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Sample records for thioredoxin metabolism enhances

  1. Inhibition of Glutathione and Thioredoxin Metabolism Enhances Sensitivity to Perifosine in Head and Neck Cancer Cells

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    Andrean L. Simons

    2009-01-01

    Full Text Available The hypothesis that the Akt inhibitor, perifosine (PER, combined with inhibitors of glutathione (GSH and thioredoxin (Trx metabolism will induce cytotoxicity via metabolic oxidative stress in human head and neck cancer (HNSCC cells was tested. PER induced increases in glutathione disulfide (%GSSG in FaDu, Cal-27, and SCC-25 HNSCCs as well as causing significant clonogenic cell killing in FaDu and Cal-27, which was suppressed by simultaneous treatment with N-acetylcysteine (NAC. An inhibitor of GSH synthesis, buthionine sulfoximine (BSO, sensitized Cal-27 and SCC-25 cells to PER-induced clonogenic killing as well as decreased total GSH and increased %GSSG. Additionally, inhibition of thioredoxin reductase activity (TrxRed with auranofin (AUR was able to induce PER sensitization in SCC-25 cells that were initially refractory to PER. These results support the conclusion that PER induces oxidative stress and clonogenic killing in HNSCC cells that is enhanced with inhibitors of GSH and Trx metabolism.

  2. Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase

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    Jayakumar, Sundarraj; Patwardhan, R.S.; Pal, Debojyoti [Radiation Biology & Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Sharma, Deepak [Radiation Biology & Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Homi Bhabha National Institute, Anushaktinagar, Mumbai 400094 (India); Sandur, Santosh K., E-mail: sskumar@barc.gov.in [Radiation Biology & Health Sciences Division, Modular Laboratories, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Homi Bhabha National Institute, Anushaktinagar, Mumbai 400094 (India)

    2016-09-09

    Dimethoxycurcumin (DIMC), a structural analogue of curcumin, has been shown to have more stability, bioavailability, and effectiveness than its parent molecule curcumin. In this paper the radiosensitizing effect of DIMC has been investigated in A549 lung cancer cells. As compared to its parent molecule curcumin, DIMC showed a very potent radiosensitizing effect as seen by clonogenic survival assay. DIMC in combination with radiation significantly increased the apoptosis and mitotic death in A549 cells. This combinatorial treatment also lead to effective elimination of cancer stem cells. Further, there was a significant increase in cellular ROS, decrease in GSH to GSSG ratio and also significant slowdown in DNA repair when DIMC was combined with radiation. In silico docking studies and in vitro studies showed inhibition of thioredoxin reductase enzyme by DIMC. Overexpression of thioredoxin lead to the abrogation of radiosensitizing effect of DIMC underscoring the role of thioredoxin reductase in radiosensitization. Our results clearly demonstrate that DIMC can synergistically enhance the cancer cell killing when combined with radiation by targeting thioredoxin system. - Highlights: • DIMC enhances radiosensitivity of cancer cells by inducing cell death. • DIMC with radiation disrupted the cellular redox and targeted cancer stem cells. • DNA repair is hampered when cells are treated with DIMC. • DIMC inhibited thioredoxin reductase in cancer cells.

  3. Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase

    International Nuclear Information System (INIS)

    Jayakumar, Sundarraj; Patwardhan, R.S.; Pal, Debojyoti; Sharma, Deepak; Sandur, Santosh K.

    2016-01-01

    Dimethoxycurcumin (DIMC), a structural analogue of curcumin, has been shown to have more stability, bioavailability, and effectiveness than its parent molecule curcumin. In this paper the radiosensitizing effect of DIMC has been investigated in A549 lung cancer cells. As compared to its parent molecule curcumin, DIMC showed a very potent radiosensitizing effect as seen by clonogenic survival assay. DIMC in combination with radiation significantly increased the apoptosis and mitotic death in A549 cells. This combinatorial treatment also lead to effective elimination of cancer stem cells. Further, there was a significant increase in cellular ROS, decrease in GSH to GSSG ratio and also significant slowdown in DNA repair when DIMC was combined with radiation. In silico docking studies and in vitro studies showed inhibition of thioredoxin reductase enzyme by DIMC. Overexpression of thioredoxin lead to the abrogation of radiosensitizing effect of DIMC underscoring the role of thioredoxin reductase in radiosensitization. Our results clearly demonstrate that DIMC can synergistically enhance the cancer cell killing when combined with radiation by targeting thioredoxin system. - Highlights: • DIMC enhances radiosensitivity of cancer cells by inducing cell death. • DIMC with radiation disrupted the cellular redox and targeted cancer stem cells. • DNA repair is hampered when cells are treated with DIMC. • DIMC inhibited thioredoxin reductase in cancer cells.

  4. Susceptibility of human head and neck cancer cells to combined inhibition of glutathione and thioredoxin metabolism.

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    Arya Sobhakumari

    Full Text Available Increased glutathione (GSH and thioredoxin (Trx metabolism are mechanisms that are widely implicated in resistance of cancer cells to chemotherapy. The current study determined if simultaneous inhibition of GSH and Trx metabolism enhanced cell killing of human head and neck squamous cell carcinoma (HNSCC cells by a mechanism involving oxidative stress. Inhibition of GSH and Trx metabolism with buthionine sulfoximine (BSO and auranofin (AUR, respectively, induced significant decreases in clonogenic survival compared to either drug alone in FaDu, Cal-27 and SCC-25 HNSCC cells in vitro and in vivo in Cal-27 xenografts. BSO+AUR significantly increased glutathione and thioredoxin oxidation and suppressed peroxiredoxin activity in vitro. Pre-treatment with N-acetylcysteine completely reversed BSO+AUR-induced cell killing in FaDu and Cal-27 cells, while catalase and selenium supplementation only inhibited BSO+AUR-induced cell killing in FaDu cells. BSO+AUR decreased caspase 3/7 activity in HNSCC cells and significantly reduced the viability of both Bax/Bak double knockout (DKO and DKO-Bax reconstituted hematopoietic cells suggesting that necrosis was involved. BSO+AUR also significantly sensitized FaDu, Cal-27, SCC-25 and SQ20B cells to cell killing induced by the EGFR inhibitor Erlotinib in vitro. These results support the conclusion that simultaneous inhibition of GSH and Trx metabolism pathways induces oxidative stress and clonogenic killing in HNSCCs and this strategy may be useful in sensitizing HNSCCs to EGFR inhibitors.

  5. JS-K, a Nitric Oxide Prodrug, Has Enhanced Cytotoxicity in Colon Cancer Cells with Knockdown of Thioredoxin Reductase 1

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    Edes, Kornelia; Cassidy, Pamela; Shami, Paul J.; Moos, Philip J.

    2010-01-01

    Background The selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted. Methodology The role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured. Conclusions/Significance In cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner. PMID:20098717

  6. The role of thioredoxin h in protein metabolism during wheat (Triticum aestivum L.) seed germination.

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    Guo, Hongxiang; Wang, Shaoxin; Xu, Fangfang; Li, Yongchun; Ren, Jiangping; Wang, Xiang; Niu, Hongbin; Yin, Jun

    2013-06-01

    Thioredoxin h can regulate the redox environment in the cell and play an important role in the germination of cereals. In the present study, the thioredoxin s antisense transgenic wheat with down-regulation of thioredoxin h was used to study the role of thioredoxin h in protein metabolism during germination of wheat seeds, and to explore the mechanism of the thioredoxin s antisense transgenic wheat seeds having high resistance to pre-harvest sprouting. The qRT-PCR results showed that the expression of protein disulfide isomerase in the thioredoxin s antisense transgenic wheat was up-regulated, which induced easily forming glutenin macropolymers and the resistance of storage proteins to degradation. The expression of serine protease inhibitor was also up-regulated in transgenic wheat, which might be responsible for the decreased activity of thiocalsin during the germination. The expression of WRKY6 in transgenic wheat was down-regulated, which was consistent with the decreased activity of glutamine oxoglutarate aminotransferase. In transgenic wheat, the activities of glutamate dehydrogenase, glutamic pyruvic transaminase and glutamic oxaloacetic transaminase were down-regulated, indicating that the metabolism of amino acid was lower than that in wild-type wheat during seed germination. A putative model for the role of thioredoxin h in protein metabolism during wheat seed germination was proposed and discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  7. Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

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    Davidson, John F.; Fox, Richard; Harris, Dawn D.; Lyons-Abbott, Sally; Loeb, Lawrence A.

    2003-01-01

    Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20–50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq...

  8. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

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    Bedarida, Tatiana; Domingues, Alison; Baron, Stephanie; Ferreira, Chrystophe; Vibert, Francoise; Cottart, Charles-Henry; Paul, Jean-Louis; Escriou, Virginie; Bigey, Pascal; Gaussem, Pascale; Leguillier, Teddy; Nivet-Antoine, Valerie

    2018-06-01

    Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biologic functions, the contribution of endothelial TXNIP has not been well defined. To investigate the endothelial function of TXNIP, we generated a TXNIP knockout mouse on the Cdh5-cre background (TXNIP fl/fl cdh5 cre ). Control (TXNIP fl/fl ) and TXNIP fl/fl cdh5 cre mice were fed a high protein-low carbohydrate (HP-LC) diet for 3 mo to induce metabolic stress. We found that TXNIP fl/fl and TXNIP fl/fl cdh5 cre mice on an HP-LC diet displayed impaired glucose tolerance and dyslipidemia concretizing the metabolic stress induced. We evaluated the impact of this metabolic stress on mice with reduced endothelial TXNIP expression with regard to arterial structure and function. TXNIP fl/fl cdh5 cre mice on an HP-LC diet exhibited less endothelial dysfunction than littermate mice on an HP-LC diet. These mice were protected from decreased aortic medial cell content, impaired aortic distensibility, and increased plasminogen activator inhibitor 1 secretion. This protective effect came with lower oxidative stress and lower inflammation, with a reduced NLRP3 inflammasome expression, leading to a decrease in cleaved IL-1β. We also show the major role of TXNIP in inflammation with a knockdown model, using a TXNIP-specific, small interfering RNA included in a lipoplex. These findings demonstrate a key role for endothelial TXNIP in arterial impairments induced by metabolic stress, making endothelial TXNIP a potential therapeutic target.-Bedarida, T., Domingues, A., Baron, S., Ferreira, C., Vibert, F., Cottart, C.-H., Paul, J.-L., Escriou, V., Bigey, P., Gaussem, P., Leguillier, T., Nivet-Antoine, V. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

  9. The role of thioredoxin reductase 1 in melanoma metabolism and metastasis.

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    Cassidy, Pamela B; Honeggar, Matthew; Poerschke, Robyn L; White, Karen; Florell, Scott R; Andtbacka, Robert H I; Tross, Joycelyn; Anderson, Madeleine; Leachman, Sancy A; Moos, Philip J

    2015-11-01

    Although significant progress has been made in targeted and immunologic therapeutics for melanoma, many tumors fail to respond, and most eventually progress when treated with the most efficacious targeted combination therapies thus far identified. Therefore, alternative approaches that exploit distinct melanoma phenotypes are necessary to develop new approaches for therapeutic intervention. Tissue microarrays containing human nevi and melanomas were used to evaluate levels of the antioxidant protein thioredoxin reductase 1 (TR1), which was found to increase as a function of disease progression. Melanoma cell lines revealed metabolic differences that correlated with TR1 levels. We used this new insight to design a model treatment strategy that creates a synthetic lethal interaction wherein targeting TR1 sensitizes melanoma to inhibition of glycolytic metabolism, resulting in a decrease in metastases in vivo. This approach holds the promise of a new clinical therapeutic strategy, distinct from oncoprotein inhibition. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Methylseleninic acid (MSA) inhibits 17β-estradiol-induced cell growth in breast cancer T47D cells via enhancement of the antioxidative thioredoxin/ thioredoxin reductase system.

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    Okuno, Tomofumi; Miura, Kiyoshi; Sakazaki, Fumitoshi; Nakamuro, Katsuhiko; Ueno, Hitoshi

    2012-01-01

    The purpose of this study was to clarify the cell growth inhibitory mechanism of human breast cancer cells caused by selenium (Se) compounds. In the presence of 17β-estradiol (E(2)) at physiological concentrations, growth of estrogen receptor α (ERα)-positive T47D cells was markedly inhibited by 1 × 10(-6) mol/L methylseleninic acid (MSA) with no Se related toxicity.Under conditions where cell growth was inhibited, MSA decreased ERα mRNA levels and subsequent protein levels; further decreasing expression of estrogen-responsive finger protein (Efp) which is a target gene product of ERα and promotes G2/M progression of the cell cycle. Therefore, the decline in Efp expression is presumed to be involved in G2 arrest. Coincidentally, the antioxidative thioredoxin/ thioredoxin reductase (Trx/TrxR) system in cells was enhanced by the synergistic action of E(2) and MSA. It has been reported that ROS-induced oxidative stress enhanced ERα expression. E(2) increased production of intracellular ROS in T47D cells. Meanwhile, MSA significantly decreased E(2)-induced ROS accumulation. From these results, activation of the Trx/TrxR system induced by the coexistence of MSA and E(2) suppresses oxidative stress and decreases expression of ERα, and finally induces the growth arrest of T47D cells through disruption of ERα signaling.

  11. Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

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    Poerschke, Robyn L.; Moos, Philip J.

