dutasteride are currently investigating the role of near-maximal DHT suppression in the settings of prostate cancer risk reduction and expectant management of localized prostate cancer. PMID:18471794

  1. Glutathione reductase gsr-1 is an essential gene required for Caenorhabditis elegans early embryonic development.

    PubMed

    Mora-Lorca, José Antonio; Sáenz-Narciso, Beatriz; Gaffney, Christopher J; Naranjo-Galindo, Francisco José; Pedrajas, José Rafael; Guerrero-Gómez, David; Dobrzynska, Agnieszka; Askjaer, Peter; Szewczyk, Nathaniel J; Cabello, Juan; Miranda-Vizuete, Antonio

    2016-07-01

    Glutathione is the most abundant thiol in the vast majority of organisms and is maintained in its reduced form by the flavoenzyme glutathione reductase. In this work, we describe the genetic and functional analysis of the Caenorhabditis elegans gsr-1 gene that encodes the only glutathione reductase protein in this model organism. By using green fluorescent protein reporters we demonstrate that gsr-1 produces two GSR-1 isoforms, one located in the cytoplasm and one in the mitochondria. gsr-1 loss of function mutants display a fully penetrant embryonic lethal phenotype characterized by a progressive and robust cell division delay accompanied by an aberrant distribution of interphasic chromatin in the periphery of the cell nucleus. Maternally expressed GSR-1 is sufficient to support embryonic development but these animals are short-lived, sensitized to chemical stress, have increased mitochondrial fragmentation and lower mitochondrial DNA content. Furthermore, the embryonic lethality of gsr-1 worms is prevented by restoring GSR-1 activity in the cytoplasm but not in mitochondria. Given the fact that the thioredoxin redox systems are dispensable in C. elegans, our data support a prominent role of the glutathione reductase/glutathione pathway in maintaining redox homeostasis in the nematode. PMID:27117030

  2. Inhibitory effects of Colocasia esculenta (L.) Schott constituents on aldose reductase.

    PubMed

    Li, Hong Mei; Hwang, Seung Hwan; Kang, Beom Goo; Hong, Jae Seung; Lim, Soon Sung

    2014-01-01

    The goal of this study was to determine the rat lens aldose reductase-inhibitory effects of 95% ethanol extracts from the leaves of C. esculenta and, its organic solvent soluble fractions, including the dichloromethane (CH2Cl2), ethyl acetate (EtOAc), n-butanol (BuOH) and water (H2O) layers, using dl-glyceraldehyde as a substrate. Ten compounds, namely tryptophan (1), orientin (2), isoorientin (3), vitexin (4), isovitexin (5), luteolin-7-O-glucoside (6), luteolin-7-O-rutinoside (7), rosmarinic acid (8), 1-O-feruloyl-d-glucoside (9) and 1-O-caffeoyl-d-glucoside (10) were isolated from the EtOAc and BuOH fractions of C. esculenta. The structures of compounds 1-10 were elucidated by spectroscopic methods and comparison with previous reports. All the isolates were subjected to an in vitro bioassay to evaluate their inhibitory activity against rat lens aldose reductase. Among tested compounds, compounds 2 and 3 significantly inhibited rat lens aldose reductase, with IC50 values of 1.65 and 1.92 μM, respectively. Notably, the inhibitory activity of orientin was 3.9 times greater than that of the positive control, quercetin (4.12 μM). However, the isolated compounds showed only moderate ABTS+ [2,29-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] activity. These results suggest that flavonoid derivatives from Colocasia esculenta (L.) Schott represent potential compounds for the prevention and/or treatment of diabetic complications. PMID:25255750

  3. Modification of dinitrogenase reductase in the cyanobacterium Anabaena variabilis due to C starvation and ammonia.

    PubMed

    Ernst, A; Reich, S; Böger, P

    1990-02-01

    In the heterocystous cyanobacterium Anabaena variabilis, a change in nitrogenase activity and concomitant modification of dinitrogenase reductase (the Fe protein of nitrogenase) was induced either by NH4Cl at pH 10 (S. Reich and P. Böger, FEMS Microbiol. Lett. 58:81-86, 1989) or by cessation of C supply resulting from darkness, CO2 limitation, or inhibition of photosystem II activity. Modification induced by both C limitation and NH4Cl was efficiently prevented by anaerobic conditions. Under air, endogenously stored glycogen and added fructose protected against modification triggered by C limitation but not by NH4Cl. With stored glycogen present, dark modification took place after inhibition of respiration by KCN. Reactivation of inactivated nitrogenase and concomitant demodification of dinitrogenase reductase occurred after restoration of diazotrophic growth conditions. In previously C-limited cultures, reactivation was also observed in the dark after addition of fructose (heterotrophic growth) and under anaerobiosis upon reillumination in the presence of a photosynthesis inhibitor. The results indicate that modification of dinitrogenase reductase develops as a result of decreased carbohydrate-supported reductant supply of the heterocysts caused by C limitation or by increased diversion of carbohydrates towards ammonia assimilation. Apparently, a product of N assimilation such as glutamine is not necessary for modification. The increase of oxygen concentration in the heterocysts is a plausible consequence of all treatments causing Fe protein modification. PMID:2105302

  4. Aldose Reductase Inhibitory Activity of Compounds from  Zea mays L.

    PubMed Central

    Kim, Tae Hyeon; Kim, Jin Kyu; Kang, Young-Hee; Lee, Jae-Yong; Kang, Il Jun; Lim, Soon Sung

    2013-01-01

    Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1–7) and 5 anthocyanins (compound 8–12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC50, 4.78 μM). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. PMID:23586057

  5. The Effectiveness of Crataegus orientalis M Bieber. (Hawthorn) Extract Administration in Preventing Alveolar Bone Loss in Rats with Experimental Periodontitis

    PubMed Central

    Hatipoğlu, Mükerrem; Sağlam, Mehmet; Köseoğlu, Serhat; Köksal, Ekrem; Keleş, Ali; Esen, Hacı Hasan

    2015-01-01

    The purpose of this animal study was to evaluate the effects of hawthorn (Crataeus orientalis M Bieber.) extract on serum oxidative status and alveolar bone loss in experimental periodontitis. Twenty-seven Wistar rats were assigned to one of the following groups: non- ligated+placebo (saline) (NL, n = 9), ligature only+placebo (saline) (LO, n = 9), and ligature and treated with hawthorn extract in saline (H, n = 9) (100 mg/kg orogastrically, once a day for 11 days). Periodontitis was induced by submerging a 4/0 silk ligature in the sulcus of the mandibular right first molars of rats, and the animals were sacrificed after 11 days. Micro-CT examinations were performed for linear and volumetric parameter assessment of alveolar bone. Periodontal tissues were histopathologically examined to assess the differences among the study groups. Levels of serum total antioxidant status (TAS)/total oxidant status (TOS), and oxidative stress index (OSI) were also analyzed. Alveolar bone loss was significantly reduced by hawthorn administration compared to LO group (p<0.05). The number of inflammatory cells and osteoclasts in the LO group was significantly higher than that of the NL and H groups (p< 0.05). The number of osteoblasts in the LO and H groups was significantly higher than that of the NL group (p<0.05). TOS and OSI levels were significantly reduced in H group compared to LO group (P <0.05) and TAS levels were similar in H and NL group (p< 0.05). Hawthorn extract showed inhibitory effect on periodontal inflammation and alveolar bone loss by regulating TAS, TOS and OSI levels in periodontal disease in rats when administered systemically. PMID:26030160

  6. The Effectiveness of Crataegus orientalis M Bieber. (Hawthorn) Extract Administration in Preventing Alveolar Bone Loss in Rats with Experimental Periodontitis.

    PubMed

    Hatipoğlu, Mükerrem; Sağlam, Mehmet; Köseoğlu, Serhat; Köksal, Ekrem; Keleş, Ali; Esen, Hacı Hasan

    2015-01-01

    The purpose of this animal study was to evaluate the effects of hawthorn (Crataeus orientalis M Bieber.) extract on serum oxidative status and alveolar bone loss in experimental periodontitis. Twenty-seven Wistar rats were assigned to one of the following groups: non- ligated+placebo (saline) (NL, n = 9), ligature only+placebo (saline) (LO, n = 9), and ligature and treated with hawthorn extract in saline (H, n = 9) (100 mg/kg orogastrically, once a day for 11 days). Periodontitis was induced by submerging a 4/0 silk ligature in the sulcus of the mandibular right first molars of rats, and the animals were sacrificed after 11 days. Micro-CT examinations were performed for linear and volumetric parameter assessment of alveolar bone. Periodontal tissues were histopathologically examined to assess the differences among the study groups. Levels of serum total antioxidant status (TAS)/total oxidant status (TOS), and oxidative stress index (OSI) were also analyzed. Alveolar bone loss was significantly reduced by hawthorn administration compared to LO group (p<0.05). The number of inflammatory cells and osteoclasts in the LO group was significantly higher than that of the NL and H groups (p< 0.05). The number of osteoblasts in the LO and H groups was significantly higher than that of the NL group (p<0.05). TOS and OSI levels were significantly reduced in H group compared to LO group (P <0.05) and TAS levels were similar in H and NL group (p< 0.05). Hawthorn extract showed inhibitory effect on periodontal inflammation and alveolar bone loss by regulating TAS, TOS and OSI levels in periodontal disease in rats when administered systemically. PMID:26030160

  7. Modulation of haemostatic function and prevention of experimental thrombosis by red wine in rats: a role for increased nitric oxide production

    PubMed Central

    Wollny, Tomasz; Aiello, Luca; Di Tommaso, Donata; Bellavia, Vincenzo; Rotilio, Domenico; Donati, Maria Benedetta; de Gaetano, Giovanni; Iacoviello, Licia

    1999-01-01

    The effects of ethyl alcohol and wine (red and white) on haemostatic parameters and experimental thrombosis were studied in rats; NO was evaluated as a possible mediator of these effects. We found that red wine (12% alcohol) supplementation (8.4±0.4 ml d−1 in drinking water, for 10 days) induced a marked prolongation of ‘template' bleeding time (BT) (258±13 vs 132±13 s in controls; P<0.001), a decrease in platelet adhesion to fibrillar collagen (11.6±1.0 vs 32.2±1.3%; P<0.01) and a reduction in thrombus weight (1.45±0.33 vs 3.27±0.39 mg; P<0.01). Alcohol-free red wine showed an effect similar to red wine. In contrast, neither ethyl alcohol (12%) nor white wine (12% alcohol) affected these systems. All these effects were also observed after red wine i.v. injection (1 ml kg−1 of 1 : 4 dilution) 15 min before the experiments. The effects of red wine were prevented by the NO inhibitor, Nωnitro-L-arginine-methyl ester (L-NAME). L-arginine, not D-arginine, reversed the effect of L-NAME on red wine infusion. Red wine injection induced a 3 fold increase in total radical-trapping antioxidant parameter values of rat plasma with respect to controls, while white wine and alcohol did not show any effect. Our study provides evidence that red wine modulates primary haemostasis and prevents experimental thrombosis in rats, independently of its alcohol content, by a NO-mediated mechanism. PMID:10401566

  8. Infusion of Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate-Conjugated MOG35–55-Coupled Spleen Cells Effectively Prevents and Reverses Experimental Autoimmune Encephalomyelitis in Mice

    PubMed Central

    Zhang, Lanfang; Guo, Yixian; Xia, Chang-Qing

    2015-01-01

    In this study, we have evaluated our recently developed method for antigen-cell coupling using sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) heterobifunctional crosslinker in prevention and reversal of experimental autoimmune encephalomyelitis (EAE). We demonstrate that infusion of MOG35–55-coupled spleen cells (MOG-SP) significantly prevents and reverses EAE. Further studies show that the protected animals exhibit significantly delayed EAE upon EAE reinduction. Moreover, adoptive transfer of CD4+ T cells from the protected mice to naïve syngeneic mice renders the recipient mice resistant to EAE induction. Unexpectedly, CD4+ T cell proliferation is similar upon ex vivo stimulation by MOG35–55 amongst all groups. However, further analysis of those proliferating CD4+ T cells shows remarkable differences in Foxp3+ regulatory T cells (70% in MOG-SP groups versus 10–25% in control groups) and in IL-17+ cells (2-3% in MOG-SP groups versus 6–9% in control groups). In addition, we discover that MOG-SP treatment also significantly attenuates MOG35–55-responding IFN-γ-producing Th1 cells. These findings suggest that MOG-SP treatment induces EAE protective MOG35–55-specific regulatory T cells and suppresses EAE pathogenic Th17 and Th1 cells. Our study provides a novel approach for antigen-based EAE immunotherapy, which can potentially be translated into clinical application for immunotherapy of multiple sclerosis. PMID:26258148

  9. Aldose reductase inhibition improves nerve conduction velocity in diabetic patients.

    PubMed

    Judzewitsch, R G; Jaspan, J B; Polonsky, K S; Weinberg, C R; Halter, J B; Halar, E; Pfeifer, M A; Vukadinovic, C; Bernstein, L; Schneider, M; Liang, K Y; Gabbay, K H; Rubenstein, A H; Porte, D

    1983-01-20

    To assess the potential role of polyol-pathway activity in diabetic neuropathy, we measured the effects of sorbinil--a potent inhibitor of the key polyol-pathway enzyme aldose reductase--on nerve conduction velocity in 39 stable diabetics in a randomized, double-blind, cross-over trial. During nine weeks of treatment with sorbinil (250 mg per day), nerve conduction velocity was greater than during a nine-week placebo period for all three nerves tested: the peroneal motor nerve (mean increase [+/- S.E.M.], 0.70 +/- 0.24 m per second, P less than 0.008), the median motor nerve (mean increase, 0.66 +/- 0.27, P less than 0.005), and the median sensory nerve (mean increase, 1.16 +/- 0.50, P less than 0.035). Conduction velocity for all three nerves declined significantly within three weeks after cessation of the drug. These effects of sorbinil were not related to glycemic control, which was constant during the study. Although the effect of sorbinil in improving nerve conduction velocity in diabetics was small, the findings suggest that polyol-pathway activity contributes to slowed nerve conduction in diabetics. The clinical applicability of these observations remains to be determined, but they encourage further exploration of this approach to the treatment or prevention of diabetic neuropathy. PMID:6401351

  10. A high-throughput assay format for determination of nitrate reductase and nitrite reductase enzyme activities

    SciTech Connect

    McNally, N.; Liu, Xiang Yang; Choudary, P.V.

    1997-01-01

    The authors describe a microplate-based high-throughput procedure for rapid assay of the enzyme activities of nitrate reductase and nitrite reductase, using extremely small volumes of reagents. The new procedure offers the advantages of rapidity, small sample size-nanoliter volumes, low cost, and a dramatic increase in the throughput sample number that can be analyzed simultaneously. Additional advantages can be accessed by using microplate reader application software packages that permit assigning a group type to the wells, recording of the data on exportable data files and exercising the option of using the kinetic or endpoint reading modes. The assay can also be used independently for detecting nitrite residues/contamination in environmental/food samples. 10 refs., 2 figs.

  11. Transcripts of anthocyanidin reductase and leucoanthocyanidin reductase and measurement of catechin and epicatechin in tartary buckwheat.

    PubMed

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, Yeji; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions. PMID:24605062

  12. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

    PubMed Central

    Kim, Yeon Bok; Thwe, Aye Aye; Kim, YeJi; Li, Xiaohua; Cho, Jin Woong; Park, Phun Bum; Valan Arasu, Mariadhas; Abdullah Al-Dhabi, Naif; Kim, Sun-Ju; Suzuki, Tastsuro; Hyun Jho, Kwang; Park, Sang Un

    2014-01-01

    Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs) such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions. PMID:24605062

  13. Berberine inhibits androgen synthesis by interaction with aldo-keto reductase 1C3 in 22Rv1 prostate cancer cells

    PubMed Central

    Tian, Yuantong; Zhao, Lijing; Wang, Ye; Zhang, Haitao; Xu, Duo; Zhao, Xuejian; Li, Yi; Li, Jing

    2016-01-01

    Aldo-keto reductase family 1 member C3 has recently been regarded as a potential therapeutic target in castrate-resistant prostate cancer. Herein, we investigated whether berberine delayed the progression of castrate-resistant prostate cancer by reducing androgen synthesis through the inhibition of Aldo-keto reductase family 1 member C3. Cell viability and cellular testosterone content were measured in prostate cancer cells. Aldo-keto reductase family 1 member C3 mRNA and protein level were detected by RT-PCR and Western bolt analyses, respectively. Computer analysis with AutoDock Tools explored the molecular interaction of berberine with Aldo-keto reductase family 1 member C3. We found that berberine inhibited 22Rv1 cells proliferation and decreased cellular testosterone formation in a dose-dependent manner. Berberine inhibited Aldo-keto reductase family 1 member C3 enzyme activity, rather than influenced mRNA and protein expressions. Molecular docking study demonstrated that berberine could enter the active center of Aldo-keto reductase family 1 member C3 and form π-π interaction with the amino-acid residue Phe306 and Phe311. In conclusion, the structural interaction of berberine with Aldo-keto reductase family 1 member C3 is attributed to the suppression of Aldo-keto reductase family 1 member C3 enzyme activity and the inhibition of 22Rv1 prostate cancer cell growth by decreasing the intracellular androgen synthesis. Our result provides the experimental basis for the design, research, and development of AKR1C3 inhibitors using berberine as the lead compound. PMID:26698234

  14. Berberine inhibits androgen synthesis by interaction with aldo-keto reductase 1C3 in 22Rv1 prostate cancer cells.

    PubMed

    Tian, Yuantong; Zhao, Lijing; Wang, Ye; Zhang, Haitao; Xu, Duo; Zhao, Xuejian; Li, Yi; Li, Jing

    2016-01-01

    Aldo-keto reductase family 1 member C3 has recently been regarded as a potential therapeutic target in castrate-resistant prostate cancer. Herein, we investigated whether berberine delayed the progression of castrate-resistant prostate cancer by reducing androgen synthesis through the inhibition of Aldo-keto reductase family 1 member C3. Cell viability and cellular testosterone content were measured in prostate cancer cells. Aldo-keto reductase family 1 member C3 mRNA and protein level were detected by RT-PCR and Western bolt analyses, respectively. Computer analysis with AutoDock Tools explored the molecular interaction of berberine with Aldo-keto reductase family 1 member C3. We found that berberine inhibited 22Rv1 cells proliferation and decreased cellular testosterone formation in a dose-dependent manner. Berberine inhibited Aldo-keto reductase family 1 member C3 enzyme activity, rather than influenced mRNA and protein expressions. Molecular docking study demonstrated that berberine could enter the active center of Aldo-keto reductase family 1 member C3 and form p-p interaction with the amino-acid residue Phe306 and Phe311. In conclusion, the structural interaction of berberine with Aldo-keto reductase family 1 member C3 is attributed to the suppression of Aldo-keto reductase family 1 member C3 enzyme activity and the inhibition of 22Rv1 prostate cancer cell growth by decreasing the intracellular androgen synthesis. Our result provides the experimental basis for the design, research, and development of AKR1C3 inhibitors using berberine as the lead compound. PMID:26698234

  15. Ribonucleotide reductase metallocofactor: assembly, maintenance and inhibition

    PubMed Central

    ZHANG, Caiguo; LIU, Guoqi; HUANG, Mingxia

    2014-01-01

    Ribonucleotide reductase (RNR) supplies cellular deoxyribonucleotide triphosphates (dNTP) pools by converting ribonucleotides to the corresponding deoxy forms using radical-based chemistry. Eukaryotic RNR comprises α and β subunits: α contains the catalytic and allosteric sites; β houses a diferric-tyrosyl radical cofactor (FeIII2-Y•) that is required to initiates nucleotide reduction in α. Cells have evolved multi-layered mechanisms to regulate RNR level and activity in order to maintain the adequate sizes and ratios of their dNTP pools to ensure high-fidelity DNA replication and repair. The central role of RNR in nucleotide metabolism also makes it a proven target of chemotherapeutics. In this review, we discuss recent progress in understanding the function and regulation of eukaryotic RNRs, with a focus on studies revealing the cellular machineries involved in RNR metallocofactor biosynthesis and its implication in RNR-targeting therapeutics. PMID:24899886

  16. Dynamics of trimethoprim bound to dihydrofolate reductase

    SciTech Connect

    Searle, M.S.; Forster, M.J.; Birdsall, B.; Roberts, G.C.K.; Feeney, J.; Cheung, H.T.A.; Kompis, I.; Geddes, A.J. )

    1988-06-01

    The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, the authors report the use of {sup 1}H and {sup 13}C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its target, dihydrofolate reductase. {sup 13}C relaxation measurements reveal dihedral angle changes of {plus minus}25{degree} to {plus minus}35{degree} on the subnanosecond time scale, while {sup 13}C line-shape analysis demonstrates dihedral angle changes of at least {plus minus}65{degree} on the millisecond time scale. {sup 1}H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.

  17. Nitrite Reductase Activity in Engineered Azurin Variants.

    PubMed

    Berry, Steven M; Strange, Jacob N; Bladholm, Erika L; Khatiwada, Balabhadra; Hedstrom, Christine G; Sauer, Alexandra M

    2016-05-01

    Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis. PMID:27055058

  18. Molecular evolution of nitrate reductase genes.

    PubMed

    Zhou, J; Kleinhofs, A

    1996-04-01

    To understand the evolutionary mechanisms and relationships of nitrate reductases (NRs), the nucleotide sequences encoding 19 nitrate reductase (NR) genes from 16 species of fungi, algae, and higher plants were analyzed. The NR genes examined show substantial sequence similarity, particularly within functional domains, and large variations in GC content at the third codon position and intron number. The intron positions were different between the fungi and plants, but conserved within these groups. The overall and nonsynonymous substitution rates among fungi, algae, and higher plants were estimated to be 4.33 x 10(-10) and 3.29 x 10(-10) substitutions per site per year. The three functional domains of NR genes evolved at about one-third of the rate of the N-terminal and the two hinge regions connecting the functional domains. Relative rate tests suggested that the nonsynonymous substitution rates were constant among different lineages, while the overall nucleotide substitution rates varied between some lineages. The phylogenetic trees based on NR genes correspond well with the phylogeny of the organisms determined from systematics and other molecular studies. Based on the nonsynonymous substitution rate, the divergence time of monocots and dicots was estimated to be about 340 Myr when the fungi-plant or algae-higher plant divergence times were used as reference points and 191 Myr when the rice-barley divergence time was used as a reference point. These two estimates are consistent with other estimates of divergence times based on these reference points. The lack of consistency between these two values appears to be due to the uncertainty of the reference times. PMID:8642612

  19. The cytochrome bd respiratory oxygen reductases

    PubMed Central

    Borisov, Vitaliy B.; Gennis, Robert B.; Hemp, James; Verkhovsky, Michael I.

    2011-01-01

    Summary Cytochrome bd is a respiratory quinol:O2 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O2 and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O2-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O2, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated. PMID:21756872

  20. The 5α-reductase inhibitor Dutasteride but not Finasteride protects dopamine neurons in the MPTP mouse model of Parkinson's disease.

    PubMed

    Litim, Nadhir; Bourque, Mélanie; Al Sweidi, Sara; Morissette, Marc; Di Paolo, Thérèse

    2015-10-01

    Finasteride and Dutasteride are 5α-reductase inhibitors used in the clinic to treat endocrine conditions and were recently found to modulate brain dopamine (DA) neurotransmission and motor behavior. We investigated if Finasteride and Dutasteride have a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) male mice as a model of Parkinson's disease (PD). Experimental groups included saline treated controls and mice treated with saline, Finasteride (5 and 12.5 mg/kg) or Dutasteride (5 and 12.5 mg/kg) for 5 days before and 5 days after MPTP administration (4 MPTP injections, 6.5 mg/kg on day 5 inducing a moderate DA depletion) and then they were euthanized. MPTP administration decreased striatal DA contents measured by HPLC while serotonin contents remained unchanged. MPTP mice treated with Dutasteride 5 and 12.5 mg/kg had higher striatal DA and metabolites (DOPAC and HVA) contents with a decrease of metabolites/DA ratios compared to saline-treated MPTP mice. Finasteride had no protective effect on striatal DA contents. Tyrosine hydroxylase (TH) mRNA levels measured by in situ hybridization in the substantia nigra pars compacta were unchanged. Dutasteride at 12.5 mg/kg reduced the effect of MPTP on specific binding to striatal DA transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) measured by autoradiography. MPTP reduced compared to controls plasma testosterone (T) and dihydrotestosterone (DHT) concentrations measured by liquid chromatography-tandem mass spectrometry; Dutasteride and Finasteride increased plasma T levels while DHT levels remained low. In summary, our results showed that a 5α-reductase inhibitor, Dutasteride has neuroprotective activity preventing in male mice the MPTP-induced loss of several dopaminergic markers. PMID:26006269

  1. Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase

    PubMed Central

    Erb, Tobias J.; Brecht, Volker; Fuchs, Georg; Müller, Michael; Alber, Birgit E.

    2009-01-01

    Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO2. Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis. PMID:19458256

  2. The Effectiveness and Sustainability of a Universal School-Based Programme for Preventing Depression in Chinese Adolescents: A Follow-Up Study Using Quasi-Experimental Design

    PubMed Central

    Lai, Eliza S. Y.; Kwok, Chi-Leung; Wong, Paul W. C.; Fu, King-Wa; Law, Yik-Wa; Yip, Paul S. F.

    2016-01-01

    Background A pilot study about the effectiveness of a universal school-based programme, “The Little Prince is Depressed”, for preventing depression in Chinese adolescents in Hong Kong was conducted and reported previously. This study used a larger sample to examine the effectiveness and sustainability of the programme. Methods This study used quasi-experimental design. Twelve schools enrolled in “The Little Prince is Depressed” programme either as an intervention or a control condition. The intervention schools carried out the 12-session programme in two phases: the professional-led first phase and the teacher-led second phase. All participants were required to complete a questionnaire at three time points measuring their (1) depressive, anxiety, and stress levels; (2) knowledge of mental health; (3) attitudes towards mental illness; (4) perceived social support; and (5) help-seeking behaviours. Results A total of 3,391 students participated in the study. The level of depressive symptoms did not reduce significantly at post-intervention; however, a delayed effect was observed at follow-up assessment for the participants of the teacher-led group in reducing anxiety and stress levels. Also, the knowledge of mental health and attitudes towards mental illness of the intervention-group participants significantly improved at post-test, and the outcomes were maintained at 4 to 5 months after the intervention in both the professional-led and the teacher-led conditions (p<.05). A preference among schoolchildren for whom to seek help from was identified. Conclusions The universal depression prevention programme was effective in enhancing knowledge of mental health and promoting a more positive attitude towards mental illness among adolescents in Hong Kong. In particular, the teacher-led group showed better outcomes than the professional-led group in reducing students’ anxiety and stress at follow-up period. The programme can achieve sustainability in schools if

  3. Solubilization and Resolution of the Membrane-Bound Nitrite Reductase from Paracoccus Halodenitrificans into Nitrite and Nitric Oxide Reductases

    NASA Technical Reports Server (NTRS)

    Grant, Michael A.; Cronin, Sonja E.; Hochstein, Lawrence I.

    1984-01-01

    Membranes prepared from Paracoccus halodenitrificans reduced nitrite or nitric oxide to nitrous oxide. Extraction of these membranes with the detergent CHAPSO [3-(3-Chlolamidoporopyldimethylammonio)-1-(2- hydroxy-1-propanesulfonate)], followed by ammonium sulfate fractionation of the solubilized proteins, resulted in the separation of nitrite and nitric oxide reductase activities. The fraction containing nitrite reductase activity spectrally resembled a cd-type cytochrome. Several cytochromes were detected in the nitric oxide reductase fraction. Which, if any, of these cytochromes is associated with the reduction of nitric oxide is not clear at this time.

  4. COMPARISON OF THE METHYL REDUCTASE GENES AND GENE PRODUCTS

    EPA Science Inventory

    The DNA sequences encoding component C of methyl coenzyme M reductase (mcr genes) in Methanothermus fervidus, Methanobacterium thermoautotrophicum, Methanococcus vannielii, and Methanosarcina barkeri have been published. omparisons of transcription initiation and termination site...

  5. Structural features of the ribonucleotide reductase of Aujeszky's disease virus.

    PubMed

    Kaliman, A V; Boldogköi, Z; Fodor, I

    1994-01-01

    A gene construct of the Aujeszky's disease virus (ADV) genome was prepared and the DNA fragment encoding the ribonucleotide reductase was structurally characterized. We determined the entire DNA sequence of two adjacent open reading frames of the ribonucleotide reductase genes with the intergenic sequence of nine base pairs. From the sequence analysis we predict that Aujeszky's disease virus encodes a ribonucleotide reductase which comprises two polypeptides--large and small subunits, with sizes of 835 and 303 amino acids, respectively. Nucleotide and amino acid sequences of the large and small subunits of the Aujeszky's disease virus ribonucleotide reductase have been compared with that of other herpesviruses, and structural features of both proteins have been characterized. PMID:7810419

  6. Glucose and blood pressure lowering effects of Pycnogenol® are inefficient to prevent prolongation of QT interval in experimental diabetic cardiomyopathy.

    PubMed

    Jankyova, Stanislava; Kmecova, Jana; Cernecka, Hana; Mesarosova, Lucia; Musil, Peter; Brnoliakova, Zuzana; Kyselovic, Jan; Babal, Pavel; Klimas, Jan

    2012-08-15

    Diabetic cardiomyopathy shows ECG alterations related to cardiac repolarization and manifested by increased duration of QT interval. Although the mechanism is unknown, it is widely believed that the reduction of hyperglycaemia might prevent such alterations. To test this hypothesis, we used the standardized extract of French pine bark - Pycnogenol(®) (PYC) with hypoglycaemic and antioxidant properties in 8-9 week old rats with experimentally (streptozotocin) induced diabetes mellitus (DM). PYC was administered orally for 6 weeks in three different doses (10, 20, and 50 mg/kg b.w., resp.). Experimental DM was manifested by hyperglycaemia (four to six-fold increase in plasma glucose concentration; p<0.05) and significantly increased mean arterial blood pressure (by 19%; p<0.05) measured using catheterization of carotid artery in vivo. Both abnormalities were dose-dependently reduced by PYC. In addition, diabetic cardiomyopathy was associated with a significant increase in left ventricular weight to body weight ratio (by 21%; p<0.05) and a significant decrease of the width of cardiomyocytes (by 23%; p<0.05) indicating cardiac edema on the one side, and hypotrophy of cardiomyocytes on the other. Both of these changes were not affected by PYC. Consequently to metabolic and hemodynamic alterations, significant prolongation of QT interval (by 20%; p<0.05) was present in diabetic rats, however, PYC failed to correct it. Conclusively, PYC fails to correct QT prolongation in spite of dose-dependent reduction of glycaemia and high blood pressure in streptozotocin-induced diabetic cardiomyopathy. PMID:22749577

  7. Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.

    PubMed Central

    Rensing, C; Kües, U; Stahl, U; Nies, D H; Friedrich, B

    1992-01-01

    The gene merA coding for bacterial mercuric ion reductase was cloned under the control of the yeast promoter for alcohol dehydrogenase I in the yeast-Escherichia coli shuttle plasmid pADH040-2 and transformed into Saccharomyces cerevisiae AH22. The resulting transformant harbored stable copies of the merA-containing hybrid plasmid, displayed a fivefold increase in the MIC of mercuric chloride, and synthesized mercuric ion reductase activity. Images PMID:1735719

  8. Comparative anatomy of the aldo-keto reductase superfamily.

    PubMed Central

    Jez, J M; Bennett, M J; Schlegel, B P; Lewis, M; Penning, T M

    1997-01-01

    The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short

  9. Purification and characterization of assimilatory nitrite reductase from Candida utilis.

    PubMed Central

    Sengupta, S; Shaila, M S; Rao, G R

    1996-01-01

    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems. PMID:8694757

  10. The REDUCE trial: chemoprevention in prostate cancer using a dual 5alpha-reductase inhibitor, dutasteride.

    PubMed

    Musquera, Mireia; Fleshner, Neil E; Finelli, Antonio; Zlotta, Alexandre R

    2008-07-01

    Dutasteride, a dual 5alpha-reductase inhibitor, is used in the treatment of benign prostatic hyperplasia (BPH). It reduces serum prostate-specific antigen levels by approximately 50% at 6 months and total prostate volume by 25% after 2 years. Randomized placebo-controlled trials in BPH patients have shown the efficacy of dutasteride in symptomatic relief, improvements in quality of life and peak urinary flow rate. Side effects occurring with dutasteride are decreased libido, erectile dysfunction, ejaculation disorders and gynecomastia. Preliminary data from placebo-controlled BPH trials have shown a decrease in the detection of prostate cancer in patients treated with dutasteride, although these studies were not designed to look at this issue. Dutasteride differs from finasteride in that it inhibits both isoenzymes of 5alpha-reductase, type I and type II. The landmark Prostate Cancer Prevention Trial at the end of the 7-year study demonstrated a 24.8% reduction in the incidence of prostate cancer in the finasteride group compared with placebo. However, a 25.5% increase in the prevalence of high-grade Gleason tumors has been observed, the clinical significance of which has been debated. Preliminary data suggest a decrease in prostate cancer incidence in dutasteride-treated patients and demonstrate type I alphareductase enzyme expression in prostate cancer. As a result, dutasteride is being investigated for prostate cancer prevention in the ongoing Reduction by Dutasteride of Prostate Cancer Events (REDUCE) trial, which is discussed here. PMID:18588452

  11. Low apparent aldose reductase activity produced by monosaccharide autoxidation.

    PubMed Central

    Wolff, S P; Crabbe, M J

    1985-01-01

    Low apparent aldose reductase activity, as measured by NADPH oxidation, can be produced by the spontaneous autoxidation of monosaccharides. NADPH is oxidized to metabolically active NADP+ in a solution of autoxidizing DL-glyceraldehyde at rates of up to 15 X 10(-4) A340/min. The close parallelism between the effects of buffer salt type and concentration, monosaccharide structure and temperature activation on autoxidation and NADPH oxidation imply that autoxidation is a prerequisite for the NADPH oxidation, probably via the hydroperoxy radical. Nucleotide-binding proteins enhanced NADPH oxidation induced by DL-glyceraldehyde, up to 10.6-fold with glucose-6-phosphate dehydrogenase. Glutathione reductase-catalysed NADPH oxidation in the presence of autoxidizing monosaccharide showed many characteristics of the aldose reductase reaction. Aldose reductase inhibitors acted as antioxidants in inhibiting this NADPH oxidation. These results indicate that low apparent aldose reductase activities may be due to artifacts of monosaccharide autoxidation, and could provide an explanation for the non-linear steady-state kinetics observed with DL-glyceraldehyde and aldose reductase. PMID:2985042

  12. Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase.

    PubMed

    Sun, Qi-An; Su, Dan; Novoselov, Sergey V; Carlson, Bradley A; Hatfield, Dolph L; Gladyshev, Vadim N

    2005-11-01

    Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes. PMID:16262253

  13. Effects of thioredoxin reductase-1 deletion on embryogenesis and transcriptome

    PubMed Central

    Bondareva, Alla A.; Capecchi, Mario R.; Iverson, Sonya V.; Li, Yan; Lopez, Nathan I.; Lucas, Olivier; Merrill, Gary F.; Prigge, Justin R.; Siders, Ashley M.; Wakamiya, Maki; Wallin, Stephanie L.; Schmidt, Edward E.

    2007-01-01

    Thioredoxin reductases (Txnrd)1 maintain intracellular redox homeostasis in most organisms. Metazoans Txnrds also participate in signal transduction. Mouse embryos homozygous for a targeted null mutation of the txnrd1 gene, encoding the cytosolic thioredoxin reductase, were viable at embryonic day 8.5 (E8.5) but not at E9.5. Histology revealed that txnrd1−/− cells were capable of proliferation and differentiation; however, mutant embryos were smaller than wild-type littermates and failed to gastrulate. In situ marker gene analyses indicated primitive streak mesoderm did not form. Microarray analyses on E7.5 txnrd−/− and txnrd+/+ littermates showed similar mRNA levels for peroxiredoxins, glutathione reductases, mitochondrial Txnrd2, and most markers of cell proliferation. Conversely, mRNAs encoding sulfiredoxin, IGF-binding protein 1, carbonyl reductase 3, glutamate cysteine ligase, glutathione S-transferases, and metallothioneins were more abundant in mutants. Many gene expression responses mirrored those in thioredoxin reductase 1-null yeast; however mice exhibited a novel response within the peroxiredoxin catalytic cycle. Thus, whereas yeast induce peroxiredoxin mRNAs in response to thioredoxin reductase disruption, mice induced sulfiredoxin mRNA. In summary, Txnrd1 was required for correct patterning of the early embryo and progression to later development. Conserved responses to Txnrd1 disruption likely allowed proliferation and limited differentiation of the mutant embryo cells. PMID:17697936

  14. An overview on 5alpha-reductase inhibitors.

    PubMed

    Aggarwal, Saurabh; Thareja, Suresh; Verma, Abhilasha; Bhardwaj, Tilak Raj; Kumar, Manoj

    2010-02-01

    Benign prostatic hyperplasia (BPH) is the noncancerous proliferation of the prostate gland associated with benign prostatic obstruction and lower urinary tract symptoms (LUTS) such as frequency, hesitancy, urgency, etc. Its prevalence increases with age affecting around 70% by the age of 70 years. High activity of 5alpha-reductase enzyme in humans results in excessive dihydrotestosterone levels in peripheral tissues and hence suppression of androgen action by 5alpha-reductase inhibitors is a logical treatment for BPH as they inhibit the conversion of testosterone to dihydrotestosterone. Finasteride (13) was the first steroidal 5alpha-reductase inhibitor approved by U.S. Food and Drug Administration (USFDA). In human it decreases the prostatic DHT level by 70-90% and reduces the prostatic size. Dutasteride (27) another related analogue has been approved in 2002. Unlike Finasteride, Dutasteride is a competitive inhibitor of both 5alpha-reductase type I and type II isozymes, reduced DHT levels >90% following 1 year of oral administration. A number of classes of non-steroidal inhibitors of 5alpha-reductase have also been synthesized generally by removing one or more rings from the azasteroidal structure or by an early non-steroidal lead (ONO-3805) (261). In this review all categories of inhibitors of 5alpha-reductase have been covered. PMID:19879888

  15. Regulation of the Neurospora crassa assimilatory nitrate reductase.

    PubMed Central

    Ketchum, P A; Zeeb, D D; Owens, M S

    1977-01-01

    Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase from Neurospora crassa was purified and found to be stimulated by certain amino acids, citrate, and ethylenediaminetetraacetic acid (EDTA). Stimulation by citrate and the amino acids was dependent upon the prior removal of EDTA from the enzyme preparations, since low quantities of EDTA resulted in maximal stimulation. Removal of EDTA from enzyme preparations by dialysis against Chelex-containing buffer resulted in a loss of nitrate reductase activity. Addition of alanine, arginine, glycine, glutamine, glutamate, histidine, tryptophan, and citrate restored and stimulated nitrate reductase activity from 29- to 46-fold. The amino acids tested altered the Km of NADPH-nitrate reductase for NADPH but did not significantly change that for nitrate. The Km of nitrate reductase for NADPH increased with increasing concentrations of histidine but decreased with increasing concentrations of glutamine. Amino acid modulation of NADPH-nitrate reductase activity is discussed in relation to the conservation of energy (NADPH) by Neurospora when nitrate is the nitrogen source. PMID:19423

  16. Preventing stroke

    MedlinePlus

    Stroke - prevention; CVA - prevention; cerebral vascular accident - prevention; TIA - prevention, transient ischemic attack - prevention ... Clinical Cardiology; Council on Functional Genomics and ... Council on Hypertension. Guidelines for the primary prevention ...