    2010-01-01

    Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction. PMID:20920480

  12. The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation.

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    Muri, Jonathan; Heer, Sebastian; Matsushita, Mai; Pohlmeier, Lea; Tortola, Luigi; Fuhrer, Tobias; Conrad, Marcus; Zamboni, Nicola; Kisielow, Jan; Kopf, Manfred

    2018-05-10

    The thioredoxin-1 (Trx1) system is an important contributor to cellular redox balance and is a sensor of energy and glucose metabolism. Here we show critical c-Myc-dependent activation of the Trx1 system during thymocyte and peripheral T-cell proliferation, but repression during T-cell quiescence. Deletion of thioredoxin reductase-1 (Txnrd1) prevents expansion the CD4 - CD8 - thymocyte population, whereas Txnrd1 deletion in CD4 + CD8 + thymocytes does not affect further maturation and peripheral homeostasis of αβT cells. However, Txnrd1 is critical for expansion of the activated T-cell population during viral and parasite infection. Metabolomics show that TrxR1 is essential for the last step of nucleotide biosynthesis by donating reducing equivalents to ribonucleotide reductase. Impaired availability of 2'-deoxyribonucleotides induces the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal function of the Trx1 system in metabolic reprogramming of thymic and peripheral T cells and provide a rationale for targeting Txnrd1 in T-cell leukemia.

  13. Overexpression of chloroplast NADPH-dependent thioredoxin reductase in Arabidopsis enhances leaf growth and elucidates in-vivo function of reductase and thioredoxin domains

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    Jouni eToivola

    2013-10-01

    Full Text Available Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC contains both reductase (NTRd and thioredoxin (TRXd domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive thioredoxin domain, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modelling of the 3-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protected pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for

  14. Combined inhibition of glycolysis, the pentose cycle, and thioredoxin metabolism selectively increases cytotoxicity and oxidative stress in human breast and prostate cancer

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    Ling Li

    2015-04-01

    Full Text Available Inhibition of glycolysis using 2-deoxy-d-glucose (2DG, 20 mM, 24–48 h combined with inhibition of the pentose cycle using dehydroepiandrosterone (DHEA, 300 µM, 24–48 h increased clonogenic cell killing in both human prostate (PC-3 and DU145 and human breast (MDA-MB231 cancer cells via a mechanism involving thiol-mediated oxidative stress. Surprisingly, when 2DG+DHEA treatment was combined with an inhibitor of glutathione (GSH synthesis (l-buthionine sulfoximine; BSO, 1 mM that depleted GSH>90% of control, no further increase in cell killing was observed during 48 h exposures. In contrast, when an inhibitor of thioredoxin reductase (TrxR activity (Auranofin; Au, 1 µM, was combined with 2DG+DHEA or DHEA-alone for 24 h, clonogenic cell killing was significantly increased in all three human cancer cell lines. Furthermore, enhanced clonogenic cell killing seen with the combination of DHEA+Au was nearly completely inhibited using the thiol antioxidant, N-acetylcysteine (NAC, 20 mM. Redox Western blot analysis of PC-3 cells also supported the conclusion that thioredoxin-1 (Trx-1 oxidation was enhanced by treatment DHEA+Au and inhibited by NAC. Importantly, normal human mammary epithelial cells (HMEC were not as sensitive to 2DG, DHEA, and Au combinations as their cancer cell counterparts (MDA-MB-231. Overall, these results support the hypothesis that inhibition of glycolysis and pentose cycle activity, combined with inhibition of Trx metabolism, may provide a promising strategy for selectively sensitizing human cancer cells to oxidative stress-induced cell killing.

  15. Thioredoxin-albumin fusion protein prevents copper enhanced zinc-induced neurotoxicity via its antioxidative activity.

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    Tanaka, Ken-Ichiro; Shimoda, Mikako; Chuang, Victor T G; Nishida, Kento; Kawahara, Masahiro; Ishida, Tatsuhiro; Otagiri, Masaki; Maruyama, Toru; Ishima, Yu

    2018-01-15

    Zinc (Zn) is a co-factor for a vast number of enzymes, and functions as a regulator for immune mechanism and protein synthesis. However, excessive Zn release induced in pathological situations such as stroke or transient global ischemia is toxic. Previously, we demonstrated that the interaction of Zn and copper (Cu) is involved in the pathogenesis of Alzheimer's disease and vascular dementia. Furthermore, oxidative stress has been shown to play a significant role in the pathogenesis of various metal ions induced neuronal death. Thioredoxin-Albumin fusion (HSA-Trx) is a derivative of thioredoxin (Trx), an antioxidative protein, with improved plasma retention and stability of Trx. In this study, we examined the effect of HSA-Trx on Cu 2+ /Zn 2+ -induced neurotoxicity. Firstly, HSA-Trx was found to clearly suppress Cu 2+ /Zn 2+ -induced neuronal cell death in mouse hypothalamic neuronal cells (GT1-7 cells). Moreover, HSA-Trx markedly suppressed Cu 2+ /Zn 2+ -induced ROS production and the expression of oxidative stress related genes, such as heme oxygenase-1. In contrast, HSA-Trx did not affect the intracellular levels of both Cu 2+ and Zn 2+ after Cu 2+ /Zn 2+ treatment. Finally, HSA-Trx was found to significantly suppress endoplasmic reticulum (ER) stress response induced by Cu 2+ /Zn 2+ treatment in a dose dependent manner. These results suggest that HSA-Trx counteracted Cu 2+ /Zn 2+ -induced neurotoxicity by suppressing the production of ROS via interfering the related gene expressions, in addition to the highly possible radical scavenging activity of the fusion protein. Based on these findings, HSA-Trx has great potential as a promising therapeutic agent for the treatment of refractory neurological diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. The relationship of thioredoxin-1 and cisplatin resistance: its impact on ROS and oxidative metabolism in lung cancer cells.

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    Wangpaichitr, Medhi; Sullivan, Elizabeth J; Theodoropoulos, George; Wu, Chunjing; You, Min; Feun, Lynn G; Lampidis, Theodore J; Kuo, Macus T; Savaraj, Niramol

    2012-03-01

    Elimination of cisplatin-resistant lung cancer cells remains a major obstacle. We have shown that cisplatin-resistant tumors have higher reactive oxygen species (ROS) levels and can be exploited for targeted therapy. Here, we show that increased secretion of the antioxidant thioredoxin-1 (TRX1) resulted in lowered intracellular TRX1 and contributed to higher ROS in cisplatin-resistant tumors in vivo and in vitro. By reconstituting TRX1 protein in cisplatin-resistant cells, we increased sensitivity to cisplatin but decreased sensitivity to elesclomol (ROS inducer). Conversely, decreased TRX1 protein in parental cells reduced the sensitivity to cisplatin but increased sensitivity to elesclomol. Cisplatin-resistant cells had increased endogenous oxygen consumption and mitochondrial activity but decreased lactic acid production. They also exhibited higher levels of argininosuccinate synthetase (ASS) and fumarase mRNA, which contributed to oxidative metabolism (OXMET) when compared with parental cells. Restoring intracellular TRX1 protein in cisplatin-resistant cells resulted in lowering ASS and fumarase mRNAs, which in turn sensitized them to arginine deprivation. Interestingly, cisplatin-resistant cells also had significantly higher basal levels of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Overexpressing TRX1 lowered ACC and FAS proteins expressions in cisplatin-resistant cells. Chemical inhibition and short interfering RNA of ACC resulted in significant cell death in cisplatin-resistant compared with parental cells. Conversely, TRX1 overexpressed cisplatin-resistant cells resisted 5-(tetradecyloxy)-2-furoic acid (TOFA)-induced death. Collectively, lowering TRX1 expression through increased secretion leads cisplatin-resistant cells to higher ROS production and increased dependency on OXMET. These changes raise an intriguing therapeutic potential for future therapy in cisplatin-resistant lung cancer.

  17. Seed thioredoxin h

    DEFF Research Database (Denmark)

    Hägglund, Per; Finnie, Christine; Yano, Hiroyuki

    2016-01-01

    , for example chloroplastic f- and m-type thioredoxins involved in regulation of the Calvin-Benson cycle. The cytosolic h-type thioredoxins act as key regulators of seed germination and are recycled by NADPH-dependent thioredoxin reductase. The present review on thioredoxin h systems in plant seeds focuses...

  18. Expression, purification and molecular structure modeling of thioredoxin (Trx) and thioredoxin reductase (TrxR) from Acidithiobacillus ferrooxidans.

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    Wang, Yiping; Zhang, Xiaojian; Liu, Qing; Ai, Chenbing; Mo, Hongyu; Zeng, Jia

    2009-07-01

    The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.

  19. Thioredoxin-linked redox control of metabolism in Methanocaldococcus jannaschii, an evolutionarily deeply-rooted hyperthermophilic methanogenic archaeon

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    Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. We have investigated this aspect in methanarchaea. These ancient methanogens produce methane almost exclusively from H2 plus CO2 carried approxima...

  20. Deficiency of thioredoxin binding protein-2 (TBP-2 enhances TGF-β signaling and promotes epithelial to mesenchymal transition.

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    So Masaki

    Full Text Available Transforming growth factor beta (TGF-β has critical roles in regulating cell growth, differentiation, apoptosis, invasion and epithelial-mesenchymal transition (EMT of various cancer cells. TGF-β-induced EMT is an important step during carcinoma progression to invasion state. Thioredoxin binding protein-2 (TBP-2, also called Txnip or VDUP1 is downregulated in various types of human cancer, and its deficiency results in the earlier onset of cancer. However, it remains unclear how TBP-2 suppresses the invasion and metastasis of cancer.In this study, we demonstrated that TBP-2 deficiency increases the transcriptional activity in response to TGF-β and also enhances TGF-β-induced Smad2 phosphorylation levels. Knockdown of TBP-2 augmented the TGF-β-responsive expression of Snail and Slug, transcriptional factors related to TGF-β-mediated induction of EMT, and promoted TGF-β-induced spindle-like morphology consistent with the depletion of E-Cadherin in A549 cells.Our results indicate that TBP-2 deficiency enhances TGF-β signaling and promotes TGF-β-induced EMT. The control of TGF-β-induced EMT is critical for the inhibition of the invasion and metastasis. Thus TBP-2, as a novel regulatory molecule of TGF-β signaling, is likely to be a prognostic indicator or a potential therapeutic target for preventing tumor progression.

  1. Tricksy business : Transcriptome analysis reveals the involvement of thioredoxin a in redox homeostasis, oxidative stress, sulfur metabolism, and cellular differentiation in Bacillus subtilis

    NARCIS (Netherlands)

    Smits, Wiep; Dubois, Jean-Yves; Bron, S; van Dijl, J.M; Kuipers, O.P.

    Thioredoxins are important thiol-reactive proteins. Most knowledge about this class of proteins is derived from proteome studies, and little is known about the global transcriptional response of cells to various thioredoxin levels. In Bacillus subtilis, thioredoxin A is encoded by trxA and is

  2. Thioredoxin from Escherichia coli

    International Nuclear Information System (INIS)

    Holmgren, A.; Ohlsson, I.; Grankvist, M.L.

    1978-01-01

    A competition radioimmunoassay for Escherichia coli thioredoxin using 125 I-labeled thioredoxin-S 2 and a double antibody technique was developed. The method permits determination of picomole amounts of thioredoxin in crude cell extracts and was used to study the localization of thioredoxin cell fractions. E. coli B was calculated to have approximately 10,000 copies of thioredoxin per cell mainly located in the soluble fraction after separation of the membrane and soluble fractions by gentle lysis and centrifugation. E. coli B tsnC mutants which are defective in the replication of phage T7 DNA in vivo and in vitro were examined for their content of thioredoxin. E. coli B tsnC 7004 contained no detectable level of thioredoxin in cell-free extracts examined under a variety of conditions. The results strongly suggest that tsnC 7004 is a nonsense or deletion mutant. Two other E. coli tsnC mutants, 7007 and 7008, contained detectable levels of thioredoxin in crude extracts as measured by thioredoxin reductase and gave similar immunoprecipitation reactions as the parent strain B/1. By radioimmunoassay incompletely cross-reacting material was present in both strains. These results show that tsnC 7007 and 7008 belong to a type of thioredoxin mutants with missence mutations in the thioredoxin gene affecting the function of thioredoxin as subunit in phage T7 DNA polymerase

  3. Thioredoxin and evolution

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    Buchanan, B. B.

    1991-01-01

    Comparisons of primary structure have revealed significant homology between the m type thioredoxins of chloroplasts and the thioredoxins from a variety of bacteria. Chloroplast thioredoxin f, by comparison, remains an enigma: certain residues are invariant with those of the other thioredoxins, but a phylogenetic relationship to bacterial or m thioredoxins seems distant. Knowledge of the evolutionary history of thioredoxin f is, nevertheless, of interest because of its role in photosynthesis. Therefore, we have attempted to gain information on the evolutionary history of chloroplast thioredoxin f, as well as m. Our goal was first to establish the utility of thioredoxin as a phylogenetic marker, and, if found suitable, to deduce the evolutionary histories of the chloroplast thioredoxins. To this end, we have constructed phylogenetic (minimal replacement) trees using computer analysis. The results show that the thioredoxins of bacteria and animals fall into distinct phylogenetic groups - the bacterial group resembling that derived from earlier 16s RNA analysis and the animal group showing a cluster consistent with known relationships. The chloroplast thioredoxins show a novel type of phylogenetic arrangement: one m type aligns with its counterpart of eukaryotic algae, cyanobacteria and other bacteria, whereas the second type (f type) tracks with animal thioredoxin. The results give new insight into the evolution of photosynthesis.

  4. Truncated thioredoxin (Trx-80) promotes pro-inflammatory macrophages of the M1 phenotype and enhances atherosclerosis.