  17. Prevention of antipsychotic-induced hyperglycaemia by vitamin D: a data mining prediction followed by experimental exploration of the molecular mechanism.

    PubMed

    Nagashima, Takuya; Shirakawa, Hisashi; Nakagawa, Takayuki; Kaneko, Shuji

    2016-01-01

    Atypical antipsychotics are associated with an increased risk of hyperglycaemia, thus limiting their clinical use. This study focused on finding the molecular mechanism underlying antipsychotic-induced hyperglycaemia. First, we searched for drug combinations in the FDA Adverse Event Reporting System (FAERS) database wherein a coexisting drug reduced the hyperglycaemia risk of atypical antipsychotics, and found that a combination with vitamin D analogues significantly decreased the occurrence of quetiapine-induced adverse events relating diabetes mellitus in FAERS. Experimental validation using mice revealed that quetiapine acutely caused insulin resistance, which was mitigated by dietary supplementation with cholecalciferol. Further database analysis of the relevant signalling pathway and gene expression predicted quetiapine-induced downregulation of Pik3r1, a critical gene acting downstream of insulin receptor. Focusing on the phosphatidylinositol 3-kinase (PI3K) signalling pathway, we found that the reduced expression of Pik3r1 mRNA was reversed by cholecalciferol supplementation in skeletal muscle, and that insulin-stimulated glucose uptake into C2C12 myotube was inhibited in the presence of quetiapine, which was reversed by concomitant calcitriol in a PI3K-dependent manner. Taken together, these results suggest that vitamin D coadministration prevents antipsychotic-induced hyperglycaemia and insulin resistance by upregulation of PI3K function. PMID:27199286

  18. The schistosome glutathione S-transferase P28GST, a unique helminth protein, prevents intestinal inflammation in experimental colitis through a Th2-type response with mucosal eosinophils.

    PubMed

    Driss, V; El Nady, M; Delbeke, M; Rousseaux, C; Dubuquoy, C; Sarazin, A; Gatault, S; Dendooven, A; Riveau, G; Colombel, J F; Desreumaux, P; Dubuquoy, L; Capron, M

    2016-03-01

    Intestinal helminth parasites are potent inducers of T helper type 2 (Th2) response and have a regulatory role, notably on intestinal inflammation. As infection with schistosomes is unlikely to provide a reliable treatment of inflammatory bowel diseases, we have investigated the beneficial effect of a schistosome enzymatic protein, the 28-kDa glutathione S-transferase (P28GST), on the modulation of disease activity and immune responses in experimental colitis. Our results showed that immunization with recombinant P28GST is at least as efficient as established schistosome infection to reduce colitis lesions and expression of pro-inflammatory cytokines. Considering underlying mechanisms, the decrease of inflammatory parameters was associated with the polarization of the immune system toward a Th2 profile, with local and systemic increases of interleukin (IL)-13 and IL-5. Dense eosinophil infiltration was observed in the colons of P28GST-immunized rats and mice. Depletion of eosinophils by treatment with an anti-Siglec-F monoclonal antibody and use of IL-5-deficient mice led to the loss of therapeutic effect, suggesting the crucial role for eosinophils in colitis prevention by P28GST. These findings reveal that immunization with P28GST, a unique recombinant schistosome enzyme, ameliorates intestinal inflammation through eosinophil-dependent modulation of harmful type 1 responses, representing a new immuno-regulatory strategy against inflammatory bowel diseases. PMID:26174763

  19. The schistosome glutathione S-transferase P28GST, a unique helminth protein, prevents intestinal inflammation in experimental colitis through a Th2-type response with mucosal eosinophils

    PubMed Central

    Driss, V; El Nady, M; Delbeke, M; Rousseaux, C; Dubuquoy, C; Sarazin, A; Gatault, S; Dendooven, A; Riveau, G; Colombel, J F; Desreumaux, P; Dubuquoy, L; Capron, M

    2016-01-01

    Intestinal helminth parasites are potent inducers of T helper type 2 (Th2) response and have a regulatory role, notably on intestinal inflammation. As infection with schistosomes is unlikely to provide a reliable treatment of inflammatory bowel diseases, we have investigated the beneficial effect of a schistosome enzymatic protein, the 28-kDa glutathione S-transferase (P28GST), on the modulation of disease activity and immune responses in experimental colitis. Our results showed that immunization with recombinant P28GST is at least as efficient as established schistosome infection to reduce colitis lesions and expression of pro-inflammatory cytokines. Considering underlying mechanisms, the decrease of inflammatory parameters was associated with the polarization of the immune system toward a Th2 profile, with local and systemic increases of interleukin (IL)-13 and IL-5. Dense eosinophil infiltration was observed in the colons of P28GST-immunized rats and mice. Depletion of eosinophils by treatment with an anti-Siglec-F monoclonal antibody and use of IL-5-deficient mice led to the loss of therapeutic effect, suggesting the crucial role for eosinophils in colitis prevention by P28GST. These findings reveal that immunization with P28GST, a unique recombinant schistosome enzyme, ameliorates intestinal inflammation through eosinophil-dependent modulation of harmful type 1 responses, representing a new immuno-regulatory strategy against inflammatory bowel diseases. PMID:26174763

  20. Prevention of antipsychotic-induced hyperglycaemia by vitamin D: a data mining prediction followed by experimental exploration of the molecular mechanism

    PubMed Central

    Nagashima, Takuya; Shirakawa, Hisashi; Nakagawa, Takayuki; Kaneko, Shuji

    2016-01-01

    Atypical antipsychotics are associated with an increased risk of hyperglycaemia, thus limiting their clinical use. This study focused on finding the molecular mechanism underlying antipsychotic-induced hyperglycaemia. First, we searched for drug combinations in the FDA Adverse Event Reporting System (FAERS) database wherein a coexisting drug reduced the hyperglycaemia risk of atypical antipsychotics, and found that a combination with vitamin D analogues significantly decreased the occurrence of quetiapine–induced adverse events relating diabetes mellitus in FAERS. Experimental validation using mice revealed that quetiapine acutely caused insulin resistance, which was mitigated by dietary supplementation with cholecalciferol. Further database analysis of the relevant signalling pathway and gene expression predicted quetiapine-induced downregulation of Pik3r1, a critical gene acting downstream of insulin receptor. Focusing on the phosphatidylinositol 3-kinase (PI3K) signalling pathway, we found that the reduced expression of Pik3r1 mRNA was reversed by cholecalciferol supplementation in skeletal muscle, and that insulin-stimulated glucose uptake into C2C12 myotube was inhibited in the presence of quetiapine, which was reversed by concomitant calcitriol in a PI3K-dependent manner. Taken together, these results suggest that vitamin D coadministration prevents antipsychotic-induced hyperglycaemia and insulin resistance by upregulation of PI3K function. PMID:27199286

  1. The Imperial College Cambridge Manchester (ICCAM) platform study: An experimental medicine platform for evaluating new drugs for relapse prevention in addiction. Part A: Study description.

    PubMed

    Paterson, Louise M; Flechais, Remy S A; Murphy, Anna; Reed, Laurence J; Abbott, Sanja; Boyapati, Venkataramana; Elliott, Rebecca; Erritzoe, David; Ersche, Karen D; Faluyi, Yetunde; Faravelli, Luca; Fernandez-Egea, Emilio; Kalk, Nicola J; Kuchibatla, Shankar S; McGonigle, John; Metastasio, Antonio; Mick, Inge; Nestor, Liam; Orban, Csaba; Passetti, Filippo; Rabiner, Eugenii A; Smith, Dana G; Suckling, John; Tait, Roger; Taylor, Eleanor M; Waldman, Adam D; Robbins, Trevor W; Deakin, J F William; Nutt, David J; Lingford-Hughes, Anne R

    2015-09-01

    Drug and alcohol dependence are global problems with substantial societal costs. There are few treatments for relapse prevention and therefore a pressing need for further study of brain mechanisms underpinning relapse circuitry. The Imperial College Cambridge Manchester (ICCAM) platform study is an experimental medicine approach to this problem: using functional magnetic resonance imaging (fMRI) techniques and selective pharmacological tools, it aims to explore the neuropharmacology of putative relapse pathways in cocaine, alcohol, opiate dependent, and healthy individuals to inform future drug development. Addiction studies typically involve small samples because of recruitment difficulties and attrition. We established the platform in three centres to assess the feasibility of a multisite approach to address these issues. Pharmacological modulation of reward, impulsivity and emotional reactivity were investigated in a monetary incentive delay task, an inhibitory control task, and an evocative images task, using selective antagonists for µ-opioid, dopamine D3 receptor (DRD3) and neurokinin 1 (NK1) receptors (naltrexone, GSK598809, vofopitant/aprepitant), in a placebo-controlled, randomised, crossover design. In two years, 609 scans were performed, with 155 individuals scanned at baseline. Attrition was low and the majority of individuals were sufficiently motivated to complete all five sessions (n=87). We describe herein the study design, main aims, recruitment numbers, sample characteristics, and explain the test hypotheses and anticipated study outputs. PMID:26246443

  2. Prostate Cancer Prevention: Concepts and Clinical Trials.

    PubMed

    Hamilton, Zachary; Parsons, J Kellogg

    2016-04-01

    Prevention is an important treatment strategy for diminishing prostate cancer morbidity and mortality and is applicable to both early- and late-stage disease. There are three basic classifications of cancer prevention: primary (prevention of incident disease), secondary (identification and treatment of preclinical disease), and tertiary (prevention of progression or recurrence). Based on level I evidence, 5-alpha reductase inhibitors (5-ARIs) should be considered in selected men to prevent incident prostate cancer. Level I evidence also supports the consideration of dutasteride, a 5-ARI, for tertiary prevention in active surveillance and biochemical recurrence patients. Vitamins and supplements, including selenium or vitamin E, have not been proven in clinical trials to prevent prostate cancer and in the case of Vitamin E has been found to increase the risk of incident prostate cancer. Ongoing and future trials may further elucidate the role of diet and immunotherapy for prevention of prostate cancer. PMID:26957512

  3. Glutathione Reductase Is Essential for Host Defense against Bacterial Infection

    PubMed Central

    Yan, Jing; Ralston, Melissa M.; Meng, Xiaomei; Bongiovanni, Kathleen D.; Jones, Amanda L.; Benndorf, Rainer; Nelin, Leif D.; Frazier, W. Joshua; Rogers, Lynette K.; Smith, Charles V.; Liu, Yusen

    2013-01-01

    Glutathione reductase (Gsr)1 catalyzes the reduction of glutathione disulfide to glutathione, a major cellular antioxidant. We have recently shown that Gsr is essential for host defense against the Gram-negative bacteria Escherichia coli in a mouse model of sepsis. While we have demonstrated that Gsr is required for sustaining the oxidative burst and the development of neutrophil extracellular traps, the role of Gsr in other phagocytic functions remains unclear. It is also unclear whether Gsr-deficient mice exhibit host defense defects against Gram-positive bacteria. In the present study, we characterized the effects of Gsr deficiency on the innate immune responses to a Gram-positive bacterium, group B Streptococcus, and to the Gram-negative bacterial cell wall component lipopolysaccharide (LPS). We found that like, E. coli, group B Streptococcus resulted in a substantially more robust cytokine response and a markedly higher morbidity and mortality in Gsr-deficient mice than in wildtype mice. The increased morbidity and mortality were associated with greater bacterial burden in the Gsr-deficient mice. Interestingly, Gsr-deficient mice did not exhibit a greater sensitivity to LPS than did wildtype mice. Analysis of the neutrophils of Gsr-deficient mice revealed impaired phagocytosis. In response to thioglycollate stimulation, Gsr-deficient mice mobilized far fewer phagocytes, including neutrophils, macrophages, and eosinophils, into their peritoneal cavities than did wildtype mice. The defective phagocyte mobilization is associated with profound oxidation and aggregation of ascitic proteins, particularly albumin. Our results indicate that the oxidative defense mechanism mediated by Gsr is required for an effective innate immune response against bacteria, likely by preventing phagocyte dysfunction due to oxidative damage. PMID:23623936

  4. Diversity in Overall Activity Regulation of Ribonucleotide Reductase*

    PubMed Central

    Jonna, Venkateswara Rao; Crona, Mikael; Rofougaran, Reza; Lundin, Daniel; Johansson, Samuel; Brännström, Kristoffer; Sjöberg, Britt-Marie; Hofer, Anders

    2015-01-01

    Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives. PMID:25971975

  5. Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa

    PubMed Central

    Lu, Wanxiang; Yang, Li; Karim, Abdul; Luo, Keming

    2013-01-01

    Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (−)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus. PMID:23741362

  6. A Quasi-Experimental Study Analyzing the Effectiveness of Portable High-Efficiency Particulate Absorption Filters in Preventing Infections in Hematology Patients during Construction

    PubMed Central

    Özen, Mehmet; Yılmaz, Gülden; Coşkun, Belgin; Topçuoğlu, Pervin; Öztürk, Bengi; Gündüz, Mehmet; Atilla, Erden; Arslan, Önder; Özcan, Muhit; Demirer, Taner; İlhan, Osman; Konuk, Nahide; Balık, İsmail; Gürman, Günhan; Akan, Hamdi

    2016-01-01

    Objective: The increased risk of infection for patients caused by construction and renovation near hematology inpatient clinics is a major concern. The use of high-efficiency particulate absorption (HEPA) filters can reduce the risk of infection. However, there is no standard protocol indicating the use of HEPA filters for patients with hematological malignancies, except for those who have undergone allogeneic hematopoietic stem cell transplantation. This quasi-experimental study was designed to measure the efficacy of HEPA filters in preventing infections during construction. Materials and Methods: Portable HEPA filters were placed in the rooms of patients undergoing treatment for hematological malignancies because of large-scale construction taking place near the hematology clinic. The rates of infection during the 6 months before and after the installation of the portable HEPA filters were compared. A total of 413 patients were treated during this 1-year period. Results: There were no significant differences in the antifungal prophylaxis and treatment regimens between the groups. The rates of infections, clinically documented infections, and invasive fungal infections decreased in all of the patients following the installation of the HEPA filters. When analyzed separately, the rates of invasive fungal infections were similar before and after the installation of HEPA filters in patients who had no neutropenia or long neutropenia duration. HEPA filters were significantly protective against infection when installed in the rooms of patients with acute lymphocytic leukemia, patients who were undergoing consolidation treatment, and patients who were neutropenic for 1-14 days. Conclusion: Despite the advent of construction and the summer season, during which environmental Aspergillus contamination is more prevalent, no patient or patient subgroup experienced an increase in fungal infections following the installation of HEPA filters. The protective effect of HEPA

  7. Oroxyloside prevents dextran sulfate sodium-induced experimental colitis in mice by inhibiting NF-κB pathway through PPARγ activation.

    PubMed

    Wang, Xiaoping; Sun, Yang; Zhao, Yue; Ding, Youxiang; Zhang, Xiaobo; Kong, Lingyi; Li, Zhiyu; Guo, Qinglong; Zhao, Li

    2016-04-15

    Oroxyloside, as a metabolite of oroxylin A, may harbor various beneficial bioactivities which have rarely been reported in the previous studies. Here we established the dextran sulfate sodium (DSS)-induced experimental colitis and evaluated the anti-inflammatory effect of oroxyloside in vivo. As a result, oroxyloside attenuated DSS-induced body weight loss, colon length shortening and colonic pathological damage. Furthermore, oroxyloside inhibited inflammatory cell infiltration and decreased myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) activities as well. The production of pro-inflammatory cytokines in serum and colon was also significantly reduced by oroxyloside. We unraveled the underlying mechanisms that oroxyloside inhibited NF-κB pathway by activating Peroxisome Proliferator-Activated Receptor γ (PPARγ) to attenuate DSS-induced colitis. Moreover, we investigated the anti-inflammatory effect and mechanisms of oroxyloside in the mouse macrophage cell line RAW264.7 and bone marrow derived macrophages (BMDM). Oroxyloside decreased several LPS-induced inflammatory cytokines, including IL-1β, IL-6 and TNF-α in RAW264.7 and BMDM. We also found that oroxyloside inhibited LPS-induced activation of NF-κB signaling pathway via activating PPARγ in RAW 264.7 and BMDM. Docking study showed that oroxyloside could bind with PPARγ. GW9662, the inhibitor of PPARγ, and PPARγ siRNA transfection blocked the effect of oroxyloside on PPARγ activation. Our study suggested that oroxyloside prevented DSS-induced colitis by inhibiting NF-κB pathway through PPARγ activation. Therefore, oroxyloside may be a promising and effective agent for inflammatory bowel disease (IBD). PMID:26947454

  8. Acute treatment with valproic acid and l-thyroxine ameliorates clinical signs of experimental autoimmune encephalomyelitis and prevents brain pathology in DA rats.

    PubMed

    Castelo-Branco, Gonçalo; Stridh, Pernilla; Guerreiro-Cacais, André Ortlieb; Adzemovic, Milena Z; Falcão, Ana Mendanha; Marta, Monica; Berglund, Rasmus; Gillett, Alan; Hamza, Kedir Hussen; Lassmann, Hans; Hermanson, Ola; Jagodic, Maja

    2014-11-01

    Multiple sclerosis (MS) is the most common chronic inflammatory demyelinating disease of the central nervous system (CNS) in young adults. Chronic treatments with histone deacetylase inhibitors (HDACis) have been reported to ameliorate experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, by targeting immune responses. We have recently shown that the HDAC inhibition/knockdown in the presence of thyroid hormone (T3) can also promote oligodendrocyte (OL) differentiation and expression of myelin genes in neural stem cells (NSCs) and oligodendrocyte precursors (OPCs). In this study, we found that treatment with an HDACi, valproic acid (VPA), and T3, alone or in combination, directly affects encephalitogenic CD4+ T cells. VPA, but not T3, compromised their proliferation, while both molecules reduced the frequency of IL-17-producing cells. Transfer of T3, VPA and VPA/T3 treated encephalitogenic CD4+ T cells into naïve rats induced less severe EAE, indicating that the effects of these molecules are persistent and do not require their maintenance after the initial stimuli. Thus, we investigated the effect of acute treatment with VPA and l-thyroxine (T4), a precursor of T3, on myelin oligodendrocyte glycoprotein-induced EAE in Dark Agouti rats, a close mimic of MS. We found that a brief treatment after disease onset led to sustained amelioration of EAE and prevention of inflammatory demyelination in the CNS accompanied with a higher expression of myelin-related genes in the brain. Furthermore, the treatment modulated immune responses, reduced the number of CD4+ T cells and affected the Th1 differentiation program in the brain. Our data indicate that an acute treatment with VPA and T4 after the onset of EAE can produce persistent clinically relevant therapeutic effects by limiting the pathogenic immune reactions while promoting myelin gene expression. PMID:25149263

  9. DNA damage induction of ribonucleotide reductase.

    PubMed Central

    Elledge, S J; Davis, R W

    1989-01-01

    RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidase activity in yeast strains containing the RNR2-lacZ fusion was inducible in response to DNA-damaging agents (UV light, 4-nitroquinoline-1-oxide [4-NQO], and methyl methanesulfonate [MMS]) and agents that block DNA replication (hydroxyurea [HU] and methotrexate) but not heat shock. When MATa cells were arrested in G1 by alpha-factor, RNR2 mRNA was still inducible by DNA damage, indicating that the observed induction can occur outside of S phase. In addition, RNR2 induction was not blocked by the presence of cycloheximide and is therefore likely to be independent of protein synthesis. A mutation, rnr2-314, was found to confer hypersensitivity to HU and increased sensitivity to MMS. In rnr2-314 mutant strains, the DNA damage stress response was found to be partially constitutive as well as hypersensitive to induction by HU but not MMS. The induction properties of RNR2 were examined in a rad4-2 mutant background; in this genetic background, RNR2 was hypersensitive to induction by 4-NQO but not MMS. Induction of the RNR2-lacZ fusion in a RAD(+) strain in response to 4-NQO was not enhanced by the presence of an equal number of rad4-2 cells that lacked the fusion, implying that the DNA damage stress response in cell autonomous. Images PMID:2513480

  10. Anti-HMG-CoA Reductase, Antioxidant, and Anti-Inflammatory Activities of Amaranthus viridis Leaf Extract as a Potential Treatment for Hypercholesterolemia.

    PubMed

    Salvamani, Shamala; Gunasekaran, Baskaran; Shukor, Mohd Yunus; Shaharuddin, Noor Azmi; Sabullah, Mohd Khalizan; Ahmad, Siti Aqlima

    2016-01-01

    Inflammation and oxidative stress are believed to contribute to the pathology of several chronic diseases including hypercholesterolemia (elevated levels of cholesterol in blood) and atherosclerosis. HMG-CoA reductase inhibitors of plant origin are needed as synthetic drugs, such as statins, which are known to cause adverse effects on the liver and muscles. Amaranthus viridis (A. viridis) has been used from ancient times for its supposedly medically beneficial properties. In the current study, different parts of A. viridis (leaf, stem, and seed) were evaluated for potential anti-HMG-CoA reductase, antioxidant, and anti-inflammatory activities. The putative HMG-CoA reductase inhibitory activity of A. viridis extracts at different concentrations was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as substrate. A. viridis leaf extract revealed the highest HMG-CoA reductase inhibitory effect at about 71%, with noncompetitive inhibition in Lineweaver-Burk plot analysis. The leaf extract showed good inhibition of hydroperoxides, 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and ferric ion radicals in various concentrations. A. viridis leaf extract was proven to be an effective inhibitor of hyaluronidase, lipoxygenase, and xanthine oxidase enzymes. The experimental data suggest that A. viridis leaf extract is a source of potent antioxidant and anti-inflammatory agent and may modulate cholesterol metabolism by inhibition of HMG-CoA reductase. PMID:27051453

  11. Anti-HMG-CoA Reductase, Antioxidant, and Anti-Inflammatory Activities of Amaranthus viridis Leaf Extract as a Potential Treatment for Hypercholesterolemia

    PubMed Central

    Salvamani, Shamala; Gunasekaran, Baskaran; Shukor, Mohd Yunus; Shaharuddin, Noor Azmi; Sabullah, Mohd Khalizan

    2016-01-01

    Inflammation and oxidative stress are believed to contribute to the pathology of several chronic diseases including hypercholesterolemia (elevated levels of cholesterol in blood) and atherosclerosis. HMG-CoA reductase inhibitors of plant origin are needed as synthetic drugs, such as statins, which are known to cause adverse effects on the liver and muscles. Amaranthus viridis (A. viridis) has been used from ancient times for its supposedly medically beneficial properties. In the current study, different parts of A. viridis (leaf, stem, and seed) were evaluated for potential anti-HMG-CoA reductase, antioxidant, and anti-inflammatory activities. The putative HMG-CoA reductase inhibitory activity of A. viridis extracts at different concentrations was determined spectrophotometrically by NADPH oxidation, using HMG-CoA as substrate. A. viridis leaf extract revealed the highest HMG-CoA reductase inhibitory effect at about 71%, with noncompetitive inhibition in Lineweaver-Burk plot analysis. The leaf extract showed good inhibition of hydroperoxides, 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and ferric ion radicals in various concentrations. A. viridis leaf extract was proven to be an effective inhibitor of hyaluronidase, lipoxygenase, and xanthine oxidase enzymes. The experimental data suggest that A. viridis leaf extract is a source of potent antioxidant and anti-inflammatory agent and may modulate cholesterol metabolism by inhibition of HMG-CoA reductase. PMID:27051453

  12. Purification and properties of the NADH reductase component of alkene monooxygenase from Mycobacterium strain E3.

    PubMed Central

    Weber, F J; van Berkel, W J; Hartmans, S; de Bont, J A

    1992-01-01

    Alkene monooxygenase, a multicomponent enzyme system which catalyzes the epoxidation of short-chain alkenes, is induced in Mycobacterium strain E3 when it is grown on ethene. We purified the NADH reductase component of this enzyme system to homogeneity. Recovery of the enzyme was 19%, with a purification factor of 920-fold. The enzyme is a monomer with a molecular mass of 56 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is yellow-red with absorption maxima at 384, 410, and 460 nm. Flavin adenine dinucleotide (FAD) was identified as a prosthetic group at a FAD-protein ratio of 1:1. Tween 80 prevented irreversible dissociation of FAD from the enzyme during chromatographic purification steps. Colorimetric analysis revealed 2 mol each of iron and acid-labile sulfide, indicating the presence of a [2Fe-2S] cluster. The presence of this cluster was confirmed by electron paramagnetic resonance spectroscopy (g values at 2.011, 1.921, and 1.876). Anaerobic reduction of the reductase by NADH resulted in formation of a flavin semiquinone. Images PMID:1315734

  13. Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism: epidemiology, metabolism and the associated diseases.

    PubMed

    Liew, Siaw-Cheok; Gupta, Esha Das

    2015-01-01

    The Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism is associated with various diseases (vascular, cancers, neurology, diabetes, psoriasis, etc) with the epidemiology of the polymorphism of the C677T that varies dependent on the geography and ethnicity. The 5,10-Methylenetetrahydrofolate reductase (MTHFR) locus is mapped on chromosome 1 at the end of the short arm (1p36.6). This enzyme is important for the folate metabolism which is an integral process for cell metabolism in the DNA, RNA and protein methylation. The mutation of the MTHFR gene which causes the C677T polymorphism is located at exon 4 which results in the conversion of valine to alanine at codon 222, a common polymorphism that reduces the activity of this enzyme. The homozygous mutated subjects have higher homocysteine levels while the heterozygous mutated subjects have mildly raised homocysteine levels compared with the normal, non-mutated controls. Hyperhomocysteinemia is an emerging risk factor for various cardiovascular diseases and with the increasing significance of this polymorphism in view of the morbidity and mortality impact on the patients, further prevention strategies and nutritional recommendations with the supplementation of vitamin B12 and folic acid which reduces plasma homocysteine level would be necessary as part of future health education. This literature review therefore focuses on the recent evidence-based reports on the associations of the MTHFR C677T polymorphism and the various diseases globally. PMID:25449138

  14. Glutathione reductase 2 maintains the function of photosystem II in Arabidopsis under excess light.

    PubMed

    Ding, Shunhua; Jiang, Rui; Lu, Qingtao; Wen, Xiaogang; Lu, Congming

    2016-06-01

    Glutathione reductase plays a crucial role in the elimination of H(2)O(2) molecules via the ascorbate-glutathione cycle. In this study, we used transgenic Arabidopsis plants with decreased glutathione reductase 2 (GR2) levels to investigate whether this GR2 activity protects the photosynthetic machinery under excess light. The transgenic plants were highly sensitive to excess light and accumulated high levels of H(2)O(2). Photosystem II (PSII) activity was significantly decreased in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements demonstrated inhibition of electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants. Immunoblot and blue native gel analysis showed that the levels of PSII proteins and PSII complexes were decreased in transgenic plants. Analyses of the repair of photodamaged PSII and in vivo pulse labeling of thylakoid proteins showed that the repair of photodamaged PSII is inhibited due to the inhibition of the synthesis of the D1 protein de novo in transgenic plants. Taken together, our results suggest that under excess light conditions, GR2 plays an important role in maintaining both the function of the acceptor side of PSII and the repair of photodamaged PSII by preventing the accumulation of H(2)O(2). In addition, our results provide details of the role of H(2)O(2) in vivo accumulation in photoinhibition in plants. PMID:26906429

  15. Bee Venom Promotes Hair Growth in Association with Inhibiting 5α-Reductase Expression.

    PubMed

    Park, Seeun; Erdogan, Sedef; Hwang, Dahyun; Hwang, Seonwook; Han, Eun Hye; Lim, Young-Hee

    2016-06-01

    Alopecia is an important issue that can occur in people of all ages. Recent studies show that bee venom can be used to treat certain diseases including rheumatoid arthritis, neuralgia, and multiple sclerosis. In this study, we investigated the preventive effect of bee venom on alopecia, which was measured by applying bee venom (0.001, 0.005, 0.01%) or minoxidil (2%) as a positive control to the dorsal skin of female C57BL/6 mice for 19 d. Growth factors responsible for hair growth were analyzed by quantitative real-time PCR and Western blot analysis using mice skins and human dermal papilla cells (hDPCs). Bee venom promoted hair growth and inhibited transition from the anagen to catagen phase. In both anagen phase mice and dexamethasone-induced catagen phase mice, hair growth was increased dose dependently compared with controls. Bee venom inhibited the expression of SRD5A2, which encodes a type II 5α-reductase that plays a major role in the conversion of testosterone into dihydrotestosterone. Moreover, bee venom stimulated proliferation of hDPCs and several growth factors (insulin-like growth factor 1 receptor (IGF-1R), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)2 and 7) in bee venom-treated hDPCs dose dependently compared with the control group. In conclusion, bee venom is a potentially potent 5α-reductase inhibitor and hair growth promoter. PMID:27040904

  16. Inhibition of Vibrio harveyi bioluminescence by cerulenin: In vivo evidence for covalent modification of the reductase enzyme involved in aldehyde synthesis

    SciTech Connect

    Byers, D.M. ); Meighen, E.A. )

    1989-07-01

    Bacterial bioluminescence is very sensitive to cerulenin, a fungal antibiotic which is known to inhibit fatty acid synthesis. When Vibrio harveyi cells pretreated with cerulenin were incubated with ({sup 3}H)myristic acid in vivo, acylation of the 57-kilodalton reductase subunit of the luminescence-specific fatty acid reductase complex was specifically inhibited. Light emission of wild-type V. harveyi was 20-fold less sensitive to cerulenin at low concentrations (10{mu}g/ml) than that of the dark mutant strain M17, which requires exogenous myristic acid for luminescence because of a defective transferase subunit. The sensitivity of myristic acid-stimulated luminescence in the mutant strain M17 exceeded that of phospholipid synthesis from ({sup 14}C)acetate, whereas uptake and incorporation of exogenous ({sup 14}C)myristic acid into phospholipids was increased by cerulenin. The reductase subunit could be labeled by incubating M17 cells with ({sup 3}H)tetrahydrocerulenin; this labeling was prevented by preincubation with either unlabeled cerulenin or myristic acid. Labeling of the reductase subunit with ({sup 3}H)tetrahydrocerulenin was also noted in an aldehyde-stimulated mutant (A16) but not in wild-type cells or in another aldehyde-stimulated mutant (M42) in which ({sup 3}H)myristoyl turnover at the reductase subunit was found to be defective. These results indicate that (i) cerulenin specifically and covalently inhibits the reductase component of aldehyde synthesis, (ii) this enzyme is partially protected from cerulenin inhibition in the wild-type strain in vivo, and (iii) two dark mutants which exhibit similar luminescence phenotypes (mutants A16 and M42) are blocked at different stages of fatty acid reduction.

  17. Oral Activated Charcoal Prevents Experimental Cerebral Malaria in Mice and in a Randomized Controlled Clinical Trial in Man Did Not Interfere with the Pharmacokinetics of Parenteral Artesunate

    PubMed Central

    Alexander, Neal D.; Aziz, Naveed; Owens, Benjamin M. J.; Kaur, Harparkash; Jasseh, Momodou; Muangnoicharoen, Sant; Sumariwalla, Percy F.; Warhurst, David C.; Ward, Stephen A.; Conway, David J.; Ulloa, Luis; Tracey, Kevin J.; Foxwell, Brian M. J.; Kaye, Paul M.; Walther, Michael

    2010-01-01

    Background Safe, cheap and effective adjunct therapies preventing the development of, or reducing the mortality from, severe malaria could have considerable and rapid public health impact. Oral activated charcoal (oAC) is a safe and well tolerated treatment for acute poisoning, more recently shown to have significant immunomodulatory effects in man. In preparation for possible efficacy trials in human malaria, we sought to determine whether oAC would i) reduce mortality due to experimental cerebral malaria (ECM) in mice, ii) modulate immune and inflammatory responses associated with ECM, and iii) affect the pharmacokinetics of parenteral artesunate in human volunteers. Methods/Principal Findings We found that oAC provided significant protection against P. berghei ANKA-induced ECM, increasing overall survival time compared to untreated mice (p<0.0001; hazard ratio 16.4; 95% CI 6.73 to 40.1). Protection from ECM by oAC was associated with reduced numbers of splenic TNF+ CD4+ T cells and multifunctional IFNγ+TNF+ CD4+ and CD8+ T cells. Furthermore, we identified a whole blood gene expression signature (68 genes) associated with protection from ECM. To evaluate whether oAC might affect current best available anti-malarial treatment, we conducted a randomized controlled open label trial in 52 human volunteers (ISRCTN NR. 64793756), administering artesunate (AS) in the presence or absence of oAC. We demonstrated that co-administration of oAC was safe and well-tolerated. In the 26 subjects further analyzed, we found no interference with the pharmacokinetics of parenteral AS or its pharmacologically active metabolite dihydroartemisinin. Conclusions/Significance oAC protects against ECM in mice, and does not interfere with the pharmacokinetics of parenteral artesunate. If future studies succeed in establishing the efficacy of oAC in human malaria, then the characteristics of being inexpensive, well-tolerated at high doses and requiring no sophisticated storage would make o

  18. Linezolid compared with vancomycin for the prevention of methicillin-resistant Staphylococcus aureus or Staphylococcus epidermidis vascular graft infection in rats: A randomized, controlled, experimental study

    PubMed Central

    Sacar, Suzan; Sacar, Mustafa; Kaleli, Ilknur; Toprak, Semra; Cevahir, Nural; Teke, Zafer; Asan, Ali; Sahin, Barbaros; Baltalarli, Ahmet; Turgut, Huseyin

    2007-01-01

    Background: Graft infections are severe complications of vascular surgery that may result in amputation or mortality. Staphylococci are the most frequent cause of vascular graft infections. Objective: In this study we assessed the prophylactic efficacy of linezolid in comparison with vancomycin in preventing prosthetic vascular graft infection due to methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant Staphylococcus epidermidis (MRSE). Methods: This randomized, controlled, experimental study using healthy adult (aged >5 months) male Wistar rats was conducted in the research laboratory of the Pamukkale University, Denizli, Turkey. The study consisted of an uncontaminated control group and 3 groups for both staphylococcal strains: a contaminated group that did not receive any antibiotic prophylaxis; a contaminated group that received preoperative intraperitoneal (IP) prophylaxis with vancomycin; and a contaminated group that received preoperative IP prophylaxis with linezolid. All rats received a vascular Dacron graft placed inside a subcutaneous pocket created on the right side of the median line. Sterile saline solution (1 mL), to which MRSA or MRSE at a concentration of 2 × 107 colony-forming units per milliliter had been added, was inoculated onto the graft surface using a tuberculin syringe to fill the pocket. The grafts were explanted 7 days after implantation and assessed by quantitative culture. Results: Seventy rats (mean [SD]weight, 323.7 [17.9]g; mean [SD]age, 5.98 [0.64] months) were evenly divided between the 7 groups. Statistical analysis of the quantitative graft culture suggested that both vancomycin and linezolid were effective in significantly inhibiting bacterial growth when compared with the untreated contaminated groups (all, P < 0.001). However, a statistically significant difference was not observed between the bacteria count in the vancomycin and linezolid prophylaxis groups. When a comparison was made between the

  19. Mechanistic studies with solubilized rat liver steroid 5 alpha-reductase: Elucidation of the kinetic mechanism

    SciTech Connect

    Levy, M.A.; Brandt, M.; Greway, A.T. )

    1990-03-20

    A solubilized preparation of steroid 5 alpha-reductase from rat liver has been used in studies focused toward an understanding of the kinetic mechanism associated with enzyme catalysis. From the results of analyses with product and dead-end inhibitors, a preferentially ordered binding of substrates and release of products from the surface of the enzyme is proposed. The observations from these experiments were identical with those using the steroid 5 alpha-reductase activity associated with rat liver microsomes. The primary isotope effects on steady-state kinetic parameters when (4S-2H)NADPH was used also were consistent with an ordered kinetic mechanism. Normal isotope effects were observed for all three kinetic parameters (Vm/Km for both testosterone and NADPH and Vm) at all substrate concentrations used experimentally. Upon extrapolation to infinite concentration of testosterone, the isotope effect on Vm/Km for NADPH approached unity, indicating that the nicotinamide dinucleotide phosphate is the first substrate binding to and the second product released from the enzyme. The isotope effects on Vm/Km for testosterone at infinite concentration of cofactor and on Vm were 3.8 +/- 0.5 and 3.3 +/- 0.4, respectively. Data from the pH profiles of these three steady-state parameters and the inhibition constants (1/Ki) of competitive inhibitors versus both substrates indicate that the binding of nicotinamide dinucleotide phosphate involves coordination of its anionic 2'-phosphate to a protonated enzyme-associated base with an apparent pK near 8.0. From these results, relative limits have been placed on several of the internal rate constants used to describe the ordered mechanism of the rat liver steroid 5 alpha-reductase.

  20. The inhibitory activity of aldose reductase in vitro by constituents of Garcinia mangostana Linn.

    PubMed

    Fatmawati, Sri; Ersam, Taslim; Shimizu, Kuniyoshi

    2015-01-15

    We investigated aldose reductase inhibition of Garcinia mangostana Linn. from Indonesia. Dichloromethane extract of the root bark of this tree was found to demonstrate an IC50 value of 11.98 µg/ml for human aldose reductase in vitro. From the dichloromethane fraction, prenylated xanthones were isolated as potent human aldose reductase inhibitors. We discovered 3-isomangostin to be most potent against aldose reductase, with an IC50 of 3.48 µM. PMID:25636870

  1. Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.

    PubMed

    Siméone, Roxane; Constant, Patricia; Guilhot, Christophe; Daffé, Mamadou; Chalut, Christian

    2007-07-01

    Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis. PMID:17468241

  2. The 5 Alpha-Reductase Isozyme Family: A Review of Basic Biology and Their Role in Human Diseases

    PubMed Central

    Azzouni, Faris; Godoy, Alejandro; Li, Yun; Mohler, James

    2012-01-01

    Despite the discovery of 5 alpha-reduction as an enzymatic step in steroid metabolism in 1951, and the discovery that dihydrotestosterone is more potent than testosterone in 1968, the significance of 5 alpha-reduced steroids in human diseases was not appreciated until the discovery of 5 alpha-reductase type 2 deficiency in 1974. Affected males are born with ambiguous external genitalia, despite normal internal genitalia. The prostate is hypoplastic, nonpalpable on rectal examination and approximately 1/10th the size of age-matched normal glands. Benign prostate hyperplasia or prostate cancer does not develop in these patients. At puberty, the external genitalia virilize partially, however, secondary sexual hair remains sparse and male pattern baldness and acne develop rarely. Several compounds have been developed to inhibit the 5 alpha-reductase isozymes and they play an important role in the prevention and treatment of many common diseases. This review describes the basic biochemical properties, functions, tissue distribution, chromosomal location, and clinical significance of the 5 alpha-reductase isozyme family. PMID:22235201

  3. Mycobacterium tuberculosis Thioredoxin Reductase Is Essential for Thiol Redox Homeostasis but Plays a Minor Role in Antioxidant Defense.

    PubMed

    Lin, Kan; O'Brien, Kathryn M; Trujillo, Carolina; Wang, Ruojun; Wallach, Joshua B; Schnappinger, Dirk; Ehrt, Sabine

    2016-06-01

    Mycobacterium tuberculosis (Mtb) must cope with exogenous oxidative stress imposed by the host. Unlike other antioxidant enzymes, Mtb's thioredoxin reductase TrxB2 has been predicted to be essential not only to fight host defenses but also for in vitro growth. However, the specific physiological role of TrxB2 and its importance for Mtb pathogenesis remain undefined. Here we show that genetic inactivation of thioredoxin reductase perturbed several growth-essential processes, including sulfur and DNA metabolism and rapidly killed and lysed Mtb. Death was due to cidal thiol-specific oxidizing stress and prevented by a disulfide reductant. In contrast, thioredoxin reductase deficiency did not significantly increase susceptibility to oxidative and nitrosative stress. In vivo targeting TrxB2 eradicated Mtb during both acute and chronic phases of mouse infection. Deliberately leaky knockdown mutants identified the specificity of TrxB2 inhibitors and showed that partial inactivation of TrxB2 increased Mtb's susceptibility to rifampicin. These studies reveal TrxB2 as essential thiol-reducing enzyme in Mtb in vitro and during infection, establish the value of targeting TrxB2, and provide tools to accelerate the development of TrxB2 inhibitors. PMID:27249779

  4. Mycobacterium tuberculosis Thioredoxin Reductase Is Essential for Thiol Redox Homeostasis but Plays a Minor Role in Antioxidant Defense

    PubMed Central

    Lin, Kan; O'Brien, Kathryn M.; Trujillo, Carolina; Wang, Ruojun; Wallach, Joshua B.; Schnappinger, Dirk

    2016-01-01

    Mycobacterium tuberculosis (Mtb) must cope with exogenous oxidative stress imposed by the host. Unlike other antioxidant enzymes, Mtb’s thioredoxin reductase TrxB2 has been predicted to be essential not only to fight host defenses but also for in vitro growth. However, the specific physiological role of TrxB2 and its importance for Mtb pathogenesis remain undefined. Here we show that genetic inactivation of thioredoxin reductase perturbed several growth-essential processes, including sulfur and DNA metabolism and rapidly killed and lysed Mtb. Death was due to cidal thiol-specific oxidizing stress and prevented by a disulfide reductant. In contrast, thioredoxin reductase deficiency did not significantly increase susceptibility to oxidative and nitrosative stress. In vivo targeting TrxB2 eradicated Mtb during both acute and chronic phases of mouse infection. Deliberately leaky knockdown mutants identified the specificity of TrxB2 inhibitors and showed that partial inactivation of TrxB2 increased Mtb’s susceptibility to rifampicin. These studies reveal TrxB2 as essential thiol-reducing enzyme in Mtb in vitro and during infection, establish the value of targeting TrxB2, and provide tools to accelerate the development of TrxB2 inhibitors. PMID:27249779

  5. Compensatory periplasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa PAO1 in the absence of membrane nitrate reductase.

    PubMed

    Van Alst, Nadine E; Sherrill, Lani A; Iglewski, Barbara H; Haidaris, Constantine G

    2009-10-01

    Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. The inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon, and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The roles of the 2 dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expressions, were examined by use of a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 h. In contrast, the nitrate sensor-response regulator mutant DeltanarXL displayed growth arrest initially, but resumed growth after 72 h and reached the early stationary phase in liquid culture after 120 h. Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild-type P. aeruginosa PAO1, the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase. PMID:19935885

  6. Aldose reductase expression as a risk factor for cataract

    PubMed Central

    Snow, Anson; Shieh, Biehuoy; Chang, Kun-Che; Pal, Arttatrana; Lenhart, Patricia; Ammar, David; Ruzycki, Philip; Palla, Suryanarayana; Reddy, G. Bhanuprakesh; Petrash, J. Mark

    2015-01-01

    Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the

  7. Aldose reductase expression as a risk factor for cataract.

    PubMed

    Snow, Anson; Shieh, Biehuoy; Chang, Kun-Che; Pal, Arttatrana; Lenhart, Patricia; Ammar, David; Ruzycki, Philip; Palla, Suryanarayana; Reddy, G Bhanuprakesh; Petrash, J Mark

    2015-06-01

    Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis, respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the

  8. Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase reduce receptor-mediated endocytosis in opossum kidney cells.

    PubMed

    Sidaway, James E; Davidson, Robert G; McTaggart, Fergus; Orton, Terry C; Scott, Robert C; Smith, Graham J; Brunskill, Nigel J

    2004-09-01

    Renal proximal tubule cells are responsible for the reabsorption of proteins that are present in the tubular lumen. This occurs by receptor-mediated endocytosis, a process that has a requirement for some GTP-binding proteins. Statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase used for the therapeutic reduction of cholesterol-containing plasma lipoproteins. However, they can also reduce intracellular levels of isoprenoid pyrophosphates that are derived from the product of the enzyme, mevalonate, and are required for the prenylation and normal function of GTP-binding proteins. The hypothesis that inhibition of HMG-CoA reductase in renal proximal tubule cells could reduce receptor mediated-endocytosis was therefore tested. Five different statins inhibited the uptake of FITC-labeled albumin by the proximal tubule-derived opossum kidney cell line in a dose-dependent manner and in the absence of cytotoxicity. The reduction in albumin uptake was related to the degree of inhibition of HMG-CoA reductase. Simvastatin (e.g., statin) inhibited receptor-mediated endocytosis of both FITC-albumin and FITC-beta(2)-microglobulin to similar extents but without altering the binding of albumin to the cell surface. The effect on albumin endocytosis was prevented by mevalonate and by the isoprenoid geranylgeranyl pyrophosphate but not by cholesterol. Finally, evidence that the inhibitory effect of statins on endocytosis of proteins may be caused by reduced prenylation and thereby decreased function of one or more GTP-binding proteins is provided. These data establish the possibility in principle that inhibition of HMG-CoA reductase by statins in proximal tubule cells may reduce tubular protein reabsorption. PMID:15339975

  9. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase modulator: toward age- and sex-personalized medicine.

    PubMed

    Pallottini, Valentina

    2015-01-01

    Cholesterol homeostasis maintenance is regulated by a cellular feedback system that senses cholesterol amount in cellular membranes. 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMGR) plays a pivotal role in cholesterol metabolism as it is the key rate-limiting enzyme of its biosynthetic pathway; its inhibition provokes a feedback response capable of reducing plasma cholesterol content. HMGR inhibition is a keystone in the treatment and prevention of cardiovascular disease and, therefore, statins (HMGR inhibitors) are widely prescribed even though they may sometimes induce side effects. These drugs are prescribed indifferently to both man and women even if there are several well-known differences in cholesterol metabolism depending on the gender and the age. Thus, gender-related differences in cholesterol metabolism should be taken into account to identify new targets for customized pharmacological treatments for hypercholesterolemia. PMID:26135220

  10. The Lactone form of stachybotrydial: a new inhibitor of dihydrofolate reductase from stachybotrys sp. FN298.

    PubMed

    Kwon, Yun-Ju; Sohn, Mi-Jin; Kim, Hyun-Ju; Kim, Won-Gon

    2014-01-01

    Dihydrofolate reductase (DHFR) has been confirmed to be a novel target for antibacterial drug development. In this study, we determined that a fungal metabolite from Stachybotrys sp. FN298 can inhibit the DHFR of Staphylococcus aureus. Its structure was identified as a lactone form of stachybotrydial using mass spectrometry and nuclear magnetic resonance analysis. This compound inhibited S. aureus DHFR with a half-maximal inhibitory concentration of 41 µM. It also prevented the growth of S. aureus and methicillin-resistant S. aureus (MRSA) with a minimum inhibitory concentration of 32 µg·mL(-1). To our knowledge, this is the first description of a DHFR inhibitor of microbial origin. The inhibitory function of the lactone form of stachybotrydial highlights its potential for development into a new broad-spectrum antibacterial agent and as an agent against MRSA. PMID:25087962

  11. Transgenic potato plants with overexpression of dihydroflavonol reductase can serve as efficient nutrition sources.

    PubMed

    Kostyn, Kamil; Szatkowski, Michal; Kulma, Anna; Kosieradzka, Iwona; Szopa, Jan

    2013-07-10

    Potato (Solanum tuberosum) is considered to be one of the most important crops cultivated in Europe and the entire world. The tubers of the potato are characterized by rich starch and protein contents and high concentrations of antioxidants, such as vitamin C and flavonoids. Notably, the presence of the phenolic antioxidants is of high importance as they have health-related properties. They are known to reduce the incidence of atherosclerosis, prevent certain kinds of cancer, and aid with many other kinds of diseases. The aim of this study was to find the most efficient way to increase the content of phenolic antioxidants in potato tubers through transgenesis. The results showed that the most efficacious way to achieve this goal was the overexpression of the dihydroflavonol reductase gene (DFR). The produced transgenic potato plants served as a nutrition source for laboratory rats; the study has confirmed their nontoxicity and nutritional benefits on the tested animals. PMID:23692339

  12. Comparative Studies on the Induction and Inactivation of Nitrate Reductase in Corn Roots and Leaves 1

    PubMed Central

    Aslam, Muhammad; Oaks, Ann

    1976-01-01

    A comparison of induction and inactivation of nitrate reductase and two of its component activities, namely FMNH2-nitrate reductase and NO3−-induced NADH-cytochrome c reductase, was made in roots and leaves of corn (Zea mays L. var. W64A × 182E). The three activities were induced in parallel in both tissues when NO3− was supplied. WO4= suppressed the induction of NADH- and FMNH2-nitrate reductase activities in root tips and leaves. The NO3−-induced NADH-cytochrome c reductase activity showed a normal increase in roots treated with WO4=. In leaves, on the other hand, there was a marked superinduction of the NO3−-induced NADH-cytochrome c reductase in the presence of WO4=. The half-life values of NADH-nitrate reductase and FMNH2-nitrate reductase measured by removing NO3− and adding WO4= to the medium, were 4 hours in root tips and 6 hours in excised leaves. Addition of NO3− in the induction medium together with WO4= gave partial protection of NADH-nitrate reductase and FMNH2-nitrate reductase activities in both root tips and leaves with a t0.5 of 6 and 8 hours, respectively. NO3− also reduced the loss of nitrate reductase activity from mature root sections. In the presence of cycloheximide, both NADH-nitrate reductase and NO3−-induced NADH-cytochrome c reductase activities were lost at similar rates in root tips. NO3− protected the loss of NO3−-induced NADH-cytochrome c reductase to the same extent as that of NADH-nitrate reductase. PMID:16659529

  13. Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

    NASA Technical Reports Server (NTRS)

    Johnson, T. C.; Cao, R. Q.; Kung, J. E.; Buchanan, B. B.

    1987-01-01

    Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.

  14. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    NASA Astrophysics Data System (ADS)

    Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

    2015-09-01

    Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5-8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5-8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5-8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction.

  15. The Kinetics and Inhibition of the Enzyme Methemoglobin Reductase

    ERIC Educational Resources Information Center

    Splittgerber, A. G.; And Others

    1975-01-01

    Describes an undergraduate biochemistry experiment which involves the preparation and kinetics of an oxidation-reduction enzyme system, methemoglobin reductase. A crude enzyme extract is prepared and assayed spectrophotometrically. The enzyme system obeys Michaelis-Menton kinetics with respect to both substrate and the NADH cofactor. (MLH)

  16. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  17. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  18. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  19. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  20. 21 CFR 864.7375 - Glutathione reductase assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Glutathione reductase assay. 864.7375 Section 864.7375 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7375...

  1. Domain Evolution and Functional Diversification of Sulfite Reductases

    NASA Astrophysics Data System (ADS)

    Dhillon, Ashita; Goswami, Sulip; Riley, Monica; Teske, Andreas; Sogin, Mitchell

    2005-02-01

    Sulfite reductases are key enzymes of assimilatory and dissimilatory sulfur metabolism, which occur in diverse bacterial and archaeal lineages. They share a highly conserved domain "C-X5-C-n-C-X3-C" for binding siroheme and iron-sulfur clusters that facilitate electron transfer to the substrate. For each sulfite reductase cluster, the siroheme-binding domain is positioned slightly differently at the N-terminus of dsrA and dsrB, while in the assimilatory proteins the siroheme domain is located at the C-terminus. Our sequence and phylogenetic analysis of the siroheme-binding domain shows that sulfite reductase sequences diverged from a common ancestor into four separate clusters (aSir, alSir, dsr, and asrC) that are biochemically distinct; each serves a different assimilatory or dissimilatory role in sulfur metabolism. The phylogenetic distribution and functional grouping in sulfite reductase clusters (dsrA and dsrB vs. aSiR, asrC, and alSir) suggest that their functional diversification during evolution may have preceded the bacterial/archaeal divergence.

  2. The arsenic hyperaccumulating Pteris vittata expresses two arsenate reductases

    PubMed Central

    Cesaro, Patrizia; Cattaneo, Chiara; Bona, Elisa; Berta, Graziella; Cavaletto, Maria

    2015-01-01

    Enzymatic reduction of arsenate to arsenite is the first known step in arsenate metabolism in all organisms. Although the presence of one mRNA arsenate reductase (PvACR2) has been characterized in gametophytes of P. vittata, no arsenate reductase protein has been directly observed in this arsenic hyperaccumulating fern, yet. In order to assess the possible presence of arsenate reductase in P. vittata, two recombinant proteins, ACR2-His6 and Trx-His6-S-Pv2.5–8 were prepared in Escherichia coli, purified and used to produce polyclonal antibodies. The presence of these two enzymes was evaluated by qRT-PCR, immunoblotting and direct MS analysis. Enzymatic activity was detected in crude extracts. For the first time we detected and identified two arsenate reductase proteins (PvACR2 and Pv2.5–8) in sporophytes and gametophytes of P. vittata. Despite an increase of the mRNA levels for both proteins in roots, no difference was observed at the protein level after arsenic treatment. Overall, our data demonstrate the constitutive protein expression of PvACR2 and Pv2.5–8 in P. vittata tissues and propose their specific role in the complex metabolic network of arsenic reduction. PMID:26412036

  3. Activated and unactivated forms of human erythrocyte aldose reductase.

    PubMed Central

    Srivastava, S K; Hair, G A; Das, B

    1985-01-01

    Aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) has been partially purified from human erythrocytes by DEAE-cellulose (DE-52) column chromatography. This enzyme is activated severalfold upon incubation with 10 microM each glucose 6-phosphate, NADPH, and glucose. The activation of the enzyme was confirmed by following the oxidation of NADPH as well as the formation of sorbitol with glucose as substrate. The activated form of aldose reductase exhibited monophasic kinetics with both glyceraldehyde and glucose (Km of glucose = 0.68 mM and Km of glyceraldehyde = 0.096 mM), whereas the native (unactivated) enzyme exhibited biphasic kinetics (Km of glucose = 9.0 and 0.9 mM and Km of glyceraldehyde = 1.1 and 0.14 mM). The unactivated enzyme was strongly inhibited by aldose reductase inhibitors such as sorbinil, alrestatin, and quercetrin, and by phosphorylated intermediates such as ADP, glycerate 3-phosphate, glycerate 1,3-bisphosphate, and glycerate 2,3-trisphosphate. The activated form of the enzyme was less susceptible to inhibition by aldose reductase inhibitors and phosphorylated intermediates. PMID:3933003

  4. Characterization of mitochondrial thioredoxin reductase from C. elegans

    SciTech Connect

    Lacey, Brian M.; Hondal, Robert J. . E-mail: Robert.Hondal@uvm.edu

    2006-08-04

    Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a k {sub cat} of 610 min{sup -1} and a K {sub m} of 610 {mu}M using E. coli thioredoxin as substrate. The reported k {sub cat} is 25% of the k {sub cat} of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate.

  5. 5. cap alpha. -reductase activity in rat adipose tissue

    SciTech Connect

    Zyirek, M.; Flood, C.; Longcope, C.

    1987-11-01

    We measured the 5 ..cap alpha..-reductase activity in isolated cell preparations of rat adipose tissue using the formation of (/sup 3/H) dihydrotestosterone from (/sup 3/H) testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5..cap alpha..-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10/sup -8/ M), when added to the medium, caused a 90% decrease in 5..cap alpha..-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5..cap alpha..-reductase activity in each tissue studied.

  6. Effects of messaging about multiple biomedical and behavioral HIV prevention methods on intentions to use among US MSM: results of an experimental messaging study.

    PubMed

    Mustanski, Brian; Ryan, Daniel T; Sanchez, Travis; Sineath, Craig; Macapagal, Kathryn; Sullivan, Patrick S

    2014-09-01

    Combining multiple biomedical and behavioral HIV prevention approaches is a priority for at-risk populations such as men who have sex with men (MSM), and it is essential to understand how receiving messages about multiple approaches impacts attitudes and intentions for their use. We examined whether receiving combinations of different HIV prevention messages produced differences in perceived benefits and costs of condom use, and in intentions to use condoms and biomedical prevention approaches. MSM (N = 803) were recruited online and were randomly assigned to view informational messages about one, two, or four of the following prevention options: pre-exposure prophylaxis (PrEP), non-occupational post-exposure prophylaxis (nPEP), rectal microbicides, and condoms. The number of HIV prevention messages did not produce differential attitudes and intentions regarding condoms, nor did it produce changes in attitudes towards unprotected sex. Receiving multiple messages was associated with greater intentions to use PrEP and nPEP, but not rectal microbicides. PMID:24907778

  7. [REVIEW OF CLINICAL STUDIES ON COMBINATION THERAPY OF 5α-REDUCTASE INHIBITORS AND α1-BLOCKERS IN PATIENTS WITH BENIGN PROSTATIC HYPERPLASIA].

    PubMed

    Spivak, L G; Lokshin, K L; Vinarov, A Z

    2015-01-01

    The review presents the results of studies on combination therapy of 5α-reductase inhibitors and α-blockers in patients with benign prostatic hyperplasia (BPH). These data demonstrate a significant advantage of the combination therapy versus monotherapy in terms of quality of life and subjective symptoms as well as the safety, better results in the prevention of BPH progression and acute urinary retention, and reduced need for surgery. PMID:26665780

  8. Aldose reductase, ocular diabetic complications and the development of topical Kinostat(®).

    PubMed

    Kador, Peter F; Wyman, Milton; Oates, Peter J

    2016-09-01

    Diabetes mellitus (DM) is a major health problem with devastating effects on ocular health in both industrialized and developing countries. The control of hyperglycemia is critical to minimizing the impact of DM on ocular tissues because inadequate glycemic control leads to ocular tissue changes that range from a temporary blurring of vision to permanent vision loss. The biochemical mechanisms that promote the development of diabetic complications have been extensively studied. As a result, a number of prominent biochemical pathways have been identified. Among these, the two-step sorbitol pathway has been the most extensively investigated; nevertheless, it remains controversial. To date, long-term pharmacological studies in animal models of diabetes have demonstrated that the onset and development of ocular complications that include keratopathy, retinopathy and cataract can be ameliorated by the control of excess metabolic flux through aldose reductase (AR). Clinically the alleles of AR have been linked to the rapidity of onset and severity of diabetic ocular complications in diabetic patient populations around the globe. In spite of these promising preclinical and human genetic rationales, several clinical trials of varying durations with structurally diverse aldose reductase inhibitors (ARIs) have shown limited success or failure in preventing or arresting diabetic retinopathy. Despite these clinical setbacks, topical ARI Kinostat(®) promises to find a home in clinical veterinary ophthalmology where its anticipated approval by the FDA will present an alternative treatment paradigm to cataract surgery in diabetic dogs. Here, we critically review the role of AR in diabetes mellitus-linked ocular disease and highlight the development of Kinostat(®) for cataract prevention in diabetic dogs. In addition to the veterinary market, we speculate that with further safety and efficacy studies in humans, Kinostat(®) or a closely related product could have a future role

  9. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

    SciTech Connect

    Schormann, Norbert; Velu, Sadanandan E.; Murugesan, Srinivasan; Senkovich, Olga; Walker, Kiera; Chenna, Bala C.; Shinkre, Bidhan; Desai, Amar; Chattopadhyay, Debasish

    2010-09-17

    Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

  10. Regulation of nitrate reductase (NR) synthesis investigated by using mutants of Chl. sorokiniana partially NR deficient

    SciTech Connect

    Knobloch, O.; Tischner, R.

    1986-04-01

    After X-ray irradiation 13 NR and 8 nitrite reductase (NiR) deficient mutants of Chl.sorokiniana were obtained. In order to assure best experimental conditions for the characterization of the NR mutants, for which NO/sub 3//sup -/-containing medium in fact is a N-medium, they transferred wild type cells from NH/sub 4//sup +/ to NO/sub 3//sup -/ or N-medium, respectively. It turned out, that NO/sub 3//sup -/ is not necessary for starting de-novo-synthesis of NR. Therefore NR in Chlorella is a derepressible enzyme rather than an inducible one. Maximum amount of NR is present 80 min. after transfer of cells. Derepression experiments with the mutant strains characterized 3 of them as defect in Mo-co subunit of NR with best cytochrome c reductase (CCR)-activity, although xanthine oxidase (XO) was inducible. One other mutant is CCR-defect but contains intact Mo-co. The latter strain produced 4-6 times more Mo-co than the wild type, giving some evidence for an unbalanced self-regulation of NR-synthesis. Another strain lacked XO-activity, indicating a common cofactor among XO and NR as reported for other organisms.

  11. Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation.

    PubMed

    Begara-Morales, Juan C; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla, María N; López-Jaramillo, Javier; Luque, Francisco; Corpas, Francisco J; Barroso, Juan B

    2015-09-01

    The ascorbate-glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO(-)) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO(-) and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO(-). The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO(-). These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO(-) or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate-glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement of

  12. Differential molecular response of monodehydroascorbate reductase and glutathione reductase by nitration and S-nitrosylation

    PubMed Central

    Begara-Morales, Juan C.; Sánchez-Calvo, Beatriz; Chaki, Mounira; Mata-Pérez, Capilla; Valderrama, Raquel; Padilla, María N.; Luque, Francisco; Corpas, Francisco J.; Barroso, Juan B.

    2015-01-01

    The ascorbate–glutathione cycle is a metabolic pathway that detoxifies hydrogen peroxide and involves enzymatic and non-enzymatic antioxidants. Proteomic studies have shown that some enzymes in this cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) are potential targets for post-translational modifications (PMTs) mediated by nitric oxide-derived molecules. Using purified recombinant pea peroxisomal MDAR and cytosolic and chloroplastic GR enzymes produced in Escherichia coli, the effects of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO) which are known to mediate protein nitration and S-nitrosylation processes, respectively, were analysed. Although ONOO– and GSNO inhibit peroxisomal MDAR activity, chloroplastic and cytosolic GR were not affected by these molecules. Mass spectrometric analysis of the nitrated MDAR revealed that Tyr213, Try292, and Tyr345 were exclusively nitrated to 3-nitrotyrosine by ONOO–. The location of these residues in the structure of pea peroxisomal MDAR reveals that Tyr345 is found at 3.3 Å of His313 which is involved in the NADP-binding site. Site-directed mutagenesis confirmed Tyr345 as the primary site of nitration responsible for the inhibition of MDAR activity by ONOO–. These results provide new insights into the molecular regulation of MDAR which is deactivated by nitration and S-nitrosylation. However, GR was not affected by ONOO– or GSNO, suggesting the existence of a mechanism to conserve redox status by maintaining the level of reduced GSH. Under a nitro-oxidative stress induced by salinity (150mM NaCl), MDAR expression (mRNA, protein, and enzyme activity levels) was increased, probably to compensate the inhibitory effects of S-nitrosylation and nitration on the enzyme. The present data show the modulation of the antioxidative response of key enzymes in the ascorbate–glutathione cycle by nitric oxide (NO)-PTMs, thus indicating the close involvement

  13. Experimental Study on Basic Shape of Simple Device for Prevention of Inflow and Accumulation of Buoyant Refuse at the Intake of Headworks

    NASA Astrophysics Data System (ADS)

    Namihira, Atsushi; Kobayashi, Hiroyasu; Takaki, Kyoji; Goto, Masahiro

    In this research, the basic shape of the device for prevention of inflow and accumulation of buoyant refuse at the intake of headworks is investigated by hydraulic model test. As results, it is clarified that an enough result is not obtained if the prismatic bar that floats on the water is set as the device for prevention so that it may cross the intake. On the other hands, it is clarified that the inflow and accumulation of buoyant refuse at the intake is decreased greatly if the bar is set so that it may be diagonally thrust out from the upstream edge of the intake to the downstream on the river side. In this case, it is necessary to decide the length and setting angle of the bar so that it may intersect with the boundary line of the area where the refuse flow into the intake with no device for prevention.

  14. Measurement of nitrous oxide reductase activity in aquatic sediments

    SciTech Connect

    Miller, L.G.; Oremland, R.S.; Paulsen, S.

    1986-01-01

    Denitrification in aquatic sediments was measured by an N/sub 2/O reductase assay. Sediments consumed small added quantities of N/sub 2/O over short periods (a few hours). In experiments with sediment slurries, N/sub 2/O reductase activity was inhibited by 0/sub 2/, C/sub 2/H/sub 2/, heat treatment, and by high levels of nitrate (1 mM) or sulfide (10 mM). However, ambient levels of nitrate (<100 ..mu..M) did not influence activity, and moderate levels (about 150 ..mu..M) induced only a short lag before reductase activity began. Moderate levels of sulfide (<1 mM) had no effect on N/sub 2/O reductase activity. Nitrous oxide reductase displayed Michaelis-Menten kinetics in sediments from freshwater, estuarine, and alkaline-saline environments. An in situ assay was devised in which a solution of N/sub 2/O was injected into sealed glass cores containing intact sediment. Two estimates of net rates of denitrification in San Francisco Bay under approximated in situ conditions were 0.009 and 0.041 mmol of N/sub 2/O per m/sup 2/ per h. Addition of chlorate to inhibit denitrification in these intact-core experiments (to estimate gross rates of N/sub 2/O consumption) resulted in approximately a 14% upward revision of estimates of net rates. These results were comparable to an in situ estimate of 0.022 mmol of N/sub 2/O per m/sup 2/ per h made with the acetylene block assay.

  15. Nickel site of methane catalysis in the methyl reductase enzyme

    SciTech Connect

    Shelnutt, J.A.; Shiemke, A.K.; Scott, R.A.

    1988-01-01

    Methyl reductase is the enzyme of methanogenic bacteria that catalyzed the two-electron reduction of the methyl group of 2-(methylthio)ethanesulfonic acid (methyl-S-CoM) to methane and HS-CoM. The methyl group of methyl-S-CoM ultimately comes from the six-electron reduction of CO/sub 2/ by hydrogen, which also provides the reducing equivalents needed by methyl reductase. The nature of the catalytic site of methyl reductase is of current interest from the point of view of developing biomimetic C/sub 1/, chemistries directed toward methane synthesis and activation. In particular, Sandia is using molecular graphics and energy optimization techniques to design macromolecular catalysts that mimic the structure of sites of proteins that carry out C/sub 1/ chemistry. The goal is to produce catalysts whose function is the oxidation of low molecular weight hydrocarbon gases to generate liquid fuels or, alternatively, the reduction of abundant inorganic resources such as CO/sub 2/ to generate gaseous fuels. Unfortunately, the catalytic sites of many of the enzymes of interest, e.g., methyl reductase and methane monooxygenase, have not been characterized by X-ray crystallography and other structural techniques. With the goal of learning more about the structure of one of these naturally occurring sites of C/sub 1/ chemistry, we have obtained the first resonance Raman spectra of the nickel-macrocycle, called F/sub 430/, at the site of catalysis in methyl reductase. To help us structurally interpret the Raman spectra of the enzyme we have also obtained Raman spectra of solutions of the major forms of F/sub 430/ (salt-extracted and cytosol-free) at room temperature and at 77/degree/K and also, under similar solution conditions, spectra of a nickel-corphinoid derivative that is related to F/sub 430/.

  16. Characterization of recombinant glutathione reductase from the psychrophilic Antarctic bacterium Colwellia psychrerythraea.

    PubMed

    Ji, Mikyoung; Barnwell, Callie V; Grunden, Amy M

    2015-07-01

    Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k cat and k cat/K m at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems. PMID:26101017

  17. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    DOEpatents

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  18. Recominant Pinoresino-Lariciresinol Reductase, Recombinant Dirigent Protein And Methods Of Use

    DOEpatents

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki , Gang; David R. , Sarkanen; Simo , Ford; Joshua D.

    2003-10-21

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided from source species Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Linum usitatissimum, and Schisandra chinensis, which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  19. Tungstate, a Molybdate Analog Inactivating Nitrate Reductase, Deregulates the Expression of the Nitrate Reductase Structural Gene

    PubMed Central

    Deng, Mingde; Moureaux, Thérèse; Caboche, Michel

    1989-01-01

    Nitrate reductase (NR, EC 1.6.6.1) from higher plants is a homodimeric enzyme carrying a molybdenum cofactor at the catalytic site. Tungsten can be substituted for molybdenum in the cofactor structure, resulting in an inactive enzyme. When nitratefed Nicotiana tabacum plants were grown on a nutrient solution in which tungstate was substituted for molybdate, NR activity in the leaves decreased to a very low level within 24 hours while NR protein accumulated progressively to a level severalfold higher than the control after 6 days. NR mRNA level in molybdate-grown plants exhibited a considerable day-night fluctuation. However, when plants were treated with tungstate, NR mRNA level remained very high. NR activity and protein increased over a 24-hour period when nitrate was added back to N-starved molybdate-grown plants. NR mRNA level increased markedly during the first 2 hours and then decreased. In the presence of tungstate, however, the induction of NR activity by nitrate was totally abolished while high levels of NR protein and mRNA were both induced, and the high level of NR mRNA was maintained over a 10-hour period. These results suggest that the substitution of tungsten for molybdenum in NR complex leads to an overexpression of the NR structural gene. Possible mechanisms involved in this deregulation are discussed. Images Figure 2 Figure 3 Figure 5 PMID:16667015

  20. The tick salivary protein sialostatin L inhibits the Th9-derived production of the asthma-promoting cytokine interleukin-9 and is effective in the prevention of experimental asthma1

    PubMed Central

    Horka, Helena; Staudt, Valérie; Klein, Matthias; Taube, Christian; Reuter, Sebastian; Dehzad, Nina; Andersen, John F.; Kopecky, Jan; Schild, Hansjörg; Kotsyfakis, Michalis; Hoffmann, Markus; Gerlitzki, Bastian; Stassen, Michael; Bopp, Tobias; Schmitt, Edgar

    2012-01-01

    Ticks developed a multitude of different immune evasion strategies in order to obtain a blood meal. Sialostatin L is an immunosuppressive cysteine protease inhibitor present in the saliva of the hard tick Ixodes scapularis. Herein we demonstrate that sialostatin L strongly inhibits the production of IL-9 by Th9 cells. Since we could show recently that Th9-derived IL-9 is essentially involved in the induction of asthma symptoms, sialostatin L was used for the treatment of experimental asthma. Application of sialostatin L in a model of experimental asthma almost completely abrogated airway hyperresponsiveness and eosinophilia. Our data suggest that sialostatin L can prevent experimental asthma, most likely by inhibiting the IL-9 production of Th9 cells. Thus, alternative to IL-9 neutralization sialostatin L provides the basis for the development of innovative therapeutic strategies to treat asthma. PMID:22327077

  1. Future directions in cancer prevention.

    PubMed

    Umar, Asad; Dunn, Barbara K; Greenwald, Peter

    2012-12-01

    Prevention of cancer remains the most promising strategy for reducing both its incidence and the mortality due to this disease. For more than four decades, findings from epidemiology, basic research and clinical trials have informed the development of lifestyle and medical approaches to cancer prevention. These include selective oestrogen receptor modulators and aromatase inhibitors for breast cancer, the 5-α-reductase inhibitors finasteride and dutasteride for prostate cancer, and the development of vaccines for viruses that are associated with specific cancers. Future directions include genetic, proteomic and other molecular approaches for identifying pathways that are associated with cancer initiation and development, as well as refining the search for immunologically modifiable causes of cancer. PMID:23151603

  2. Purine nucleoside phosphorylase as a cytosolic arsenate reductase.

    PubMed

    Gregus, Zoltán; Németi, Balázs

    2002-11-01

    The findings of the accompanying paper (Németi and Gregus, Toxicol: Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or inosine) + phosphate <--> guanine (or hypoxanthine) + ribose-1-phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 microM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP

  3. Model of the catalytic mechanism of human aldose reductase based on quantum chemical calculations.

    SciTech Connect

    Cachau, R. C.; Howard, E. H.; Barth, P. B.; Mitschler, A. M.; Chevrier, B. C.; Lamour, V.; Joachimiak, A.; Sanishvili, R.; Van Zandt, M.; Sibley, E.; Moras, D.; Podjarny, A.; UPR de Biologie Structurale; National Cancer Inst.; Univ. Louis Pasteur; Inst. for Diabetes Discovery, Inc.

    2000-01-01

    Aldose Reductase is an enzyme involved in diabetic complications, thoroughly studied for the purpose of inhibitor development. The structure of an enzyme-inhibitor complex solved at sub-atomic resolution has been used to develop a model for the catalytic mechanism. This model has been refined using a combination of Molecular Dynamics and Quantum calculations. It shows that the proton donation, the subject of previous controversies, is the combined effect of three residues: Lys 77, Tyr 48 and His 110. Lys 77 polarises the Tyr 48 OH group, which donates the proton to His 110, which becomes doubly protonated. His 110 then moves and donates the proton to the substrate. The key information from the sub-atomic resolution structure is the orientation of the ring and the single protonafion of the His 110 in the enzyme-inhibitor complex. This model is in full agreement with all available experimental data.

  4. Aldose and aldehyde reductases : structure-function studies on the coenzyme and inhibitor-binding sites.

    SciTech Connect

    El-Kabbani, O.; Old, S. E.; Ginell, S. L.; Carper, D. A.; Biosciences Division; Monash Univ.; NIH

    1999-09-03

    PURPOSE: To identify the structural features responsible for the differences in coenzyme and inhibitor specificities of aldose and aldehyde reductases. METHODS: The crystal structure of porcine aldehyde reductase in complex with NADPH and the aldose reductase inhibitor sorbinil was determined. The contribution of each amino acid lining the coenzyme-binding site to the binding of NADPH was calculated using the Discover package. In human aldose reductase, the role of the non-conserved Pro 216 (Ser in aldehyde reductase) in the binding of coenzyme was examined by site-directed mutagenesis. RESULTS: Sorbinil binds to the active site of aldehyde reductase and is hydrogen-bonded to Trp 22, Tyr 50, His 113, and the non-conserved Arg 312. Unlike tolrestat, the binding of sorbinil does not induce a change in the side chain conformation of Arg 312. Mutation of Pro 216 to Ser in aldose reductase makes the binding of coenzyme more similar to that of aldehyde reductase. CONCLUSIONS: The participation of non-conserved active site residues in the binding of inhibitors and the differences in the structural changes required for the binding to occur are responsible for the differences in the potency of inhibition of aldose and aldehyde reductases. We report that the non-conserved Pro 216 in aldose reductase contributes to the tight binding of NADPH.

  5. Involvement of nitrate reductase in auxin-induced NO synthesis

    PubMed Central

    Erdei, L

    2008-01-01

    It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.1 Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (NG-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation. PMID:19704423

  6. Involvement of nitrate reductase in auxin-induced NO synthesis.

    PubMed

    Kolbert, Zsuzsanna; Erdei, L

    2008-11-01

    It is well known for a long time, that nitric oxide (NO) functions in variable physiological and developmental processes in plants, however the source of this signaling molecule in the diverse plant responses is very obscure.1 Although existance of nitric oxide sythase (NOS) in plants is still questionable, LNMMA (N(G)-monomethyl-L-arginine)-sensitive NO generation was observed in different plant species.2,3 In addition, nitrate reductase (NR) is confirmed to have a major role as source of NO.4,5 This multifaced molecule acts also in auxin-induced lateral root (LR) formation, since exogenous auxin enhanced NO levels in regions of Arabidopsis LR initiatives. Our results pointed out the involvement of nitrate reductase enzyme in auxin-induced NO formation. In this addendum, we speculate on auxin-induced NO production in lateral root primordial formation. PMID:19704423

  7. Methyltetrahydrofolate reductase polymorphism influences onset of Huntington's disease.

    PubMed

    Brune, N; Andrich, J; Gencik, M; Saft, C; Müller, Th; Valentin, S; Przuntek, H; Epplen, J T

    2004-01-01

    Onset of Huntington's disease (HD) negatively correlates with CAG repeat length of the HD gene, which encodes the protein huntingtin. This protein interacts with the homocysteine metabolizing enzyme cystathionine betasynthase (CBS). Objective of this study was to analyze the impact of CAG repeats, polymorphisms of various homocysteine metabolizing enzymes, like CBS, Methyltetrahydrofolate Reductase (MTHTR), Methionine Synthase Reductase (MSR) and methionine synthase (MS) on HD onset in 171 patients. The significant impact of CAG repeats on HD onset (chi2= 25.54, FG = 4, p<0.0001) with a significant correlation between both (R= -0.521, p=0.01) was obvious. HD patients with the homozygous MTHFR-1298-CC significantly (p = 0.024) earlier experienced HD symptoms. There was no influence demonstrable of CBS, MSR and MS. Determination of MTHFR polymorphisms and CAG repeats enables screening for subjects with putative early HD onset in order to study neuroprotective compounds in their efficacy to delay HD symptoms. PMID:15354395

  8. Structural Basis for Substrate Specificity in Human Monomeric Carbonyl Reductases

    PubMed Central

    El-Hawari, Yasser; Dunford, James E.; Kochan, Grazyna; Wsol, Vladimir; Martin, Hans-Joerg; Maser, Edmund; Oppermann, Udo

    2009-01-01

    Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. PMID:19841672

  9. Two fatty acyl reductases involved in moth pheromone biosynthesis.

    PubMed

    Antony, Binu; Ding, Bao-Jian; Moto, Ken'Ichi; Aldosari, Saleh A; Aldawood, Abdulrahman S

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective 'single pgFARs' produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a 'single reductase' can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  10. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A. Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

  11. Using chemical approaches to study selenoproteins - focus on thioredoxin reductases

    PubMed Central

    Hondal, Robert J.