    Science.gov (United States)

    Mahmood, Dler Faieeq Darweesh; Abderrazak, Amna; Couchie, Dominique; Lunov, Oleg; Diderot, Vimala; Syrovets, Tatiana; Slimane, Mohamed-Naceur; Gosselet, Fabien; Simmet, Thomas; Rouis, Mustapha; El Hadri, Khadija

    2013-07-01

    Vascular cells are particularly susceptible to oxidative stress that is believed to play a key role in the pathogenesis of cardiovascular disorders. Thioredoxin-1 (Trx-1) is an oxidative stress-limiting protein with anti-inflammatory and anti-apoptotic properties. In contrast, its truncated form (Trx-80) exerts pro-inflammatory effects. Here we analyzed whether Trx-80 might exert atherogenic effects by promoting macrophage differentiation into the M1 pro-inflammatory phenotype. Trx-80 at 1 µg/ml significantly attenuated the polarization of anti-inflammatory M2 macrophages induced by exposure to either IL-4 at 15 ng/ml or IL-4/IL-13 (10 ng/ml each) in vitro, as evidenced by the expression of the characteristic markers, CD206 and IL-10. By contrast, in LPS-challenged macrophages, Trx-80 significantly potentiated the differentiation into inflammatory M1 macrophages as indicated by the expression of the M1 cytokines, TNF-α and MCP-1. When Trx-80 was administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/g body weight (b.w.) challenged either with LPS at 30 µg/30 g (b.w.) or IL-4 at 500 ng/30 g (b.w.), it significantly induced the M1 phenotype but inhibited differentiation of M2 macrophages in thymus and liver. When ApoE2.Ki mice were challenged once weekly with LPS for 5 weeks, they showed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. Such effect was potentiated when mice received daily, in addition to LPS, the Trx-80. Moreover, the Trx-80 treatment led to a significantly increased aortic lesion area. The ability of Trx-80 to promote differentiation of macrophages into the classical proinflammatory phenotype may explain its atherogenic effects in cardiovascular diseases. Copyright © 2013 Wiley Periodicals, Inc.

  5. PaTrx1 and PaTrx3, Two Cytosolic Thioredoxins of the Filamentous Ascomycete Podospora anserina Involved in Sexual Development and Cell Degeneration▿ †

    Science.gov (United States)

    Malagnac, Fabienne; Klapholz, Benjamin; Silar, Philippe

    2007-01-01

    In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2, and PaTrx3 shows that PaTrx1 is the major thioredoxin involved in sulfur metabolism. Deletions have no effect on peroxide resistance; however, data show that either PaTrx1 or PaTrx3 is necessary for sexual reproduction and for the development of the crippled growth cell degeneration (CG), processes that also required the PaMpk1 mitogen-activated protein kinase (MAPK) pathway. Since PaTrx1 PaTrx3 mutants show not an enhancement but rather an impairment in CG, it seems unlikely that PaTrx1 and PaTrx3 thioredoxins participate in the inhibition of this MAPK pathway. Altogether, these results underscore a role for thioredoxins in fungal development. PMID:17933907

  6. PaTrx1 and PaTrx3, two cytosolic thioredoxins of the filamentous ascomycete Podospora anserina involved in sexual development and cell degeneration.

    Science.gov (United States)

    Malagnac, Fabienne; Klapholz, Benjamin; Silar, Philippe

    2007-12-01

    In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2, and PaTrx3 shows that PaTrx1 is the major thioredoxin involved in sulfur metabolism. Deletions have no effect on peroxide resistance; however, data show that either PaTrx1 or PaTrx3 is necessary for sexual reproduction and for the development of the crippled growth cell degeneration (CG), processes that also required the PaMpk1 mitogen-activated protein kinase (MAPK) pathway. Since PaTrx1 PaTrx3 mutants show not an enhancement but rather an impairment in CG, it seems unlikely that PaTrx1 and PaTrx3 thioredoxins participate in the inhibition of this MAPK pathway. Altogether, these results underscore a role for thioredoxins in fungal development.

  7. Enhancement of thioredoxin/glutaredoxin-mediated L-cysteine synthesis from S-sulfocysteine increases L-cysteine production in Escherichia coli

    Science.gov (United States)

    2012-01-01

    Background Escherichia coli has two L-cysteine biosynthetic pathways; one is synthesized from O-acetyl L-serine (OAS) and sulfate by L-cysteine synthase (CysK), and another is produced via S-sulfocysteine (SSC) from OAS and thiosulfate by SSC synthase (CysM). SSC is converted into L-cysteine and sulfite by an uncharacterized reaction. As thioredoxins (Trx1 and Trx2) and glutaredoxins (Grx1, Grx2, Grx3, Grx4, and NrdH) are known as reductases of peptidyl disulfides, overexpression of such reductases might be a good way for improving L-cysteine production to accelerate the reduction of SSC in E. coli. Results Because the redox enzymes can reduce the disulfide that forms on proteins, we first tested whether these enzymes catalyze the reduction of SSC to L-cysteine. All His-tagged recombinant enzymes, except for Grx4, efficiently convert SSC into L-cysteine in vitro. Overexpression of Grx1 and NrdH enhanced a 15-40% increase in the E. coliL-cysteine production. On the other hand, disruption of the cysM gene cancelled the effect caused by the overexpression of Grx1 and NrdH, suggesting that its improvement was due to the efficient reduction of SSC under the fermentative conditions. Moreover, L-cysteine production in knockout mutants of the sulfite reductase genes (ΔcysI and ΔcysJ) and the L-cysteine synthase gene (ΔcysK) each decreased to about 50% of that in the wild-type strain. Interestingly, there was no significant difference in L-cysteine production between wild-type strain and gene deletion mutant of the upstream pathway of sulfite (ΔcysC or ΔcysH). These results indicate that sulfite generated from the SSC reduction is available as the sulfur source to produce additional L-cysteine molecule. It was finally found that in the E. coliL-cysteine producer that co-overexpress glutaredoxin (NrdH), sulfite reductase (CysI), and L-cysteine synthase (CysK), there was the highest amount of L-cysteine produced per cell. Conclusions In this work, we showed that Grx1 and

  8. Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees

    Directory of Open Access Journals (Sweden)

    Alberto eGonzález

    2014-10-01

    Full Text Available In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control, with OC kappa at 1 mg mL-1, or treated with inhibitors of NAD(PH, ascorbate (ASC and glutathione (GSH syntheses and thioredoxin reductase (TRR activity, CHS-828, lycorine, buthionine sulfoximine (BSO and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS, adenosine 5´-phosphosulfate reductase (APR, involved in C, N and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism and growth in Eucalyptus trees.

  9. Oligo-carrageenan kappa increases NADPH, ascorbate and glutathione syntheses and TRR/TRX activities enhancing photosynthesis, basal metabolism, and growth in Eucalyptus trees.

    Science.gov (United States)

    González, Alberto; Moenne, Fabiola; Gómez, Melissa; Sáez, Claudio A; Contreras, Rodrigo A; Moenne, Alejandra

    2014-01-01

    In order to analyze the effect of OC kappa in redox status, photosynthesis, basal metabolism and growth in Eucalyptus globulus, trees were treated with water (control), with OC kappa at 1 mg mL(-1), or treated with inhibitors of NAD(P)H, ascorbate (ASC), and glutathione (GSH) syntheses and thioredoxin reductase (TRR) activity, CHS-828, lycorine, buthionine sulfoximine (BSO), and auranofin, respectively, and with OC kappa, and cultivated for 4 months. Treatment with OC kappa induced an increase in NADPH, ASC, and GSH syntheses, TRR and thioredoxin (TRX) activities, photosynthesis, growth and activities of basal metabolism enzymes such as rubisco, glutamine synthetase (GlnS), adenosine 5'-phosphosulfate reductase (APR), involved in C, N, and S assimilation, respectively, Krebs cycle and purine/pyrimidine synthesis enzymes. Treatment with inhibitors and OC kappa showed that increases in ASC, GSH, and TRR/TRX enhanced NADPH synthesis, increases in NADPH and TRR/TRX enhanced ASC and GSH syntheses, and only the increase in NADPH enhanced TRR/TRX activities. In addition, the increase in NADPH, ASC, GSH, and TRR/TRX enhanced photosynthesis and growth. Moreover, the increase in NADPH, ASC and TRR/TRX enhanced activities of rubisco, Krebs cycle, and purine/pyrimidine synthesis enzymes, the increase in GSH, NADPH, and TRR/TRX enhanced APR activity, and the increase in NADPH and TRR/TRX enhanced GlnS activity. Thus, OC kappa increases NADPH, ASC, and GSH syntheses leading to a more reducing redox status, the increase in NADPH, ASC, GSH syntheses, and TRR/TRX activities are cross-talking events leading to activation of photosynthesis, basal metabolism, and growth in Eucalyptus trees.

  10. Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass

    Directory of Open Access Journals (Sweden)

    Ros Joaquim

    2010-02-01

    Full Text Available Abstract Background Wine Saccharomyces cerevisiae strains, adapted to anaerobic must fermentations, suffer oxidative stress when they are grown under aerobic conditions for biomass propagation in the industrial process of active dry yeast production. Oxidative metabolism of sugars favors high biomass yields but also causes increased oxidation damage of cell components. The overexpression of the TRX2 gene, coding for a thioredoxin, enhances oxidative stress resistance in a wine yeast strain model. The thioredoxin and also the glutathione/glutaredoxin system constitute the most important defense against oxidation. Trx2p is also involved in the regulation of Yap1p-driven transcriptional response against some reactive oxygen species. Results Laboratory scale simulations of the industrial active dry biomass production process demonstrate that TRX2 overexpression increases the wine yeast final biomass yield and also its fermentative capacity both after the batch and fed-batch phases. Microvinifications carried out with the modified strain show a fast start phenotype derived from its enhanced fermentative capacity and also increased content of beneficial aroma compounds. The modified strain displays an increased transcriptional response of Yap1p regulated genes and other oxidative stress related genes. Activities of antioxidant enzymes like Sod1p, Sod2p and catalase are also enhanced. Consequently, diminished oxidation of lipids and proteins is observed in the modified strain, which can explain the improved performance of the thioredoxin overexpressing strain. Conclusions We report several beneficial effects of overexpressing the thioredoxin gene TRX2 in a wine yeast strain. We show that this strain presents an enhanced redox defense. Increased yield of biomass production process in TRX2 overexpressing strain can be of special interest for several industrial applications.

  11. Gene Gun Bombardment with DNA-Coated Golden Particles Enhanced the Protective Effect of a DNA Vaccine Based on Thioredoxin Glutathione Reductase of Schistosoma japonicum

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    Yan Cao

    2013-01-01

    Full Text Available Schistosomiasis, caused by infection with Schistosoma species, remains an important parasitic zoonosis. Thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR plays an important role in the development of the parasite and for its survival. Here we present a recombinant plasmid DNA vaccine, pVAX1/SjTGR, to estimate its protection against S. japonicum in BALB/c mice. The DNA vaccine administrated by particle bombardment induced higher protection than by intramuscular injection. All animals vaccinated with pVAX1/SjTGR developed significant specific anti-SjTGR antibodies than control groups. Moreover, animals immunized by gene gun exhibited a splenocyte proliferative response, with an increase in IFN-γ and IL-4. The recombinant plasmid administrated by gene gun achieved a medium protective efficacy of 27.83–38.83% ( of worm reduction and 40.38–44.51% ( of liver egg count reduction. It suggests that different modes of administering a DNA vaccine can influence the protective efficacy induced by the vaccine. Interestingly, from the enzymatic activity results, we found that worms obtained from pVAX1/SjTGR-vaccinated animals expressed lower enzymatic activity than the control group and the antibodies weakened the enzymatic activity of SjTGR in vitro, too. It implies that the high-level antibodies may contribute to the protective effects.

  12. New insights into the posttranslational regulation of human cytosolic thioredoxin by S-palmitoylation

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Zhiyu; Zhong, Liangwei, E-mail: liazho@ucas.ac.cn

    2015-05-15

    High level of palmitate is associated with metabolic disorders. We recently showed that enhanced level of S-palmitoylated cytosolic thioredoxin (Trx1) in mouse liver was new characteristic feature of insulin resistance. However, our understanding of the effect of S-palmitoylation on Trx1 is limited, and the tissue specificity of Trx1 S-palmitoylation is unclear. Here we show that S-palmitoylation also occurs at Cys73 of Trx1 in living endothelial cells, and the level of S-palmitoylated Trx1 undergoes regulation by insulin signaling. Trx1 prefers thiol-thioester exchange with palmitoyl-CoA to acetyl-CoA. S-palmitoylation alters conformation or secondary structure of Trx1, as well as decreases the ability of Trx1 to transfer electrons from thioredoxin reductase to S-nitrosylated protein–tyrosine phosphatase 1B and S-nitroso-glutathione. Our results demonstrate that S-palmitoylation is an important post-translational modification of human Trx1. - Highlights: • S-palmitoylation occurs at Cys73 of Trx1 in living endothelial cells. • Insulin signaling may regulate level of S-palmitoylated Trx1 in the cells. • S-palmitoylation plays significant effects on Trx1 structure and functions.

  13. Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii

    Science.gov (United States)

    Huppe, H. C.; Picaud, A.; Buchanan, B. B.; Miginiac-Maslow, M.

    1991-01-01

    The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii. The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system. Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins. In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P. Decottignies et al., 1991, Eur. J. Biochem. 198, 505-512). The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast. The NTR was purified from Chlamydomonas using thioredoxin h from the same source. Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx. 36 kDa and was specific for NADPH. Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin. Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin. Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f.

  14. Epigallocatechin-3-gallate enhances key enzymatic activities of hepatic thioredoxin and glutathione systems in selenium-optimal mice but activates hepatic Nrf2 responses in selenium-deficient mice

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    Ruixia Dong

    2016-12-01

    Full Text Available Selenium participates in the antioxidant defense mainly through a class of selenoproteins, including thioredoxin reductase. Epigallocatechin-3-gallate (EGCG is the most abundant and biologically active catechin in green tea. Depending upon the dose and biological systems, EGCG may function either as an antioxidant or as an inducer of antioxidant defense via its pro-oxidant action or other unidentified mechanisms. By manipulating the selenium status, the present study investigated the interactions of EGCG with antioxidant defense systems including the thioredoxin system comprising of thioredoxin and thioredoxin reductase, the glutathione system comprising of glutathione and glutathione reductase coupled with glutaredoxin, and the Nrf2 system. In selenium-optimal mice, EGCG increased hepatic activities of thioredoxin reductase, glutathione reductase and glutaredoxin. These effects of EGCG appeared to be not due to overt pro-oxidant action because melatonin, a powerful antioxidant, did not influence the increase. However, in selenium-deficient mice, with low basal levels of thioredoxin reductase 1, the same dose of EGCG did not elevate the above-mentioned enzymes; intriguingly EGCG in turn activated hepatic Nrf2 response, leading to increased heme oxygenase 1 and NAD(PH:quinone oxidoreductase 1 protein levels and thioredoxin activity. Overall, the present work reveals that EGCG is a robust inducer of the Nrf2 system only in selenium-deficient conditions. Under normal physiological conditions, in selenium-optimal mice, thioredoxin and glutathione systems serve as the first line defense systems against the stress induced by high doses of EGCG, sparing the activation of the Nrf2 system.