    2009-01-01

    The study of selenocysteine-containing proteins is difficult due to the problems associated with the heterologous production of these proteins. These problems are due to the intricate recoding mechanism used by cells to translate the UGA codon as a sense codon for selenocysteine. The process is further complicated by the fact that eukaryotes and prokaryotes have different UGA recoding machineries. This review focuses on chemical approaches to produce selenoproteins and study the mechanism of selenoenzymes. The use of intein-mediated peptide ligation is discussed with respect to the production of the mammalian selenoenzymes thioredoxin reductase and selenoprotein R, also known as methionine sulfoxide reductase B1. New methods for removing protecting groups from selenocysteine post-synthesis and methods for selenosulfide/diselenide formation are also reviewed. Chemical approaches have also been used to study the enzymatic mechanism of thioredoxin reductase. The approach divides the enzyme into two modules, a large protein module lacking selenocysteine and a small, synthetic selenocysteine-containing peptide. Study of this semisynthetic enzyme has revealed three distinct enzymatic pathways that depend on the properties of the substrate. The enzyme utilizes a macromolecular mechanism for protein substrates, a second mechanism for small molecule substrates and a third pathway for selenium-containing substrates such as selenocystine. PMID:19406205

  12. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  13. Degradation of endogenous bacterial cell wall polymers by the muralytic enzyme mutanolysin prevents hepatobiliary injury in genetically susceptible rats with experimental intestinal bacterial overgrowth.

    PubMed Central

    Lichtman, S N; Okoruwa, E E; Keku, J; Schwab, J H; Sartor, R B

    1992-01-01

    Jejunal self-filling blind loops with subsequent small bowel bacterial overgrowth (SBBO) induce hepatobiliary injury in genetically susceptible Lewis rats. Lesions consist of portal tract inflammation, bile duct proliferation, and destruction. To determine the pathogenesis of SBBO-induced hepatobiliary injury, we treated Lewis rats with SBBO by using several agents with different mechanisms of activity. Buffer treatment, ursodeoxycholic acid, prednisone, methotrexate, and cyclosporin A failed to prevent SBBO-induced injury as demonstrated by increased plasma aspartate aminotransferase (AST) and elevated histology scores. However, hepatic injury was prevented by mutanolysin, a muralytic enzyme whose only known activity is to split the beta 1-4 N-acetylmuramyl-N-acetylglucosamine linkage of peptidoglycan-polysaccharide (PG-PS), a bacterial cell wall polymer with potent inflammatory and immunoregulatory properties. Mutanolysin therapy started on the day blind loops were surgically created and continued for 8 wk significantly diminished AST (101 +/- 37 U/liter) and liver histology scores (2.2 +/- 2.7) compared to buffer-treated rats (228 +/- 146 U/liter, P < 0.05, 8.2 +/- 1.9, P < 0.001 respectively). Mutanolysin treatment started during the early phase of hepatic injury, 16-21 d after surgery, decreased AST in 7 of 11 rats from 142 +/- 80 to 103 +/- 24 U/liter contrasted to increased AST in 9 of 11 buffer-treated rats from 108 +/- 52 to 247 +/- 142 U/liter, P < 0.05. Mutanolysin did not change total bacterial numbers within the loop, eliminate Bacteroides sp., have in vitro antibiotic effects, or diminish mucosal PG-PS transport. However, mutanolysin treatment prevented elevation of plasma anti-PG antibodies and tumor necrosis factor-alpha (TNF alpha) levels which occurred in buffer treated rats with SBBO and decreased TNF alpha production in isolated Kupffer cells stimulated in vitro with PG-PS. Based on the preventive and therapeutic activity of this highly specific

  14. Impairment in Sulfite Reductase Leads to Early Leaf Senescence in Tomato Plants1[W][OPEN

    PubMed Central

    Yarmolinsky, Dmitry; Brychkova, Galina; Kurmanbayeva, Assylay; Bekturova, Aizat; Ventura, Yvonne; Khozin-Goldberg, Inna; Eppel, Amir; Fluhr, Robert; Sagi, Moshe

    2014-01-01

    Sulfite reductase (SiR) is an essential enzyme of the sulfate assimilation reductive pathway, which catalyzes the reduction of sulfite to sulfide. Here, we show that tomato (Solanum lycopersicum) plants with impaired SiR expression due to RNA interference (SIR Ri) developed early leaf senescence. The visual chlorophyll degradation in leaves of SIR Ri mutants was accompanied by a reduction of maximal quantum yield, as well as accumulation of hydrogen peroxide and malondialdehyde, a product of lipid peroxidation. Interestingly, messenger RNA transcripts and proteins involved in chlorophyll breakdown in the chloroplasts were found to be enhanced in the mutants, while transcripts and their plastidic proteins, functioning in photosystem II, were reduced in these mutants compared with wild-type leaves. As a consequence of SiR impairment, the levels of sulfite, sulfate, and thiosulfate were higher and glutathione levels were lower compared with the wild type. Unexpectedly, in a futile attempt to compensate for the low glutathione, the activity of adenosine-5′-phosphosulfate reductase was enhanced, leading to further sulfite accumulation in SIR Ri plants. Increased sulfite oxidation to sulfate and incorporation of sulfite into sulfoquinovosyl diacylglycerols were not sufficient to maintain low basal sulfite levels, resulting in accumulative leaf damage in mutant leaves. Our results indicate that, in addition to its biosynthetic role, SiR plays an important role in prevention of premature senescence. The higher sulfite is likely the main reason for the initiation of chlorophyll degradation, while the lower glutathione as well as the higher hydrogen peroxide and malondialdehyde additionally contribute to premature senescence in mutant leaves. PMID:24987017

  15. Interplay between cytosolic disulfide reductase systems and the Nrf2/Keap1 pathway

    PubMed Central

    Schmidt, Edward E.

    2015-01-01

    NADPH transfers reducing power from bioenergetic pathways to thioredoxin reductase-1 (TrxR1) and glutathione reductase (GR) to support essential reductive systems. Surprisingly, it was recently shown that mouse livers lacking both TrxR1 and GR (‘TR/GR-null’) can sustain redox (reduction-oxidation) homoeostasis using a previously unrecognized NADPH-independent source of reducing power fuelled by dietary methionine. The NADPH-dependent systems are robustly redundant in liver, such that disruption of either TrxR1 or GR alone does not cause oxidative stress. However, disruption of TrxR1 induces transcription factor Nrf2 (nuclear factor erythroid-derived 2-like-2) whereas disruption of GR does not. This suggests the Nrf2 pathway responds directly to the status of the thioredoxin-1 (Trx1) system. The proximal regulator of Nrf2 is Keap1 (Kelch-like ECH-associated protein-1), a cysteine (Cys)-rich protein that normally interacts transiently with Nrf2, targeting it for degradation. During oxidative stress, this interaction is stabilized, preventing degradation of newly synthesized Nrf2, thereby allowing Nrf2 accumulation. Within the Trx1 system, TrxR1 and peroxiredoxins (Prxs) contain some of the most reactive nucleophilic residues in the cell, making them likely targets for oxidants or electrophiles. We propose that Keap1 activity and therefore Nrf2 is regulated by interactions of Trx1 system enzymes with oxidants. In TR/GR-null livers, Nrf2 activity is further induced, revealing that TrxR-independent systems also repress Nrf2 and these might be induced by more extreme challenges. PMID:26551704

  16. Prevention strategies for prostate cancer.

    PubMed

    Schmitz-Dräger, B J; Lümmen, G; Bismarck, E; Fischer, C

    2012-12-01

    Through the last decade consideration of the role of vitamins and minerals in primary prevention of genitourinary tumors has dramatically changed. Despite all efforts efficacy of a specific compound has not been proven, so far. In consequence, recommendations for a use of vitamins or other supplements with the intention of prostate cancer prevention should be avoided today. In contrast, there is some evidence that life style modification might be helpful: recent investigations suggest that smoking may be involved in prostate cancer carcinogenesis. In addition, there is evidence that moderate food consumption, reduction of dairy products and an Asian or Mediterranean diet might not only prevent prostate cancer but also harbors additional beneficial effects on general health. This move from single compounds to more complex diets can be considered as a change of paradigm in prostate cancer prevention and could be the starting point of future epidemiological research. Disappointing findings with regards to nutritional cancer prevention contrast with a solid evidence concerning the efficacy of chemoprevention using 5a-reductase inhibitors: Long-term use of Finasteride and Dutasteride significantly reduces prostate cancer detection. Further candidate drugs are under investigation. However, translation of these findings into urological practice remains a matter of controversial discussion. PMID:23288209

  17. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    NASA Astrophysics Data System (ADS)

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils.

  18. Effect of dietary calcium and magnesium on experimental renal tubular deposition of calcium oxalate crystal induced by ethylene glycol administration and its prevention with phytin and citrate.

    PubMed

    Ebisuno, S; Morimoto, S; Yoshida, T; Fukatani, T; Yasukawa, S; Ohkawa, T

    1987-01-01

    Oral administration of ethylene glycol to rats, and the resultant intratubular depositions of microcrystals of calcium oxalate were studied investigating the influences of dietary calcium or magnesium and assessing the protective efficacies against the crystallizations by treatment with phytin and sodium citrate. With increase of calcium intake and consequent increase of urinary calcium excretion there was a marked increase in the amount of tubular deposit of calcium oxalate crystal and in the calcium content of renal tissue. Although magnesium deficiency accelerated renal tubular calcium oxalate deposition, the protection against the crystal formation was not observed with excessive dietary magnesium. When rats were fed a high-calcium diet supplemented with phytin, a significant inhibition of the intratubular crystallization was observed. It appeared obvious that a hypocalciuric action of phytin was attributed to the effect of the prevention. There was vigorous protection of crystal formation by treatment with sodium citrate, which correlated with the level of citrate concentration in the drinking water. PMID:3433579

  19. Possible involvement of PPARγ-associated eNOS signaling activation in rosuvastatin-mediated prevention of nicotine-induced experimental vascular endothelial abnormalities.

    PubMed

    Kathuria, Sonam; Mahadevan, Nanjaian; Balakumar, Pitchai

    2013-02-01

    Nicotine exposure via cigarette smoking and tobacco chewing is associated with vascular complications. The present study investigated the effect of rosuvastatin in nicotine (2 mg/kg/day, i.p., 4 weeks)-induced vascular endothelial dysfunction (VED) in rats. The development of VED was assessed by employing isolated aortic ring preparation and estimating aortic and serum nitrite/nitrate concentration. Further, scanning electron microscopy and hematoxylin-eosin staining of thoracic aorta were performed to assess the vascular endothelial integrity. Moreover, oxidative stress was assessed by estimating aortic superoxide anion generation and serum thiobarbituric acid-reactive substances. The nicotine administration produced VED by markedly reducing acetylcholine-induced endothelium-dependent relaxation, impairing the integrity of vascular endothelium, decreasing aortic and serum nitrite/nitrate concentration, increasing oxidative stress, and inducing lipid alteration. However, treatment with rosuvastatin (10 mg/kg/day, i.p., 4 weeks) markedly attenuated nicotine-induced vascular endothelial abnormalities, oxidative stress, and lipid alteration. Interestingly, the co-administration of peroxisome proliferator-activated receptor γ (PPARγ) antagonist, GW9662 (1 mg/kg/day, i.p., 2 weeks) submaximally, significantly prevented rosuvastatin-induced improvement in vascular endothelial integrity, endothelium-dependent relaxation, and nitrite/nitrate concentration in rats administered nicotine. However, GW9662 co-administration did not affect rosuvastatin-associated vascular anti-oxidant and lipid-lowering effects. The incubation of aortic ring, isolated from rosuvastatin-treated nicotine-administered rats, with L-NAME (100 μM), an inhibitor of nitric oxide synthase (NOS), significantly attenuated rosuvastatin-induced improvement in acetylcholine-induced endothelium-dependent relaxation. Rosuvastatin prevents nicotine-induced vascular endothelial abnormalities by activating

  20. Neonatal aerosol exposure to Bermuda grass allergen prevents subsequent induction of experimental allergic feline asthma: evidence for establishing early immunologic tolerance.

    PubMed

    Heller, M C; Lee-Fowler, T M; Liu, H; Cohn, L A; Reinero, C R

    2014-07-15

    Allergic asthma is increasing in industrialized countries, especially in children. Rodent and human studies suggest an opportunity to "prevent" asthma in the perinatal period. The aims of this study were to create a more "natural" model of feline asthma by exposing offspring of asthmatic queens to Bermuda grass allergen (BGA) by inhalation only, and to investigate maternal-fetal-infant interactions in the development of asthma. Kittens from asthmatic queens were divided into four groups: maternal exposure to aerosolized BGA during the third trimester, neonatal exposure to aerosolized BGA in the first three months of life, both maternal and neonatal exposure, or saline control. Kittens failing to achieve an asthmatic phenotype based on bronchoalveolar lavage fluid (BALF) analysis by 6 months underwent traditional sensitization: adjuvanted allergen injection, intranasal allergen, and aerosol challenges. BALF was collected at 3, 4 and 6 months, and after sensitization at 8 months, and analyzed for eosinophil counts and BGA-specific IgG and IgA. Intradermal testing (IDT) was performed at 6 and 7 months. At six months none of the kittens had airway eosinophilia, BGA-specific IgG or IgA, and were non-responsive to IDT. After sensitization, kittens receiving neonatal aerosolization failed to develop airway eosinophilia as seen in the controls. Kittens exposed to BGA aerosols, either in-utero or neonatally, continued to lack IDT response. Chronic exposure to BGA aerosols failed to induce asthma in kittens, and instead tolerized the kittens to BGA. This is the first evidence that neonatal intervention could potentially "prevent" allergic asthma in cats. PMID:24704287

  1. Current status of 5α-reductase inhibitors in prostate disease management.

    PubMed

    Kang, Dong Il; Chung, Jae Il

    2013-04-01

    The key enzyme in the androgen synthesis and androgen receptor pathways is 5α-reductase (5-AR), which occurs as three isoenzymes. Types I and II 5-ARs the most important clinically, and two different 5-AR inhibitors (5-ARIs), finasteride and dutasteride, have been developed. Several urology associations have recommended and upgraded the use of 5-ARIs for an enlarged prostate with lower urinary tract symptoms. In the Prostate Cancer Prevention Trial and the Reduction by Dutasteride of Prostate Cancer Events Trial, 5-ARIs reduced the incidence of low-grade prostate cancer. However, despite the documented reductions in the overall incidence of prostate cancer, 5-ARIs are at the center of a dispute. The American Society of Clinical Oncology (ASCO) and the American Urology Association (AUA) presented clinical guidelines for the use of 5-ARIs for chemoprevention of prostate cancer in 2008. However, ASCO/AUA has eliminated these from the main "Clinical Guidelines" in 2012, because the U.S. Food and Drug Administration denied a supplemental New Drug Application for the use of dutasteride for prostate cancer chemoprevention. The 5-ARIs can also be used to manage hemospermia and prostatic hematuria, and to prevent intraoperative bleeding, although there is insufficient evidence for a standard strategy. This review summarizes the current use of 5-ARIs for prostate disease, including benign prostate hyperplasia, prostate cancer, prostate-related bleeding, and hemospermia. PMID:23614056

  2. A flavone from Manilkara indica as a specific inhibitor against aldose reductase in vitro.

    PubMed

    Haraguchi, Hiroyuki; Hayashi, Ryosuke; Ishizu, Takashi; Yagi, Akira

    2003-09-01

    Isoaffinetin (5,7,3',4',5'-pentahydroxyflavone-6-C-glucoside) was isolated from Manilkara indica as a potent inhibitor of lens aldose reductase by bioassay-directed fractionation. This C-glucosyl flavone showed specific inhibition against aldose reductases (rat lens, porcine lens and recombinant human) with no inhibition against aldehyde reductase and NADH oxidase. Kinetic analysis showed that isoaffinetin exhibited uncompetitive inhibition against both dl-glyceraldehyde and NADPH. A structure-activity relationship study revealed that the increasing number of hydroxy groups in the B-ring contributes to the increase in aldose reductase inhibition by C-glucosyl flavones. PMID:14598214

  3. Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

    PubMed Central

    Kuhn, A; van Zyl, C; van Tonder, A; Prior, B A

    1995-01-01

    A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases. PMID:7747971

  4. Structure of the Molybdenum Site of EEcherichia Coli Trimethylamine N-Oxide Reductase

    SciTech Connect

    Zhang, L.; Nelson, K.Johnson; Rajagopalan, K.V.; George, G.N.

    2009-05-28

    We report a structural characterization of the molybdenum site of recombinant Escherichia coli trimethylamine N-oxide (TMAO) reductase using X-ray absorption spectroscopy. The enzyme active site shows considerable similarity to that of dimethyl sulfoxide (DMSO) reductase, in that, like DMSO reductase, the TMAO reductase active site can exist in multiple forms. Examination of the published crystal structure of TMAO oxidase from Shewanella massilia indicates that the postulated Mo coordination structure is chemically impossible. The presence of multiple active site structures provides a potential explanation for the anomalous features reported from the crystal structure.

  5. Combination therapies prevent the neuropathic, proinflammatory characteristics of bone marrow in streptozotocin-induced diabetic rats.

    PubMed

    Dominguez, James M; Yorek, Mark A; Grant, Maria B

    2015-02-01

    We previously showed that peripheral neuropathy of the bone marrow was associated with loss of circadian rhythmicity of stem/progenitor cell release into the circulation. Bone marrow neuropathy results in dramatic changes in hematopoiesis that lead to microvascular complications, inflammation, and reduced endothelial repair. This series of events represents early pathogenesis before development of diabetic retinopathy. In this study we characterized early alterations within the bone marrow of streptozotocin (STZ)-induced diabetic rats following treatments that prevent experimental peripheral neuropathy. We asked whether bone marrow neuropathy and the associated bone marrow pathology were reversed with treatments that prevent peripheral neuropathy. Three strategies were tested: inhibition of neutral endopeptidase, inhibition of aldose reductase plus lipoic acid supplementation, and insulin therapy with antioxidants. All strategies prevented loss of nerve conduction velocity resulting from STZ-induced diabetes and corrected the STZ-induced diabetes-associated increase of immunoreactivity of neuropeptide Y, tyrosine hydroxylase, and somatostatin. The treatments also reduced concentrations of interleukin-1β, granulocyte colony-stimulating factor, and matrix metalloproteinase 2 in STZ-induced diabetic bone marrow supernatant and decreased the expression of NADPH oxidase 2, nitric oxide synthase 2, and nuclear factor-κB1 mRNA in bone marrow progenitor cells. These therapies represent novel approaches to attenuate the diabetic phenotype within the bone marrow and may constitute an important therapeutic strategy for diabetic microvascular complications. PMID:25204979

  6. Protective role of glutathione reductase in paraquat induced neurotoxicity.

    PubMed

    Djukic, Mirjana M; Jovanovic, Marina D; Ninkovic, Milica; Stevanovic, Ivana; Ilic, Katarina; Curcic, Marijana; Vekic, Jelena

    2012-08-30

    Paraquat (PQ), a widely used herbicide is a well-known free radical producing agent. The mechanistic pathways of PQ neurotoxicity were examined by assessing oxidative/nitrosative stress markers. Focus was on the role of glutathione (GSH) cycle and to examine whether the pre-treatment with enzyme glutathione reductase (GR) could protect the vulnerable brain regions (VBRs) against harmful oxidative effect of PQ. The study was conducted on Wistar rats, randomly divided in five groups: intact-control group, (n = 8) and four experimental groups (n = 24). All tested compounds were administered intrastriatally (i.s.) in one single dose. The following parameters of oxidative status were measured in the striatum, hippocampus and cortex, at 30 min, 24 h and 7 days post treatment: superoxide anion radical (O₂·⁻), nitrate (NO₃⁻), malondialdehyde (MDA), superoxide dismutase (SOD), total GSH (tGSH) and its oxidized, disulfide form (GSSG) and glutathione peroxidase (GPx). Results obtained from the intact and the sham operated groups were not statistically different, confirming that invasive i.s. route of administration would not influence the reliability of results. Also, similar pattern of changes were observed between ipsi- and contra- lateral side of examined VBRs, indicating rapid spatial spreading of oxidative stress. Mortality of the animals (10%), within 24h, along with symptoms of Parkinsonism, after awakening from anesthesia for 2-3 h, were observed in the PQ group, only. Increased levels of O₂·⁻, NO₃⁻ and MDA, increased ratio of GSSG/GSH and considerably high activity of GPx were measured at 30 min after the treatment. Cytotoxic effect of PQ was documented by drastic drop of all measured parameters and extremely high peak of the ratio GSSG/GSH at 24th hrs after the PQ i.s. injection. In the GR+PQ group, markedly low activity of GPx and low content of NO₃⁻ (in striatum and cortex) were measured during whole experiment, while increase value was

  7. The neurosteroidogenic enzyme 5α-reductase modulates the role of D1 dopamine receptors in rat sensorimotor gating.

    PubMed

    Frau, Roberto; Mosher, Laura J; Bini, Valentina; Pillolla, Giuliano; Pes, Romina; Saba, Pierluigi; Fanni, Silvia; Devoto, Paola; Bortolato, Marco

    2016-01-01

    Neurosteroids exert diverse modulatory actions on dopamine neurotransmission and signaling. We previously documented that the enzyme 5α-reductase, which catalyzes the main rate-limiting step in neurosteroid synthesis, is required for the behavioral responses of Sprague-Dawley rats to non-selective dopaminergic agonists, such as the D1-D2 receptor agonist apomorphine. Specifically, systemic and intra-accumbal administrations of the 5α-reductase inhibitor finasteride countered apomorphine-induced deficits of sensorimotor gating, as measured by the prepulse inhibition (PPI) of the startle reflex; the classes of dopamine receptors involved in these effects, however, remain unknown. Prior rodent studies have revealed that the contributions of dopamine receptors to PPI regulation vary depending on the genetic background; thus, we analyzed the effect of finasteride on the PPI deficits induced by selective dopamine receptor agonists in Long-Evans (a strain exhibiting PPI deficits in response to both D1 and D2 receptor agonists) and Sprague-Dawley rats (which display PPI reductions following treatment with D2, and D3, but not D1 receptor agonists). In Long-Evans rats, finasteride opposed the PPI deficits induced by activation of D1, but not D2 receptors; conversely, in Sprague-Dawley rats, finasteride prevented the reductions in %PPI and accumbal dopamine extracellular levels caused by selective stimulation of D3, but not D2 receptors; however, the effects on %PPI were not confirmed by analyses on absolute PPI values. Our findings suggest that 5α-reductase modulates the effects of D1, but not D2 receptor agonists on sensorimotor gating. These data may help elucidate the role of neurosteroids in neuropsychiatric disorders featuring PPI deficits, including schizophrenia and Tourette syndrome. PMID:26415119

  8. Magnesium Deficiency and High Light Intensity Enhance Activities of Superoxide Dismutase, Ascorbate Peroxidase, and Glutathione Reductase in Bean Leaves 1

    PubMed Central

    Cakmak, Ismail; Marschner, Horst

    1992-01-01

    The influence of varied Mg supply (10-1000 micromolar) and light intensity (100-580 microeinsteins per square meter per second) on the concentrations of ascorbate (AsA) and nonprotein SH-compounds and the activities of superoxide dismutase (SOD; EC 1.15.11) and the H2O2 scavenging enzymes, AsA peroxidase (EC 1.11.1.7), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were studied in bean (Phaseolus vulgaris L.) leaves over a 13-day period. The concentrations of AsA and SH-compounds and the activities of SOD and H2O2 scavenging enzymes increased with light intensity, in particular in Mg-deficient leaves. Over the 12-day period of growth for a given light intensity, the concentrations of AsA and SH-compounds and the activities of these enzymes remained more or less constant in Mg-sufficient leaves. In contrast, in Mg-deficient leaves, a progressive increase was recorded, particularly in concentrations of AsA and activities of AsA peroxidase and glutathione reductase, whereas the activities of guaiacol peroxidase and catalase were only slightly enhanced. Partial shading of Mg-deficient leaf blades for 4 days prevented chlorosis, and the activities of the O2.− and H2O2 scavenging enzymes remained at a low level. The results demonstrate the role of both light intensity and Mg nutritional status on the regulation of O2.− and H2O2 scavenging enzymes in chloroplasts. PMID:16668779

  9. β-Glucan treatment prevents progressive burn ischaemia in the zone of stasis and improves burn healing: an experimental study in rats.

    PubMed

    Firat, Cemal; Samdanci, Emine; Erbatur, Serkan; Aytekin, Ahmet Hamdi; Ak, Muharrem; Turtay, Muhammed Gokhan; Coban, Yusuf Kenan

    2013-02-01

    Saving the zone of stasis is one of the major goals of burn specialists. Increasing the tissue tolerance to ischaemia and inhibiting inflammation have been proposed to enable salvage of this zone. After a burn, excessive inflammation, including increased vascular permeability, local tissue oedema and neutrophil activation, causes local tissue damage by triggering vascular thrombosis and blocking capillaries, resulting in tissue ischaemia and necrosis. Oxygen radicals also contribute to tissue damage after a burn. However, macrophages play a pivotal role in the response to burn. We studied β-glucan because of its many positive systemic effects that are beneficial to burn healing, including immunomodulatory effects, antioxidant effects (free-radical scavenging activity) and effects associated with the reduction of the inflammatory response. There were four test groups in this study with eight rats in each group. Group 1 was the control group, group 2 was administered a local pomade (bacitracin+neomycin sulphate), group 3 received β-glucan (50 mg kg(-1), orally) + the local pomade and group 4 received β-glucan. Burns were created using a brass comb model. Macroscopic, histopathological and statistical assessments were performed. Samples were harvested on the 3rd, 7th and 21 days for analysis. The neutrophilic infiltration into the zone of stasis was analysed on day 3. Macrophage infiltration, fibroblast proliferation, angiogenesis and re-epithelialisation ratios in the zone of stasis were analysed on days 7 and 21. The β-glucan groups (groups 3 and 4) exhibited lower neutrophil counts on the 3rd day, and macrophage infiltration, fibroblast proliferation, angiogenesis and re-epithelialisation were very high in these groups on the 7th day. In particular, re-epithelialisation on the 21st day was significantly better in the β-glucan groups. This study demonstrated that β-glucan may prevent neutrophil-dependent tissue damage and burn-induced oxidative injury through

  10. Alkyl Hydroperoxide Reductases C and D Are Major Antigens Constitutively Expressed by Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Olsen, Ingrid; Reitan, Liv J.; Holstad, Gudmund; Wiker, Harald G.

    2000-01-01

    Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum. Two antigens were present in M. avium subsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium. They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected with M. avium subsp. paratuberculosis had strong gamma interferon (IFN-γ) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-γ production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M. avium subsp. paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences. PMID:10639449

  11. A Novel C5a-neutralizing Mirror-image (l-)Aptamer Prevents Organ Failure and Improves Survival in Experimental Sepsis

    PubMed Central

    Hoehlig, Kai; Maasch, Christian; Shushakova, Nelli; Buchner, Klaus; Huber-Lang, Markus; Purschke, Werner G; Vater, Axel; Klussmann, Sven

    2013-01-01

    Complement factor C5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here, we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (l-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes (PMN) by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex (MAC). In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multiorgan failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis. PMID:23887360

  12. The Neuropilin-1 Inhibitor, ATWLPPR Peptide, Prevents Experimental Diabetes-Induced Retinal Injury by Preserving Vascular Integrity and Decreasing Oxidative Stress

    PubMed Central

    Wang, Jun; Wang, Shuaiwei; Li, Mengling; Wu, Dongdong; Liu, Fang; Yang, Ruisheng; Ji, Shaoping; Ji, Ailing; Li, Yanzhang

    2015-01-01

    Neuropilin-1 (NRP-1) is a transmembrane glycoprotein. As a VEGF co-receptor, NRP1 significantly enhances VEGFR2 signaling and promotes vascular permeability and migration. The purpose of this study was to evaluate the effects of an NRP-1 inhibitor, ATWLPPR peptide, on the early stages of diabetic retinopathy. Eight-week-old male C57BL/6 mice were divided into three groups: a Normal group, a Diabetes (DB) ATWLPPR treatment group and a DB saline group. Electroretinography (ERG), fundus fluorescence angiography (FFA) and leukostasis were examined to evaluate the retinal injury induced by diabetes at the end of the fifth week after STZ injection. Occludin expression and extravasation of albumin were measured to determine the extent of vascular injury. The oxidative stress level and the levels of inflammation-associated proteins were also assayed. The results indicated that treatment with ATWLPPR prevents the abnormal condition of ERG (amplitudes of b-wave decreased and implicit time increased) and vascular injury (occludin degradation and increase in extravasated albumin). These effects were associated with a reduction in the oxidase stress level and the expression of VEGF, GFAP, and ICAM-1. We conclude that ATWLPPR, an NRP-1 inhibitor, may reduce the early retinal damage induced by diabetes by preserving vascular integrity and decreasing the oxidative stress level. Blockade of NRP-1 may be a new therapeutic strategy for the early stages of DR. PMID:26554379

  13. Preventing Suicide

    MedlinePlus

    ... The top three methods used in suicides include firearms (49.9%), suffocation (26.7%), and poisoning (15. ... Content source: National Center for Injury Prevention and Control, Division of Violence Prevention Page maintained by: Office ...

  14. Drowning Prevention

    MedlinePlus

    ... Listen Español Text Size Email Print Share Drowning Prevention: Information for Parents Page Content Article Body Drowning ... in very cold water for lengthy periods. Drowning Prevention: Know the Warning Signs These signs may signal ...

  15. Differential Light Induction of Nitrate Reductases in Greening and Photobleached Soybean Seedlings 1

    PubMed Central

    Kakefuda, Genichi; Duke, Stanley H.; Duke, Stephen O.

    1983-01-01

    Soybean (Glycine max [L.] Merr.) seeds were imbibed and germinated with or without NO3−, tungstate, and norflurazon (San 9789). Norflurazon is a herbicide which causes photobleaching of chlorophyll by inhibiting carotenoid synthesis and which impairs normal chloroplast development. After 3 days in the dark, seedlings were placed in white light to induce extractable nitrate reductase activity. The induction of maximal nitrate reductase activity in greening cotyledons did not require NO3− and was not inhibited by tungstate. Induction of nitrate reductase activity in norflurazon-treated cotyledons had an absolute requirement for NO3− and was completely inhibited by tungstate. Nitrate was not detected in seeds or seedlings which had not been treated with NO3−. The optimum pH for cotyledon nitrate reductase activity from norflurazon-treated seedlings was at pH 7.5, and near that for root nitrate reductase activity, whereas the optimum pH for nitrate reductase activity from greening cotyledons was pH 6.5. Induction of root nitrate reductase activity was also inhibited by tungstate and was dependent on the presence of NO3−, further indicating that the isoform of nitrate reductase induced in norflurazon-treated cotyledons is the same or similar to that found in roots. Nitrate reductases with and without a NO3− requirement for light induction appear to be present in developing leaves. In vivo kinetics (light induction and dark decay rates) and in vitro kinetics (Arrhenius energies of activation and NADH:NADPH specificities) of nitrate reductases with and without a NO3− requirement for induction were quite different. Km values for NO3− were identical for both nitrate reductases. PMID:16663185

  16. The effect of copper and gallium compounds on ribonucleotide reductase

    SciTech Connect

    Narasimhan, J.

    1992-01-01

    The mode of action of copper complexes (CuL and CuKTS) and gallium compounds (gallium nitrate and citrate) in cytotoxicity was studied. The effects of these agents on the enzyme ribonucleotide reductase was investigated by monitoring the tyrosyl free radical present in the active site of the enzyme through electron spin resonance (ESR) spectroscopy. Ribonucleotide reductase, a key enzyme in cellular proliferation, consists of two subunits. M1, a dimer of molecular weight 170,000 contains the substrate and effector binding sites. M2, a dimer of molecular weight 88,000, contains non-heme iron and tyrosyl free radical essential for the activity of the enzyme. In studies using copper complexes, the cellular oxidative chemistry was examined by ESR studies on adduct formation with membranes, and oxidation of thiols. Membrane thiols were oxidized through the reduction of the ESR signal of the thiol adduct and the analysis of sulfhydryl content. Using the radiolabel [sup 59]Fe, the inhibitory action of copper thiosemicarbazones on cellular iron uptake was shown. The inhibitory action of CuL on ribonucleotide reductase was shown by the quenching of the tyrosyl free radical on the M2 subunit. The hypothesis that gallium directly interacts with the M2 subunit of the enzyme and displaces the iron from it was proven. The tyrosyl free radical signal from cell lysates was inhibited by the direct addition of gallium compounds. Gallium content in the cells was measured by a fluorimetric method, to ensure the presence of sufficient amounts of gallium to compete with the iron in the M2 subunit. The enzyme activity, measured by the conversion of [sup 14]C-CDP to the labeled deoxy CDP, was inhibited by the addition of gallium nitrate in a cell free assay system. The immunoprecipitation studies of the [sup 59]Fe labeled M2 protein using the monoclonal antibody directed against this subunit suggested that gallium releases iron from the M2 subunit.

  17. Erythropoietin and its carbamylated derivative prevent the development of experimental diabetic autonomic neuropathy in STZ-induced diabetic NOD-SCID mice.

    PubMed

    Schmidt, Robert E; Green, Karen G; Feng, Dongyan; Dorsey, Denise A; Parvin, Curtis A; Lee, Jin-Moo; Xiao, Qinlgi; Brines, Michael

    2008-01-01

    Autonomic neuropathy is a significant diabetic complication resulting in increased morbidity and mortality. Studies of autopsied diabetic patients and several rodent models demonstrate that the neuropathologic hallmark of diabetic sympathetic autonomic neuropathy in prevertebral ganglia is the occurrence of synaptic pathology resulting in distinctive dystrophic neurites ("neuritic dystrophy"). Our prior studies show that neuritic dystrophy is reversed by exogenous IGF-I administration without altering the metabolic severity of diabetes, i.e. functioning as a neurotrophic substance. The description of erythropoietin (EPO) synergy with IGF-I function and the recent discovery of EPO's multifaceted neuroprotective role suggested it might substitute for IGF-I in treatment of diabetic autonomic neuropathy. Our current studies demonstrate EPO receptor (EPO-R) mRNA in a cDNA set prepared from NGF-maintained rat sympathetic neuron cultures which decreased with NGF deprivation, a result which demonstrates clearly that sympathetic neurons express EPO-R, a result confirmed by immunohistochemistry. Treatment of STZ-diabetic NOD-SCID mice have demonstrated a dramatic preventative effect of EPO and carbamylated EPO (CEPO, which is neuroprotective but not hematopoietic) on the development of neuritic dystrophy. Neither EPO nor CEPO had a demonstrable effect on the metabolic severity of diabetes. Our results coupled with reported salutary effects of EPO on postural hypotension in a few clinical studies of EPO-treated anemic diabetic and non-diabetic patients may reflect a primary neurotrophic effect of EPO on the sympathetic autonomic nervous system, rather than a primary hematopoietic effect. These findings may represent a major clinical advance since EPO has been widely and safely used in anemic patients due to a variety of clinical conditions. PMID:17967455

  18. Aerobic Exercise Training Prevents the Onset of Endothelial Dysfunction via Increased Nitric Oxide Bioavailability and Reduced Reactive Oxygen Species in an Experimental Model of Menopause

    PubMed Central

    Braga, Viviane A. V. N.; Couto, Gisele K.; Lazzarin, Mariana C.; Rossoni, Luciana V.; Medeiros, Alessandra

    2015-01-01

    Objective Previous studies have shown that estrogen deficiency, arising in postmenopause, promotes endothelial dysfunction. This study evaluated the effects of aerobic exercise training on endothelial dependent vasodilation of aorta in ovariectomized rats, specifically investigating the role of nitric oxide (NO) and reactive oxygen species (ROS). Methods Female Wistar rats ovariectomized (OVX – n=20) or with intact ovary (SHAM – n=20) remained sedentary (OVX and SHAM) or performed aerobic exercise training on a treadmill 5 times a week for a period of 8 weeks (OVX-TRA and SHAM-TRA). In the thoracic aorta the endothelium-dependent and –independent vasodilation was assessed by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. Certain aortic rings were incubated with L-NAME to assess the NO modulation on the ACh-induced vasodilation. The fluorescence to dihydroethidium in aortic slices and plasma nitrite/nitrate concentrations were measured to evaluate ROS and NO bioavailability, respectively. Results ACh-induced vasodilation was reduced in OVX rats as compared SHAM (Rmax: SHAM: 86±3.3 vs. OVX: 57±3.0%, p<0.01). Training prevented this response in OVX-TRA (Rmax: OVX-TRA: 88±2.0%, p<0.01), while did not change it in SHAM-TRA (Rmax: SHAM-TRA: 80±2.2%, p<0.01). The L-NAME incubation abolished the differences in ACh-induced relaxation among groups. SNP-induced vasodilation was not different among groups. OVX reduced nitrite/nitrate plasma concentrations and increased ROS in aortic slices, training as effective to restore these parameters to the SHAM levels. Conclusions Exercise training, even in estrogen deficiency conditions, is able to improve endothelial dependent vasodilation in rat aorta via enhanced NO bioavailability and reduced ROS levels. PMID:25923465

  19. Methylenetetrahydrofolate Reductase C677T: Hypoplastic Left Heart and Thrombosis.

    PubMed

    Spronk, Kimberly J; Olivero, Anthony D; Haw, Marcus P; Vettukattil, Joseph J

    2015-10-01

    The incidence of congenital heart defects is higher in infants with mutation of methylenetetrahydrofolate reductase (MTHFR) gene. The MTHFR C677T gene decreases the bioavailability of folate and increases plasma homocysteine, a risk factor for thrombosis. There have been no reported cases in the literature on the clinical implications of this procoagulable state in the setting of cyanotic heart disease, which itself has prothrombotic predisposition. Two patients with hypoplastic left heart syndrome developed postoperative thrombotic complications, both were homozygous for MTHFR C677T. We present these cases and highlight the implications of MTHFR mutation in the management of complex congenital heart disease. PMID:26467879

  20. Terpenoids from Diplophyllum taxifolium with quinone reductase-inducing activity.

    PubMed

    Wang, Xiao; Zhang, Jiao-Zhen; Zhou, Jin-Chuan; Shen, Tao; Lou, Hong-Xiang

    2016-03-01

    Two new ent-prenylaromadendrane-type diterpenoids, diplotaxifols A (1) and B (2), a new ent-eudesmol, ent-eudesma-4(15),11(13)-dien-6α,12-diol (3), eight new eudesmanolides enantiomers (4-11) of the corresponding compounds from higher plants along with four known ent-eudesmanolides (12-15) were isolated from the 95% EtOH extract of Chinese liverwort Diplophyllum taxifolium. Their structures were elucidated on the basis of MS, NMR and IR spectral data, and confirmed by single-crystal X-ray diffraction analysis. The quinone reductase-inducing activity of the compounds was evaluated. PMID:26656409

  1. Asymmetric Reduction of Activated Alkenes by Pentaerythritol Tetranitrate Reductase: Specificity and Control of Stereochemical Outcome by Reaction Optimisation

    PubMed Central

    Fryszkowska, Anna; Toogood, Helen; Sakuma, Michiyo; Gardiner, John M.; Stephens, Gill M.; Scrutton, Nigel S.