  15. High-fat diet-induced changes in liver thioredoxin and thioredoxin reductase as a novel feature of insulin resistance

    Directory of Open Access Journals (Sweden)

    Huijun Qin

    2014-01-01

    Full Text Available High-fat diet (HFD can induce oxidative stress. Thioredoxin (Trx and thioredoxin reductase (TrxR are critical antioxidant proteins but how they are affected by HFD remains unclear. Using HFD-induced insulin-resistant mouse model, we show here that liver Trx and TrxR are significantly decreased, but, remarkably, the degree of their S-acylation is increased after consuming HFD. These HFD-induced changes in Trx/TrxR may reflect abnormalities of lipid metabolism and insulin signaling transduction. HFD-driven accumulation of 4-hydroxynonenal is another potential mechanism behind inactivation and decreased expression of Trx/TrxR. Thus, we propose HFD-induced impairment of liver Trx/TrxR as major contributor to oxidative stress and as a novel feature of insulin resistance.

  16. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

    Science.gov (United States)

    Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

    2002-09-01

    Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

  17. Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis.

    Science.gov (United States)

    Da, Qingen; Wang, Peng; Wang, Menglong; Sun, Ting; Jin, Honglei; Liu, Bing; Wang, Jinfa; Grimm, Bernhard; Wang, Hong-Bin

    2017-10-01

    In chloroplasts, thioredoxin (TRX) isoforms and NADPH-dependent thioredoxin reductase C (NTRC) act as redox regulatory factors involved in multiple plastid biogenesis and metabolic processes. To date, less is known about the functional coordination between TRXs and NTRC in chlorophyll biosynthesis. In this study, we aimed to explore the potential functions of TRX m and NTRC in the regulation of the tetrapyrrole biosynthesis (TBS) pathway. Silencing of three genes, TRX m1 , TRX m2 , and TRX m4 ( TRX ms ), led to pale-green leaves, a significantly reduced 5-aminolevulinic acid (ALA)-synthesizing capacity, and reduced accumulation of chlorophyll and its metabolic intermediates in Arabidopsis ( Arabidopsis thaliana ). The contents of ALA dehydratase, protoporphyrinogen IX oxidase, the I subunit of Mg-chelatase, Mg-protoporphyrin IX methyltransferase (CHLM), and NADPH-protochlorophyllide oxidoreductase were decreased in triple TRX m- silenced seedlings compared with the wild type, although the transcript levels of the corresponding genes were not altered significantly. Protein-protein interaction analyses revealed a physical interaction between the TRX m isoforms and CHLM. 4-Acetoamido-4-maleimidylstilbene-2,2-disulfonate labeling showed the regulatory impact of TRX ms on the CHLM redox status. Since CHLM also is regulated by NTRC (Richter et al., 2013), we assessed the concurrent functions of TRX m and NTRC in the control of CHLM. Combined deficiencies of three TRX m isoforms and NTRC led to a cumulative decrease in leaf pigmentation, TBS intermediate contents, ALA synthesis rate, and CHLM activity. We discuss the coordinated roles of TRX m and NTRC in the redox control of CHLM stability with its corollary activity in the TBS pathway. © 2017 American Society of Plant Biologists. All Rights Reserved.

  18. Identification of thioredoxin-interacting protein (TXNIP) as a downstream target for IGF1 action.

    Science.gov (United States)

    Nagaraj, Karthik; Lapkina-Gendler, Lena; Sarfstein, Rive; Gurwitz, David; Pasmanik-Chor, Metsada; Laron, Zvi; Yakar, Shoshana; Werner, Haim

    2018-01-30

    Laron syndrome (LS), or primary growth hormone (GH) insensitivity, is the best-characterized entity among the congenital insulin-like growth factor 1 (IGF1) deficiencies. Life-long exposure to minute endogenous IGF1 levels is linked to low stature as well as a number of endocrine and metabolic abnormalities. While elevated IGF1 is correlated with increased cancer incidence, epidemiological studies revealed that patients with LS do not develop tumors. The mechanisms associated with cancer protection in LS are yet to be discovered. Recent genomic analyses identified a series of metabolic genes that are overrepresented in patients with LS. Given the augmented expression of these genes in a low IGF1 milieu, we hypothesized that they may constitute targets for IGF1 action. Thioredoxin-interacting protein (TXNIP) plays a critical role in cellular redox control by thioredoxin. TXNIP serves as a glucose and oxidative stress sensor, being commonly silenced by genetic or epigenetic events in cancer cells. Consistent with its enhanced expression in LS, we provide evidence that TXNIP gene expression is negatively regulated by IGF1. These results were corroborated in animal studies. In addition, we show that oxidative and glucose stresses led to marked increases in TXNIP expression. Supplementation of IGF1 attenuated TXNIP levels, suggesting that IGF1 exerts its antiapoptotic effect via inhibition of TXNIP Augmented TXNIP expression in LS may account for cancer protection in this condition. Finally, TXNIP levels could be potentially useful in the clinic as a predictive or diagnostic biomarker for IGF1R-targeted therapies.

  19. Mycobacterium tuberculosis Phosphoenolpyruvate Carboxykinase Is Regulated by Redox Mechanisms and Interaction with Thioredoxin

    Czech Academy of Sciences Publication Activity Database

    Machová, Iva; Snášel, Jan; Zimmermann, M.; Laubitz, D.; Plocinski, P.; Oehlmann, W.; Singh, M.; Dostál, Jiří; Sauer, U.; Pichová, Iva

    2014-01-01

    Roč. 289, č. 19 (2014), s. 13066-13078 ISSN 0021-9258 EU Projects: European Commission(XE) 241587 - SYSTEMTB Grant - others:OPPK(CZ) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 Keywords : enzyme kinetics * hypoxia * metabolism * Mycobacterium tuberculosis * oxidation-reduction * thioredoxin * Phosphoenolpyruvate carboxykinase Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  20. Enhanced muscle glucose metabolism after exercise

    DEFF Research Database (Denmark)

    Richter, Erik; Garetto, L P; Goodman, M N

    1984-01-01

    Studies in the rat suggest that after voluntary exercise there are two phases of glycogen repletion in skeletal muscle (preceding study). In phase I glucose utilization and glycogen synthesis are enhanced both in the presence and absence of insulin, whereas in phase II only the increase in the pr......Studies in the rat suggest that after voluntary exercise there are two phases of glycogen repletion in skeletal muscle (preceding study). In phase I glucose utilization and glycogen synthesis are enhanced both in the presence and absence of insulin, whereas in phase II only the increase...... in the stimulated leg closely mimicked that observed previously after voluntary exercise on a treadmill. With no insulin added to the perfusate, glucose incorporation into glycogen was markedly enhanced in muscles that were glycogen depleted as were the uptake of 2-deoxyglucose and 3-O-methylglucose. Likewise......, the stimulation of these processes by insulin was enhanced and continued to be so 2 h later when the muscles of the stimulated leg had substantially repleted their glycogen stores. The results suggest that the increases in insulin-mediated glucose utilization and glycogen synthesis in muscle after exercise...

  1. Light Sensitivity of Lactococcus lactis Thioredoxin Reductase

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas

    The thioredoxin system has evolved in all kingdoms of life acting as a key antioxidant system in the defense against oxidative stress. The thioredoxin system utilizes reducing equivalents from NADPH to reduce protein disulfide targets. The reducing equivalents are shuttled via a flavin and redox...... active dithiol motif in thioredoxin reductase (TrxR) to reduce the small ubiquitous thioredoxin (Trx). Trx in turn regulates the protein dithiol/disulfide balance by reduction of protein disulfide targets in e.g. ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductase. The glutathione......, thus expected to rely mainly on the Trx system for thiol-disulfide control. L. lactis is an important industrial microorganism used as starter culture in the dairy production of cheese, buttermilk etc. and known to be sensitive to oxidative stress. The L. lactis TrxR (LlTrxR) is a homodimeric...

  2. The Lactococcus lactis Thioredoxin System

    DEFF Research Database (Denmark)

    Efler, Petr

    -dependent thioredoxin reductase (NTR) in order to complete its catalytic cycle. Glutathione-dependent glutaredoxin complements Trx in many organisms. This thesis focuses on disulfide reduction pathways in Lactococcus lactis, an important industrial microorganism used traditionally for cheese and buttermilk production...... caused about 30% growth inhibition at non-stressed conditions and significantly increased sensitivity to oxidants (e.g. H2O2, diamide), while deletion of trxD displayed an effect predominantly in the ΔtrxAΔtrxD mutant. The ΔtrxD mutant exhibited a significantly higher sensitivity only in case of exposure......D mutants by difference gel electrophoresis (DIGE) revealed significant changes between ΔtrxA and wt. Higher levels of several oxidative stress-related proteins (e.g. glutathione peroxidase) were observed in the ΔtrxA mutant. Proteomic analysis (pulse labeling by [35S]-L-methionine) of the ΔtrxD mutant vs...

  3. In silico analysis of Eucalyptus thioredoxins

    Directory of Open Access Journals (Sweden)

    Aulus Estevão Barbosa

    2005-01-01

    Full Text Available The Eucalyptus Genome Sequencing Project (FORESTs, an initiative from the Brazilian ONSA consortium (Organization for Nucleotide Sequencing and Analysis, has achieved the sequencing of 123.889 EST clones from 18 different cDNA libraries. We have investigated the FORESTs data set to identify EST clusters potentially encoding thioredoxins (TRX. Two types of thioredoxin families described in plants, chloroplastic (TRXm/f/x/y and cytosolic (TRXh, have been found in the transcriptome. Putative typical TRXs have been identified in fifteen clusters, four m-type, seven h-type, two f-type, one cluster for each x/y-types and one putative homologue of the TDX gene from Arabidopsis thaliana. One cluster presents an atypical active site WCMPS, different from the conserved WCGPC present in the other 15 clusters, and corresponds to a subgroup of cytosolic thioredoxins. Except in specific libraries from callus, roots, seedlings and wood tissues, thioredoxin deduced ESTs are found in all remaining libraries. According to the calculated frequencies of ESTs, chloroplastic thioredoxins are preferentially present in green tissues such as leaves whilst cytoplasmic thioredoxins are more general but demonstrate elevated frequencies in seedlings and flower tissues. TRX frequency patterns in the Eucalyptus transcriptome seem to indicate a good coherence with data from Arabidopsis thaliana gene expression.

  4. Molecular characterization of the thioredoxin system from Methanosarcina acetivorans

    OpenAIRE

    McCarver, Addison C.; Lessner, Daniel J.

    2014-01-01

    The thioredoxin system, composed of thioredoxin reductase (TrxR) and thioredoxin (Trx), is widely distributed in nature, where it serves key roles in electron transfer and in defense against oxidative stress. Although recent evidence reveals Trx homologues are almost universally present among the methane-producing archaea (methanogens), a complete thioredoxin system has not been characterized from any methanogen. We examined the phylogeny of Trx homologues among methanogens and characterized ...

  5. The Barley Grain Thioredoxin System – an Update

    Directory of Open Access Journals (Sweden)

    Per eHägglund

    2013-05-01

    Full Text Available Thioredoxin reduces disulfide bonds and play numerous important functions in plants. In cereal seeds, cytosolic h-type thioredoxin facilitates the release of energy reserves during the germination process and is recycled by NADPH-dependent thioredoxin reductase. This review presents a summary of the research conducted during the last ten years to elucidate the structure and function of the barley seed thioredoxin system at the molecular level combined with proteomic approaches to identify target proteins.

  6. New insights into the reduction systems of plastidial thioredoxins point out the unique properties of thioredoxin z from Arabidopsis.

    Science.gov (United States)

    Bohrer, Anne-Sophie; Massot, Vincent; Innocenti, Gilles; Reichheld, Jean-Philippe; Issakidis-Bourguet, Emmanuelle; Vanacker, Hélène

    2012-11-01

    In plants, thioredoxins (TRX) constitute a large protein disulphide oxidoreductase family comprising 10 plastidial members in Arabidopsis thaliana and subdivided in five types. The f- and m-types regulate enzymes involved mainly in carbon metabolism whereas the x, y, and z types have an antioxidant function. The reduction of TRXm and f in chloroplasts is performed in the light by ferredoxin:thioredoxin reductase (FTR) that uses photosynthetically reduced ferredoxin (Fd) as a reductant. The reduction system of Arabidopsis TRXx, y, and z has never been demonstrated. Recently, a gene encoding an atypical plastidial NADPH-dependent TRX reductase (NTRC) was found. In the present study, gene expression analysis revealed that both reductases are expressed in all organs of Arabidopsis and could potentially serve as electron donors to plastidial TRX. This ability was tested in vitro either with purified NTRC in presence of NADPH or with a light-driven reconstituted system comprising thylakoids and purified Fd and FTR. The results demonstrate that FTR reduces the x and y TRX isoforms but not the recently identified TRXz. Moreover, the results show that NTRC cannot be an efficient alternative reducing system, neither for TRXz nor for the other plastidial TRX. The data reveal that TRXf, m, x, and y, known as redox regulators in the chloroplast, have also the ability to reduce TRXz in vitro. Overall, the present study points out the unique properties of TRXz among plastidial TRX.

  7. PGK1 Drives Hepatocellular Carcinoma Metastasis by Enhancing Metabolic Process.

    Science.gov (United States)

    Xie, Huijun; Tong, Guihui; Zhang, Yupei; Liang, Shu; Tang, Kairui; Yang, Qinhe

    2017-07-27

    During the proliferation and metastasis, the tumor cells prefer glycolysis (Warburg effect), but its exact mechanism remains largely unknown. In this study, we demonstrated that phosphoglycerate kinase 1 (PGK1) is an important enzyme in the pathway of metabolic glycolysis. We observed a significant overexpression of PGK1 in hepatocellular carcinoma tissues, and a correlation between PGK1 expression and poor survival of hepatocellular carcinoma patients. Also, the depletion of PGK1 dramatically reduced cancer cell proliferation and metastasis, indicating an oncogenic role of PGK1 in liver cancer progression. Further experiments showed that PGK1 played an important role in MYC -induced metabolic reprogramming, which led to an enhanced Warburg effect. Our results revealed a new effect of PGK1, which can provide a new treatment strategy for hepatocellular carcinoma, as PGK1 is used to indicate the prognosis of hepatocellular carcinoma (HCC).

  8. Thioredoxin and Cancer: A Role for Thioredoxin in all States of Tumor Oxygenation

    International Nuclear Information System (INIS)

    Karlenius, Therese Christina; Tonissen, Kathryn Fay

    2010-01-01

    Thioredoxin is a small redox-regulating protein, which plays crucial roles in maintaining cellular redox homeostasis and cell survival and is highly expressed in many cancers. The tumor environment is usually under either oxidative or hypoxic stress and both stresses are known up-regulators of thioredoxin expression. These environments exist in tumors because their abnormal vascular networks result in an unstable oxygen delivery. Therefore, the oxygenation patterns in human tumors are complex, leading to hypoxia/re-oxygenation cycling. During carcinogenesis, tumor cells often become more resistant to hypoxia or oxidative stress-induced cell death and most studies on tumor oxygenation have focused on these two tumor environments. However, recent investigations suggest that the hypoxic cycling occurring within tumors plays a larger role in the contribution to tumor cell survival than either oxidative stress or hypoxia alone. Thioredoxin is known to have important roles in both these cellular responses and several studies implicate thioredoxin as a contributor to cancer progression. However, only a few studies exist that investigate the regulation of thioredoxin in the hypoxic and cycling hypoxic response in cancers. This review focuses on the role of thioredoxin in the various states of tumor oxygenation

  9. Thioredoxin system in obligate anaerobe Desulfovibrio desulfuricans: Identification and characterization of a novel thioredoxin 2.

    Science.gov (United States)

    Sarin, Ritu; Sharma, Yagya D

    2006-07-05

    Metal corroding sulfate reducing bacteria have been poorly characterized at molecular level due to difficulties pertaining to isolation and handling of anaerobes. We report here for the first time the presence and characterization of thioredoxin 2 in an obligate anaerobic dissimilatory sulfate reducing bacterium Desulfovibrio desulfuricans. In silico analysis of the D. desulfuricans genome revealed the presence of thioredoxin 1 (dstrx1), thioredoxin 2 (dstrx2) and thioredoxin reductase (dstrxR) genes. These genes were found to be actively expressed by the bacteria under the anaerobic growth conditions. We have overexpressed the anaerobic thioredoxin genes in E. coli to produce functionally active recombinant proteins. Recombinant DsTrxR recognized both DsTrx1 and DsTrx2 as its substrate. Mutation studies revealed that the activity of DsTrx2 can be completely abolished with a single amino acid mutation (C69A) in the signature motif 'WCGPC'. Furthermore, the N-terminal domain of DsTrx2 containing two extra CXXC motifs was found to have a negative regulation on its biochemical activity. In conclusion, we have shown the presence of thioredoxin 2 for the first time in an obligate anaerobe which in this anaerobe may be required for its survival under either oxidative stress conditions or metal ion hemostasis.

  10. Maneb and Paraquat-Mediated Neurotoxicity: Involvement of Peroxiredoxin/Thioredoxin System

    Science.gov (United States)

    Roede, James R.; Hansen, Jason M.; Go, Young-Mi; Jones, Dean P.

    2011-01-01

    Epidemiological and in vivo studies have demonstrated that exposure to the pesticides paraquat (PQ) and maneb (MB) increase the risk of developing Parkinson’s disease (PD) and cause dopaminergic cell loss, respectively. PQ is a well-recognized cause of oxidative toxicity; therefore, the purpose of this study was to determine if MB potentiates oxidative stress caused by PQ, thus providing a mechanism for enhanced neurotoxicity by the combination. The results show that PQ alone at a moderately toxic dose (20–30% cell death in 24 h) caused increased reactive oxygen species (ROS) generation, oxidation of mitochondrial thioredoxin-2 and peroxiredoxin-3, lesser oxidation of cytoplasmic thioredoxin-1 and peroxiredoxin-1, and no oxidation of cellular GSH/GSSG. In contrast, MB alone at a similar toxic dose resulted in no ROS generation, no oxidation of thioredoxin and peroxiredoxin, and an increase in cellular GSH after 24 h. Together, MB increased GSH and inhibited ROS production and thioredoxin/peroxiredoxin oxidation observed with PQ alone, yet resulted in more extensive (> 50%) cell death. MB treatment resulted in increased abundance of nuclear Nrf2 and mRNA for phase II enzymes under the control of Nrf2, indicating activation of cell protective responses. The results show that MB potentiation of PQ neurotoxicity does not occur by enhancing oxidative stress and suggests that increased toxicity occurs by a combination of divergent mechanisms, perhaps involving alkylation by MB and oxidation by PQ. PMID:21402726

  11. The barley grain thioredoxin system - an update

    DEFF Research Database (Denmark)

    Hägglund, Per; Björnberg, Olof; Navrot, Nicolas

    2013-01-01

    Thioredoxin (Trx) reduces disulfide bonds and play numerous important functions in plants. In cereal seeds, cytosolic h-type Trx facilitates the release of energy reserves during the germination process and is recycled by NADPH-dependent Trx reductase. This review presents a summary of the research...

  12. Enhancing microbial production of biofuels by expanding microbial metabolic pathways.

    Science.gov (United States)

    Yu, Ping; Chen, Xingge; Li, Peng

    2017-09-01

    Fatty acid, isoprenoid, and alcohol pathways have been successfully engineered to produce biofuels. By introducing three genes, atfA, adhE, and pdc, into Escherichia coli to expand fatty acid pathway, up to 1.28 g/L of fatty acid ethyl esters can be achieved. The isoprenoid pathway can be expanded to produce bisabolene with a high titer of 900 mg/L in Saccharomyces cerevisiae. Short- and long-chain alcohols can also be effectively biosynthesized by extending the carbon chain of ketoacids with an engineered "+1" alcohol pathway. Thus, it can be concluded that expanding microbial metabolic pathways has enormous potential for enhancing microbial production of biofuels for future industrial applications. However, some major challenges for microbial production of biofuels should be overcome to compete with traditional fossil fuels: lowering production costs, reducing the time required to construct genetic elements and to increase their predictability and reliability, and creating reusable parts with useful and predictable behavior. To address these challenges, several aspects should be further considered in future: mining and transformation of genetic elements related to metabolic pathways, assembling biofuel elements and coordinating their functions, enhancing the tolerance of host cells to biofuels, and creating modular subpathways that can be easily interconnected. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  13. Characterization of mitochondrial thioredoxin reductase from C. elegans

    International Nuclear Information System (INIS)

    Lacey, Brian M.; Hondal, Robert J.

    2006-01-01

    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 cat of 610 min -1 and a K m of 610 μM using E. coli thioredoxin as substrate. The reported k cat is 25% of the k 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

  14. Thioredoxin and Thioredoxin Target Proteins: From Molecular Mechanisms to Functional Significance

    Science.gov (United States)

    Lee, Samuel; Kim, Soo Min

    2013-01-01

    Abstract The thioredoxin (Trx) system is one of the central antioxidant systems in mammalian cells, maintaining a reducing environment by catalyzing electron flux from nicotinamide adenine dinucleotide phosphate through Trx reductase to Trx, which reduces its target proteins using highly conserved thiol groups. While the importance of protecting cells from the detrimental effects of reactive oxygen species is clear, decades of research in this field revealed that there is a network of redox-sensitive proteins forming redox-dependent signaling pathways that are crucial for fundamental cellular processes, including metabolism, proliferation, differentiation, migration, and apoptosis. Trx participates in signaling pathways interacting with different proteins to control their dynamic regulation of structure and function. In this review, we focus on Trx target proteins that are involved in redox-dependent signaling pathways. Specifically, Trx-dependent reductive enzymes that participate in classical redox reactions and redox-sensitive signaling molecules are discussed in greater detail. The latter are extensively discussed, as ongoing research unveils more and more details about the complex signaling networks of Trx-sensitive signaling molecules such as apoptosis signal-regulating kinase 1, Trx interacting protein, and phosphatase and tensin homolog, thus highlighting the potential direct and indirect impact of their redox-dependent interaction with Trx. Overall, the findings that are described here illustrate the importance and complexity of Trx-dependent, redox-sensitive signaling in the cell. Our increasing understanding of the components and mechanisms of these signaling pathways could lead to the identification of new potential targets for the treatment of diseases, including cancer and diabetes. Antioxid. Redox Signal. 18, 1165–1207. PMID:22607099

  15. The thioredoxin system in breast cancer cell invasion and migration

    Directory of Open Access Journals (Sweden)

    Maneet Bhatia

    2016-08-01

    Full Text Available Metastasis is the most life threatening aspect of breast cancer. It is a multi-step process involving invasion and migration of primary tumor cells with a subsequent colonization of these cells at a secondary location. The aim of the present study was to investigate the role of thioredoxin (Trx1 in the invasion and migration of breast cancer cells and to assess the strength of the association between high levels of Trx1 and thioredoxin reductase (TrxR1 expression with breast cancer patient survival. Our results indicate that the expression of both Trx1 and TrxR1 are statistically significantly increased in breast cancer patient cells compared with paired normal breast tissue from the same patient. Over-expression of Trx1 in MDA-MB-231 breast cancer cell lines enhanced cell invasion in in vitro assays while expression of a redox inactive mutant form of Trx1 (designated 1SS or the antisense mRNA inhibited cell invasion. Addition of exogenous Trx1 also enhanced cell invasion, while addition of a specific monoclonal antibody that inhibits Trx1 redox function decreased cell invasion. Over-expression of intracellular Trx1 did not increase cell migration but expression of intracellular 1SS inhibited migration. Addition of exogenous Trx1 enhanced cell migration while 1SS had no effect. Treatment with auranofin inhibited TrxR activity, cell migration and clonogenic activity of MDA-MB-231 cells, while increasing reactive oxygen species (ROS levels. Analysis of 25 independent cohorts with 5910 patients showed that Trx1 and TrxR1 were both associated with a poor patient prognosis in terms of overall survival, distant metastasis free survival and disease free survival. Therefore, targeting the Trx system with auranofin or other specific inhibitors may provide improved breast cancer patient outcomes through inhibition of cancer invasion and migration.

  16. Targeting the Thioredoxin System for Cancer Therapy.

    Science.gov (United States)

    Zhang, Junmin; Li, Xinming; Han, Xiao; Liu, Ruijuan; Fang, Jianguo

    2017-09-01

    Thioredoxin (Trx) and thioredoxin reductase (TrxR) are essential components of the Trx system which plays pivotal roles in regulating multiple cellular redox signaling pathways. In recent years TrxR/Trx have been increasingly recognized as an important modulator of tumor development, and hence targeting TrxR/Trx is a promising strategy for cancer treatment. In this review we first discuss the structural details of TrxR, the functions of the Trx system, and the rational of targeting TrxR/Trx for cancer treatment. We also highlight small-molecule TrxR/Trx inhibitors that have potential anticancer activity and review their mechanisms of action. Finally, we examine the challenges of developing TrxR/Trx inhibitors as anticancer agents and perspectives for selectively targeting TrxR/Trx. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Enhanced muscle glucose metabolism after exercise in the rat

    DEFF Research Database (Denmark)

    Garetto, L P; Richter, Erik; Goodman, M N

    1984-01-01

    glycogen was substantially repleted at the time (30 min postexercise) that glucose metabolism was examined. When rats were run at twice the previous rate (36 m/min), muscle glycogen was still substantially diminished 30 min after the run. At this time the previously noted increase in insulin sensitivity......Thirty minutes after a treadmill run, glucose utilization and glycogen synthesis in perfused rat skeletal muscle are enhanced due to an increase in insulin sensitivity (Richter et al., J. Clin. Invest. 69: 785-793, 1982). The exercise used in these studies was of moderate intensity, and muscle...... was still observed in perfused muscle; however, glucose utilization was also increased in the absence of added insulin (1.5 vs. 4.2 mumol X g-1 X h-1). In contrast 2.5 h after the run, muscle glycogen had returned to near preexercise values, and only the insulin-induced increase in glucose utilization...

  18. Electrochemical determination of thioredoxin redox states

    Czech Academy of Sciences Publication Activity Database

    Dorčák, Vlastimil; Paleček, Emil

    2009-01-01

    Roč. 81, č. 4 (2009), s. 1543-1548 ISSN 0003-2700 R&D Projects: GA AV ČR(CZ) KAN400310651; GA ČR(CZ) GA301/07/0490; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : thioredoxin redox states * constant current chronopotentiometric stripping * carbon and mercury electrodes Subject RIV: BO - Biophysics Impact factor: 5.214, year: 2009

  19. Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

    OpenAIRE

    Obiero, Josiah; Sanders, David AR

    2011-01-01

    In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR–Trx interface suggested...