    2009-01-01

    We show that pentaerythritol tetranitrate reductase (PETNR), a member of the ‘ene’ reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated α,β-unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme-substrate complexes based on the crystal complex of the PETNR with 2-cyclohexenone 4a. The optical purity of products is variable (49–99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cβ (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18–19a), an enantiodivergent course of the reduction of E/Z-forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Cα is due to non-enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation. PMID:20396613

  2. AAV8-Mediated Angiotensin-Converting Enzyme 2 Gene Delivery Prevents Experimental Autoimmune Uveitis by Regulating MAPK, NF-κB and STAT3 Pathways

    PubMed Central

    Qiu, Yiguo; Tao, Lifei; Zheng, Shijie; Lin, Ru; Fu, Xinyu; Chen, Zihe; Lei, Chunyan; Wang, Jiaming; Li, Hongwei; Li, Qiuhong; Lei, Bo

    2016-01-01

    Renin angiotensin system (RAS) is a key hormonal system which regulates the cardiovascular function and is implicated in several autoimmune diseases. With the discovery of the angiotensin-converting enzyme 2 (ACE2), a protective axis of RAS namely ACE2/Ang-(1–7)/Mas that counteracts the deleterious ACE/AngII/AT1R axis has been established. This axis is emerging as a novel target to attenuate ocular inflammation. However, the underlying molecular mechanisms remain unclear. We investigated the hypothesis that enhancing the activity of the protective axis of RAS by subretinal delivery of an AAV8 (Y733F)-ACE2 vector would protect against the ocular inflammation in experimental autoimmune uveitis (EAU) mice through regulating the local immune responses. Our studies demonstrated that increased ACE2 expression exerts protective effects on inflammation in EAU mouse by modulating ocular immune responses, including the differentiation of Th1/Th17 cells and the polarization of M1/M2 macrophages; whereas the systemic immune responses appeared not affected. These effects were mediated by activating the Ang-(1–7)/Mas and inhibiting the MAPK, NF-κB and STAT3 signaling pathways. This proof-of-concept study suggests that activation of ocular ACE2/Ang-(1–7)/Mas axis with AAV gene transfer modulates local immune responses and may be a promising, long-lasting therapeutic strategy for refractory and recurrent uveitis, as well as other inflammatory eye diseases. PMID:27558087

  3. Efficacy of two acidified chlorite postmilking teat disinfectants with sodium dodecylbenzene sulfonic acid on prevention of contagious mastitis using an experimental challenge protocol.

    PubMed

    Oura, L Y; Fox, L K; Warf, C C; Kempt, G K

    2002-01-01

    Two acidified sodium chlorite postmilking teat disinfectants were evaluated for efficacy against Staphylococcus aureus and Streptococcus agalactiae by using National Mastitis Council experimental challenge procedures. The effect of these teat dips on teat skin and teat end condition was also determined. Both dips contained 0.32% sodium chlorite, 1.32% lactic, and 2.5% glycerin. Dips differed in the amount of sodium dodecylbenzene sulfonic acid (0.53 or 0.27%) added as a surfactant. Both dips significantly reduced new intramammary infection (IMI) rates compared with undipped controls. The dip containing 0.53% dodecylbenzene sulfonic acid reduced new IMI by Staph. aureus by 72% and Strep. agalactiae by 75%. The dip containing 0.27% dodecylbenzene sulfonic acid reduced new IMI by Staph. aureus by 100% and by Strep. agalactiae by 88%. Changes in teat skin and teat end condition for treatment and control groups varied in parallel over time. Teats treated with either teat dip had higher mean teat skin and teat end scores than control teats at some weeks. However, teat skin and teat end condition did not tend to change from the start to the completion of the trial. Application of the two new postmilking teat dips was effective in reducing new IMI from contagious mastitis pathogens. (Key words: teat dip, contagious mastitis, chlorous acid) PMID:11860118

  4. In vivo and in vitro studies of the prevention of proteolipid apoprotein-induced murine experimental allergic encephalomyelitis by monoclonal antibody against L3T4.

    PubMed

    Satoh, J; Kunishita, T; Tabira, T

    1988-05-01

    The suppressive effect of anti-L3T4 monoclonal antibody (mAb) on murine experimental allergic encephalomyelitis (EAE) induced by sensitization with proteolipid apoprotein (PLP) was examined in vivo and in vitro. This mAb inhibited the antigen-specific proliferation of the encephalitogenic T cell lines but did not block the mitogen-mediated response. Serial injections of the mAb during the pre-effector phase of EAE markedly suppressed the development and relapse of the disease but this treatment initiated after appearance of clinical signs was not effective. In treated animals, L3T4+ T cells in the spleen were profoundly decreased and the antigen-specific proliferative response of spleen cells was completely suppressed. Moreover, adoptive transfer of spleen cells from the treated mice induced resistance against EAE induction in the recipients. However, no obvious evidence for antigen-specific suppressor cells was found in vitro in the L3T4- populations of spleen cells from treated mice. PMID:2451681

  5. Anti-Oxidative Effect of Myrtenal in Prevention and Treatment of Colon Cancer Induced by 1, 2-Dimethyl Hydrazine (DMH) in Experimental Animals

    PubMed Central

    Lokeshkumar, Booupathy; Sathishkumar, Venkatachalam; Nandakumar, Natarajan; Rengarajan, Thamaraiselvan; Madankumar, Arumugam; Balasubramanian, Maruthaiveeran Periyasamy

    2015-01-01

    Colon cancer is considered as the precarious forms of cancer in many developed countries, with few to no symptoms; the tumor is often diagnosed in the later stages of cancer. Monoterpenes are a major part of plant essential oils found largely in fruits, vegetables and herbs. The cellular and molecular activities show therapeutic progression that may reduce the risk of developing cancer by modulating the factors responsible for colon carcinogenesis. Colon cancer was induced with DMH with a dose of (20 mg/Kg/body weight) for 15 weeks by subcutaneous injection once in a week. Myrtenal treatment was started with (230 mg/Kg/body weight) by intragastric administration, one week prior to DMH induction and continued till the experimental period of 30 weeks. The Invivo results exhibit the elevated antioxidant and lipid peroxidation levels in DMH treated animals. The Histopathological analysis of colon tissues well supported the biochemical alterations and inevitably proves the protective role of Myrtenal. Treatment with myrtenal to cancer bearing animals resulted in a remarkable increase in the inherent antioxidants and excellent modulation in the morphological and physiological nature of the colon tissue. It is thus concluded that myrtenal exhibits excellent free radical scavenging activity and anticancer activity through the suppression of colon carcinoma in Wistar albino rats. PMID:26336588

  6. A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

    PubMed

    Milhim, Mohammed; Gerber, Adrian; Neunzig, Jens; Hannemann, Frank; Bernhardt, Rita

    2016-08-10

    Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions. PMID:27238232

  7. Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4’-sulfonyl derivative of L-methionine (dabsyl Met), the ...

  8. Evaluation of 5α-reductase inhibitory activity of certain herbs useful as antiandrogens.

    PubMed

    Nahata, A; Dixit, V K

    2014-08-01

    This study demonstrates 5α-reductase inhibitory activity of certain herbs useful in the management of androgenic disorders. Ganoderma lucidum (Curtis) P. Karst (GL), Urtica dioica Linn. (UD), Caesalpinia bonducella Fleming. (CB), Tribulus terrestris Linn. (TT), Pedalium murex Linn. (PM), Sphaeranthus indicus Linn. (SI), Cuscuta reflexa Roxb. (CR), Citrullus colocynthis Schrad. (CC), Benincasa hispida Cogn. (BH), Phyllanthus niruri Linn. (PN) and Echinops echinatus Linn. (EE) were included in the study. Petroleum ether, ethanol and aqueous extracts of these herbs were tested for their 5α-reductase inhibitory activity against the standard 5α-reductase inhibitor, finasteride. A biochemical method to determine the activity of 5α-reductase was used to evaluate the inhibition of different extracts to the enzyme. The optical density (OD) value of each sample was measured continuously with ultraviolet spectrophotometer for the reason that the substrate NADPH has a specific absorbance at 340 nm. As the enzyme 5α-reductase uses NADPH as a substrate, so in the presence of 5α-reductase inhibitor, the NADPH concentration will increase with the function of time. This method thus implicates the activity of 5α-reductase. The method proved to be extremely useful to screen the herbs for their 5α-reductase inhibitory potential. GL, UD, BH, SI and CR came out to be promising candidates for further exploring their antiandrogenic properties. PMID:23710567

  9. Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2.

    PubMed Central

    French, C E; Nicklin, S; Bruce, N C

    1996-01-01

    Pentaerythritol tetranitrate reductase, which reductively liberates nitrite from nitrate esters, is related to old yellow enzyme. Pentaerythritol tetranitrate reductase follows a ping-pong mechanism with competitive substrate inhibition by NADPH, is strongly inhibited by steroids, and is capable of reducing the unsaturated bond of 2-cyclohexen-1-one. PMID:8932320

  10. Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

    NASA Technical Reports Server (NTRS)

    Warner, R. L.; Huffaker, R. C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

  11. Adversity-induced relapse of fear: neural mechanisms and implications for relapse prevention from a study on experimentally induced return-of-fear following fear conditioning and extinction.

    PubMed

    Scharfenort, R; Menz, M; Lonsdorf, T B

    2016-01-01

    The efficacy of current treatments for anxiety disorders is limited by high relapse rates. Relapse of anxiety disorders and addiction can be triggered by exposure to life adversity, but the underlying mechanisms remain unexplored. Seventy-six healthy adults were a priori selected for the presence or absence of adverse experiences during childhood (CA) and recent past (RA; that is, past 12 months). Participants underwent fear conditioning (day 1) and fear extinction and experimental return-of-fear (ROF) induction through reinstatement (a model for adversity-induced relapse; day 2). Ratings, autonomic (skin conductance response) and neuronal activation measures (functional magnetic resonance imaging (fMRI)) were acquired. Individuals exposed to RA showed a generalized (that is, not CS- specific) fear recall and ROF, whereas unexposed individuals showed differential (that is, CS+ specific) fear recall and ROF on an autonomic level despite no group differences during fear acquisition and extinction learning. These group differences in ROF were accompanied by corresponding activation differences in brain areas known to be involved in fear processing and differentiability/generalization of ROF (that is, hippocampus). In addition, dimensional measures of RA, CA and lifetime adversity were negatively correlated with differential skin conductance responses (SCRs) during ROF and hippocampal activation. As discriminating signals of danger and safety, as well as a tendency for overgeneralization, are core features in clinically anxious populations, these deficits may specifically contribute to relapse risk following exposure to adversity, in particular to recent adversity. Hence, our results may provide first and novel insights into the possible mechanisms mediating enhanced relapse risk following exposure to (recent) adversity, which may guide the development of effective pre- and intervention programs. PMID:27434492

  12. Prevention of UVA-Induced Oxidative Damage in Human Dermal Fibroblasts by New UV Filters, Assessed Using a Novel In Vitro Experimental System

    PubMed Central

    Emanuelli, Monica; Damiani, Elisabetta

    2014-01-01

    Background UVA rays present in sunlight are able to reach the dermal skin layer generating reactive oxygen species (ROS) responsible for oxidative damage, alterations in gene expression, DNA damage, leading to cell inflammation, photo-ageing/-carcinogenesis. Sunscreens contain UV filters as active ingredients that absorb/reflect/dissipate UV radiation: their efficiency depends on their spectral profile and photostability which should then be reflected in biological protection of underlying skin. Methods A set of new UV filters was synthesized, and the most photostable one was compared to BMDBM, a widely used UVA filter. Cultured human dermal fibroblasts were exposed to UVA radiation which was filtered by a base cream containing or not UV filters placed above cell culture wells. The endpoints measured were: cell viability (MTT assay), ROS generation (DCFH-DA assay), mitochondrial function (JC-1 assay), DNA integrity (Comet assay) and gene expression (MMP-1, COL1A1) by RT-qPCR. Results The new UV filter resulted more efficient than BMDBM in preserving cell viability, mitochondrial functionality and oxidative DNA damage, despite similar inhibition levels of intracellular ROS. Moreover, expression of genes involved in dermal photoageing were positively affected by the filtering action of the tested molecules. Conclusions The experimental model proposed was able to validate the efficacy of the new UV filter, taking into account important cellular events related to UV-induced intracellular oxidative stress, often underestimated in the assessments of these compounds. General Significance The model may be used to compare the actual biological protection of commercial sunscreens and suncare products aside from their SPF and UVA-PF values. PMID:24409282

  13. Moving forward in fall prevention: an intervention to improve balance among patients in a quasi-experimental study of hospitalized patients.

    PubMed

    Villafañe, Jorge H; Pirali, Caterina; Buraschi, Riccardo; Arienti, Chiara; Corbellini, Camilo; Negrini, Stefano

    2015-12-01

    We investigated the effectiveness of three different rehabilitative programs: group exercise, individual core stability or balance training intervention with a stabilometric platform to improve balance ability in elderly hospitalized patients. We used a prospective quasi-experimental study design. Twenty-eight patients, 39.3% women [age (mean±SD) 72.4±6.5 years], known to have had at least a fall in the last 12 months, were consecutively assigned to one of the following three groups: group exercise intervention, individual core stability or balance training with a stabilometric platform (five sessions a week for 3 weeks in each group). Outcomes were collected at baseline and immediately following the intervention period. In each intervention group, patients showed improvement in balance and mobility, shown as an improvement in the three functional tests score (the Tinetti scale, the Berg Balance Scale, and the Time Up and Go test) (all, P<0.05), whereas, generally, the changes in the score of the test of the stabilometric platform (Postural Stability Test and Fall Risk Test) were not significant for all the interventions. No significant group-by-time interaction was detected for any of the intervention groups, which suggests that the groups improved in the same way. These findings indicate that participation in an exercise program can improve balance and functional mobility, which might contribute toward the reductions of the falls of elderly hospitalized patients and the subsequent fall-related costs. Functional scales might be more appropriate than an instrumental test (Postural Stability Test and Fall Risk Test of the Biodex Balance System) in detecting the functional improvement because of a rehabilitative intervention. PMID:26230947

  14. The functional nitrite reductase activity of the heme-globins

    PubMed Central

    2008-01-01

    Hemoglobin and myoglobin are among the most extensively studied proteins, and nitrite is one of the most studied small molecules. Recently, multiple physiologic studies have surprisingly revealed that nitrite represents a biologic reservoir of NO that can regulate hypoxic vasodilation, cellular respiration, and signaling. These studies suggest a vital role for deoxyhemoglobin- and deoxymyoglobin-dependent nitrite reduction. Biophysical and chemical analysis of the nitrite-deoxyhemoglobin reaction has revealed unexpected chemistries between nitrite and deoxyhemoglobin that may contribute to and facilitate hypoxic NO generation and signaling. The first is that hemoglobin is an allosterically regulated nitrite reductase, such that oxygen binding increases the rate of nitrite conversion to NO, a process termed R-state catalysis. The second chemical property is oxidative denitrosylation, a process by which the NO formed in the deoxyhemoglobin-nitrite reaction that binds to other deoxyhemes can be released due to heme oxidation, releasing free NO. Third, the reaction undergoes a nitrite reductase/anhydrase redox cycle that catalyzes the anaerobic conversion of 2 molecules of nitrite into dinitrogen trioxide (N2O3), an uncharged molecule that may be exported from the erythrocyte. We will review these reactions in the biologic framework of hypoxic signaling in blood and the heart. PMID:18596228

  15. Life with too much polyprenol–polyprenol reductase deficiency

    PubMed Central

    Gründahl, J.E.H.; Guan, Z.; Rust, S.; Reunert, J.; Müller, B.; Du Chesne, I.; Zerres, K.; Rudnik-Schöneborn, S.; Ortiz-Brüchle, N.; Häusler, M.G.; Siedlecka, J.; Swiezewska, E.; Raetz, C.R.H.; Marquardt, T.

    2012-01-01

    Congenital disorders of glycosylation (CDG) are caused by a dysfunction of glycosylation, an essential step in the manufacturing process of glycoproteins. This paper focuses on a 6-year-old patient with a new type of CDG-I caused by a defect of the steroid 5α reductase type 3 gene (SRD5A3). The clinical features were psychomotor retardation, pathological nystagmus, slight muscular hypotonia and microcephaly. SRD5A3 was recently identified encoding the polyprenol reductase, an enzyme catalyzing the final step of the biosynthesis of dolichol, which is required for the assembly of the glycans needed for N-glycosylation. Although an early homozygous stop-codon (c.57G> A [W19X]) with no functional protein was found in the patient, about 70% of transferrin (Tf) was correctly glycosylated. Quantification of dolichol and unreduced polyprenol in the patient's fibroblasts demonstrated a high polyprenol/dolichol ratio with normal amounts of dolichol, indicating that high polyprenol levels might compete with dolichol for the initiation of N-glycan assembly but without supporting normal glycosylation and that there must be an alternative pathway for dolichol biosynthesis. PMID:22304929

  16. Optimisation of nitrate reductase enzyme activity to synthesise silver nanoparticles.

    PubMed

    Khodashenas, Bahareh; Ghorbani, Hamid Reza

    2016-06-01

    Today, the synthesis of silver nanoparticles (Ag NPs) is very common since it has many applications in different areas. The synthesis of these nanoparticles is done by means of physical, chemical, or biological methods. However, due to its inexpensive and environmentally friendly features, the biological method is more preferable. In the present study, using nitrate reductase enzyme available in the Escherichia coli (E. coli) bacterium, the biosynthesis of Ag NPs was investigated. In addition, the activity of the nitrate reductase enzyme was optimised by changing its cultural conditions, and the effects of silver nitrate (AgNO(3)) concentration and enzyme amount on nanoparticles synthesis were studied. Finally, the produced nanoparticles were studied using ultraviolet -visible (UV-Vis) spectrophotometer, dynamic light scattering technique, and transmission electron microscopy. UV-Visible spectrophotometric study showed the characteristic peak for Ag NPs at wavelength 405-420 nm for 1 mM metal precursor solution (AgNO(3)) with 1, 5, 10, and 20 cc supernatant and 435 nm for 0.01M AgNO(3) with 20 cc supernatant. In this study, it was found that there is a direct relationship between the AgNO(3) concentration and the size of produced Ag NPs. PMID:27256897

  17. New pteridine substrates for dihydropteridine reductase and horseradish peroxidase.

    PubMed Central

    Armarego, W L; Ohnishi, A; Taguchi, H

    1986-01-01

    The oxidation of 4,5-diaminopyrimidin-6(1H)-one, 5,6,7,8-tetrahydropteridin-4(3H)-one, its 6-methyl and cis-6,7-dimethyl derivatives, and 6-methyl- and cis-6-7-dimethyl-5,6,7,8-tetrahydropterins, by horseradish peroxidase/H2O2 is enzymic and follows Michaelis-Menten kinetics, and its Km and kcat. values were determined. This oxidation of 5,6,7,8-tetrahydropterins produces quinonoid dihydropterins of established structure, and they are known to be specific substrates for dihydropteridine reductase. By analogy the peroxidase/H2O2 oxidation of the 5,6,7,8-tetrahydropteridin-4(3H)-ones should produce similar quinonoid dihydro species. The quinonoid species derived from 5,6,7,8-tetrahydropteridin-4(3H)-one and its 6-methyl and cis-6,7-dimethyl derivatives are shown to be viable substrates for human brain dihydropteridine reductase, and apparent Km and Vmax. values are reported. PMID:3718470

  18. Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase.

    PubMed

    Davenport, Susie; Le Lay, Pascaline; Sanchez-Tamburrrino, Juan Pablo

    2015-12-01

    Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants. PMID:26447683

  19. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

    Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

    2016-01-01

    Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

  20. Molecular cloning and characterization of Schistosoma japonicum aldose reductase.

    PubMed

    Liu, Jian; Wang, Jipeng; Wang, Shuqi; Xu, Bin; Liu, Xiufeng; Wang, Xiaoning; Hu, Wei

    2013-02-01

    Antioxidant defense is an essential mechanism for schistosomes to cope with damage from host immune-generated reactive oxygen species. The evaluation of the effects of aldose reductase, an important enzyme that may be involved in this system, has long been neglected. In the present study, aldose reductase of Schistosoma japonicum (SjAR) was cloned and characterized. The activity of SjAR was assessed in vitro and was suppressed by the reported inhibitor, epalrestat. RT-PCR analysis revealed that SjAR was expressed at each of the development stages analyzed with increased levels in cercariae. The results also showed that SjAR was expressed at higher levels in adult male worms than in adult female worms. Indirect enzyme-linked immunosorbent assay and western blot analysis indicated that the purified recombinant SjAR (rSjAR) protein displayed a significant level of antigenicity. Immunolocalization analysis revealed that SjAR was mainly distributed in the gynecophoral canal of adult male worms. BALB/c mice immunized with rSjAR induced a 32.91 % worm reduction compared to the adjuvant group (P < 0.01). Moreover, a 28.27 % reduction in egg development in the liver (P > 0.05) and a 42.75 % reduction in egg development in the fecal samples (P < 0.05) were also observed. These results suggested that SjAR may be a potential new drug target or vaccine candidate for schistosomes. PMID:23160889

  1. Evolution Alters the Enzymatic Reaction Coordinate of Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    How evolution has affected enzyme function is a topic of great interest in the field of biophysical chemistry. Evolutionary changes from Escherichia coli dihydrofolate reductase (ecDHFR) to human dihydrofolate reductase (hsDHFR) have resulted in increased catalytic efficiency and an altered dynamic landscape in the human enzyme. Here, we show that a subpicosecond protein motion is dynamically coupled to hydride transfer catalyzed by hsDHFR but not ecDHFR. This motion propagates through residues that correspond to mutational events along the evolutionary path from ecDHFR to hsDHFR. We observe an increase in the variability of the transition states, reactive conformations, and times of barrier crossing in the human system. In the hsDHFR active site, we detect structural changes that have enabled the coupling of fast protein dynamics to the reaction coordinate. These results indicate a shift in the DHFR family to a form of catalysis that incorporates rapid protein dynamics and a concomitant shift to a more flexible path through reactive phase space. PMID:25369552

  2. Azotobacter vinelandii NADPH:ferredoxin reductase cloning, sequencing, and overexpression.

    PubMed

    Isas, J M; Yannone, S M; Burgess, B K

    1995-09-01

    Azotobacter vinelandii ferredoxin I (AvFdI) controls the expression of another protein that was originally designated Protein X. Recently we reported that Protein X is a NADPH-specific flavoprotein that binds specifically to FdI (Isas, J.M., and Burgess, B.K. (1994) J. Biol. Chem. 269, 19404-19409). The gene encoding this protein has now been cloned and sequenced. Protein X is 33% identical and has an overall 53% similarity with the fpr gene product from Escherichia coli that encodes NADPH:ferredoxin reductase. On the basis of this similarity and the similarity of the physical properties of the two proteins, we now designate Protein X as A. vinelandii NADPH:ferredoxin reductase and its gene as the fpr gene. The protein has been overexpressed in its native background in A. vinelandii by using the broad host range multicopy plasmid, pKT230. In addition to being regulated by FdI, the fpr gene product is overexpressed when A. vinelandii is grown under N2-fixing conditions even though the fpr gene is not preceded by a nif specific promoter. By analogy to what is known about fpr expression in E. coli, we propose that FdI may exert its regulatory effect on fpr by interacting with the SoxRS regulon. PMID:7673160

  3. SORGOdb: Superoxide Reductase Gene Ontology curated DataBase

    PubMed Central

    2011-01-01

    Background Superoxide reductases (SOR) catalyse the reduction of superoxide anions to hydrogen peroxide and are involved in the oxidative stress defences of anaerobic and facultative anaerobic organisms. Genes encoding SOR were discovered recently and suffer from annotation problems. These genes, named sor, are short and the transfer of annotations from previously characterized neelaredoxin, desulfoferrodoxin, superoxide reductase and rubredoxin oxidase has been heterogeneous. Consequently, many sor remain anonymous or mis-annotated. Description SORGOdb is an exhaustive database of SOR that proposes a new classification based on domain architecture. SORGOdb supplies a simple user-friendly web-based database for retrieving and exploring relevant information about the proposed SOR families. The database can be queried using an organism name, a locus tag or phylogenetic criteria, and also offers sequence similarity searches using BlastP. Genes encoding SOR have been re-annotated in all available genome sequences (prokaryotic and eukaryotic (complete and in draft) genomes, updated in May 2010). Conclusions SORGOdb contains 325 non-redundant and curated SOR, from 274 organisms. It proposes a new classification of SOR into seven different classes and allows biologists to explore and analyze sor in order to establish correlations between the class of SOR and organism phenotypes. SORGOdb is freely available at http://sorgo.genouest.org/index.php. PMID:21575179

  4. Recessive congenital methaemoglobinaemia: cytochrome b(5) reductase deficiency.

    PubMed

    Percy, Melanie J; Lappin, Terry R

    2008-05-01

    Some 60 years ago, Quentin Gibson reported the first hereditary disorder involving an enzyme when he deduced that familial methaemoglobinaemia was caused by an enzymatic lesion associated with the glycolysis pathway in red blood cells. This disorder, now known as recessive congenital methaemoglobinaemia (RCM), is caused by NADH-cytochrome b5 reductase (cb(5)r) deficiency. Two distinct clinical forms, types I and II, have been recognized, both characterized by cyanosis from birth. In type II, the cyanosis is accompanied by neurological impairment and reduced life expectancy. Cytochrome b(5) reductase is composed of one FAD and one NADH binding domain linked by a hinge region. It is encoded by the CYB5R3 (previously known as DIA1) gene and more than 40 mutations have been described, some of which are common to both types of RCM. Mutations associated with type II tend to cause incorrect splicing, disruption of the active site or truncation of the protein. At present the description of the sequence variants of cb(5)r in the literature is confusing, due to the use of two conventions which differ by one codon position. Herein we propose a new system for nomenclature of cb(5)r based on recommendations of the Human Genome Variation Society. The development of a heterologous expression system has allowed the impact of naturally occurring variants of cb(5)r to be assessed and has provided insight into the function of cb(5)r. PMID:18318771

  5. Retrospective approach to methylenetetrahydrofolate reductase mutations in children.

    PubMed

    Özer, Işıl; Özçetin, Mustafa; Karaer, Hatice; Kurt, Semiha G; Şahin, Şemsettin

    2011-07-01

    Methylenetetrahydrofolate reductase reduces methyltetrahydrofolate, a cosubstrate in the remethylation of homocysteine, from methylenetetrahydrofolate. Congenital defects, hematologic tumors, and intrauterine growth retardation can occur during childhood. This study evaluated clinical and laboratory treatment approaches in children diagnosed with methylenetetrahydrofolate reductase mutations. Our group included 23 boys and 14 girls, aged 103.4 ± 70.8 months S.D. Clinical findings of patients and homocysteine, vitamin B12, folate, hemogram, electroencephalography, cranial magnetic resonance imaging, and echocardiography data were evaluated in terms of treatment approach. Our patients' findings included vitamin B12 at 400.4 ± 224.6 pg/mL S.D. (normal range, 300-700 pg/mL), folate at 10.1 ± 4.5 ng/mL S.D. (normal range, 1.8-9 ng/mL), and homocysteine at 8.4 ± 4.7 μmol/L S.D. (normal range, 5.5-17 μmol/L). Eighty-eight percent of patients demonstrated clinical findings. In comparisons involving categorical variables between groups, χ(2) tests were used. No relationship was evident between mutation type, laboratory data, and clinical severity. All mothers who had MTHFR mutations and had babies with sacral dimples had taken folate supplements during pregnancy. To avoid the risk of neural tube defects, pregnant women with a MTHFR mutation may require higher than normally recommended doses of folic acid supplementation for optimum health. PMID:21723457

  6. The Role of Thioredoxin Reductases in Brain Development

    PubMed Central

    Soerensen, Jonna; Jakupoglu, Cemile; Beck, Heike; Förster, Heidi; Schmidt, Jörg; Schmahl, Wolfgang; Schweizer, Ulrich

    2008-01-01

    The thioredoxin-dependent system is an essential regulator of cellular redox balance. Since oxidative stress has been linked with neurodegenerative disease, we studied the roles of thioredoxin reductases in brain using mice with nervous system (NS)-specific deletion of cytosolic (Txnrd1) and mitochondrial (Txnrd2) thioredoxin reductase. While NS-specific Txnrd2 null mice develop normally, mice lacking Txnrd1 in the NS were significantly smaller and displayed ataxia and tremor. A striking patterned cerebellar hypoplasia was observed. Proliferation of the external granular layer (EGL) was strongly reduced and fissure formation and laminar organisation of the cerebellar cortex was impaired in the rostral portion of the cerebellum. Purkinje cells were ectopically located and their dendrites stunted. The Bergmann glial network was disorganized and showed a pronounced reduction in fiber strength. Cerebellar hypoplasia did not result from increased apoptosis, but from decreased proliferation of granule cell precursors within the EGL. Of note, neuron-specific inactivation of Txnrd1 did not result in cerebellar hypoplasia, suggesting a vital role for Txnrd1 in Bergmann glia or neuronal precursor cells. PMID:18350150

  7. Synthesis and metabolism of inhibitors of ribonucleotide reductase

    SciTech Connect

    Smith, F.T.

    1985-01-01

    In an effort to prepare more effective inhibitors of ribo-nucleotide reductase a series of 2-substituted-4,6-dihydroxypyrimidines was prepared via the appropriately substituted benzamidine. None of the compounds exhibited in vivo activity against L1210 leukemia. No further testing was performed. In order to investigate the metabolism of 3,4-dihydroxybenzohydroxamic acid, a known inhibitor of ribonucleotide reductase, radiolabeled 3,4-dihydroxybenzohydroxamic acid was synthesized by a modification of the procedure of Pichat and Tostain. /sup 14/C-3,4-Dihydroxybenzoic acid was converted to the methyl ester and subsequently reacted with hydroxylamine to give the hydroxamic acid. /sup 14/C-3,4-Dihydroxybenzohydroxamic acid was given i.p. to Sprague-Dawley rats. Excretion occurred mainly (72%) via the urine. HPLC coupled with GC/MS analyses showed that the compound was excreted mainly unchanged. The compound was metabolized to 3,4-dihydroxybenzamide, 4-methoxy-3-hydroxybenzohydroxamic acid, and 4-hydroxy-3-methoxybenzohydroxamic acid. HPLC analysis also showed the lack of formation of any glucuronide or sulfate conjugates through either the hydroxamic acid or catechol functionalities.

  8. A mutant of barley lacking NADH-hydroxypyruvate reductase

    SciTech Connect

    Blackwell, R.; Lea, P. )

    1989-04-01

    A mutant of barley, LaPr 88/29, deficient in peroxisomal NADH-hydroxypyruvate reductase (HPR) activity has been identified. Compared to the wild type the activities of NADH-HPR and NADPH-HPR were severely reduced but the mutant was still capable of fixing CO{sub 2} at rates equivalent to 75% of that of the wild type in air. Although lacking an enzyme in the main photorespiratory pathway, there appeared to be little disruption to photorespiratory metabolism as ammonia release, CO{sub 2} efflux and {sup 14}CO{sub 2} release from L-(U-{sup 14}C) serine were similar in both mutant and wild type. LaPr 88/29 has been used to show that NADH-glyoxylate reductase (GR) and NADH-HPR are probably not catalyzed by the same enzyme in barley and that over 80% of the NADPH-HPR activity is due to the NADH-HPR enzyme. Immunological studies, using antibodies raised against spinach HPR, have shown that the NADH-dependent enzyme protein is absent in LaPr 88/29 but there appears to be enhanced synthesis of the NADPH-dependent enzyme protein.

  9. Carbonyl reductase of dog liver: purification, properties, and kinetic mechanism.

    PubMed

    Hara, A; Nakayama, T; Deyashiki, Y; Kariya, K; Sawada, H

    1986-01-01

    A carbonyl reductase has been extracted into 0.5 M KCl from dog liver and purified to apparent homogeneity by a three-step procedure consisting of chromatography on CM-Sephadex, Matrex green A, and Sephadex G-100 in high-ionic-strength buffers. The enzyme is a dimer composed of two identical subunits of molecular weight 27,000. The pH optimum is 5.5 and the isoelectric point of the enzyme is 9.3. The enzyme reduces aromatic ketones and aldehydes; the aromatic ketones with adjacent medium alkyl chains are the best substrates. Quinones, ketosteroids, prostaglandins, and aliphatic carbonyl compounds are poor or inactive substrates for the enzyme. As a cofactor the enzyme utilizes NADPH, the pro-S hydrogen atom of which is transferred to the substrate. Two moles of NADPH bind to one mole of the enzyme molecule, causing a blue shift and enhancement of the cofactor fluorescence. The reductase reaction is reversible and the equilibrium constant determined at pH 7.0 is 12.8. Steady-state kinetic measurements in both directions suggest that the reaction proceeds through a di-iso ordered bi-bi mechanism. PMID:3511844

  10. Prostate Cancer Cells Differ in Testosterone Accumulation, Dihydrotestosterone Conversion, and Androgen Receptor Signaling Response to Steroid 5α-Reductase Inhibitors

    PubMed Central

    Wu, Yue; Godoy, Alejandro; Azzouni, Faris; Wilton, John H.; Ip, Clement; Mohler, James L.

    2014-01-01

    BACKGROUND Blocking 5α-reductase-mediated testosterone conversion to dihydrotestosterone (DHT) with finasteride or dutasteride is the driving hypothesis behind two prostate cancer prevention trials. Factors affecting intracellular androgen levels and the androgen receptor (AR) signaling axis need to be examined systematically in order to fully understand the outcome of interventions using these drugs. METHODS The expression of three 5α-reductase isozymes, as determined by immunohistochemistry and qRT-PCR, was studied in five human prostate cancer cell lines. Intracellular testosterone and DHT were analyzed using mass spectrometry. A luciferase reporter assay and AR-regulated genes were used to evaluate the modulation of AR activity. RESULTS Prostate cancer cells were capable of accumulating testosterone to a level 15–50 times higher than that in the medium. The profile and expression of 5α-reductase isozymes did not predict the capacity to convert testosterone to DHT. Finasteride and dutasteride were able to depress testosterone uptake in addition to lowering intracellular DHT. The inhibition of AR activity following drug treatment often exceeded the expected response due to reduced availability of DHT. The ability to maintain high intracellular testosterone might compensate for the shortage of DHT. CONCLUSIONS The biological effect of finasteride or dutasteride appears to be complex and may depend on the interplay of several factors, which include testosterone turnover, enzymology of DHT production, ability to use testosterone and DHT interchangeably, and propensity of cells for off-target AR inhibitory effect. PMID:23813697

  11. Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

    PubMed

    Lygidakis, Antonios; Karuppiah, Vijaykumar; Hoeven, Robin; Ní Cheallaigh, Aisling; Leys, David; Gardiner, John M; Toogood, Helen S; Scrutton, Nigel S

    2016-08-01

    Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases. PMID:27411040

  12. Natural variation in arsenate tolerance identifies an arsenate reductase in Arabidopsis thaliana.

    PubMed

    Sánchez-Bermejo, Eduardo; Castrillo, Gabriel; del Llano, Bárbara; Navarro, Cristina; Zarco-Fernández, Sonia; Martinez-Herrera, Dannys Jorge; Leo-del Puerto, Yolanda; Muñoz, Riansares; Cámara, Carmen; Paz-Ares, Javier; Alonso-Blanco, Carlos; Leyva, Antonio

    2014-01-01

    The enormous amount of environmental arsenic was a major factor in determining the biochemistry of incipient life forms early in the Earth's history. The most abundant chemical form in the reducing atmosphere was arsenite, which forced organisms to evolve strategies to manage this chemical species. Following the great oxygenation event, arsenite oxidized to arsenate and the action of arsenate reductases became a central survival requirement. The identity of a biologically relevant arsenate reductase in plants nonetheless continues to be debated. Here we identify a quantitative trait locus that encodes a novel arsenate reductase critical for arsenic tolerance in plants. Functional analyses indicate that several non-additive polymorphisms affect protein structure and account for the natural variation in arsenate reductase activity in Arabidopsis thaliana accessions. This study shows that arsenate reductases are an essential component for natural plant variation in As(V) tolerance. PMID:25099865

  13. Inhibition of rat steroid 5 alpha-reductase (isozyme 1) by suramin.

    PubMed

    Taylor, M F; Bhattacharyya, A K; Collins, D C

    1995-07-01

    In this study, we show the inhibition of rat steroid 5 alpha-reductase (isozyme 1) by suramin. The enzyme activity decreased in a dose-dependent manner as suramin concentrations increased with the calculated drug dose required for 50% inhibition (at 5 microM testosterone and 200 microM NADPH) being 13 microM. Suramin showed non-competitive inhibition of 5 alpha-reductase with respect to testosterone (KT1 = 2.4 microM) and competitive inhibition with respect to NADPH (KiNADPH = 220 nM). Furthermore, suramin and NADP+, but not NAD+, protected 5 alpha-reductase from labeling by 2-azido-NADP+, a photoactive probe which has recently been used to identify the NADPH binding domain of 5 alpha-reductase. These results suggest that suramin inhibits rat steroid 5 alpha-reductase (isozyme 1) at the level of NADPH binding to the enzyme. PMID:7482629

  14. Phenotypic assessment of pulmonary hypertension using high-resolution echocardiography is feasible in neonatal mice with experimental bronchopulmonary dysplasia and pulmonary hypertension: a step toward preventing chronic obstructive pulmonary disease.

    PubMed

    Reynolds, Corey L; Zhang, Shaojie; Shrestha, Amrit Kumar; Barrios, Roberto; Shivanna, Binoy

    2016-01-01

    Bronchopulmonary dysplasia (BPD) and chronic obstructive pulmonary disease (COPD) are chronic lung diseases of human infants and adults, respectively, that are characterized by alveolar simplification. One-third of the infants with severe BPD develop pulmonary hypertension (PH). More importantly, PH increases morbidity and mortality in BPD patients. Additionally, COPD is a common respiratory morbidity in former BPD patients. The lack of an appropriate small animal model wherein echocardiography (Echo) can demonstrate PH is one of the major barriers to understand the molecular mechanisms of the disease and, thereby, develop rational therapies to prevent and/or treat PH in BPD patients. Thus, the goal of this study was to establish a model of experimental BPD and PH and investigate the feasibility of Echo to diagnose PH in neonatal mice. Since hyperoxia-induced oxidative stress and inflammation contributes to the development of BPD with PH, we tested the hypothesis that exposure of newborn C57BL/6J mice to 70% O2 (hyperoxia) for 14 days leads to lung oxidative stress, inflammation, alveolar and pulmonary vascular simplification, pulmonary vascular remodeling, and Echo evidence of PH. Hyperoxia exposure caused lung oxidative stress and inflammation as evident by increased malondialdehyde adducts and inducible nitric oxide synthase, respectively. Additionally, hyperoxia exposure caused growth restriction, alveolar and pulmonary vascular simplification, and pulmonary vascular remodeling. At 14 days of age, Echo of these mice demonstrated that hyperoxia exposure decreased pulmonary acceleration time (PAT) and PAT/ejection time ratio and increased right ventricular free wall thickness, which are indicators of significant PH. Thus, we have demonstrated the feasibility of Echo to phenotype PH in neonatal mice with experimental BPD with PH, which can aid in discovery of therapies to prevent and/or treat BPD with PH and its sequelae such as COPD in humans. PMID:27478373

  15. Phenotypic assessment of pulmonary hypertension using high-resolution echocardiography is feasible in neonatal mice with experimental bronchopulmonary dysplasia and pulmonary hypertension: a step toward preventing chronic obstructive pulmonary disease

    PubMed Central

    Reynolds, Corey L; Zhang, Shaojie; Shrestha, Amrit Kumar; Barrios, Roberto; Shivanna, Binoy

    2016-01-01

    Bronchopulmonary dysplasia (BPD) and chronic obstructive pulmonary disease (COPD) are chronic lung diseases of human infants and adults, respectively, that are characterized by alveolar simplification. One-third of the infants with severe BPD develop pulmonary hypertension (PH). More importantly, PH increases morbidity and mortality in BPD patients. Additionally, COPD is a common respiratory morbidity in former BPD patients. The lack of an appropriate small animal model wherein echocardiography (Echo) can demonstrate PH is one of the major barriers to understand the molecular mechanisms of the disease and, thereby, develop rational therapies to prevent and/or treat PH in BPD patients. Thus, the goal of this study was to establish a model of experimental BPD and PH and investigate the feasibility of Echo to diagnose PH in neonatal mice. Since hyperoxia-induced oxidative stress and inflammation contributes to the development of BPD with PH, we tested the hypothesis that exposure of newborn C57BL/6J mice to 70% O2 (hyperoxia) for 14 days leads to lung oxidative stress, inflammation, alveolar and pulmonary vascular simplification, pulmonary vascular remodeling, and Echo evidence of PH. Hyperoxia exposure caused lung oxidative stress and inflammation as evident by increased malondialdehyde adducts and inducible nitric oxide synthase, respectively. Additionally, hyperoxia exposure caused growth restriction, alveolar and pulmonary vascular simplification, and pulmonary vascular remodeling. At 14 days of age, Echo of these mice demonstrated that hyperoxia exposure decreased pulmonary acceleration time (PAT) and PAT/ejection time ratio and increased right ventricular free wall thickness, which are indicators of significant PH. Thus, we have demonstrated the feasibility of Echo to phenotype PH in neonatal mice with experimental BPD with PH, which can aid in discovery of therapies to prevent and/or treat BPD with PH and its sequelae such as COPD in humans. PMID:27478373

  16. Glutathione reductase: Comparison of steady-state and rapid reaction primary kinetic isotope effects exhibited by the yeast, spinach, and Escherichia coli enzymes

    SciTech Connect

    Vanoni, M.A.; Wong, K.K.; Ballou, D.P.; Blanchard, J.S. )

    1990-06-19

    Kinetic parameters for NADPH and NADH have been determined at pH 8.1 for spinach, yeast, and E. coli glutathione reductases. NADPH exhibited low Km values for all enzymes (3-6 microM), while the Km values for NADH were 100 times higher (approximately 400 microM). Under our experimental conditions, the percentage of maximal velocities with NADH versus those measured with NADPH were 18.4, 3.7, and 0.13% for the spinach, yeast, and E. coli enzymes, respectively. Primary deuterium kinetic isotope effects were independent of GSSG concentration between Km and 15Km levels, supporting a ping-pong kinetic mechanism. For each of the three enzymes, NADPH yielded primary deuterium kinetic isotope effects on Vmax only, while NADH exhibited primary deuterium kinetic isotope effects on both V and V/K. The magnitude of DV/KNADH at pH 8.1 is 4.3 for the spinach enzyme, 2.7 for the yeast enzyme, and 1.6 for the E. coli glutathione reductase. The experimentally determined values of TV/KNADH of 7.4, 4.2, and 2.2 for the spinach, yeast, and E. coli glutathione reductases agree well with those calculated from the corresponding DV/KNADH using the Swain-Schaad expression. This suggests that the intrinsic primary kinetic isotope effect on NADH oxidation is fully expressed. In order to confirm this conclusion, single-turnover experiments have been performed. The measured primary deuterium kinetic isotope effects on the enzyme reduction half-reaction using NADH match those measured in the steady state for each of the three glutathione reductases.

  17. Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint.

    PubMed

    Turner, Glenn W; Davis, Edward M; Croteau, Rodney B

    2012-06-01

    Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined. PMID:22170164

  18. Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

    PubMed Central

    Cohen, G; Yanko, M; Mislovati, M; Argaman, A; Schreiber, R; Av-Gay, Y; Aharonowitz, Y

    1993-01-01

    The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated. Images PMID:8349555

  19. Mycobacterial Dihydrofolate Reductase Inhibitors Identified Using Chemogenomic Methods and In Vitro Validation

    PubMed Central

    Mugumbate, Grace; Abrahams, Katherine A.; Cox, Jonathan A. G.; Papadatos, George; van Westen, Gerard; Lelièvre, Joël; Calus, Szymon T.; Loman, Nicholas J.; Ballell, Lluis; Barros, David; Overington, John P.; Besra, Gurdyal S.

    2015-01-01

    The lack of success in target-based screening approaches to the discovery of antibacterial agents has led to reemergence of phenotypic screening as a successful approach of identifying bioactive, antibacterial compounds. A challenge though with this route is then to identify the molecular target(s) and mechanism of action of the hits. This target identification, or deorphanization step, is often essential in further optimization and validation studies. Direct experimental identification of the molecular target of a screening hit is often complex, precisely because the properties and specificity of the hit are not yet optimized against that target, and so many false positives are often obtained. An alternative is to use computational, predictive, approaches to hypothesize a mechanism of action, which can then be validated in a more directed and efficient manner. Specifically here we present experimental validation of an in silico prediction from a large-scale screen performed against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. The two potent anti-tubercular compounds studied in this case, belonging to the tetrahydro-1,3,5-triazin-2-amine (THT) family, were predicted and confirmed to be an inhibitor of dihydrofolate reductase (DHFR), a known essential Mtb gene, and already clinically validated as a drug target. Given the large number of similar screening data sets shared amongst the community, this in vitro validation of these target predictions gives weight to computational approaches to establish the mechanism of action (MoA) of novel screening hit. PMID:25799414

  20. Hydrogen-Bonded Networks Along and Bifurcation of the E-Pathway in Quinol: Fumarate Reductase

    SciTech Connect

    Herzog, Elena; Gu, Wei; Juhnke, Hanno D.; Haas, Alexander H.; Mantele, Werner; Simon, Jorg; Helms, Volkhard H.; Lancaster , C. Roy D.

    2012-09-19

    The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu- C180 and heme bD ring C (bD-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the bD-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the bD-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme bD ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.

  1. Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways

    SciTech Connect

    Zeng, Ke-Wu; Li, Jun; Dong, Xin; Wang, Ying-Hong; Ma, Zhi-Zhong; Jiang, Yong; Jin, Hong-Wei; Tu, Peng-Fei

    2013-11-15

    Aldose reductase (AR) has a key role in several inflammatory diseases: diabetes, cancer and cardiovascular diseases. Therefore, AR inhibition seems to be a useful strategy for anti-inflammation therapy. In the central nervous system (CNS), microglial over-activation is considered to be a central event in neuroinflammation. However, the effects of AR inhibition in CNS inflammation and its underlying mechanism of action remain unknown. In the present study, we found that FMHM (a naturally derived AR inhibitor from the roots of Polygala tricornis Gagnep.) showed potent anti-neuroinflammatory effects in vivo and in vitro by inhibiting microglial activation and expression of inflammatory mediators. Mechanistic studies showed that FMHM suppressed the activity of AR-dependent phospholipase C/protein kinase C signaling, which further resulted in downstream inactivation of the IκB kinase/IκB/nuclear factor-kappa B (NF-κB) inflammatory pathway. Therefore, AR inhibition-dependent NF-κB inactivation negatively regulated the transcription and expression of various inflammatory genes. AR inhibition by FMHM exerted neuroprotective effects in lipopolysaccharide-induced neuron–microglia co-cultures. These findings suggested that AR is a potential target for neuroinflammation inhibition and that FMHM could be an effective agent for treating or preventing neuroinflammatory diseases. - Highlights: • FMHM is a natural-derived aldose reductase (AR) inhibitor. • FMHM inhibits various neuroinflammatory mediator productions in vitro and in vivo. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent NF-κB pathway. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent MAPK pathway. • FMHM protects neurons against inflammatory injury in microglia-neuron co-cultures.

  2. Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene.

    PubMed

    Sun, Yu; Gao, Xiaoshu; Li, Qiyun; Zhang, Qingqi; Xu, Zhengkai

    2006-08-01

    Nitrate reductase (NR) catalyzes NAD (P) H dependent reduction of nitrate to nitrite. Transformation systems have been established in several species of green algae by nitrate reductase gene functional complementation. In this report, an endogenous NR cDNA (3.4 kb) and a genomic fragment (14.6 kb) containing the NR gene (DvNIA1) were isolated from the D. viridis cDNA and genomic libraries respectively. Southern blot and Northern blot analyses showed that this gene exists as a single copy in D. viridis and is induced by nitrate. To obtain a NR defective mutant as a recipient strain, D. viridis cells were treated with a chemical mutagen and then cultured on a chlorate-containing plate to enrich chlorate tolerant mutants. Southern analysis showed that one isolate, B14, had a deletion in the DvNIA1 gene region. Using electroporation conditions determined in this laboratory, plasmid pDVNR containing the intact DvNIA1 gene has been electroporated into the defective mutant B14. Strains retaining a nitrate assimilation phenotype were obtained from nitrate plates after spreading the electroporated cells. In some individual strains, transcription of the introduced gene was detected. NR activity in these strains was slightly higher than that in the defective B14 cell, but excretion of nitrite into culture media was almost as high as that of the wild-type cell. Possible episomal presence of the introduced DNA in D. viridis is discussed. PMID:16797881

  3. Polymorphisms in the methylene tetrahydrofolate reductase and methionine synthase reductase genes and their correlation with unexplained recurrent spontaneous abortion susceptibility.

    PubMed

    Zhu, L

    2015-01-01

    We aimed to explore the correlation between unexplained recurrent spontaneous abortion and polymorphisms in the methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes. A case control study was conducted in 118 patients with unexplained recurrent spontaneous abortion (abortion group) and 174 healthy women (control group). The genetic material was extracted from the oral mucosal epithelial cells obtained from all subjects. The samples were subjected to fluorescence quantitative PCR to detect the single nucleotide polymorphisms (SNPs) in the MTHFR (C677T and A1298C) and MTRR (A66G) gene loci. The distribution frequency (18/118, 15.3%) of the MTHFR 677TT genotype was significantly higher in the abortion group (χ2 = 11.006, P = 0.004) than in the control group (2/174, 1.1%); on the other hand, the distribution frequency of the MTHFR A1298C genotype did not significantly differ between the abortion and control groups (χ(2) = 0.441, P = 0.507). The distribution frequency of the MTRR A66G genotype was also significantly higher in the abortion group (14/118, 11.9%; χ(2) = 10.503, P = 0.005) than in the control group (8/174, 4.6%). The MTHFR C677T and MTRR A66G polymorphisms are significantly correlated with the occurrence of spontaneous abortion. PMID:26345779

  4. Peach MYB7 activates transcription of the proanthocyanidin pathway gene encoding leucoanthocyanidin reductase, but not anthocyanidin reductase

    PubMed Central

    Zhou, Hui; Lin-Wang, Kui; Liao, Liao; Gu, Chao; Lu, Ziqi; Allan, Andrew C.; Han, Yuepeng

    2015-01-01

    Proanthocyanidins (PAs) are a group of natural phenolic compounds that have a great effect on both flavor and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs) via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase and anthocyanidin reductase. Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5) via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants. PMID:26579158

  5. The nitrate-sensing NasST system regulates nitrous oxide reductase and periplasmic nitrate reductase in Bradyrhizobium japonicum.

    PubMed

    Sánchez, Cristina; Itakura, Manabu; Okubo, Takashi; Matsumoto, Takashi; Yoshikawa, Hirofumi; Gotoh, Aina; Hidaka, Masafumi; Uchida, Takafumi; Minamisawa, Kiwamu

    2014-10-01

    The soybean endosymbiont Bradyrhizobium japonicum is able to scavenge the greenhouse gas N2O through the N2O reductase (Nos). In previous research, N2O emission from soybean rhizosphere was mitigated by B. japonicum Nos(++) strains (mutants with increased Nos activity). Here, we report the mechanism underlying the Nos(++) phenotype. Comparative analysis of Nos(++) mutant genomes showed that mutation of bll4572 resulted in Nos(++) phenotype. bll4572 encodes NasS, the nitrate (NO3(-))-sensor of the two-component NasST regulatory system. Transcriptional analyses of nosZ (encoding Nos) and other genes from the denitrification process in nasS and nasST mutants showed that, in the absence of NO3(-) , nasS mutation induces nosZ and nap (periplasmic nitrate reductase) via nasT. NO3(-) addition dissociated the NasS-NasT complex in vitro, suggesting the release of the activator NasT. Disruption of nasT led to a marked decrease in nosZ and nap transcription in cells incubated in the presence of NO3(-). Thus, although NasST is known to regulate the NO3(-)-mediated response of NO3(-) assimilation genes in bacteria, our results show that NasST regulates the NO3(-) -mediated response of nosZ and napE genes, from the dissimilatory denitrification pathway, in B. japonicum. PMID:24947409

  6. The Protein Oxidation Repair Enzyme Methionine Sulfoxide Reductase A Modulates Aβ Aggregation and Toxicity In Vivo

    PubMed Central

    Minniti, Alicia N.; Arrazola, Macarena S.; Bravo-Zehnder, Marcela; Ramos, Francisca; Inestrosa, Nibaldo C.

    2015-01-01

    Abstract Aims: To examine the role of the enzyme methionine sulfoxide reductase A-1 (MSRA-1) in amyloid-β peptide (Aβ)-peptide aggregation and toxicity in vivo, using a Caenorhabditis elegans model of the human amyloidogenic disease inclusion body myositis. Results: MSRA-1 specifically reduces oxidized methionines in proteins. Therefore, a deletion of the msra-1 gene was introduced into transgenic C. elegans worms that express the Aβ-peptide in muscle cells to prevent the reduction of oxidized methionines in proteins. In a constitutive transgenic Aβ strain that lacks MSRA-1, the number of amyloid aggregates decreases while the number of oligomeric Aβ species increases. These results correlate with enhanced synaptic dysfunction and mislocalization of the nicotinic acetylcholine receptor ACR-16 at the neuromuscular junction (NMJ). Innovation: This approach aims at modulating the oxidation of Aβ in vivo indirectly by dismantling the methionine sulfoxide repair system. The evidence presented here shows that the absence of MSRA-1 influences Aβ aggregation and aggravates locomotor behavior and NMJ dysfunction. The results suggest that therapies which boost the activity of the Msr system could have a beneficial effect in managing amyloidogenic pathologies. Conclusion: The absence of MSRA-1 modulates Aβ-peptide aggregation and increments its deleterious effects in vivo. Antioxid. Redox Signal. 22, 48–62. PMID:24988428

  7. Studies of dihydrofolate reductase and methotrexate transport utilizing the technique of photoaffinity labeling

    SciTech Connect

    Price, E.M.

    1987-01-01

    The methotrexate (MTX) transport mechanism in murine L1210 leukemia cells appears to involve a temperature- and energy-dependent, carrier-mediated, saturable process. Although the intracellular target for MTX, dihydrofolate reductase (DHFR), has been well characterized, the protein(s) involved in MTX uptake have yet to be unequivocally identified. In order to characterize the MTX transport system of L1210 cells, photoaffinity analogues of the drug have been synthesized. They are the azidosalicylyl derivatives of lysine and ornithine analogues of MTX, and their iodinated counterparts. All of the compounds were potent inhibitors of DHFR. When the L1210 DHFR:APA-(/sup 125/I)ASA-Lys complex was irradiated with long-wave UV light, the photoaffinity analogue modified the enzyme with high efficiency. This reaction was specific as an excess of MTX prevented the covalent incorporation of the probe into DHFR. Cyanogen bromide digestion of the labeled enzyme, followed by sequence analysis of the singularly labeled peptide revealed that the enzyme was labeled in a region that encompasses Lys 63, Asn 64 and Arg 65 of native DHRF.

  8. 5,10 Methylenetetrahydrofolate reductase genetic polymorphism as a risk factor for neural tube defects

    SciTech Connect

    Ou, C.Y.; Brown, V.K.; Khoury, M.J.

    1996-06-28

    Persons with a thermolabile form of the enzyme 5,10 methylenetetrahydrofolate reductase (MTHFR) have reduced enzyme activity and increased plasma homocysteine which can be lowered by supplemental folic acid. Thermolability of the enzyme has recently been shown to be caused by a common mutation (677C{sup {r_arrow}}T) in the MTHFR gene. We studied 41 fibroblast cultures from NTD-affected fetuses and compared their genotypes with those of 109 blood specimens from individuals in the general population. 677C{sup {r_arrow}}T homozygosity was associated with a 7.2 fold increased risk for NTDs (95% confidence interval: 1.8-30.3; p value: 0.001). These preliminary data suggest that the 677C{sup {r_arrow}}T polymorphism of the MTHFR gene is a risk factor for spina bifida and anencephaly that may provide a partial biologic explanation for why folic acid prevents these types of NTD. 13 refs., 1 fig., 1 tab.

  9. Thioredoxin reductase 1 upregulates MCP-1 release in human endothelial cells

    SciTech Connect

    Liu, Zhen-Bo; Shen, Xun

    2009-09-04

    To know if thioredoxin reductase 1 (TrxR1) plays a role in antioxidant defense mechanisms against atherosclerosis, effect of TrxR1 on expression/release of monocyte chemoattractant protein (MCP-1) was investigated in activated human endothelial-like EAhy926 cells. The MCP-1 release and expression, cellular generation of reactive oxygen species (ROS), nuclear translocation and DNA-binding activity of NF-{kappa}B subunit p65 were assayed in cells either overexpressing recombinant TrxR1 or having their endogenous TrxR1 knocked down. It was found that overexpression of TrxR1 enhanced, while knockdown of TrxR1 reduced MCP-1 release and expression. Upregulation of MCP-1 by TrxR1 was associated with increasing generation of intracellular ROS generation, enhanced nuclear translocation and DNA-binding activity of NF-{kappa}B. Assay using NF-{kappa}B reporter revealed that TrxR1 upregulated transcriptional activity of NF-{kappa}B. This study suggests that TrxR1 enhances ROS generation, NF-{kappa}B activity and subsequent MCP-1 expression in endothelial cells, and may promote rather than prevent vascular endothelium from forming atherosclerotic plaque.

  10. New small-molecule inhibitors of dihydrofolate reductase inhibit Streptococcus mutans.

    PubMed

    Zhang, Qiong; Nguyen, Thao; McMichael, Megan; Velu, Sadanandan E; Zou, Jing; Zhou, Xuedong; Wu, Hui

    2015-08-01

    Streptococcus mutans is a major aetiological agent of dental caries. Formation of biofilms is a key virulence factor of S. mutans. Drugs that inhibit S. mutans biofilms may have therapeutic potential. Dihydrofolate reductase (DHFR) plays a critical role in regulating the metabolism of folate. DHFR inhibitors are thus potent drugs and have been explored as anticancer and antimicrobial agents. In this study, a library of analogues based on a DHFR inhibitor, trimetrexate (TMQ), an FDA-approved drug, was screened and three new analogues that selectively inhibited S. mutans were identified. The most potent inhibitor had a 50% inhibitory concentration (IC50) of 454.0±10.2nM for the biofilm and 8.7±1.9nM for DHFR of S. mutans. In contrast, the IC50 of this compound for human DHFR was ca. 1000nM, a >100-fold decrease in its potency, demonstrating the high selectivity of the analogue. An analogue that exhibited the least potency for the S. mutans biofilm also had the lowest activity towards inhibiting S. mutans DHFR, further indicating that inhibition of biofilms is related to reduced DHFR activity. These data, along with docking of the most potent analogue to the modelled DHFR structure, suggested that the TMQ analogues indeed selectively inhibited S. mutans through targeting DHFR. These potent and selective small molecules are thus promising lead compounds to develop new effective therapeutics to prevent and treat dental caries. PMID:26022931

  11. Screening natural products for inhibitors of quinone reductase-2 using ultrafiltration LC-MS

    PubMed Central

    Choi, Yongsoo; Jermihov, Katherine; Nam, Sang-Jip; Sturdy, Megan; Maloney, Katherine; Qiu, Xi; Chadwick, Lucas R.; Main, Matthew; Chen, Shao-Nong; Mesecar, Andrew D.; Farnsworth, Norman R.; Pauli, Guido F.; Fenical, William; Pezzuto, John M.; van Breemen, Richard R.

    2011-01-01

    Inhibitors of quinone reductase-2 (NQO2; QR-2) can have anti-malarial activity and anti-tumor activities or can function as chemoprevention agents by preventing the metabolic activation of toxic quinones such as menadione. To expedite the search for new natural product inhibitors of QR-2, we developed a screening assay based on ultrafiltration liquid chromatography-mass spectrometry that is compatible with complex samples such as bacterial or botanical extracts. Human QR-2 was prepared recombinantly, and the known QR-2 inhibitor, resveratrol, was used as a positive control and as a competitive ligand to eliminate false positives. Ultrafiltration LC-MS screening of extracts of marine sediment bacteria resulted in the discovery of tetrangulol methyl ether as an inhibitor of QR-2. When applied to the screening of hop extracts from the botanical, Humulus lupulus L., xanthohumol and xanthohumol D were identified as ligands of QR-2. Inhibition of QR-2 by these ligands was confirmed using a functional enzyme assay. Furthermore, binding of xanthohumol and xanthohumol D to the active site of QR-2 were confirmed using X-ray crystallography. Ultrafiltration LC-MS was shown to be a useful assay for the discovery of inhibitors of QR-2 in complex matrices such as extracts of bacteria and botanicals. PMID:21192729

  12. Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo

    PubMed Central

    Cotruvo, Joseph A.; Stubbe, JoAnne

    2015-01-01

    Incorporation of metallocofactors essential for the activity of many enyzmes is a major mechanism of posttranslational modification. The cellular machinery required for these processes in the case of mono- and dinuclear nonheme iron and manganese cofactors has remained largely elusive. In addition, many metallocofactors can be converted to inactive forms, and pathways for their repair have recently come to light. The class I ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides and require dinuclear metal clusters for activity: an FeIIIFeIII-tyrosyl radical (Y•) cofactor (class Ia), a MnIIIMnIII-Y• cofactor (class Ib), and a MnIVFeIII cofactor (class Ic). The class Ia, Ib, and Ic RNRs are structurally homologous and contain almost identical metal coordination sites. Recent progress in our under-standing of the mechanisms by which the cofactor of each of these RNRs is generated in vitro and in vivo and by which the damaged cofactors are repaired is providing insight into how nature prevents mismetallation and orchestrates active cluster formation in high yields. PMID:21456967

  13. Preventing Rejection

    MedlinePlus

    ... Drug Assistance Lifestyle Changes Back to Work or School Physical Changes Relationship Changes Pregnancy Precautions Fertility Labor & Delivery Breastfeeding Risks Cancer Types Risk Factors Prevention & Early Detection ...

  14. Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617.

    PubMed

    Prudêncio, M; Pereira, A S; Tavares, P; Besson, S; Cabrito, I; Brown, K; Samyn, B; Devreese, B; Van Beeumen, J; Rusnak, F; Fauque, G; Moura, J J; Tegoni, M; Cambillau, C; Moura, I

    2000-04-11

    The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family. PMID:10747777

  15. Free-floating thrombus of the carotid artery with a homozygous methylenetetrahydrofolate reductase gene mutation: a case report.

    PubMed

    Colak, Necmettin; Nazli, Yunus; Kosehan, Dilek; Alpay, Mehmet Fatih; Cakir, Omer

    2013-02-01

    Free-floating thrombus (FFT) of the carotid artery is a rare condition of currently unknown etiology. We describe a symptomatic patient with an FFT in the left common carotid artery. A duplex ultrasonography scan showed the presence of a mobile floating thrombus moving in cyclical motion with the cardiac cycles in the left common carotid artery. During emergency surgery, an FFT was seen at this location and removed. No underlying wall defect was seen at the time of surgery. In a genetic screening test, TT homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T genetic polymorphisms was detected. The patient recovered uneventfully, with no neurogical events. Lifelong anticoagulant therapy was recommended. An aggressive surgical approach is recommended in the patient to prevent embolic episodes. PMID:22101856

  16. A novel NADPH:(bound) NADP+ reductase and NADH:(bound) NADP+ transhydrogenase function in bovine liver catalase.

    PubMed

    Gaetani, Gian F; Ferraris, Anna M; Sanna, Paola; Kirkman, Henry N

    2005-02-01

    Many catalases have the shared property of containing bound NADPH and being susceptible to inactivation by their own substrate, H2O2. The presence of additional (unbound) NADPH effectively prevents bovine liver and human erythrocytic catalase from becoming compound II, the reversibly inactivated state of catalase, and NADP+ is known to be generated in the process. The function of the bound NADPH, which is tightly bound in bovine liver catalase, has been unknown. The present study with bovine liver catalase and [14C]NADPH and [14C]NADH revealed that unbound NADPH or NADH are substrates for an internal reductase and transhydrogenase reaction respectively; the unbound NADPH or NADH cause tightly bound NADP+ to become NADPH without becoming tightly bound themselves. This and other results provide insight into the function of tightly bound NADPH. PMID:15456401

  17. Molecular recognition in the interaction of chloroplast 2-Cys peroxiredoxin with NADPH-thioredoxin reductase C (NTRC) and thioredoxin x.

    PubMed

    Bernal-Bayard, Pilar; Ojeda, Valle; Hervás, Manuel; Cejudo, Francisco J; Navarro, José A; Velázquez-Campoy, Adrián; Pérez-Ruiz, Juan M

    2014-11-28

    In addition to the standard NADPH thioredoxin reductases (NTRs), plants hold a plastidic NTR (NTRC), with a thioredoxin module fused at the C-terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs). The interaction of NTRC and chloroplastic thioredoxin x with 2-Cys Prxs has been confirmed in vivo, by bimolecular fluorescence complementation (BiFC) assays, and in vitro, by isothermal titration calorimetry (ITC) experiments. In comparison with thioredoxin x, NTRC interacts with 2-Cys Prx with higher affinity, both the thioredoxin and NTR domains of NTRC contributing significantly to this interaction, as demonstrated by using the NTR and thioredoxin modules of the enzyme expressed separately. The presence of the thioredoxin domain seems to prevent the interaction of NTRC with thioredoxin x. PMID:25448674

  18. Thermal Stabilization of Dihydrofolate Reductase Using Monte Carlo Unfolding Simulations and Its Functional Consequences

    PubMed Central

    Whitney, Anna; Shakhnovich, Eugene I.

    2015-01-01

    Design of proteins with desired thermal properties is important for scientific and biotechnological applications. Here we developed a theoretical approach to predict the effect of mutations on protein stability from non-equilibrium unfolding simulations. We establish a relative measure based on apparent simulated melting temperatures that is independent of simulation length and, under certain assumptions, proportional to equilibrium stability, and we justify this theoretical development with extensive simulations and experimental data. Using our new method based on all-atom Monte-Carlo unfolding simulations, we carried out a saturating mutagenesis of Dihydrofolate Reductase (DHFR), a key target of antibiotics and chemotherapeutic drugs. The method predicted more than 500 stabilizing mutations, several of which were selected for detailed computational and experimental analysis. We find a highly significant correlation of r = 0.65–0.68 between predicted and experimentally determined melting temperatures and unfolding denaturant concentrations for WT DHFR and 42 mutants. The correlation between energy of the native state and experimental denaturation temperature was much weaker, indicating the important role of entropy in protein stability. The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive. However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR. By combining stabilizing mutations predicted by our method, we created a highly stable catalytically active E. coli DHFR mutant with measured denaturation temperature 7.2°C higher than WT. Prediction results for DHFR and several other proteins indicate that computational approaches based on unfolding simulations are useful as a general technique to discover stabilizing

  19. Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

    PubMed Central

    Kwak, Young Hak; Lee, Dong Seok; Kim, Han Bok

    2003-01-01

    The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5α and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5α NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5α NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The Km values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 μM, respectively. The Vmax values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each Vmax/Km value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30°C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI). PMID:12902220

  20. Computational simulation of the effect of quantum chemical parameters on the molecular docking of HMG-CoA reductase drugs

    NASA Astrophysics Data System (ADS)

    Atlam, Faten M.; Awad, Mohamed K.; El-Bastawissy, Eman A.

    2014-10-01

    Density functional theory (B3LYP-6-31G(d)) was performed to study the effect of molecular and electronic structures, of 2-cyclopropyl-4-thiophenyl-quinoline mevalonolactones as potential hypocholesterolemic inhibitors, on their biological activities and discuss the correlation between the inhibition efficiency and quantum chemical parameters. Molecular docking was performed to investigate the mode of interactions between the investigated inhibitors and the active sites of the target Hydroxymethylglutaryl-Coenzyme A(HMG-CoA) reductase. The results could suggest further structural modifications to discover more potent and selective HMG-CoA reductase inhibitors. The catalytic active sites of HMGR have a positive electrostatic potential which is complemented with a negative electrostatic potential of the investigated drugs to form a stabilized complex. The presence of lipophobic groups, such as quinoline nucleus, cyclopropyl and substituted thiophenyl groups as well as a lactone moiety, is important for binding to the active sites. A good correlation between the experimental and theoretical data confirms that the quantum chemical methods and molecular docking studies are successful tools for enriching screening experiments aimed at the discovery of novel bioactive compounds.

  1. Preventative Maintenance.

    ERIC Educational Resources Information Center

    Migliorino, James

    Boards of education must be convinced that spending money up front for preventive maintenance will, in the long run, save districts' tax dollars. A good program of preventive maintenance can minimize disruption of service; reduce repair costs, energy consumption, and overtime; improve labor productivity and system equipment reliability; handle…

  2. Preventing Falls

    MedlinePlus

    ... from osteoporosis. Lower-body strength exercises and balance exercises can help you prevent falls and avoid the disability that may result from falling. Here are some fall prevention tips from Go4Life : l Have your eyes and hearing tested often. Always wear your glasses when you ...

  3. Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

    PubMed Central

    Snape, J R; Walkley, N A; Morby, A P; Nicklin, S; White, G F

    1997-01-01

    Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively. PMID:9401040

  4. Ascorbate free radical reductase mRNA levels are induced by wounding.

    PubMed Central

    Grantz, A A; Brummell, D A; Bennett, A B

    1995-01-01

    A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide- and NAD(P)H-binding proteins but did not possess typical eukaryotic targeting sequences, suggesting that it encodes a cytosolic form of AFR reductase. Low-stringency genomic DNA gel blot analysis indicated that a single nuclear gene encoded this enzyme. Total ascorbate contents were greatest in leaves, with decreasing amounts in stems and roots and relatively constant levels in all stages of fruit. AFR reductase activity was inversely correlated with total ascorbate content, whereas the relative abundance of AFR reductase mRNA was directly correlated with enzyme activity in tissues examined. AFR reductase mRNA abundance increased dramatically in response to wounding, a treatment that is known to also induce ascorbate-dependent prolyl hydroxylation required for the accumulation of hydroxyproline-rich glycoproteins. In addition, AFR reductase may contribute to maintaining levels of ascorbic acid for protection against wound-induced free radical-mediated damage. Collectively, the results suggest that AFR reductase activity is regulated at the level of mRNA abundance by low ascorbate contents or by factors that promote ascorbate utilization. PMID:7784511

  5. Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.

    PubMed Central

    Benveniste, I; Lesot, A; Hasenfratz, M P; Durst, F

    1989-01-01

    Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals. Images Fig. 5. PMID:2499315

  6. Steroid 5β-Reductase from Leaves of Vitis vinifera: Molecular Cloning, Expression, and Modeling.

    PubMed

    Ernst, Mona; Munkert, Jennifer; Campa, Manuela; Malnoy, Mickael; Martens, Stefan; Müller-Uri, Frieder

    2015-11-25

    A steroid 5β-reductase gene corresponding to the hypothetical protein LOC100247199 from leaves of Vitis vinifera (var. 'Chardonnay') was cloned and overexpressed in Escherichia coli. The recombinant protein showed 5β-reductase activity when progesterone was used as a substrate. The reaction was stereoselective, producing only 5β-products such as 5β-pregnane-3,20-dione. Other small substrates (terpenoids and enones) were also accepted as substrates, indicating the highly promiscuous character of the enzyme class. Our results show that the steroid 5β-reductase gene, encoding an orthologous enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in leaves of the cardenolide-free plant V. vinifera. We emphasize the fact that, on some occasions, different reductases (e.g., progesterone 5β-reductase and monoterpenoid reductase) can also use molecules that are similar to the final products as a substrate. Therefore, in planta, the different reductases may contribute to the immense number of diverse small natural products finally leading to the flavor of wine. PMID:26537436

  7. Melatonin Reduces Cataract Formation and Aldose Reductase Activity in Lenses of Streptozotocin-induced Diabetic Rat

    PubMed Central

    Khorsand, Marjan; Akmali, Masoumeh; Sharzad, Sahab; Beheshtitabar, Mojtaba

    2016-01-01

    Background: The relationship between the high activity of aldose reductase (AR) and diabetic cataract formation has been previously investigated. The purpose of the present study was to determine the preventing effect of melatonin on streptozotocin (STZ)-induced diabetic cataract in rats. Methods: 34 adult healthy male Sprague-Dawely rats were divided into four groups. Diabetic control and diabetic+melatonin received a single dose of STZ (50 mg/kg, intraperitoneally), whereas the normal control and normal+melatonin received vehicle. The melatonin groups were gavaged with melatonin (5 mg/kg) daily for a period of 8 weeks, whereas the rats in the normal control and diabetic control groups received only the vehicle. The rats’ eyes were examined every week and cataract formation scores (0-4) were determined by slit-lamp microscope. At the end of the eighth week, the rats were sacrificed and markers of the polyol pathway and antioxidative (Glutathione, GSH) in their lens were determined. The levels of blood glucose, HbA1c and plasma malondialdhyde (MDA), as a marker of lipid peroxidation, were also measured. Results: Melatonin prevented STZ-induced hyperglycemia by decreased blood glucose and HbA1c levels. Slit lamp examination indicated that melatonin delayed cataract progression in diabetic rats. The results revealed that melatonin feeding increased the GSH levels, decreased the activities of AR and sorbitol dehydrogenase (SDH) and sorbitol formation in catractous lenses as well as plasma MDA content. Conclusion: In summary, for the first time we demonstrated that melatonin delayed the formation and progression of cataract in diabetic rat lenses. PMID:27365552

  8. Divergent evolution of protein conformational dynamics in dihydrofolate reductase.

    PubMed

    Bhabha, Gira; Ekiert, Damian C; Jennewein, Madeleine; Zmasek, Christian M; Tuttle, Lisa M; Kroon, Gerard; Dyson, H Jane; Godzik, Adam; Wilson, Ian A; Wright, Peter E

    2013-11-01

    Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about the evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, Escherichia coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary-sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics following a pattern of divergent evolution that is tuned by cellular environment. PMID:24077226

  9. Structure of a bacterial homologue of vitamin K epoxide reductase

    SciTech Connect

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A.

    2010-03-19

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

  10. Go Green: The Anti-Inflammatory Effects of Biliverdin Reductase

    PubMed Central

    Wegiel, Barbara; Otterbein, Leo E.

    2012-01-01

    Biliverdin (BV) has emerged as a cytoprotective and important anti-inflammatory molecule. Conversion of BV to bilirubin (BR) is catalyzed by biliverdin reductase (BVR) and is required for the downstream signaling and nuclear localization of BVR. Recent data by others and us make clear that BVR is a critical regulator of innate immune responses resulting from acute insult and injury and moreover, that a lack of BVR results in an enhanced proinflammatory phenotype. In macrophages, BVR is regulated by its substrate BV which leads to activation of the PI3K–Akt-IL-10 axis and inhibition of TLR4 expression via direct binding of BVR to the TLR4 promoter. In this review, we will summarize recent findings on the role of BVR and the bile pigments in inflammation in context with its activity as an enzyme, receptor, and transcriptional regulator. PMID:22438844

  11. Genetic Evidence for a Molybdopterin-Containing Tellurate Reductase

    PubMed Central

    Theisen, Joanne; Zylstra, Gerben J.

    2013-01-01

    The genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction in Escherichia coli K-12. The results show that mutants deleted of the moaA, moaB, moaE, or mog gene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of the modB or modC gene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction in E. coli involves a molybdoenzyme. PMID:23475618

  12. Pulse radiolysis studies on superoxide reductase from Treponema pallidum.

    PubMed

    Nivière, V; Lombard, M; Fontecave, M; Houée-Levin, C

    2001-05-25

    Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species. PMID:11377434

  13. Divergent evolution of protein conformational dynamics in dihydrofolate reductase

    PubMed Central

    Bhabha, Gira; Ekiert, Damian C.; Jennewein, Madeleine; Zmasek, Christian M.; Tuttle, Lisa M.; Kroon, Gerard; Dyson, H. Jane; Godzik, Adam; Wilson, Ian A.; Wright, Peter E.

    2013-01-01

    Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, E. coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics, following a pattern of divergent evolution that is tuned by the cellular environment. PMID:24077226

  14. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

    PubMed

    Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A

    2006-05-19

    Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins. PMID:16600295

  15. Thioredoxin reductase 1 suppresses adipocyte differentiation and insulin responsiveness

    PubMed Central

    Peng, Xiaoxiao; Giménez-Cassina, Alfredo; Petrus, Paul; Conrad, Marcus; Rydén, Mikael; Arnér, Elias S. J.