  20. Crystallization and diffraction analysis of thioredoxin reductase from Streptomyces coelicolor

    International Nuclear Information System (INIS)

    Koháryová, Michaela; Brynda, Jiří; Řezáčová, Pavlína; Kollárová, Marta

    2011-01-01

    Thioredoxin reductase from S. coelicolor was crystallized and diffraction data were collected to 2.4 Å resolution. Thioredoxin reductases are homodimeric flavoenzymes that catalyze the transfer of electrons from NADPH to oxidized thioredoxin substrate. Bacterial thioredoxin reductases represent a promising target for the development of new antibiotics. Recombinant thioredoxin reductase TrxB from Streptomyces coelicolor was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from cryocooled crystals to 2.4 Å resolution using a synchrotron-radiation source. The crystals belonged to the primitive monoclinic space group P2 1 , with unit-cell parameters a = 82.9, b = 60.6, c = 135.4 Å, α = γ = 90.0, β = 96.5°

  1. Comparable Enhanced Prothrombogenesis in Simple Central Obesity and Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Noor Shafina Mohd Nor

    2018-01-01

    Full Text Available Objective. There is limited data comparing prothrombogenic or fibrinolysis biomarkers (tissue plasminogen activator (tPA and plasminogen activator inhibitor-1 (PAI-1 simultaneously in subjects with Metabolic Syndrome (MS, simple central obesity without MS (COB and normal controls (NC. We investigated the concentrations of fibrinolysis biomarkers in subjects with MS, COB and NC. Methods. A cross-sectional study involving 503 drug naive subjects (163 males, aged 30–65 years old (mean age ± SD = 47.4 ± 8.3 years divided into MS, COB and NC groups. COB was defined as central obesity (waist circumference (WC males ≥90 cm, females ≥80 cm in the absence of MS according to the International Diabetes Federation 2006. Fasting blood levels of tPA and PAI-1were analyzed. Results. MS and COB had significantly higher concentration of all biomarkers compared to NC. The MS group had significantly higher concentration of tPA and PAI-1 compared to COB. WC and HDL-c had significant correlation with all biomarkers (tPA p<0.001, PAI-1 p<0.001. Fasting plasma glucose and diastolic blood pressure were independent predictors after correcting for confounding factors. Conclusion. Central obesity with or without MS both demonstrated enhanced prothrombogenesis. This suggests that simple obesity possibly increases the risk of coronary artery disease in part, via increased susceptibility to thrombogenesis.

  2. Expression of the thioredoxin-thioredoxin reductase system in the inflamed joints of patients with rheumatoid arthritis

    NARCIS (Netherlands)

    Maurice, M. M.; Nakamura, H.; Gringhuis, S.; Okamoto, T.; Yoshida, S.; Kullmann, F.; Lechner, S.; van der Voort, E. A.; Leow, A.; Versendaal, J.; Muller-Ladner, U.; Yodoi, J.; Tak, P. P.; Breedveld, F. C.; Verweij, C. L.

    1999-01-01

    OBJECTIVE: To examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases. METHODS: Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay.

  3. Thioredoxin Cross-Linking by Nitrogen Mustard in Lung Epithelial Cells: Formation of Multimeric Thioredoxin/Thioredoxin Reductase Complexes and Inhibition of Disulfide Reduction

    OpenAIRE

    Jan, Yi-Hua; Heck, Diane E.; Casillas, Robert P.; Laskin, Debra L.; Laskin, Jeffrey D.

    2015-01-01

    The thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (TrxR), is a major cellular disulfide reduction system important in antioxidant defense. TrxR is a target of mechlorethamine (methylbis(2-chloroethyl)amine; HN2), a bifunctional alkylating agent that covalently binds to selenocysteine/cysteine residues in the redox centers of the enzyme, leading to inactivation and toxicity. Mammalian Trx contains two catalytic cysteines; herein, we determined if HN2 also targets Tr...

  4. Nitrate-containing beetroot enhances myocyte metabolism and mitochondrial content

    Science.gov (United States)

    Vaughan, Roger A.; Gannon, Nicholas P.; Carriker, Colin R.

    2015-01-01

    Beetroot (甜菜 tián cài) juice consumption is of current interest for improving aerobic performance by acting as a vasodilator and possibly through alterations in skeletal muscle metabolism and physiology. This work explored the effects of a commercially available beetroot supplement on metabolism, gene expression, and mitochondrial content in cultured myocytes. C2C12 myocytes were treated with various concentrations of the beetroot supplement for various durations. Glycolytic metabolism and oxidative metabolism were quantified via measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured using quantitative reverse transcription–polymerase chain reaction, and mitochondrial content was assessed with flow cytometry and confocal microscopy. Cells treated with beetroot exhibited significantly increased oxidative metabolism, concurrently with elevated metabolic gene expression including peroxisome proliferator-activated receptor gamma coactivator-1 alpha, nuclear respiratory factor 1, mitochondrial transcription factor A, and glucose transporter 4, leading to increased mitochondrial biogenesis. Our data show that treatment with a beetroot supplement increases basal oxidative metabolism. Our observations are also among the first to demonstrate that beetroot extract is an inducer of metabolic gene expression and mitochondrial biogenesis. These observations support the need for further investigation into the therapeutic and pharmacological effects of nitrate-containing supplements for health and athletic benefits. PMID:26870674

  5. Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

    Science.gov (United States)

    Metcalfe, Clive; Ramasubramoni, Anjana; Pula, Giordano; Harper, Matthew T; Mundell, Stuart J; Coxon, Carmen H

    2016-01-01

    Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

  6. Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

    Directory of Open Access Journals (Sweden)

    Clive Metcalfe

    Full Text Available Thioredoxin (Trx is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12 to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase. In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb. This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.

  7. From Proteomics to Structural Studies of Cytosolic/Mitochondrial-Type Thioredoxin Systems in Barley Seeds

    DEFF Research Database (Denmark)

    Shahpiri, Azar; Svensson, Birte; Finnie, Christine

    2009-01-01

    Thioredoxins (Trx) are ubiquitous proteins that participate in thiol disulfide reactions via two active site cysteine residues, allowing Trx to reduce disulfide bonds in target proteins. Recent progress in proteome analysis has resulted in identification of a wide range of potential target proteins...... for Trx, indicating that Trx plays a key role in several aspects of cell metabolism. In contrast to other organisms, plants contain multiple forms of Trx that are classified based on their primary structures and sub-cellular localization. The reduction of cytosolic and mitochondrial types of Trx...

  8. Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering

    DEFF Research Database (Denmark)

    Asadollahi, Mohammadali; Maury, Jerome; Patil, Kiran Raosaheb

    2009-01-01

    A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene as the modeling framework...

  9. The effects of acrolein on peroxiredoxins, thioredoxins, and thioredoxin reductase in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Myers, Charles R.; Myers, Judith M.

    2009-01-01

    Inhalation is a common form of exposure to acrolein, a toxic reactive volatile aldehyde that is a ubiquitous environmental pollutant. Bronchial epithelial cells would be directly exposed to inhaled acrolein. The thioredoxin (Trx) system is essential for the maintenance of cellular thiol redox balance, and is critical for cell survival. Normally, thioredoxin reductase (TrxR) maintains the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins in the reduced state, and the thioredoxins keep the peroxiredoxins (Prx) reduced, thereby supporting their peroxidase function. The effects of acrolein on TrxR, Trx and Prx in human bronchial epithelial (BEAS-2B) cells were determined. A 30-min exposure to 5 μM acrolein oxidized both Trx1 and Trx2, although significant effects were noted for Trx1 at even lower acrolein concentrations. The effects on Trx1 and Trx2 could not be reversed by treatment with disulfide reductants. TrxR activity was inhibited 60% and >85% by 2.5 and 5 μM acrolein, respectively. The endogenous electron donor for TrxR, NADPH, could not restore its activity, and activity did not recover in cells during a 4-h acrolein-free period in complete medium. The effects of acrolein on TrxR and Trx therefore extend beyond the duration of exposure. While there was a strong correlation between TrxR inhibition and Trx1 oxidation, the irreversible effects on Trx1 suggest direct effects of acrolein rather than loss of reducing equivalents from TrxR. Trx2 did not become oxidized until ≥90% of TrxR was inhibited, but irreversible effects on Trx2 also suggest direct effects of acrolein. Prx1 (cytosolic) and Prx3 (mitochondrial) shifted to a largely oxidized state only when >90 and 100% of their respective Trxs were oxidized. Prx oxidation was readily reversed with a disulfide reductant, suggesting that Prx oxidation resulted from lack of reducing equivalents from Trx and not direct reaction with acrolein. The effects of acrolein on the thioredoxin system and

  10. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    International Nuclear Information System (INIS)

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

    1987-01-01

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [ 14 C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined

  11. Thioredoxin, thioredoxin reductase, and alpha-crystallin revive inactivated glyceraldehyde 3-phosphate dehydrogenase in human aged and cataract lens extracts.

    Science.gov (United States)

    Yan, Hong; Lou, Marjorie F; Fernando, M Rohan; Harding, John J

    2006-10-02

    To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without alpha-crystallin can revive inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both the cortex and nucleus of human aged clear and cataract lenses. The lens cortex (including capsule-epithelium) and the nucleus were separated from human aged clear and cataract lenses (grade II and grade IV) with similar average age. The activity of GAPDH in the water-soluble fraction after incubation with or without Trx or/and TrxR for 60 min at 30 degrees C was measured spectrophotometrically. In addition, the effect of a combination of Trx/TrxR and bovine lens alpha-crystallin was investigated. GAPDH activity was lower in the nucleus of clear lenses than in the cortex, and considerably diminished in the cataractous lenses, particularly in the nucleus of cataract lenses grade IV. Trx and TrxR were able to revive the activity of GAPDH markedly in both the cortex and nucleus of the clear and cataract lenses. The percentage increase of activity in the cortex of the clear lenses was less than that of the nucleus in the presence of Trx and TrxR, whereas it was opposite in the cataract lenses. The revival of activity in both the cortex and nucleus from the cataract lenses grade II was higher than that of the grade IV. Moreover, Trx alone, but not TrxR, efficiently enhanced GAPDH activity. The combination of Trx and TrxR had greater effect than that of either alone. In addition, alpha(L)-crystallin enhanced the activity in the cortex of cataract grade II with Trx and TrxR present. However, it failed to provide a statistically significant increase of activity in the nucleus. This is the first evidence to show that mammalian Trx and TrxR are able to revive inactivated GAPDH in human aged clear and cataract lenses, and alpha-crystallin helped this effect. The inactivation of GAPDH during aging and cataract development must be caused in part by disulphide formation and in part by

  12. Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

    Science.gov (United States)

    Martínez-González, J J; Guevara-Flores, A; Rendón, J L; Sosa-Peinado, A; Del Arenal Mena, I P

    2015-04-01

    Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1μM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  13. Deletion of thioredoxin reductase and effects of selenite and selenate toxicity in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Christopher J Boehler

    Full Text Available Thioredoxin reductase-1 (TRXR-1 is the sole selenoprotein in C. elegans, and selenite is a substrate for thioredoxin reductase, so TRXR-1 may play a role in metabolism of selenium (Se to toxic forms. To study the role of TRXR in Se toxicity, we cultured C. elegans with deletions of trxr-1, trxr-2, and both in axenic media with increasing concentrations of inorganic Se. Wild-type C. elegans cultured for 12 days in Se-deficient axenic media grow and reproduce equivalent to Se-supplemented media. Supplementation with 0-2 mM Se as selenite results in inverse, sigmoidal response curves with an LC50 of 0.20 mM Se, due to impaired growth rather than reproduction. Deletion of trxr-1, trxr-2 or both does not modulate growth or Se toxicity in C. elegans grown axenically, and (75Se labeling showed that TRXR-1 arises from the trxr-1 gene and not from bacterial genes. Se response curves for selenide (LC50 0.23 mM Se were identical to selenite, but selenate was 1/4(th as toxic (LC50 0.95 mM Se as selenite and not modulated by TRXR deletion. These nutritional and genetic studies in axenic media show that Se and TRXR are not essential for C. elegans, and that TRXR alone is not essential for metabolism of inorganic Se to toxic species.

  14. Role of metabolic stress for enhancing muscle adaptations: Practical applications

    OpenAIRE

    de Freitas, Marcelo Conrado; Gerosa-Neto, Jose; Zanchi, Nelo Eidy; Lira, Fabio Santos; Rossi, Fabr?cio Eduardo

    2017-01-01

    Metabolic stress is a physiological process that occurs during exercise in response to low energy that leads to metabolite accumulation [lactate, phosphate inorganic (Pi) and ions of hydrogen (H+)] in muscle cells. Traditional exercise protocol (i.e., Resistance training) has an important impact on the increase of metabolite accumulation, which influences hormonal release, hypoxia, reactive oxygen species (ROS) production and cell swelling. Changes in acute exercise routines, such as intensit...

  15. Thioredoxin 1 regulation of protein S-desulfhydration

    Directory of Open Access Journals (Sweden)

    Youngjun Ju

    2016-03-01

    Full Text Available The importance of H2S in biology and medicine has been widely recognized in recent years, and protein S-sulfhydration is proposed to mediate the direct actions of H2S bioactivity in the body. Thioredoxin 1 (Trx1 is an important reducing enzyme that cleaves disulfides in proteins and acts as an S-denitrosylase. The regulation of Trx1 on protein S-sulfhydration is unclear. Here we showed that Trx1 facilitates protein S-desulfhydration. Overexpression of Trx1 attenuated the basal level and H2S-induced protein S-sulfhydration by direct interaction with S-sulfhydrated proteins, i.e., glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase. In contrast, knockdown of Trx1 mRNA expression by short interfering RNA or blockage of Trx1 redox activity with PX12 or 2,4-dinitrochlorobenzene enhanced protein S-sulfhydration. Mutation of cysteine-32 but not cysteine-35 in the Trp–Cys32–Gly–Pro–Cys35 motif eliminated the binding of Trx1 with S-sulfhydrated proteins and abolished the S-desulfhydrating effect of Trx1. All these data suggest that Trx1 acts as an S-desulfhydrase.