    2016-01-01

    Recently thioredoxin reductase 1 (TrxR1), encoded by Txnrd1, was suggested to modulate glucose and lipid metabolism in mice. Here we discovered that TrxR1 suppresses insulin responsiveness, anabolic metabolism and adipocyte differentiation. Immortalized mouse embryonic fibroblasts (MEFs) lacking Txnrd1 (Txnrd1−/−) displayed increased metabolic flux, glycogen storage, lipogenesis and adipogenesis. This phenotype coincided with upregulated PPARγ expression, promotion of mitotic clonal expansion and downregulation of p27 and p53. Enhanced Akt activation also contributed to augmented adipogenesis and insulin sensitivity. Knockdown of TXNRD1 transcripts accelerated adipocyte differentiation also in human primary preadipocytes. Furthermore, TXNRD1 transcript levels in subcutaneous adipose tissue from 56 women were inversely associated with insulin sensitivity in vivo and lipogenesis in their isolated adipocytes. These results suggest that TrxR1 suppresses anabolic metabolism and adipogenesis by inhibition of intracellular signaling pathways downstream of insulin stimulation. PMID:27346647

  16. 5-Alpha-Reductase Inhibitors and Combination Therapy.

    PubMed

    Füllhase, Claudius; Schneider, Marc P

    2016-08-01

    By inhibiting the conversion from testosterone to dihydrotestosterone 5-Alpha reductase inhibitors (5ARIs) are able to hinder prostatic growth, shrink prostate volumes, and improve BPH-related LUTS. 5ARIs are particularly beneficial for patients with larger prostates (>30-40ml). Generally the side effects of 5ARI treatment are mild, and according to the FORTA classification 5ARIs are suitable for frail elderly. 5ARI / alpha-blocker (AB) combination therapy showed the best symptomatic outcome and risk reduction for clinical progression. Combining Phosphodieseterase type 5 inhbibitors (PDE5Is) with 5ARIs counteracts the negative androgenic sexual side effects of 5ARIs, and simultaneously combines their synergistic effects on LUTS. PMID:27476125

  17. Substrate specificity of an aflatoxin-metabolizing aldehyde reductase.

    PubMed Central

    Ellis, E M; Hayes, J D

    1995-01-01

    The enzyme from rat liver that reduces aflatoxin B1-dialdehyde exhibits a unique catalytic specificity distinct from that of other aldo-keto reductases. This enzyme, designated AFAR, displays high activity towards dicarbonyl-containing compounds with ketone groups on adjacent carbon atoms; 9,10-phenanthrenequinone, acenaphthenequinone and camphorquinone were found to be good substrates. Although AFAR can also reduce aromatic and aliphatic aldehydes such as succinic semialdehyde, it is inactive with glucose, galactose and xylose. The enzyme also exhibits low activity towards alpha,beta-unsaturated carbonyl-containing compounds. Determination of the apparent Km reveals that AFAR has highest affinity for 9,10-phenanthrenequinone and succinic semialdehyde, and low affinity for glyoxal and DL-glyceraldehyde. PMID:8526867

  18. Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation 1

    PubMed Central

    Laliberté, Gilles; Hellebust, Johan A.

    1989-01-01

    Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent Km values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments. PMID:16667157

  19. Evidence for a hexaheteromeric methylenetetrahydrofolate reductase in Moorella thermoacetica.

    PubMed

    Mock, Johanna; Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Thauer, Rudolf K

    2014-09-01

    Moorella thermoacetica can grow with H₂ and CO₂, forming acetic acid from 2 CO₂ via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown. PMID:25002540

  20. Fatty acyl-CoA reductases of birds

    PubMed Central

    2011-01-01

    Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413

  1. The modulation of carbonyl reductase 1 by polyphenols.

    PubMed

    Boušová, Iva; Skálová, Lenka; Souček, Pavel; Matoušková, Petra

    2015-01-01

    Carbonyl reductase 1 (CBR1), an enzyme belonging to the short-chain dehydrogenases/reductases family, has been detected in all human tissues. CBR1 catalyzes the reduction of many xenobiotics, including important drugs (e.g. anthracyclines, nabumetone, bupropion, dolasetron) and harmful carbonyls and quinones. Moreover, it participates in the metabolism of a number of endogenous compounds and it may play a role in certain pathologies. Plant polyphenols are not only present in many human food sources, but are also a component of many popular dietary supplements and herbal medicines. Many studies reviewed herein have demonstrated the potency of certain flavonoids, stilbenes and curcuminoids in the inhibition of the activity of CBR1. Interactions of these polyphenols with transcriptional factors, which regulate CBR1 expression, have also been reported in several studies. As CBR1 plays an important role in drug metabolism as well as in the protection of the organism against potentially harmful carbonyls, the modulation of its expression/activity may have significant pharmacological and/or toxicological consequences. Some polyphenols (e.g. luteolin, apigenin and curcumin) have been shown to be very potent CBR1 inhibitors. The inhibition of CBR1 seems useful regarding the increased efficacy of anthracycline therapy, but it may cause the worse detoxification of reactive carbonyls. Nevertheless, all known information about the interactions of polyphenols with CBR1 have only been based on the results of in vitro studies. With respect to the high importance of CBR1 and the frequent consumption of polyphenols, in vivo studies would be very helpful for the evaluation of risks/benefits of polyphenol interactions with CBR1. PMID:26415702

  2. Characterization of the nitric oxide reductase from Thermus thermophilus

    PubMed Central

    Schurig-Briccio, Lici A.; Venkatakrishnan, Padmaja; Hemp, James; Bricio, Carlos; Berenguer, José; Gennis, Robert B.

    2013-01-01

    Nitrous oxide (N2O) is a powerful greenhouse gas implicated in climate change. The dominant source of atmospheric N2O is incomplete biological dentrification, and the enzymes responsible for the release of N2O are NO reductases. It was recently reported that ambient emissions of N2O from the Great Boiling Spring in the United States Great Basin are high, and attributed to incomplete denitrification by Thermus thermophilus and related bacterial species [Hedlund BP, et al. (2011) Geobiology 9(6)471–480]. In the present work, we have isolated and characterized the NO reductase (NOR) from T. thermophilus. The enzyme is a member of the cNOR family of enzymes and belongs to a phylogenetic clade that is distinct from previously examined cNORs. Like other characterized cNORs, the T. thermophilus cNOR consists of two subunits, NorB and NorC, and contains a one heme c, one Ca2+, a low-spin heme b, and an active site consisting of a high-spin heme b and FeB. The roles of conserved residues within the cNOR family were investigated by site-directed mutagenesis. The most important and unexpected result is that the glutamic acid ligand to FeB is not essential for function. The E211A mutant retains 68% of wild-type activity. Mutagenesis data and the pattern of conserved residues suggest that there is probably not a single pathway for proton delivery from the periplasm to the active site that is shared by all cNORs, and that there may be multiple pathways within the T. thermophilus cNOR. PMID:23858452

  3. Isolation and characterization of nitric oxide reductase from Paracoccus halodenitrificans.

    PubMed

    Sakurai, N; Sakurai, T

    1997-11-11

    Nitric oxide reductase was isolated from the membrane fraction of a denitrifying bacterium, Paracoccus halodenitrificans, in the presence of n-dodecyl beta-D-maltoside. A relatively simple and effective procedure to purify NO reductase using DEAE-Toyopearl and hydroxyapatite (ceramic) chromatographies has been developed. The enzyme consisted of two subunits with molecular masses of 20 and 42 kDa associated with the c-type heme and two b-type hemes, respectively. The optical and magnetic circular dichroism (MCD) spectra of the oxidized (as isolated) and reduced enzymes indicated that the heme c is in the low-spin state and the hemes b are in the high- and low-spin states. The EPR spectrum also showed the presence of the split high-spin component (g perpendicular = 6.6, 6.0) and two low spin components (gz,y,x = 2.96, 2.26, 1.46, gz = 3.59). Although the presence of an extra iron was suggested from atomic absorption spectroscopy, a non-heme iron could not be detected by colorimetric titrations using ferene and 2-(5-nitro-2-pyridylazo)- 5-(N-propyl-N-sulfopropylamino)phenolate (PAPS). One of the extra signals at g = 4.3 and 2.00 might come from a non-heme iron, while they may originate from an adventitious iron and a certain nonmetallic radical, respectively. When CO acted on the reduced enzyme, both of the low-spin hemes were not affected, and when NO acted on the reduced enzyme, the optical and MCD spectra were of a mixture of the oxidized and reduced enzymes. Consequently, the reduction of NO was supposed to take place at the high-spin heme b. The heme c and the low-spin heme b centers were considered to function as electron mediators during the intermolecular and intramolecular processes. PMID:9374857

  4. Bacteriophage T4 Virion Baseplate Thymidylate Synthetase and Dihydrofolate Reductase

    PubMed Central

    Kozloff, L. M.; Lute, M.; Crosby, L. K.

    1977-01-01

    Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td+, whereas particles produced after infection with td− had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from tdts to td+ simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both tdts and frdts genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frdts mutants to frd+ not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles. PMID:894793

  5. Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

    PubMed Central

    Mock, Johanna; Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

    2014-01-01

    Moorella thermoacetica can grow with H2 and CO2, forming acetic acid from 2 CO2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown. PMID:25002540

  6. Nitrite and Nitrous Oxide Reductase Regulation by Nitrogen Oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106

    PubMed Central

    Sabaty, Monique; Schwintner, Carole; Cahors, Sandrine; Richaud, Pierre; Verméglio, Andre

    1999-01-01

    We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N2O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N2O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase. PMID:10498715

  7. Nitrite and nitrous oxide reductase regulation by nitrogen oxides in Rhodobacter sphaeroides f. sp. denitrificans IL106.

    PubMed

    Sabaty, M; Schwintner, C; Cahors, S; Richaud, P; Verméglio, A

    1999-10-01

    We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N(2)O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N(2)O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase. PMID:10498715

  8. Virtual screening of plant derived compounds for aldose reductase inhibition using molecular docking.

    PubMed

    Muppalaneni, Naresh Babu; Rao, Allam Appa

    2012-01-01

    The role of the aldose reductase in type 2 diabetes is widely described. Therefore, it is of interest to identify plant derived compounds to inhibit its activity. We studied the protein-ligand interaction of 267 compounds from different parts of seven plants (Allium sativum, Coriandrum sativum, Dacus carota, Murrayyakoneigii, Eucalyptus, Calendula officinalis and Lycopersicon esculentum) with aldose reductase as the target protein. Molecular docking and re-scoring of top ten compounds (using GOLD, AutoDock Vina, eHiTS, PatchDock and MEDock) followed by rank-sum technique identified compound allium38 with high binding affinity for aldose reductase. PMID:23275691

  9. Cloning and characterization of the methyl coenzyme M reductase genes from Methanobacterium thermoautotrophicum.

    PubMed Central

    Bokranz, M; Bäumner, G; Allmansberger, R; Ankel-Fuchs, D; Klein, A

    1988-01-01

    The genes coding for methyl coenzyme M reductase were cloned from a genomic library of Methanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, alpha, beta, and gamma. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes. Images PMID:2448287

  10. Transcriptional regulation of human and murine short-chain dehydrogenase/reductases (SDRs) - an in silico approach.

    PubMed

    Ebert, Bettina; Kisiela, Michael; Maser, Edmund

    2016-05-01

    Numerous physiological functions of the body are controlled by endogenous (e.g. steroids, retinoids, lipid mediators) or exogenous molecules (e.g. drugs, xenobiotics) that bind to transcription factors (TF). The biosynthesis and catabolism of these signaling molecules depend, apart from CYPs, on enzymes belonging to the short-chain dehydrogenase/reductase (SDR) superfamily. Moreover, the contribution of SDRs to the metabolism of therapeutic drugs and xenobiotics is increasingly recognized. However, only scarce information exists regarding the transcriptional regulation of most SDR proteins. This work aims to illustrate the role of nuclear receptors (NR) and TF related to oxidative stress, inflammation, hypoxia, and xenobiotics in the regulation of selected human and murine SDRs that play crucial roles in steroid, retinoid, eicosanoid, fatty acid, and xenobiotic metabolism. These include, for example, 17β-hydroxysteroid dehydrogenases, retinol dehydrogenases, and carbonyl reductases. Because existing experimental data are limited, an in silico analysis (TRANSFAC(®) Professional database) of the 5'-upstream sequences for putative response elements was performed. Experimental and in silico data suggest that pharmaceutical, environmental, or dietary NR ligands may alter SDR-mediated retinoid, steroid, and xenobiotic metabolism, likely affecting basic cellular events like energy expenditure, cell proliferation/differentiation, or aging processes. Also, some SDRs are possibly induced by their own substrates. Further experimental work is urgently needed to fully understand the NR-mediated transcriptional regulation of SDRs. This is essential for deducing their possible involvement in drug side effects and will help to identify new substrates and further physiological functions of these SDRs. PMID:27362327

  11. An ethoxyquin-inducible aldehyde reductase from rat liver that metabolizes aflatoxin B1 defines a subfamily of aldo-keto reductases.

    PubMed Central

    Ellis, E M; Judah, D J; Neal, G E; Hayes, J D

    1993-01-01

    Protection of liver against the toxic and carcinogenic effects of aflatoxin B1 (AFB1) can be achieved through the induction of detoxification enzymes by chemoprotectors such as the phenolic antioxidant ethoxyquin. We have cloned and sequenced a cDNA encoding an aldehyde reductase (AFB1-AR), which is expressed in rat liver in response to dietary ethoxyquin. Expression of the cDNA in Escherichia coli and purification of the recombinant enzyme reveals that the protein exhibits aldehyde reductase activity and is capable of converting the protein-binding dialdehyde form of AFB1-dihydrodiol to the nonbinding dialcohol metabolite. We show that the mRNA encoding this enzyme is markedly elevated in the liver of rats fed an ethoxyquin-containing diet, correlating with acquisition of resistance to AFB1. AFB1-AR represents the only carcinogen-metabolizing aldehyde reductase identified to date that is induced by a chemoprotector. Alignment of the amino acid sequence of AFB1-AR with other known and putative aldehyde reductases shows that it defines a subfamily within the aldo-keto reductase superfamily. Images Fig. 2 Fig. 3 Fig. 4 PMID:8234296

  12. Advances in pharmacological strategies for the prevention of cataract development

    PubMed Central

    Gupta, S K; Selvan, V Kalai; Agrawal, S S; Saxena, Rohit

    2009-01-01

    Cataractous-opacification of the lens is one of the leading causes of blindness in India. The situation can be managed by surgical removal of the cataractous lens. Various pharmacological strategies have been proposed for the prevention and treatment of cataract. Information on possible benefits of putative anticataract agents comes from a variety of approaches, ranging from laboratory experiments, both in vitro and in vivo, to epidemiological studies in patients. This review deals with the various mechanisms, and possible pharmacological interventions for the prevention of cataract. The article also reviews research on potential anticataractous agents, including aldose reductase inhibitors, glutathione boosters, antiglycating agents, vitamins and various drugs from indigenous sources. PMID:19384010

  13. A biophysically based mathematical model for the catalytic mechanism of glutathione reductase.

    PubMed

    Pannala, Venkat R; Bazil, Jason N; Camara, Amadou K S; Dash, Ranjan K

    2013-12-01

    Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) using NADPH as the reducing cofactor, and thereby maintains a constant GSH level in the system. GSH scavenges superoxide (O2(*-)) and hydroxyl radicals (OH) nonenzymatically or by serving as an electron donor to several enzymes involved in reactive oxygen species (ROS) detoxification. In either case, GSH oxidizes to GSSG and is subsequently regenerated by the catalytic action of GR. Although the GR kinetic mechanism has been extensively studied under various experimental conditions with variable substrates and products, the catalytic mechanism has not been studied in terms of a mechanistic model that accounts for the effects of the substrates and products on the reaction kinetics. The aim of this study is therefore to develop a comprehensive mathematical model for the catalytic mechanism of GR. We use available experimental data on GR kinetics from various species/sources to develop the mathematical model and estimate the associated model parameters. The model simulations are consistent with the experimental observation that GR operates via both ping-pong and sequential branching mechanisms based on relevant concentrations of its reaction substrate GSSG. Furthermore, we show the observed pH-dependent substrate inhibition of GR activity by GSSG and bimodal behavior of GR activity with pH. The model presents a unique opportunity to understand the effects of products on the kinetics of GR. The model simulations show that under physiological conditions, where both substrates and products are present, the flux distribution depends on the concentrations of both GSSG and NADP(+), with ping-pong flux operating at low levels and sequential flux dominating at higher levels. The kinetic model of GR may serve as a key module for the development of integrated models for ROS-scavenging systems to understand protection of cells under normal and oxidative stress

  14. A community-based intervention for primary prevention of cardiovascular diseases in the slums of Nairobi: the SCALE UP study protocol for a prospective quasi-experimental community-based trial

    PubMed Central

    2013-01-01

    Background The burden of cardiovascular disease is rising in sub-Saharan Africa with hypertension being the main risk factor. However, context-specific evidence on effective interventions for primary prevention of cardiovascular diseases in resource-poor settings is limited. This study aims to evaluate the feasibility and cost-effectiveness of one such intervention—the “Sustainable model for cardiovascular health by adjusting lifestyle and treatment with economic perspective in settings of urban poverty”. Methods/Design Design: A prospective quasi-experimental community-based intervention study. Setting: Two slum settlements (Korogocho and Viwandani) in Nairobi, Kenya. Study population: Adults aged 35 years and above in the two communities. Intervention: The intervention community (Korogocho) will be exposed to an intervention package for primary prevention of cardiovascular disease that comprises awareness campaigns, household screening for cardiovascular diseases risk factors, and referral and treatment of people with high cardiovascular diseases risk at a primary health clinic. The control community (Viwandani) will continue accessing the usual standard of care for primary prevention of cardiovascular diseases in Kenya. Data: Demographic and socioeconomic data; anthropometric and clinical measurements including blood pressure. Population-based data will be collected at the baseline and endline—12 months after implementing the intervention. These data will be collected from a random sample of 1,610 adults aged 35 years and above in the intervention and control sites at both baseline and endline. Additionally, operational (including cost) and clinic-based data will be collected on an ongoing basis. Main outcomes: (1) A positive difference in the change in the proportion of the intervention versus control study populations that are at moderate or high risk of cardiovascular disease; (2) a difference in the change in mean systolic blood pressure in the

  15. Topical application of preparations containing DNA repair enzymes prevents ultraviolet-induced telomere shortening and c-FOS proto-oncogene hyperexpression in human skin: an experimental pilot study.

    PubMed

    Emanuele, Enzo; Altabas, Velimir; Altabas, Karmela; Berardesca, Enzo

    2013-09-01

    The exposure to ultraviolet radiation (UVR) is one of the most important risk factors for skin aging and increases the risk of malignant transformation. Telomere shortening and an altered expression of the proto-oncogene c-FOS are among the key molecular mechanisms associated with photoaging and tumorigenesis. Photolyase from A. nidulans and endonuclease from M. luteus are xenogenic DNA repair enzymes which can reverse the molecular events associated with skin aging and carcinogenosis caused by UVR exposure. Therefore, the purpose of this study was to investigate whether the topical application of preparations containing DNA repair enzymes may prevent UVR-induced acute telomere shortening and FOS gene hyperexpression in human skin biopsies. Twelve volunteers (Fitzpatrick skin types I and II) were enrolled for this experimental study, and six circular areas (10 mm diameter) were marked out on the nonexposed lower back of each participant. One site was left untreated (site 1: negative control), whereas the remaining five sites (designated sites 2-6) were exposed to solar-simulated UVR at 3 times the MED on four consecutive days. Site 2 received UVR only (site 2: positive control), whereas the following products were applied to sites 3-6, respectively: vehicle (moisturizer base cream; applied both 30 minutes before and immediately after each irradiation; site 3); a traditional sunscreen (SS, SPF 50) 30 minutes before irradiation and a vehicle immediately after irradiation (site 4); a SS 30 minutes before irradiation and an endonuclease preparation immediately after irradiation (site 5); a SS plus photolyase 30 minutes before irradiation and an endonuclease preparation immediately after irradiation (site 6). Skin biopsies were taken 24 h after the last irradiation. The degree of telomere shortening and c-FOS gene expression were measured in all specimens. Strikingly, the combined use of a SS plus photolyase 30 minutes before irradiation and an endonuclease preparation

  16. A theoretical multiscale treatment of protein-protein electron transfer: The ferredoxin/ferredoxin-NADP(+) reductase and flavodoxin/ferredoxin-NADP(+) reductase systems.

    PubMed

    Saen-Oon, Suwipa; Cabeza de Vaca, Israel; Masone, Diego; Medina, Milagros; Guallar, Victor

    2015-12-01

    In the photosynthetic electron transfer (ET) chain, two electrons transfer from photosystem I to the flavin-dependent ferredoxin-NADP(+) reductase (FNR) via two sequential independent ferredoxin (Fd) electron carriers. In some algae and cyanobacteria (as Anabaena), under low iron conditions, flavodoxin (Fld) replaces Fd as single electron carrier. Extensive mutational studies have characterized the protein-protein interaction in FNR/Fd and FNR/Fld complexes. Interestingly, even though Fd and Fld share the interaction site on FNR, individual residues on FNR do not participate to the same extent in the interaction with each of the protein partners, pointing to different electron transfer mechanisms. Despite of extensive mutational studies, only FNR/Fd X-ray structures from Anabaena and maize have been solved; structural data for FNR/Fld remains elusive. Here, we present a multiscale modelling approach including coarse-grained and all-atom protein-protein docking, the QM/MM e-Pathway analysis and electronic coupling calculations, allowing for a molecular and electronic comprehensive analysis of the ET process in both complexes. Our results, consistent with experimental mutational data, reveal the ET in FNR/Fd proceeding through a bridge-mediated mechanism in a dominant protein-protein complex, where transfer of the electron is facilitated by Fd loop-residues 40-49. In FNR/Fld, however, we observe a direct transfer between redox cofactors and less complex specificity than in Fd; more than one orientation in the encounter complex can be efficient in ET. PMID:26385068

  17. Metabolism of the Synthetic Progestogen Norethynodrel by Human Ketosteroid Reductases of the Aldo-Keto Reductase Superfamily

    PubMed Central

    Jin, Yi; Duan, Ling; Chen, Mo; Penning, Trevor M; Kloosterboer, Helenius J.

    2012-01-01

    Human ketosteroid reductases of the aldo-keto reductase (AKR) superfamily, i.e. AKR1C1-4, are implicated in the biotransformation of synthetic steroid hormones. Norethynodrel (NOR, 17α-ethynyl-17β-hydroxy-estra-5(10)-en-3-one), the progestin component of the first marketed oral contraceptive, is known to undergo rapid and extensive metabolism to 3α- and 3β-hydroxy metabolites. The ability of the four human AKR1C enzymes to catalyze the metabolism of NOR has now been characterized. AKR1C1 and AKR1C2 almost exclusively converted NOR to 3β-hydroxy NOR, while AKR1C3 gave 3β-hydroxy NOR as the main product and AKR1C4 predominantly formed 3α-hydroxy NOR. Individual AKR1C enzymes also displayed distinct kinetic properties in the reaction of NOR. In contrast, norethindrone (NET), the Δ4-isomer of NOR and the most commonly used synthetic progestin, was not a substrate for the AKR1C enzymes. NOR is also structurally identical to the hormone replacement therapeutic tibolone (TIB), except TIB has a methyl group at the 7α-position. Product profiles and kinetic parameters for the reduction of NOR catalyzed by each individual AKR1C isoform were identical to those for the reduction of TIB catalyzed by the respective isoform. These data suggest that the presence of the 7α-methyl group has a minimal effect on the stereochemical outcome of the reaction and kinetic behavior of each enzyme. Results indicate a role of AKR1C in the hepatic and peripheral metabolism of NOR to 3α- and 3β-hydroxy NOR and provide insights into the differential pharmacological properties of NOR, NET and TIB. PMID:22210085

  18. Methylenetetrahydrofolate reductase and methionine synthase reductase gene polymorphisms and protection from microvascular complications in adolescents with type 1 diabetes.

    PubMed

    Wiltshire, Esko J; Mohsin, Fauzia; Chan, Albert; Donaghue, Kim C

    2008-08-01

    Folate status has been associated with endothelial dysfunction in adolescents with type 1 diabetes, and elevated total plasma homoocyst(e)ine (tHcy) is a risk for vascular disease in the non-diabetic population. Polymorphisms in genes involved in folate and homocysteine metabolism are implicated in vascular disease. We aimed to determine whether polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes are risk factors for early microvascular disease in a large group of adolescents with type 1 diabetes. Four hundred and eighty adolescents were screened annually for retinopathy and microalbuminuria for a median of 4 yr. Molecular analysis for the polymorphisms 677C-->T, 1298A-->C in MTHFR, and 66A-->G in MTRR was performed. The MTRR 66GG genotype reduced the risk for elevated albumin excretion rate (AER) (OR 0.47, CI 0.25, 0.88, p = 0.018) and showed a trend to reduced risk for microalbuminuria (OR 0.27, CI 0.06-1.21, p = 0.09). Survival without elevated AER was increased with the MTRR 66GG genotype (12.4 vs. 9.7 yr, p = 0.04) and with the MTHFR 1298CC genotype (15.2 vs. 10.2 yr, p = 0.007). Conversely, survival without retinopathy was reduced with the MTHFR 677TT and MTRR 66GG combined genotype (6.2 vs. 10.2 yr, p = 0.015). The MTRR 66GG and MTHFR 1298 CC genotypes may confer protection against early nephropathy, possibly because they are associated with lower tHcy. The MTHFR 677 TT was only related to earlier onset retinopathy in combination with MTRR 66GG. PMID:18774994

  19. Residues Controlling Facial Selectivity in an Alkene Reductase and Semirational Alterations to Create Stereocomplementary Variants

    PubMed Central

    2015-01-01

    A systematic saturation mutagenesis campaign was carried out on an alkene reductase from Pichia stipitis (OYE 2.6) to develop variants with reversed stereoselectivities. Wild-type OYE 2.6 reduces three representative Baylis–Hillman adducts to the corresponding S products with almost complete stereoselectivities and good catalytic efficiencies. We created and screened 13 first-generation, site-saturation mutagenesis libraries, targeting residues found near the bound substrate. One variant (Tyr78Trp) showed high R selectivity toward one of the three substrates, but no change (cyclohexenone derivative) and no catalytic activity (acrylate derivative) for the other two. Subsequent rounds of mutagenesis retained the Tyr78Trp mutation and explored other residues that impacted stereoselectivity when altered in a wild-type background. These efforts yielded double and triple mutants that possessed inverted stereoselectivities for two of the three substrates (conversions >99% and at least 91% ee (R)). To understand the reasons underlying the stereochemical changes, we solved crystal structures of two key mutants: Tyr78Trp and Tyr78Trp/Ile113Cys, the latter with substrate partially occupying the active site. By combining these experimental data with modeling studies, we have proposed a rationale that explains the impacts of the most useful mutations. PMID:25068071

  20. Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure-Activity Relationships

    SciTech Connect

    J Beierlein; N Karri; A Anderson

    2011-12-31

    Several antifolates, including trimethoprim (TMP) and a series of propargyl-linked analogues, bind dihydrofolate reductase from Bacillus anthracis (BaDHFR) with lower affinity than is typical in other bacterial species. To guide lead optimization for BaDHFR, we explored a new approach to determine structure-activity relationships whereby the enzyme is altered and the analogues remain constant, essentially reversing the standard experimental design. Active site mutants of the enzyme, Ba(F96I)DHFR and Ba(Y102F)DHFR, were created and evaluated with enzyme inhibition assays and crystal structures. The affinities of the antifolates increase up to 60-fold with the Y102F mutant, suggesting that interactions with Tyr 102 are critical for affinity. Crystal structures of the enzymes bound to TMP and propargyl-linked inhibitors reveal the basis of TMP resistance and illuminate the influence of Tyr 102 on the lipophilic linker between the pyrimidine and aryl rings. Two new inhibitors test and validate these conclusions and show the value of the technique for providing new directions during lead optimization.

  1. A novel nitrite biosensor based on conductometric electrode modified with cytochrome c nitrite reductase composite membrane.

    PubMed

    Zhang, Zhiqiang; Xia, Siqing; Leonard, Didier; Jaffrezic-Renault, Nicole; Zhang, Jiao; Bessueille, François; Goepfert, Yves; Wang, Xuejiang; Chen, Ling; Zhu, Zhiliang; Zhao, Jianfu; Almeida, M Gabriela; Silveira, Célia M

    2009-02-15

    A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples. PMID:18804367

  2. Premature and ectopic anthocyanin formation by silencing of anthocyanidin reductase in strawberry (Fragaria × ananassa).

    PubMed

    Fischer, Thilo C; Mirbeth, Beate; Rentsch, Judith; Sutter, Corina; Ring, Ludwig; Flachowsky, Henryk; Habegger, Ruth; Hoffmann, Thomas; Hanke, Magda-Viola; Schwab, Wilfried

    2014-01-01

    Strawberry (Fragaria × ananassa) is a fruit crop with a distinct biphasic flavonoid biosynthesis. Whereas, in the immature receptacle, high levels of proanthocyanidins accumulate, which are associated with herbivore deterrence and pathogen defense, the prominent color-giving anthocyanins are primarily produced in ripe 'fruits' helping to attract herbivores for seed dispersal. Here, constitutive experimental down-regulation of one branch of proanthocyanidin biosynthesis was performed. As a result, the proportion of epicatechin monomeric units within the proanthocyanidin polymer chains was reduced, but this was not the case for the epicatechin starter unit. Shortened chain lengths of proanthocyanidins were also observed. All enzymatic activities for the production of color-giving anthocyanins were already present in unripe fruits at levels allowing a striking red anthocyanin phenotype in unripe fruits of the RNAi silencing lines. An immediately recognizable phenotype was also observed for the stigmata of flowers, which is another epicatechin-forming tissue. Thus, the down-regulation of anthocyanidin reductase (ANR) induced a redirection of the proanthocyanidin pathway, leading to premature and ectopic anthocyanin biosynthesis via enzymatic glycosylation as the alternative pathway. This redirection is also seen in flavonol biosynthesis, which is paralleled by higher pollen viability in silencing lines. ANRi transgenic lines of strawberry provide a versatile tool for the study of the biological functions of proanthocyanidins. PMID:24117941

  3. Prevention and early detection of prostate cancer.

    PubMed

    Cuzick, Jack; Thorat, Mangesh A; Andriole, Gerald; Brawley, Otis W; Brown, Powel H; Culig, Zoran; Eeles, Rosalind A; Ford, Leslie G; Hamdy, Freddie C; Holmberg, Lars; Ilic, Dragan; Key, Timothy J; La Vecchia, Carlo; Lilja, Hans; Marberger, Michael; Meyskens, Frank L; Minasian, Lori M; Parker, Chris; Parnes, Howard L; Perner, Sven; Rittenhouse, Harry; Schalken, Jack; Schmid, Hans-Peter; Schmitz-Dräger, Bernd J; Schröder, Fritz H; Stenzl, Arnulf; Tombal, Bertrand; Wilt, Timothy J; Wolk, Alicja

    2014-10-01

    Prostate cancer is a common malignancy in men and the worldwide burden of this disease is rising. Lifestyle modifications such as smoking cessation, exercise, and weight control offer opportunities to reduce the risk of developing prostate cancer. Early detection of prostate cancer by prostate-specific antigen (PSA) screening is controversial, but changes in the PSA threshold, frequency of screening, and the use of other biomarkers have the potential to minimise the overdiagnosis associated with PSA screening. Several new biomarkers for individuals with raised PSA concentrations or those diagnosed with prostate cancer are likely to identify individuals who can be spared aggressive treatment. Several pharmacological agents such as 5α-reductase inhibitors and aspirin could prevent development of prostate cancer. In this Review, we discuss the present evidence and research questions regarding prevention, early detection of prostate cancer, and management of men either at high risk of prostate cancer or diagnosed with low-grade prostate cancer. PMID:25281467

  4. Preventing falls

    MedlinePlus

    ... worsened. Improving your vision will help reduce falls. Images ... for preventing falls in older people living in the community. Cochrane Database of Systematic Reviews 2009, Issue 2. Art. No.: ...

  5. Preventing Influenza

    MedlinePlus

    ... spread in respiratory droplets distributed by coughing and sneezing, they readily spread from person to person. Additionally, ... and nose with a tissue when coughing or sneezing, you may help prevent those around you from ...

  6. Dengue Prevention

    MedlinePlus

    ... Compartir This photograph shows a mother applying mosquito repellent to her child's skin in order to prevent ... the lights are on. To protect yourself, use repellent on your skin while indoors or out. When ...

  7. Redesigning alcohol dehydrogenases/reductases for more efficient biosynthesis of enantiopure isomers.

    PubMed

    Zhang, Rongzhen; Xu, Yan; Xiao, Rong

    2015-12-01

    Alcohol dehydrogenases/reductases predominantly catalyze the asymmetric biosynthesis of optically pure stereoisomers because of their unique chiral constitutions. The enantioselectivities of alcohol dehydrogenases/reductases are substrate- and cofactor-dependent, and therefore they usually catalyze specific reactions with high enantioselectivity under physiological conditions; this may not be suitable for asymmetric biosynthesis with non-natural substrates or non-natural cofactors, and under nonphysiological conditions. It is therefore necessary to modify alcohol dehydrogenases/reductases using various redesigning tools such as directed evolution and rational design, and their combinations, as well as engineering enzyme modules for more efficient production of "non-natural" products. In this article, progress in these aspects of alcohol dehydrogenase/reductase design is reviewed, and future challenges are discussed. PMID:26320091

  8. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line

    SciTech Connect

    Kaufman, R.J.; Schimke, R.T.

    1981-12-01

    During stepwise increases in the methotrexate concentration in culture medium, the authors selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). The authors studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

  9. 5α-reductase inhibitors, antiviral and anti-tumor activities of some steroidal cyanopyridinone derivatives.

    PubMed

    Al-Mohizea, Abdullah M; Al-Omar, Mohamed A; Abdalla, Mohamed M; Amr, Abdel-Galil E

    2012-01-01

    We herein report the 5α-reductase inhibitors, antiviral and anti-tumor activities of some synthesized heterocyclic cyanopyridone and cyanothiopyridone derivatives fused with steroidal structure. Initially the acute toxicity of the compounds was assayed via the determination of their LD(50). All the compounds, except 3b, were interestingly less toxic than the reference drug (Prednisolone(®)). Seventeen heterocyclic derivatives containing a cyanopyridone or cyanothiopyridone rings fused to a steroidal moiety were synthesized and screened for their 5α-reductase inhibitors, antiviral and anti-tumor activities comparable to that of Anastrozole, Bicalutamide, Efavirenz, Capravirine, Ribavirin, Oseltamivir and Amantadine as the reference drugs. Some of the compounds exhibited better 5α-reductase inhibitors, antiviral and anti-tumor activities than the reference drugs. The detailed 5α-reductase inhibitors, antiviral and anti-tumor activities of the synthesized compounds were reported. PMID:22057085

  10. Implications and problems in analysing cytotoxic activity of hydroxyurea in combination with a potential inhibitor of ribonucleotide reductase.

    PubMed

    Nocentini, G; Barzi, A; Franchetti, P

    1990-01-01

    The cytotoxicity of hydroxyurea in combination with 2.2'-bipyridyl-6-carbothioamide (a potential inhibitor of ribonucleotide reductase) on P388 murine leukemia is reported. Synergistic activity was studied using various interpretations of the isobologram method and the combination index method. We evaluated the pros and cons of these methods and their overall usefulness. In our opinion, to obtain all possible information from a compound association, it is important to choose a formally correct method that (a) can quantitatively evaluate synergism or antagonism, (b) may offer the possibility of averaging final results, (c) needs a minimal amount of experimental data, and (d) is rapid. Moreover, we emphasize both the utility of testing at least three molar ratios of compound association and the importance of carefully choosing the fractional inhibition used in calculating the combination effect. Such evaluation of drug combinations gives information essential to the preparation of new anticancer drug regimens and to the early assessment of biochemical interactions. PMID:2208576

  11. Design and synthesis of hepatoselective, pyrrole-based HMG-CoA reductase inhibitors.

    PubMed

    Pfefferkorn, Jeffrey A; Song, Yuntao; Sun, Kuai-Lin; Miller, Steven R; Trivedi, Bharat K; Choi, Chulho; Sorenson, Roderick J; Bratton, Larry D; Unangst, Paul C; Larsen, Scott D; Poel, Toni-Jo; Cheng, Xue-Min; Lee, Chitase; Erasga, Noe; Auerbach, Bruce; Askew, Valerie; Dillon, Lisa; Hanselman, Jeffrey C; Lin, Zhiwu; Lu, Gina; Robertson, Andrew; Olsen, Karl; Mertz, Thomas; Sekerke, Catherine; Pavlovsky, Alexander; Harris, Melissa S; Bainbridge, Graeme; Caspers, Nicole; Chen, Huifen; Eberstadt, Matthias

    2007-08-15

    This manuscript describes the design and synthesis of a series of pyrrole-based inhibitors of HMG-CoA reductase for the treatment of hypercholesterolemia. Analogs were optimized using structure-based design and physical property considerations resulting in the identification of 44, a hepatoselective HMG-CoA reductase inhibitor with excellent acute and chronic efficacy in a pre-clinical animal models. PMID:17574412

  12. Studies on WF-3681, a novel aldose reductase inhibitor. I. Taxonomy, fermentation, isolation and characterization.

    PubMed

    Nishikawa, M; Tsurumi, Y; Namiki, T; Yoshida, K; Okuhara, M

    1987-10-01

    WF-3681 was isolated from a cultured filtrate of Chaetomella raphigera as a novel inhibitor of aldose reductase. It was extracted with ethyl acetate and then purified with silica gel chromatography. Its molecular formula was determined to be C13H12O5 by elemental analysis and high resolution electron impact mass spectrometry. IC50 of WF-3681 was 2.5 X 10(-7) M for partially purified aldose reductase of rabbit lens. PMID:3119547

  13. Isolation of Assimilatory- and Dissimilatory-Type Sulfite Reductases from Desulfovibrio vulgaris

    PubMed Central

    Lee, Jin-Po; LeGall, Jean; Peck, Harry D.