  16. Enhanced Biological Phosphorus Removal : Metabolic Insights and Salinity Effects

    NARCIS (Netherlands)

    Welles, L.

    2015-01-01

    Enhanced biological phosphorus removal (EBPR) is a biological process for efficient phosphate removal from wastewaters through intracellular storage of polyphosphate by polyphosphate-accumulating organisms (PAO) and subsequent removal of PAO from the system through wastage of sludge. In comparison

  17. The effect of charge-introduction mutations on E. coli thioredoxin stability.

    Science.gov (United States)

    Perez-Jimenez, Raul; Godoy-Ruiz, Raquel; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2005-04-01

    Technological applications of proteins are often hampered by their low-stability and, consequently, the development of procedures for protein stabilization is of considerable biotechnological interest. Here, we use simple electrostatics to determine positions in E. coli thioredoxin at which mutations that introduce new charged residues are expected to lead to stability enhancement. We also obtain the corresponding mutants and characterize their stability using differential scanning calorimetry. The results are interpreted in terms of the accessibility in the native structure of the mutated residues and the potential effect of the mutations on the residual structure of the denatured state.

  18. How thioredoxin dissociates its mixed disulfide.

    Directory of Open Access Journals (Sweden)

    Goedele Roos

    2009-08-01

    Full Text Available The dissociation mechanism of the thioredoxin (Trx mixed disulfide complexes is unknown and has been debated for more than twenty years. Specifically, opposing arguments for the activation of the nucleophilic cysteine as a thiolate during the dissociation of the complex have been put forward. As a key model, the complex between Trx and its endogenous substrate, arsenate reductase (ArsC, was used. In this structure, a Cys29(Trx-Cys89(ArsC intermediate disulfide is formed by the nucleophilic attack of Cys29(Trx on the exposed Cys82(ArsC-Cys89(ArsC in oxidized ArsC. With theoretical reactivity analysis, molecular dynamics simulations, and biochemical complex formation experiments with Cys-mutants, Trx mixed disulfide dissociation was studied. We observed that the conformational changes around the intermediate disulfide bring Cys32(Trx in contact with Cys29(Trx. Cys32(Trx is activated for its nucleophilic attack by hydrogen bonds, and Cys32(Trx is found to be more reactive than Cys82(ArsC. Additionally, Cys32(Trx directs its nucleophilic attack on the more susceptible Cys29(Trx and not on Cys89(ArsC. This multidisciplinary approach provides fresh insights into a universal thiol/disulfide exchange reaction mechanism that results in reduced substrate and oxidized Trx.

  19. Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine

    International Nuclear Information System (INIS)

    Kim, Moon-Jung; Lee, Byung Cheon; Hwang, Kwang Yeon; Gladyshev, Vadim N.; Kim, Hwa-Young

    2015-01-01

    Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. 75 Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher K m than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec. - Highlights: • The first characterization of a selenoprotein Trx is presented. • The selenoenzyme Trx exhibits 10-fold higher catalytic activity than Cys homologues. • Se utilization in Trx is primarily due to the catalytic advantage provided by Sec residue

  20. Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Moon-Jung [Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717 (Korea, Republic of); Lee, Byung Cheon [Division of Genetics, Department of Medicine, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA 02115 (United States); Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Hwang, Kwang Yeon [Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Gladyshev, Vadim N. [Division of Genetics, Department of Medicine, Brigham and Women' s Hospital, Harvard Medical School, Boston, MA 02115 (United States); Kim, Hwa-Young, E-mail: hykim@ynu.ac.kr [Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717 (Korea, Republic of)

    2015-06-12

    Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. {sup 75}Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher K{sub m} than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec. - Highlights: • The first characterization of a selenoprotein Trx is presented. • The selenoenzyme Trx exhibits 10-fold higher catalytic activity than Cys homologues. • Se utilization in Trx is primarily due to the catalytic advantage provided by Sec residue.

  1. Functional characterization of thioredoxin 3 (TRX-3), a Caenorhabditis elegans intestine-specific thioredoxin.

    Science.gov (United States)

    Jiménez-Hidalgo, María; Kurz, Cyril Léopold; Pedrajas, José Rafael; Naranjo-Galindo, Francisco José; González-Barrios, María; Cabello, Juan; Sáez, Alberto G; Lozano, Encarnación; Button, Emma L; Veal, Elizabeth A; Fierro-González, Juan Carlos; Swoboda, Peter; Miranda-Vizuete, Antonio

    2014-03-01

    Thioredoxins are a class of evolutionarily conserved proteins that have been demonstrated to play a key role in many cellular processes involving redox reactions. We report here the genetic and biochemical characterization of Caenorhabditis elegans TRX-3, the first metazoan thioredoxin with an intestine-specific expression pattern. By using green fluorescent protein reporters we have found that TRX-3 is expressed in both the cytoplasm and the nucleus of intestinal cells, with a prominent localization at the apical membrane. Although intestinal function, reproductive capacity, longevity, and resistance of trx-3 loss-of-function mutants to many stresses are indistinguishable from those of wild-type animals, we have observed a slight reduction in size and a minor reduction in the defecation cycle timing of trx-3 mutants. Interestingly, trx-3 is induced upon infection by Photorhabdus luminescens and Candida albicans, and TRX-3 overexpression provides a modest protection against these pathogens. Together, our data indicate that TRX-3 function in the intestine is dispensable for C. elegans development but may be important to fight specific bacterial and fungal infections. © 2013 Elsevier Inc. All rights reserved.

  2. The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

    2017-01-01

    The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...

  3. Enhancing the Sweetness of Yoghurt through Metabolic Remodeling of Carbohydrate Metabolism in Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

    Science.gov (United States)

    Sørensen, Kim I; Curic-Bawden, Mirjana; Junge, Mette P; Janzen, Thomas; Johansen, Eric

    2016-06-15

    Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are used in the fermentation of milk to produce yoghurt. These species normally metabolize only the glucose moiety of lactose, secreting galactose and producing lactic acid as the main metabolic end product. We used multiple serial selection steps to isolate spontaneous mutants of industrial strains of S. thermophilus and L. delbrueckii subsp. bulgaricus that secreted glucose rather than galactose when utilizing lactose as a carbon source. Sequencing revealed that the S. thermophilus strains had mutations in the galKTEM promoter, the glucokinase gene, and genes encoding elements of the glucose/mannose phosphotransferase system (PTS). These strains metabolize galactose but are unable to phosphorylate glucose internally or via the PTS. The L. delbrueckii subsp. bulgaricus mutants had mutations in genes of the glucose/mannose PTS and in the pyruvate kinase gene. These strains cannot grow on exogenous glucose but are proficient at metabolizing internal glucose released from lactose by β-galactosidase. The resulting strains can be combined to ferment milk, producing yoghurt with no detectable lactose, moderate levels of galactose, and high levels of glucose. Since glucose tastes considerably sweeter than either lactose or galactose, the sweetness of the yoghurt is perceptibly enhanced. These strains were produced without the use of recombinant DNA technology and can be used for the industrial production of yoghurt with enhanced intrinsic sweetness and low residual levels of lactose. Based on a good understanding of the physiology of the lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, we were able, by selecting spontaneously occurring mutants, to change dramatically the metabolic products secreted into the growth medium. These mutants consume substantially more of the lactose, metabolize some of the galactose, and secrete the remaining galactose

  4. Enhanced Biological Phosphorus Removal: Metabolic Insights and Salinity Effects

    OpenAIRE

    Welles, L.

    2015-01-01

    Enhanced biological phosphorus removal (EBPR) is a biological process for efficient phosphate removal from wastewaters through intracellular storage of polyphosphate by polyphosphate-accumulating organisms (PAO) and subsequent removal of PAO from the system through wastage of sludge. In comparison to physical and chemical phosphorus removal processes, the biological process has several advantages such as high removal efficiency, low cost, and no chemical sludge production, but disturbances an...

  5. The Function of Thioredoxin-Binding Protein-2 (TBP-2 in Different Diseases

    Directory of Open Access Journals (Sweden)

    Jianghua Hu

    2018-01-01

    Full Text Available Thioredoxin-binding protein-2 (TBP-2 has an important role in the redox system, but it plays a different role in many different diseases (e.g., various cancers, diabetes mellitus (DM, cardiovascular disease, and cataracts by influencing cell proliferation, differentiation, apoptosis, autophagy, and metabolism. Distinct transcription factors (TFs stimulated by different factors combine with binding sites or proteins to upregulate or downregulate TBP-2 expression, in order to respond to the change in the internal environment. Most research disclosed that the main function of TBP-2 is associating with thioredoxin (Trx to inhibit the antioxidant capacity of Trx. Furthermore, the TBP-2 located in tissues, whether normal or abnormal, has the ability to cause the dysfunctioning of cells and even death through different pathways, such as shortening the cell cycle and inducing apoptosis or autophagy. Through these studies, we found that TBP-2 promoted the development of diseases which are involved in inflammatory and oxidative damage. To a certain extent, we believe that there is some hidden connection between the biological functions which TBP-2 participates in and some distinct diseases. This review presents only a summary of the roles that TBP-2 plays in cancer, DM, cataracts, and so on, as well as its universal mechanisms. Further investigations are needed for the cell signaling pathways of the effects caused by TBP-2. A greater understanding of the mechanisms of TBP-2 could produce potential new targets for the treatment of diseases, including cancer and diabetes, cardiovascular disease, and cataracts.

  6. Metabolome strategy against Edwardsiella tarda infection through glucose-enhanced metabolic modulation in tilapias.

    Science.gov (United States)

    Peng, Bo; Ma, Yan-Mei; Zhang, Jian-Ying; Li, Hui

    2015-08-01

    Edwardsiella tarda causes fish disease and great economic loss. However, metabolic strategy against the pathogen remains unexplored. In the present study, GC-MS based metabolomics was used to investigate the metabolic profile from tilapias infected by sublethal dose of E. tarda. The metabolic differences between the dying group and survival group allow the identification of key pathways and crucial metabolites during infections. More importantly, those metabolites may modulate the survival-related metabolome to enhance the anti-infective ability. Our data showed that tilapias generated two different strategies, survival-metabolome and death-metabolome, to encounter EIB202 infection, leading to differential outputs of the survival and dying. Glucose was the most crucial biomarker, which was upregulated and downregulated in the survival and dying groups, respectively. Exogenous glucose by injection or oral administration enhanced hosts' ability against EIB202 infection and increased the chances of survival. These findings highlight that host mounts the metabolic strategy to cope with bacterial infection, from which crucial biomarkers may be identified to enhance the metabolic strategy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Enhancing gold recovery from electronic waste via lixiviant metabolic engineering in Chromobacterium violaceum

    Science.gov (United States)

    Tay, Song Buck; Natarajan, Gayathri; Rahim, Muhammad Nadjad bin Abdul; Tan, Hwee Tong; Chung, Maxey Ching Ming; Ting, Yen Peng; Yew, Wen Shan

    2013-01-01

    Conventional leaching (extraction) methods for gold recovery from electronic waste involve the use of strong acids and pose considerable threat to the environment. The alternative use of bioleaching microbes for gold recovery is non-pollutive and relies on the secretion of a lixiviant or (bio)chemical such as cyanide for extraction of gold from electronic waste. However, widespread industrial use of bioleaching microbes has been constrained by the limited cyanogenic capabilities of lixiviant-producing microorganisms such as Chromobacterium violaceum. Here we show the construction of a metabolically-engineered strain of Chromobacterium violaceum that produces more (70%) cyanide lixiviant and recovers more than twice as much gold from electronic waste compared to wild-type bacteria. Comparative proteome analyses suggested the possibility of further enhancement in cyanogenesis through subsequent metabolic engineering. Our results demonstrated the utility of lixiviant metabolic engineering in the construction of enhanced bioleaching microbes for the bioleaching of precious metals from electronic waste. PMID:23868689

  8. A remediation performance model for enhanced metabolic reductive dechlorination of chloroethenes in fractured clay till

    DEFF Research Database (Denmark)

    Manoli, Gabriele; Chambon, Julie C.; Bjerg, Poul L.

    2012-01-01

    A numerical model of metabolic reductive dechlorination is used to describe the performance of enhanced bioremediation in fractured clay till. The model is developed to simulate field observations of a full scale bioremediation scheme in a fractured clay till and thereby to assess remediation...

  9. A novel twist on molecular interactions between thioredoxin and nicotinamide adenine dinucleotide phosphate-dependent thioredoxin reductase

    DEFF Research Database (Denmark)

    Kirkensgaard, Kristine Groth; Hägglund, Per; Shahpiri, Azar

    2013-01-01

    The ubiquitous disulfide reductase thioredoxin (Trx) regulates several important biological processes such as seed germination in plants. Oxidized cytosolic Trx is regenerated by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) in a multistep transfer...... dinucleotide (FAD)-binding domain of HvNTR2 to strongly affect the interaction with Trx. In particular, Trp42 and Met43 play key roles for recognition of the endogenous HvTrxh2. Trx from Arabidopsis thaliana is also efficiently recycled by HvNTR2 but turnover in this case appears to be less dependent...

  10. Integration between anticipatory blocking and redox signaling by the peroxiredoxin/thioredoxin/thioredoxin-reductase system.