    1973-01-01

    Bisulfite reductase (desulfoviridin) and an assimilatory sulfite reductase have been purified from extracts of Desulfovibrio vulgaris. The bisulfite reductase has absorption maxima at 628, 580, 408, 390, and 279 nm, and a molecular weight of 226,000 by sedimentation equilibrium, and was judged to be free of other proteins by disk electrophoresis and ultracentrifugation. On gels, purified bisulfite reductase exhibited two green bands which coincided with activity and protein. The enzyme appears to be a tetramer but was shown to have two different types of subunits having molecular weights of 42,000 and 50,000. The chromophore did not form an alkaline ferrohemochromogen, was not reduced with dithionite or borohydride, and did not form a spectrally visible complex with CO. The assimilatory sulfite reductase has absorption maxima at 590, 545, 405 and 275 nm and a molecular weight of 26,800, and appears to consist of a single polypeptide chain as it is not dissociated into subunits by sodium dodecyl sulfate. By disk electrophoresis, purified sulfite reductase exhibited a single greenish-brown band which coincided with activity and protein. The sole product of the reduction was sulfide, and the chromophore was reduced by borohydride in the presence of sulfite. Carbon monoxide reacted with the reduced chromophore but it did not form a typical pyridine ferrohemochromogen. Thiosulfate, trithionate, and tetrathionate were not reduced by either enzyme preparation. In the presence of 8 M urea, the spectrum of bisulfite reductase resembles that of the sulfite reductase, thus suggesting a chemical relationship between the two chromophores. Images PMID:4725615

  14. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    PubMed Central

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils. PMID:24435070

  15. Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers.

    PubMed Central

    Menting, J G; Cornish, E; Scopes, R K

    1994-01-01

    NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P. hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies. Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts. Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase. The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively. We believe that the purified enzyme is a P. hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4). PMID:7991686

  16. "Subversive" substrates for the enzyme trypanothione disulfide reductase: alternative approach to chemotherapy of Chagas disease.

    PubMed Central

    Henderson, G B; Ulrich, P; Fairlamb, A H; Rosenberg, I; Pereira, M; Sela, M; Cerami, A

    1988-01-01

    The trypanosomatid flavoprotein disulfide reductase, trypanothione reductase, is shown to catalyze one-electron reduction of suitably substituted naphthoquinone and nitrofuran derivatives. A number of such compounds have been chemically synthesized, and a structure-activity relationship has been established; the enzyme is most active with compounds that contain basic functional groups in side-chain residues. The reduced products are readily reoxidized by molecular oxygen and thus undergo classical enzyme-catalyzed redox cycling. In addition to their ability to act as substrates for trypanothione reductase, the compounds are also shown to effectively inhibit enzymatic reduction of the enzyme's physiological substrate, trypanothione disulfide. Under aerobic conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions the nitrofuran compounds cause irreversible inactivation of the enzyme. When tested for biological activity against Trypanosoma cruzi trypomastigotes, many of the test compounds were trypanocidal, and this activity correlated with their relative ability to act as substrates for trypanothione reductase. The activity of the enzyme with these redox-cycling derivatives constitutes a subversion of its normal antioxidant role within the cell. For this reason these compounds may be termed "subversive" substrates for trypanothione reductase. PMID:3135548

  17. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network.

    PubMed

    Couto, Narciso; Wood, Jennifer; Barber, Jill

    2016-06-01

    In this review article we examine the role of glutathione reductase in the regulation, modulation and maintenance of cellular redox homoeostasis. Glutathione reductase is responsible for maintaining the supply of reduced glutathione; one of the most abundant reducing thiols in the majority of cells. In its reduced form, glutathione plays key roles in the cellular control of reactive oxygen species. Reactive oxygen species act as intracellular and extracellular signalling molecules and complex cross talk between levels of reactive oxygen species, levels of oxidised and reduced glutathione and other thiols, and antioxidant enzymes such as glutathione reductase determine the most suitable conditions for redox control within a cell or for activation of programmed cell death. Additionally, we discuss the translation and expression of glutathione reductase in a number of organisms including yeast and humans. In yeast and human cells, a single gene expresses more than one form of glutathione reductase, destined for residence in the cytoplasm or for translocation to different organelles; in plants, however, two genes encoding this protein have been described. In general, insects and kinetoplastids (a group of protozoa, including Plasmodia and Trypanosoma) do not express glutathione reductase or glutathione biosynthetic enzymes. Instead, they express either the thioredoxin system or the trypanothione system. The thioredoxin system is also present in organisms that have the glutathione system and there may be overlapping functions with cross-talk between the two systems. Finally we evaluate therapeutic targets to overcome oxidative stress associated cellular disorders. PMID:26923386

  18. X-ray structure of trypanothione reductase from Crithidia fasciculata at 2. 4- angstrom resolution

    SciTech Connect

    Kuriyan, J.; Xiangpeng Kong; Krishna, T.S.R.; Murgolo, N.J.; Field, H.; Cerami, A.; Henderson, G.B. ); Sweet, R.M. )

    1991-10-01

    Trypanosomes and related protozoan parasites lack glutathione reductase and possess instead a closely related enzyme that serves as the reductant of a bis(glutathione)-spermidien conjugate, trypanothione. The human and parasite enzymes have mutually exclusive substrate specificities, providing a route for the design of therapeutic agents by specific inhibition of the parasite enzyme. The authors report here the three-dimensional structure of trypanothione reductase from Crithidia fasciculata and show that it closely resembles the structure of human glutathione reductase. In particular, the core structure surrounding the catalytic machinery is almost identical in the two enzymes. However, significant differences are found at the substrate binding sites. A cluster of basic residues in glutathione reductase is replaced by neutral, hydrophobic, or acidic residues in trypanothione reductase, consistent with the nature of the spermidine linkage and the change in overall charge of the substrate from {minus}2 to +1, respectively. The binding site is more open in trypanothione reductase due to rotations of about 4{degree} in the domains that form in site, with relative shifts of as much as 2-3 {angstrom} in residues that can interact with potential inhibitors and complement previous modeling and mutagenesis studies on the two enzymes.

  19. Photoaffinity labelling of nuclear steroid 5 alpha-reductase of rat ventral prostate.

    PubMed

    Enderle-Schmitt, U; Seitz, J; Aumüller, G

    1989-09-01

    In order to get more information on the molecular structure of the rat prostatic 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3:1.22) a systematic photoaffinity labelling study has been performed. To irreversibly freeze the status quo of interaction, either testosterone, the physiological ligand, or diazo-MAPD (21-diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione), a specific 5 alpha-reductase inhibitor, was irradiated with isolated nuclei or with purified nuclear membranes or with solubilized nuclear membrane proteins and checked for optimal labelling conditions. The principal substances covalently labelled were phospholipids and at a minor ratio proteins. Analysis by SDS-PAGE and autoradiofluorography revealed two labelled polypeptides with molecular weights of 20 kDa and 26 kDa. The following evidence indicates that these polypeptides might be derived from the enzyme 5 alpha-reductase: both proteins are labelled only when specific ligands for 5 alpha-reductase are used; binding can be reduced by the addition of an excess of unlabelled ligand; enzyme activity is irreversibly suppressed when irradiated in the presence of these ligands; only subcellular fractions containing 5 alpha-reductase reveal the labelled proteins; in all 5 alpha-reductase containing preparations with increasing specific activity, independent of the polypeptide pattern, the same proteins are labelled. PMID:2779229

  20. Molecular cloning, expression and catalytic activity of a human AKR7 member of the aldo-keto reductase superfamily: evidence that the major 2-carboxybenzaldehyde reductase from human liver is a homologue of rat aflatoxin B1-aldehyde reductase.

    PubMed Central

    Ireland, L S; Harrison, D J; Neal, G E; Hayes, J D

    1998-01-01

    The masking of charged amino or carboxy groups by N-phthalidylation and O-phthalidylation has been used to improve the absorption of many drugs, including ampicillin and 5-fluorouracil. Following absorption of such prodrugs, the phthalidyl group is hydrolysed to release 2-carboxybenzaldehyde (2-CBA) and the pharmaceutically active compound; in humans, 2-CBA is further metabolized to 2-hydroxymethylbenzoic acid by reduction of the aldehyde group. In the present work, the enzyme responsible for the reduction of 2-CBA in humans is identified as a homologue of rat aflatoxin B1-aldehyde reductase (rAFAR). This novel human aldo-keto reductase (AKR) has been cloned from a liver cDNA library, and together with the rat protein, establishes the AKR7 family of the AKR superfamily. Unlike its rat homologue, human AFAR (hAFAR) appears to be constitutively expressed in human liver, and is widely expressed in extrahepatic tissues. The deduced human and rat protein sequences share 78% identity and 87% similarity. Although the two AKR7 proteins are predicted to possess distinct secondary structural features which distinguish them from the prototypic AKR1 family of AKRs, the catalytic- and NADPH-binding residues appear to be conserved in both families. Certain of the predicted structural features of the AKR7 family members are shared with the AKR6 beta-subunits of voltage-gated K+-channels. In addition to reducing the dialdehydic form of aflatoxin B1-8,9-dihydrodiol, hAFAR shows high affinity for the gamma-aminobutyric acid metabolite succinic semialdehyde (SSA) which is structurally related to 2-CBA, suggesting that hAFAR could function as both a SSA reductase and a 2-CBA reductase in vivo. This hypothesis is supported in part by the finding that the major peak of 2-CBA reductase activity in human liver co-purifies with hAFAR protein. PMID:9576847

  1. Probing the Electrostatics of Active Site Microenvironments along the Catalytic Cycle for Escherichia coli Dihydrofolate Reductase

    PubMed Central

    2015-01-01

    Electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions. These electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle. Herein, the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of Escherichia coli dihydrofolate reductase (ecDHFR) by incorporation of thiocyanate probes at two site-specific locations in the active site. The electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical/molecular mechanical (QM/MM) calculations. Changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile (CN) vibrational stretching frequencies and 13C NMR chemical shifts. These environmental changes arise from protein conformational rearrangements during catalysis. The QM/MM calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step, which spans the closed and occluded conformations of the enzyme. Analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen-bonding interactions. The electric fields along the CN axes of the probes are decomposed into contributions from specific residues, ligands, and solvent molecules that make up the microenvironments around the probes. Moreover, calculation of the electric field along the hydride donor–acceptor axis, along with decomposition of this field into specific contributions, indicates that the cofactor and substrate, as well as the enzyme, impose a substantial electric field that facilitates hydride transfer. Overall, experimental and theoretical data provide evidence for

  2. A tale of two trials: The impact of 5α-reductase inhibition on prostate cancer (Review)

    PubMed Central

    LACY, JOHN M.; KYPRIANOU, NATASHA

    2014-01-01

    The use of 5α-reductase inhibitors (5α-RIs) as prostate cancer chemoprevention agents is controversial. Two large randomized trials, the Prostate Cancer Prevention Trial (PCPT) and the Reduction by Dutasteride of Prostate Cancer Events (REDUCE) Trial, have both shown a decreased incidence of prostate cancer in patients administered with 5α-RIs. Both studies showed, however, an increased risk of higher-grade prostate cancer. Numerous studies have since analyzed the inherent biases in these landmark studies and have used mathematical modeling to estimate the true incidence of prostate cancer and the risk for high-grade prostate cancer in patients undergoing 5α-RI treatment. All primary publications associated with the PCPT and REDUCE studies were reviewed in detail. Pertinent references from the above publications were assessed and a literature search of all published articles associated with PCPT, REDUCE or 5α-RIs as chemopreventative agents through October 2013 was performed using Pubmed/Medline. PCPT and REDUCE both showed a significant decrease in the incidence of prostate cancer following the administration of 5α-reductase inhibitor, as compared with placebo, suggesting that 5α-RIs may be effective agents for prostate cancer chemoprevention. Inherent biases in the design of these two studies may have caused an artificial increase in the number of high-grade cancers reported. Mathematical models, that integrated data from these trials, revealed neither an increased nor decreased risk of high-grade disease when taking these biases into consideration. Moderately strong evidence exists that 5α-RIs may reduce the risk of prostate cancer. PCPT and REDUCE showed a decreased prevalence of prostate cancer in patients taking 5α-RIs. Urologists should have a working knowledge of these studies and discuss with patients the risks and benefits of 5α-RI treatment. Further studies to evaluate the cost-effectiveness of chemoprevention with 5α-RIs and appropriate

  3. A tale of two trials: The impact of 5α-reductase inhibition on prostate cancer (Review).

    PubMed

    Lacy, John M; Kyprianou, Natasha

    2014-10-01

    The use of 5α-reductase inhibitors (5α-RIs) as prostate cancer chemoprevention agents is controversial. Two large randomized trials, the Prostate Cancer Prevention Trial (PCPT) and the Reduction by Dutasteride of Prostate Cancer Events (REDUCE) Trial, have both shown a decreased incidence of prostate cancer in patients administered with 5α-RIs. Both studies showed, however, an increased risk of higher-grade prostate cancer. Numerous studies have since analyzed the inherent biases in these landmark studies and have used mathematical modeling to estimate the true incidence of prostate cancer and the risk for high-grade prostate cancer in patients undergoing 5α-RI treatment. All primary publications associated with the PCPT and REDUCE studies were reviewed in detail. Pertinent references from the above publications were assessed and a literature search of all published articles associated with PCPT, REDUCE or 5α-RIs as chemopreventative agents through October 2013 was performed using Pubmed/Medline. PCPT and REDUCE both showed a significant decrease in the incidence of prostate cancer following the administration of 5α-reductase inhibitor, as compared with placebo, suggesting that 5α-RIs may be effective agents for prostate cancer chemoprevention. Inherent biases in the design of these two studies may have caused an artificial increase in the number of high-grade cancers reported. Mathematical models, that integrated data from these trials, revealed neither an increased nor decreased risk of high-grade disease when taking these biases into consideration. Moderately strong evidence exists that 5α-RIs may reduce the risk of prostate cancer. PCPT and REDUCE showed a decreased prevalence of prostate cancer in patients taking 5α-RIs. Urologists should have a working knowledge of these studies and discuss with patients the risks and benefits of 5α-RI treatment. Further studies to evaluate the cost-effectiveness of chemoprevention with 5α-RIs and appropriate

  4. Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

    PubMed Central

    Cha, Mee-Kyung; Bae, Yoo-Jeen; Kim, Kyu-Jeong; Park, Byung-Joon; Kim, Il-Han

    2015-01-01

    AIM: To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria. METHODS: Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined. RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis

  5. Inhibitory effect on in vitro LDL oxidation and HMG Co-A reductase activity of the liquid-liquid partitioned fractions of Hericium erinaceus (Bull.) Persoon (lion's mane mushroom).

    PubMed

    Rahman, Mohammad Azizur; Abdullah, Noorlidah; Aminudin, Norhaniza

    2014-01-01

    Oxidation of low-density lipoprotein (LDL) has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A) reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and aqueous residue (AQ). The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins) for the formation of conjugated diene (CD) at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL) of thiobarbituric acid reactive substances (TBARS) at 1 mg/mL. It also mostly inhibited (59.91%) the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases. PMID:24959591

  6. Inhibitory Effect on In Vitro LDL Oxidation and HMG Co-A Reductase Activity of the Liquid-Liquid Partitioned Fractions of Hericium erinaceus (Bull.) Persoon (Lion's Mane Mushroom)

    PubMed Central

    Aminudin, Norhaniza

    2014-01-01

    Oxidation of low-density lipoprotein (LDL) has been strongly suggested as the key factor in the pathogenesis of atherosclerosis. Mushrooms have been implicated in having preventive effects against chronic diseases due especially to their antioxidant properties. In this study, in vitro inhibitory effect of Hericium erinaceus on LDL oxidation and the activity of the cholesterol biosynthetic key enzyme, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG Co-A) reductase, was evaluated using five liquid-liquid solvent fractions consisting of methanol : dichloromethane (M : DCM), hexane (HEX), dichloromethane (DCM), ethyl acetate (EA), and aqueous residue (AQ). The hexane fraction showed the highest inhibition of oxidation of human LDL as reflected by the increased lag time (100 mins) for the formation of conjugated diene (CD) at 1 µg/mL and decreased production (68.28%, IC50 0.73 mg/mL) of thiobarbituric acid reactive substances (TBARS) at 1 mg/mL. It also mostly inhibited (59.91%) the activity of the HMG Co-A reductase at 10 mg/mL. The GC-MS profiling of the hexane fraction identified the presence of myconutrients: inter alia, ergosterol and linoleic acid. Thus, hexane fraction of Hericium erinaceus was found to be the most potent in vitro inhibitor of both LDL oxidation and HMG Co-A reductase activity having therapeutic potential for the prevention of oxidative stress-mediated vascular diseases. PMID:24959591

  7. Severe scoliosis in a patient with severe methylenetetrahydrofolate reductase deficiency.

    PubMed

    Munoz, Tatiana; Patel, Jinesh; Badilla-Porras, Ramses; Kronick, Jonathan; Mercimek-Mahmutoglu, Saadet

    2015-01-01

    Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare autosomal recessively inherited inborn error of folate metabolism. We report a new patient with severe MTHFR deficiency who presented at age 4 months with early onset severe scoliosis associated with severe hypotonia. Markedly decreased MTHFR enzyme activity (0.3 nmoles CHO/mg protein/h; reference range>9) and compound heterozygous mutations (c. 1304T>C; p.Phe435Ser and c.1539dup; p.Glu514Argfs∗24) in the MTHFR gene confirmed the diagnosis. She was treated with vitamin B12, folic acid and betaine supplementation and showed improvements in her developmental milestones and hypotonia. To the best of our knowledge, this is the first patient with MTHFR deficiency reported with severe early onset scoliosis. Despite the late diagnosis and treatment initiation, she showed favorable short-term neurodevelopmental outcome. This case suggests that homocysteine measurement should be included in the investigations of patients with developmental delay, hypotonia and scoliosis within first year of life prior to organizing genetic investigations. PMID:24726568

  8. 5alpha-reductase: history and clinical importance.

    PubMed

    Marks, Leonard S

    2004-01-01

    The treatment of men with symptomatic benign prostatic hyperplasia (BPH) has shifted dramatically from surgery to drug therapy over the past decade. The revolution in BPH treatment began with the discovery of congenital 5alpha-reductase (5AR) deficiency, leading to the appreciation of 2 different androgenic hormones: testosterone, which mediates overt masculinization in the adult male, and dihydrotestosterone (DHT), which mediates prostatic growth, acne, facial beard, and male pattern baldness. Inhibition of DHT in adults results in prostatic shrinkage and symptomatic relief in many men, without the side effects seen with conventional androgen-deprivation therapy. The 5AR inhibitor drugs (finasteride and the dual inhibitor, dutasteride) are able to ablate the accumulation of intraprostatic DHT, the mechanism most responsible for prostate growth and maintenance. Not only may these drugs relieve symptoms, but they may also alter the natural history of the BPH process. Future indications for the 5ARI drugs could include chemoprevention of prostate cancer, prophylaxis of BPH-related complications, and treatment of BPH-associated hematuria. PMID:16985920

  9. Glyoxalase 1 and glutathione reductase 1 regulate anxiety in mice.

    PubMed

    Hovatta, Iiris; Tennant, Richard S; Helton, Robert; Marr, Robert A; Singer, Oded; Redwine, Jeffrey M; Ellison, Julie A; Schadt, Eric E; Verma, Inder M; Lockhart, David J; Barlow, Carrolee

    2005-12-01

    Anxiety and fear are normal emotional responses to threatening situations. In human anxiety disorders--such as panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, social phobia, specific phobias and generalized anxiety disorder--these responses are exaggerated. The molecular mechanisms involved in the regulation of normal and pathological anxiety are mostly unknown. However, the availability of different inbred strains of mice offers an excellent model system in which to study the genetics of certain behavioural phenotypes. Here we report, using a combination of behavioural analysis of six inbred mouse strains with quantitative gene expression profiling of several brain regions, the identification of 17 genes with expression patterns that correlate with anxiety-like behavioural phenotypes. To determine if two of the genes, glyoxalase 1 and glutathione reductase 1, have a causal role in the genesis of anxiety, we performed genetic manipulation using lentivirus-mediated gene transfer. Local overexpression of these genes in the mouse brain resulted in increased anxiety-like behaviour, while local inhibition of glyoxalase 1 expression by RNA interference decreased the anxiety-like behaviour. Both of these genes are involved in oxidative stress metabolism, linking this pathway with anxiety-related behaviour. PMID:16244648

  10. Catalytic Cycle of Human Glutathione Reductase Near 1 Å Resolution

    SciTech Connect

    Berkholz, Donald S.; Faber, H. Richard; Savvides, Savvas N.; Karplus, P. Andrew

    2008-09-08

    Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 {angstrom} that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity -- a net deviation of 53 deg. across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.

  11. Isoquinoline alkaloids from Tinospora cordifolia inhibit rat lens aldose reductase.

    PubMed

    Patel, Mayurkumar B; Mishra, Shrihari

    2012-09-01

    The inhibitory activity of Tinospora cordifolia stem-derived alkaloids was evaluated against lens aldose reductase (AR) isolated from male Wistar rats. Anticataract potential of the alkaloids of T. cordifolia was evaluated in vitro in rat lenses, considering the activity of normal rat lenses as 100%. The biologically active constituents of T. cordifolia extract were characterized as the isoquinoline alkaloids, jatrorrhizine, palmatine and magnoflorine, by spectral analysis. The inhibitory effects varied with all chemicals and concentrations used. The inhibitory concentration (IC₅₀) values of jatrorrhizine, palmatine and magnoflorine are 3.23, 3.45 and 1.25 µg/mL respectively. The concentration of maximum activity was selected for its effect on galactose-induced polyol accumulation in vitro. The percentage inhibition of galactose-induced polyol accumulation was 62.6, 58.8 and 27.7% in the presence of jatrorrhizine, palmatine and magnoflorine, respectively. Magnoflorine may be useful as lead compounds and new agents for AR inhibition. PMID:22294283

  12. Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

    PubMed Central

    Prunotto, Marco; Carnevali, Maria Luisa; Candiano, Giovanni; Murtas, Corrado; Bruschi, Maurizio; Corradini, Emilia; Trivelli, Antonella; Magnasco, Alberto; Petretto, Andrea; Santucci, Laura; Mattei, Silvia; Gatti, Rita; Scolari, Francesco; Kador, Peter; Allegri, Landino

    2010-01-01

    Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti–aldose reductase (AR) and anti–manganese superoxide dismutase (SOD2) IgG4 in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG4 from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG4 and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression. PMID:20150532

  13. Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

    PubMed

    Chu, Y X; Chen, H R; Wu, A Z; Cai, R; Pan, J S

    2015-01-01

    Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside. PMID:25966276

  14. Optical observation of correlated motions in dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2015-03-01

    Enzyme function relies on its structural flexibility to make conformational changes for substrate binding and product release. An example of a metabolic enzyme where such structural changes are vital is dihydrofolate reductase (DHFR). DHFR is essential in both prokaryotes and eukaryotes for the nucleotide biosynthesis by catalyzing the reduction of dihydrofolate to tetrahydrofolate. NMR dynamical measurements found large amplitude fast dynamics that could indicate rigid-body, twisting-hinge motion for ecDHFR that may mediate flux. The role of such long-range correlated motions in function was suggested by the observed sharp decrease in enzyme activity for the single point mutation G121V, which is remote from active sites. This decrease in activity may be caused by the mutation interfering with the long-range intramolecular vibrations necessary for rapid access to functional configurations. We use our new technique of crystal anisotropy terahertz microscopy (CATM), to observe correlated motions in ecDHFR crystals with the bonding of NADPH and methotrexate. We compare the measured intramolecular vibrational spectrum with calculations using normal mode analysis.

  15. Analysis of enzyme-catalyzed nucleotide modification by aldose reductase

    SciTech Connect

    Grimshaw, C.E.

    1987-05-01

    Homogeneous bovine kidney aldose reductase catalyzes two reactions in addition to the normal aldehyde-dependent oxidation of NADPH. First, adduct formation between the oxidized nucleotide and the oxidized substrate is observed during turnover due to initial formation of a reversible E:NADP/sup +/:R-CHO ternary complex, which subsequently reacts to give the covalent complex (E:NADP/sup +/-R-CHO). The reaction is enzyme-catalyzed with substantial enhancement of both the pseudo-first order rate constant and the overall K/sub eq/ relative to the reaction with free NADP/sup +/ in aqueous buffer. Analysis of the concentration dependence and time-course for reversible dead-end and covalent complex formation are described for several aldehyde and nucleotide substrates. Non-linear time courses for aldehyde reduction and substrate inhibition by the aldehyde substrate in initial velocity studies are completely accounted for by this mechanism, thereby eliminating a simple Dalziel-type explanation for the substrate activation by aldehyde which is also observed. Second, enzyme-catalyzed oxidation of NADPH occurs in the absence of aldehyde substrate with a rate equal to .03% of V/sub max/ for the normal reduction of glyceraldehyde. By 500 MHz /sup 1/H-NMR, the enzyme-catalyzed oxidation of (4-/sup 2/H)NADPH appears to be greater than 95% stereospecific. Spectroscopic evidence for a similar oxidation reaction is observed for the covalent E:NADP/sup +/-R-CHO adduct with glyceraldehyde, but not with glycolaldehyde.

  16. Loop interactions during catalysis by dihydrofolate reductase from Moritella profunda.

    PubMed

    Behiry, Enas M; Evans, Rhiannon M; Guo, Jiannan; Loveridge, E Joel; Allemann, Rudolf K

    2014-07-29

    Dihydrofolate reductase (DHFR) is often used as a model system to study the relation between protein dynamics and catalysis. We have studied a number of variants of the cold-adapted DHFR from Moritella profunda (MpDHFR), in which the catalytically important M20 and FG loops have been altered, and present a comparison with the corresponding variants of the well-studied DHFR from Escherichia coli (EcDHFR). Mutations in the M20 loop do not affect the actual chemical step of transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate 7,8-dihydrofolate in the catalytic cycle in either enzyme; they affect the steady state turnover rate in EcDHFR but not in MpDHFR. Mutations in the FG loop also have different effects on catalysis by the two DHFRs. Despite the two enzymes most likely sharing a common catalytic cycle at pH 7, motions of these loops, known to be important for progression through the catalytic cycle in EcDHFR, appear not to play a significant role in MpDHFR. PMID:25014120

  17. Correlated Protein Motion Measurements of Dihydrofolate Reductase Crystals

    NASA Astrophysics Data System (ADS)

    Xu, Mengyang; Niessen, Katherine; Pace, James; Cody, Vivian; Markelz, Andrea

    2014-03-01

    We report the first direct measurements of the long range structural vibrational modes in dihydrofolate reductase (DHFR). DHFR is a universal housekeeping enzyme that catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetra-hydrofolate, with the aid of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). This crucial enzymatic role as the target for anti-cancer [methotrexate (MTX)], and other clinically useful drugs, has made DHFR a long-standing target of enzymological studies. The terahertz (THz) frequency range (5-100 cm-1), corresponds to global correlated protein motions. In our lab we have developed Crystal Anisotropy Terahertz Microscopy (CATM), which directly measures these large scale intra-molecular protein vibrations, by removing the relaxational background of the solvent and residue side chain librational motions. We demonstrate narrowband features in the anisotropic absorbance for mouse DHFR with the ligand binding of NADPH and MTX single crystals as well as Escherichia coli DHFR with the ligand binding of NADPH and MTX single crystals. This work is supported by NSF grant MRI2 grant DBI2959989.

  18. Structural basis and mechanism of enoyl reductase inhibition by triclosan.

    PubMed

    Stewart, M J; Parikh, S; Xiao, G; Tonge, P J; Kisker, C

    1999-07-23

    The enoyl-acyl carrier protein reductase (ENR) is involved in bacterial fatty acid biosynthesis and is the target of the antibacterial diazaborine compounds and the front-line antituberculosis drug isoniazid. Recent studies suggest that ENR is also the target for the broad-spectrum biocide triclosan. The 1.75 A crystal structure of EnvM, the ENR from Escherichia coli, in complex with triclosan and NADH reveals that triclosan binds specifically to EnvM. These data provide a molecular mechanism for the antibacterial activity of triclosan and substantiate the hypothesis that its activity results from inhibition of a specific cellular target rather than non-specific disruption of the bacterial cell membrane. This has important implications for the emergence of drug-resistant bacteria, since triclosan is an additive in many personal care products such as toothpastes, mouthwashes and soaps. Based on this structure, rational design of triclosan derivatives is possible which might be effective against recently identified triclosan-resistant bacterial strains. PMID:10398587

  19. A second target of benzamide riboside: dihydrofolate reductase.

    PubMed

    Roussel, Breton; Johnson-Farley, Nadine; Kerrigan, John E; Scotto, Kathleen W; Banerjee, Debabrata; Felczak, Krzysztof; Pankiewicz, Krzysztof W; Gounder, Murugesan; Lin, HongXia; Abali, Emine Ercikan; Bertino, Joseph R

    2012-11-01

    Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth. PMID:22954684

  20. Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine.

    PubMed

    O'Riordan, Avril A; Morales, Veronica Athie; Mulligan, Linda; Faheem, Nazia; Windle, Henry J; Kelleher, Dermot P

    2012-06-01

    Helicobacter pylori (H. pylori) is the most important etiological agent of chronic active gastritis, peptic ulcer disease and gastric cancer. The aim of this study was to evaluate the efficacy of alkyl hydroperoxide reductase (AhpC) and mannosylated AhpC (mAhpC) as candidate vaccines in the C57BL/6J mouse model of H. pylori infection. Recombinant AhpC was cloned, over-expressed and purified in an unmodified form and was also engineered to incorporate N and C-terminal mannose residues when expressed in the yeast Pichia pastoris. Mice were immunized systemically and mucosally with AhpC and systemically with mAhpC prior to challenge with H. pylori. Serum IgG responses to AhpC were determined and quantitative culture was used to determine the efficacy of vaccination strategies. Systemic prophylactic immunization with AhpC/alum and mAhpC/alum conferred protection against infection in 55% and 77.3% of mice, respectively. Mucosal immunization with AhpC/cholera toxin did not protect against infection and elicited low levels of serum IgG in comparison with systemic immunization. These data support the use of AhpC as a potential vaccine candidate against H. pylori infection. PMID:22512976

  1. Solvent effects on catalysis by Escherichia coli dihydrofolate reductase.

    PubMed

    Loveridge, E Joel; Tey, Lai-Hock; Allemann, Rudolf K

    2010-01-27

    Hydride transfer catalyzed by dihydrofolate reductase (DHFR) has been described previously within an environmentally coupled model of hydrogen tunneling, where protein motions control binding of substrate and cofactor to generate a tunneling ready conformation and modulate the width of the activation barrier and hence the reaction rate. Changes to the composition of the reaction medium are known to perturb protein motions. We have measured kinetic parameters of the reaction catalyzed by DHFR from Escherichia coli in the presence of various cosolvents and cosolutes and show that the dielectric constant, but not the viscosity, of the reaction medium affects the rate of reaction. Neither the primary kinetic isotope effect on the reaction nor its temperature dependence were affected by changes to the bulk solvent properties. These results are in agreement with our previous report on the effect of solvent composition on catalysis by DHFR from the hyperthermophile Thermotoga maritima. However, the effect of solvent on the temperature dependence of the kinetic isotope effect on hydride transfer catalyzed by E. coli DHFR is difficult to explain within a model, in which long-range motions couple to the chemical step of the reaction, but may indicate the existence of a short-range promoting vibration or the presence of multiple nearly isoenergetic conformational substates of enzymes with similar but distinct catalytic properties. PMID:20047317

  2. Molecular Cloning of Complementary DNA Encoding Maize Nitrite Reductase

    PubMed Central

    Lahners, Kristine; Kramer, Vance; Back, Eduard; Privalle, Laura; Rothstein, Steven

    1988-01-01

    Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666376

  3. Chromate reductase activity in Streptomyces sp. MC1.

    PubMed

    Polti, Marta A; Amoroso, María J; Abate, Carlos M

    2010-02-01

    Biological transformation of Cr(VI) to Cr(III) by enzymatic reduction may provide a less costly and more environmentally friendly approach to remediation. In a previous report a Cr(VI) resistant actinomycete strain, Streptomyces sp. MC1, was able to reduce Cr(VI) present in a synthetic medium, soil extract and soil samples. This is the first time optimal conditions such as pH, temperature, growth phase and electron donor have been elucidated in vitro for Cr(VI) reduction by a streptomycete. Chromate reductase of Streptomyces sp. MC1 is a constitutive enzyme which was mainly associated with biomass and required NAD(P)H as an electron donor. It was active over a broad temperature (19-39 degrees C) and pH (5-8) range, and optimum conditions were 30 degrees C and pH 7. The enzyme was present in supernatant, pellet and cell free extract. Bioremediation with the enzyme was observed in non-compatible cell reproduction systems, conditions frequently found in contaminated environments. PMID:20339215

  4. Crystal structure studies on sulfur oxygenase reductase from Acidianus tengchongensis

    SciTech Connect

    Li Mei; Chen Zhiwei; Zhang Pingfeng; Pan Xiaowei; Jiang Chengying; An Xiaomin; Liu Shuangjiang; Chang Wenrui

    2008-05-09

    Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.

  5. 5α-Reductase: History and Clinical Importance

    PubMed Central

    Marks, Leonard S

    2004-01-01

    The treatment of men with symptomatic benign prostatic hyperplasia (BPH) has shifted dramatically from surgery to drug therapy over the past decade. The revolution in BPH treatment began with the discovery of congenital 5α-reductase (5AR) deficiency, leading to the appreciation of 2 different androgenic hormones: testosterone, which mediates overt masculinization in the adult male, and dihydrotestosterone (DHT), which mediates prostatic growth, acne, facial beard, and male pattern baldness. Inhibition of DHT in adults results in prostatic shrinkage and symptomatic relief in many men, without the side effects seen with conventional androgen-deprivation therapy. The 5AR inhibitor drugs (finasteride and the dual inhibitor, dutasteride) are able to ablate the accumulation of intraprostatic DHT, the mechanism most responsible for prostate growth and maintenance. Not only may these drugs relieve symptoms, but they may also alter the natural history of the BPH process. Future indications for the 5ARI drugs could include chemoprevention of prostate cancer, prophylaxis of BPH-related complications, and treatment of BPH-associated hematuria. PMID:16985920

  6. Reductive activation of E. coli respiratory nitrate reductase.

    PubMed

    Ceccaldi, Pierre; Rendon, Julia; Léger, Christophe; Toci, René; Guigliarelli, Bruno; Magalon, Axel; Grimaldi, Stéphane; Fourmond, Vincent

    2015-10-01

    Over the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major "low-pH" and "high-pH" Mo(V) EPR signatures correspond to species in acid-base equilibrium. PMID:26073890

  7. Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase

    PubMed Central

    Chatelle, Claire; Kraemer, Stéphanie; Ren, Guoping; Chmura, Hannah; Marechal, Nils; Boyd, Dana; Roggemans, Caroline; Ke, Na; Riggs, Paul; Bardwell, James

    2015-01-01

    Abstract Aims: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. Results: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. Innovation and Conclusions: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization. Antioxid. Redox Signal. 23, 945–957. PMID:26191605

  8. Imexon enhances gemcitabine cytotoxicity by inhibition of ribonucleotide reductase

    PubMed Central

    Roman, Nicholas O.; Samulitis, Betty K.; Wisner, Lee; Landowski, Terry H.; Dorr, Robert T.

    2010-01-01

    Purpose Gemcitabine (GEM) is currently the standard first line treatment for pancreatic cancer; however, the overall survival of patients with this disease remains poor. Imexon is a pro-oxidant small molecule which produced a high response rate in combination with GEM in a phase I trial in pancreatic cancer. In this study, we investigate the combination of GEM with a novel redox-active agent, imexon, in vitro and in vivo. Methods Median effect analysis was used for in vitro combination cytotoxicity. The effect of imexon on GEM metabolism and uptake into cells and into DNA and effects on ribonucleotide reductase (RNR) were examined in vitro. The pharmacokinetics and antitumor efficacy of the imexon/GEM combination was evaluated in mouse models. Results In three human pancreatic cancer lines, there was additivity for the imexon/GEM combination. There was significantly greater efficacy for the drug combination in Panc-1 xenograft tumors. A pharmacokinetic study in mice showed a near doubling in the AUC of imexon when GEM was co-administered, with no effect of imexon on GEM's pharmacokinetic disposition. In vitro, imexon did not alter GEM's metabolism or uptake into DNA, but significantly inhibited RNR, and this effect was greater when combined with GEM. Conclusions These results suggest that the interaction between imexon and GEM may be due to complimentary inhibition of RNR plus an enhanced exposure to imexon when the GEM is administered in vivo. This combination is currently being tested in a randomized phase II trial in pancreatic cancer. PMID:20339847

  9. Regulation and degradation of HMGCo-A reductase.

    PubMed

    Panda, T; Devi, V Amutha

    2004-12-01

    The enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) controls the biosynthesis of cholesterol. Hypercholesterolemia and atherosclerosis are critical health risk factors. One way of controlling these risk factors is to manipulate regulation as well as degradation of HMGR. At present, a class of compounds called statins, which are HMGR inhibitors, are used for the treatment of hypercholesterolemia. However, statins suffer major setbacks as their use produces more adverse reactions than the desirable one of inhibiting the enzyme. Genetically engineered forms of HMGR are also studied in primitive life forms like bacteria, but detailed investigation of this enzyme in human systems is certainly required. Extensive studies have been made on the regulatory aspects of this enzyme, but no breakthrough is conspicuous in the clinical background to find an alternative treatment for hypercholesterolemia. The immediate need is to find an alternate way of regulating degradation of the enzyme. This review presents the importance of regulation and degradation of the HMGR enzyme in different systems to gain possible insight into alternative schemes for regulating this enzyme and, if these exist, the feasibility of extending them same to studies in mammalian systems. A high degree of similarity exists between mammalian and yeast HMGR. Detailed studies reported on the regulation and degradation of the yeast enzyme also throw more light on the mammalian system, leading to a better understanding of ways of controlling hypercholesterolemia. PMID:15558272

  10. Mutation Update and Review of Severe Methylenetetrahydrofolate Reductase Deficiency.

    PubMed

    Froese, D Sean; Huemer, Martina; Suormala, Terttu; Burda, Patricie; Coelho, David; Guéant, Jean-Louis; Landolt, Markus A; Kožich, Viktor; Fowler, Brian; Baumgartner, Matthias R

    2016-05-01

    Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is caused by mutations in the MTHFR gene and results in hyperhomocysteinemia and varying severity of disease, ranging from neonatal lethal to adult onset. Including those described here, 109 MTHFR mutations have been reported in 171 families, consisting of 70 missense mutations, 17 that primarily affect splicing, 11 nonsense mutations, seven small deletions, two no-stop mutations, one small duplication, and one large duplication. Only 36% of mutations recur in unrelated families, indicating that most are "private." The most common mutation is c.1530A>G (numbered from NM_005957.4, p.Lys510 = ) causing a splicing defect, found in 13 families; the most common missense mutation is c.1129C>T (p.Arg377Cys) identified in 10 families. To increase disease understanding, we report enzymatic activity, detected mutations, and clinical onset information (early, <1 year; or late, >1 year) for all published patients available, demonstrating that patients with early onset have less residual enzyme activity than those presenting later. We also review animal models, diagnostic approaches, clinical presentations, and treatment options. This is the first large review of mutations in MTHFR, highlighting the wide spectrum of disease-causing mutations. PMID:26872964