    Science.gov (United States)

    Selvaggio, Gianluca; Coelho, Pedro M B M; Salvador, Armindo

    2014-10-01

    Cells are occasionally exposed to high H2O2 concentrations, often preceding exposure to other electrophylic compounds. Both H2O2 and these compounds can irreversibly modify protein thiols, with deleterious consequences. Induction of enzymatic defenses against those agents is too slow to avoid significant damage. Cells may solve this conundrum by reversibly "blocking" the thiols once H2O2 concentrations begin to increase. We term this mechanism "anticipatory blocking" because it acts in anticipation of irreversible damage upon detection of early signs of stress. Here we examine the design requirements for the Peroxiredoxin/Thioredoxin/Thioredoxin-Reductase/Protein-Dithiol System (PTTRDS) to effectively integrate H2O2 signaling and anticipatory blocking of protein dithiols as disulfides, and we compared them to the designs found in cells. To that effect, we developed a minimal model of the PTTRDS, and we defined a set of quantitative performance criteria that embody the requirements for (a) efficient scavenging capacity, (b) low NADPH consumption, (c) effective signal propagation, and (d) effective anticipatory blocking. We then sought the design principles (relationships among rate constants and species concentrations) that warrant fulfillment of all these criteria. Experimental data indicates that the design of the PTTRDS in human erythrocytes fulfills these principles and thus accomplishes effective integration between anticipatory blocking, antioxidant protection and redox signaling. A more general analysis suggests that the same principles hold in a wide variety of cell types and organisms. We acknowledge grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) financed by FEDER through the "Programa Operacional Factores de Competitividade, COMPETE" and by national funds through "FCT, Fundação para a Ciência e a Tecnologia". Copyright © 2014. Published by Elsevier Inc.

  11. Molecular cloning and characteristic analysis of a thioredoxin from ...

    African Journals Online (AJOL)

    Sequence comparison and phylogenetic tree analysis confirmed NmTrx as a distinct member of thioredoxin. Real-time quantitative polymerase chain reaction (PCR) revealed a significantly higher expression of NmTrx transcript in the adult stage compared with the egg and oncomiracidium stages. In the egg and adult ...

  12. Anti-Inflammatory Thioredoxin Family Proteins for Medicare, Healthcare and Aging Care

    Directory of Open Access Journals (Sweden)

    Junji Yodoi

    2017-09-01

    Full Text Available Human thioredoxin (TRX is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys-, which is induced by biological stress due to oxidative damage, metabolic dysfunction, chemicals, infection/inflammation, irradiation, or hypoxia/ischemia-reperfusion. Our research has demonstrated that exogenous TRX is effective in a wide variety of inflammatory diseases, including viral pneumonia, acute lung injury, gastric injury, and dermatitis, as well as in the prevention and amelioration of food allergies. Preclinical and clinical studies using recombinant TRX (rhTRX are now underway. We have also identified substances that induce the expression of TRX in the body, in vegetables and other plant ingredients. Skincare products are being developed that take advantage of the anti-inflammatory and anti-allergic action of TRX. Furthermore, we are currently engaged in the highly efficient production of pure rhTRX in several plants, such as lettuce, grain and rice.

  13. Ferredoxin-thioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation

    Energy Technology Data Exchange (ETDEWEB)

    Droux, M.; Miginiac-Maslow, M.; Jacquot, J.P.; Gadal, P.; Crawford, N.A.; Kosower, N.S.; Buchanan, B.B.

    1987-07-01

    The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with (/sup 14/C)iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.

  14. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

    Science.gov (United States)

    Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and

  15. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1

    Directory of Open Access Journals (Sweden)

    Matthew J. Randall

    2013-01-01

    Full Text Available Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal. Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1, a critical enzyme involved in regulation of thioredoxin (Trx-mediated redox signaling, by alkylation at its selenocysteine (Sec residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1–30 μM resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases

  16. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1☆☆☆

    Science.gov (United States)

    Randall, Matthew J.; Spiess, Page C.; Hristova, Milena; Hondal, Robert J.; van der Vliet, Albert

    2013-01-01

    Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1–30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated1 kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK

  17. Metabolism

    Science.gov (United States)

    ... Are More Common in People With Type 1 Diabetes Metabolic Syndrome Your Child's Weight Healthy Eating Endocrine System Blood Test: Basic Metabolic Panel (BMP) Activity: Endocrine System Growth Disorders Diabetes Center Thyroid Disorders Your Endocrine System Movie: Endocrine ...

  18. A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro.

    Science.gov (United States)

    Zhang, Xiaoqian; Lu, Juan; He, Bin; Tang, Lingling; Liu, Xiaoli; Zhu, Danhua; Cao, Hongcui; Wang, Yingjie; Li, Lanjuan

    2017-01-01

    Cell encapsulation provides a three-dimensional support by incorporating isolated cells into microcapsules with the goal of simultaneously maintaining cell survival and function, as well as providing active transport for a bioreactor in vitro similarly to that observed in vivo. However, the biotra-nsformation and metabolic functions of the encapsulated cells are not satisfactory for clinical applications. For this purpose, in this study, hepatoma-derived Huh7 cells/C3A cells were treated with 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous non-toxic ligand for aryl hydrocarbon receptor, in monolayer cultures and on microspheres. The mRNA and protein levels, as well as the metabolic activities of drug metabolizing enzymes, albumin secretion and urea synthesis were determined. When the Huh7 and C3A cells cultured in a monolayer on two‑dimensional surfaces, ITE enhanced the protein levels and the metabolic activities of the major cytochrome P450 (CYP450) enzymes, CYP1A1, CYP1A2, CYP3A4 and CYP1B1, and slightly increased albumin secretion and urea synthesis. Moreover, when cultured on microspheres, ITE also substantially increased the protein levels and metabolic activities of CYP1A1, CYP1A2, CYP3A4 and CYP1B1 in both liver cell lines. On the whole, our findings indicate that ITE enhances the enzymatic activities of major CYP450 enzymes and the metabolic functions of liver cells cultured in monolayer or on microspheres, indicating that it may be utilized to improve the functions of hepatocytes. Thus, it may be used in the future for the treatment of liver diseases.

  19. A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro

    Science.gov (United States)

    Zhang, Xiaoqian; Lu, Juan; He, Bin; Tang, Lingling; Liu, Xiaoli; Zhu, Danhua; Cao, Hongcui; Wang, Yingjie; Li, Lanjuan

    2017-01-01

    Cell encapsulation provides a three-dimensional support by incorporating isolated cells into microcapsules with the goal of simultaneously maintaining cell survival and function, as well as providing active transport for a bioreactor in vitro similarly to that observed in vivo. However, the biotransformation and metabolic functions of the encapsulated cells are not satisfactory for clinical applications. For this purpose, in this study, hepatoma-derived Huh7 cells/C3A cells were treated with 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous non-toxic ligand for aryl hydrocarbon receptor, in monolayer cultures and on microspheres. The mRNA and protein levels, as well as the metabolic activities of drug metabolizing enzymes, albumin secretion and urea synthesis were determined. When the Huh7 and C3A cells cultured in a monolayer on two-dimensional surfaces, ITE enhanced the protein levels and the metabolic activities of the major cytochrome P450 (CYP450) enzymes, CYP1A1, CYP1A2, CYP3A4 and CYP1B1, and slightly increased albumin secretion and urea synthesis. Moreover, when cultured on microspheres, ITE also substantially increased the protein levels and metabolic activities of CYP1A1, CYP1A2, CYP3A4 and CYP1B1 in both liver cell lines. On the whole, our findings indicate that ITE enhances the enzymatic activities of major CYP450 enzymes and the metabolic functions of liver cells cultured in monolayer or on microspheres, indicating that it may be utilized to improve the functions of hepatocytes. Thus, it may be used in the future for the treatment of liver diseases. PMID:27959388

  20. Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

    DEFF Research Database (Denmark)

    Maeda, Kenji; Hägglund, Per; Finnie, Christine

    2006-01-01

    Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure...... of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays...... a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares...

  1. The Human Thioredoxin System: Modifications and Clinical Applications

    Directory of Open Access Journals (Sweden)

    Seyed Isaac Hashemy

    2011-03-01

    Full Text Available The thioredoxin system, comprising thioredoxin (Trx, thioredoxin reductase (TrxR and NADPH, is one of the major cellular antioxidant systems, implicated in a large and growing number of biological functions. Trx acts as an oxidoreductase via a highly conserved dithiol/disulfide motif located in the active site (-Trp-Cys-Gly-Pro-Cys-Lys-. Different factors are involved in the regulation of Trx activity, including its expression level, localization, protein-protein interactions, post-translational modifications and some chemical inhibitors. Mammalian TrxRs are selenoproteins which have a –Cys-Val-Asn-Val-Gly-Cys- N-terminal active site, as well as a C-terminal selenium-containing active site. Besides two Cys-residues in the redox-regulatory domain of cytosolic Trx (Trx1, human Trx1 has three additional Cys-residues. Post-translational modifications of human Trx1 which are involved in the regulation of its activity can happen via modification of Cys-residues including thiol oxidation, glutathionylation and S-nitrosylation or via modification of other amino acid residues such as nitration of Tyr-49. Because of the numerous functions of the thioredoxin system, its inhibition (mainly happens via the targeting TrxR can result in major cellular consequences, which are potentially pro-oxidant in nature, leading to cell death via necrosis or apoptosis if overexpression of Trx and other antioxidative enzymes can not recuperate cell response. Considering this feature, several anticancer drugs have been used which can inhibit TrxR. Elevated levels of Trx and/or TrxR have been reported in many different human malignancies, positively correlated with aggressive tumor growth and poor prognosis. Moreover, anti-oxidative and anti-apoptotic effects of Trx are reasons to study its clinical application as a drug.

  2. Metabolism of chlorobiphenyls by a variant biphenyl dioxygenase exhibiting enhanced activity toward dibenzofuran

    International Nuclear Information System (INIS)

    Viger, Jean-François; Mohammadi, Mahmood; Barriault, Diane; Sylvestre, Michel

    2012-01-01

    Highlights: ► Burkholderia xenovorans LB400 biphenyl dioxygenase (BphAE LB400 ) metabolizes PCBs. ► Asn338Gln/Leu409Phe double mutation speeds up electron transfer of enzyme reaction. ► We tested how the mutations affect the PCB-degrading abilities of BphAE LB400 variants. ► The same mutations also broaden the PCB substrate range of BphAE LB400 variants. -- Abstract: The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE LB400 ) catalyzes the dihydroxylation of biphenyl and of several polychlorinated biphenyls (PCBs) but it poorly oxidizes dibenzofuran. In this work we showed that BphAE RR41 , a variant which was previously found to metabolize dibenzofuran more efficiently than its parent BphAE LB400 , metabolized a broader range of PCBs than BphAE LB400 . Hence, BphAE RR41 was able to metabolize 2,6,2′,6′-, 3,4,3′,5′- and 2,4,3′,4′-tetrachlorobiphenyl that BphAE LB400 is unable to metabolize. BphAE RR41 was obtained by changing Thr335Phe336Asn338Ile341Leu409 of BphAE LB400 to Ala335Met336Gln338Val341Phe409. Site-directed mutagenesis was used to create combinations of each substitution, in order to assess their individual contributions. Data show that the same Asn338Glu/Leu409Phe substitution that enhanced the ability to metabolize dibenzofuran resulted in a broadening of the PCB substrates range of the enzyme. The role of these substitutions on regiospecificities toward selected PCBs is also discussed.

  3. Metabolism of chlorobiphenyls by a variant biphenyl dioxygenase exhibiting enhanced activity toward dibenzofuran

    Energy Technology Data Exchange (ETDEWEB)

    Viger, Jean-Francois; Mohammadi, Mahmood; Barriault, Diane [Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, Laval, Quebec, Canada H4K 1C2 (Canada); Sylvestre, Michel, E-mail: Michel.Sylvestre@iaf.inrs.ca [Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, Laval, Quebec, Canada H4K 1C2 (Canada)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Burkholderia xenovorans LB400 biphenyl dioxygenase (BphAE{sub LB400}) metabolizes PCBs. Black-Right-Pointing-Pointer Asn338Gln/Leu409Phe double mutation speeds up electron transfer of enzyme reaction. Black-Right-Pointing-Pointer We tested how the mutations affect the PCB-degrading abilities of BphAE{sub LB400} variants. Black-Right-Pointing-Pointer The same mutations also broaden the PCB substrate range of BphAE{sub LB400} variants. -- Abstract: The biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE{sub LB400}) catalyzes the dihydroxylation of biphenyl and of several polychlorinated biphenyls (PCBs) but it poorly oxidizes dibenzofuran. In this work we showed that BphAE{sub RR41}, a variant which was previously found to metabolize dibenzofuran more efficiently than its parent BphAE{sub LB400}, metabolized a broader range of PCBs than BphAE{sub LB400}. Hence, BphAE{sub RR41} was able to metabolize 2,6,2 Prime ,6 Prime -, 3,4,3 Prime ,5 Prime - and 2,4,3 Prime ,4 Prime -tetrachlorobiphenyl that BphAE{sub LB400} is unable to metabolize. BphAE{sub RR41} was obtained by changing Thr335Phe336Asn338Ile341Leu409 of BphAE{sub LB400} to Ala335Met336Gln338Val341Phe409. Site-directed mutagenesis was used to create combinations of each substitution, in order to assess their individual contributions. Data show that the same Asn338Glu/Leu409Phe substitution that enhanced the ability to metabolize dibenzofuran resulted in a broadening of the PCB substrates range of the enzyme. The role of these substitutions on regiospecificities toward selected PCBs is also discussed.

  4. Metabolism

    Science.gov (United States)

    ... lin), which signals cells to increase their anabolic activities. Metabolism is a complicated chemical process, so it's not ... how those enzymes or hormones work. When the metabolism of body chemicals is ... Hyperthyroidism (pronounced: hi-per-THIGH-roy-dih-zum). Hyperthyroidism ...

  5. Characterization of Metabolic, Diffusion, and Perfusion Properties in GBM: Contrast-Enhancing versus Non-Enhancing Tumor.

    Science.gov (United States)

    Autry, Adam; Phillips, Joanna J; Maleschlijski, Stojan; Roy, Ritu; Molinaro, Annette M; Chang, Susan M; Cha, Soonmee; Lupo, Janine M; Nelson, Sarah J

    2017-12-01

    Although the contrast-enhancing (CE) lesion on T 1 -weighted MR images is widely used as a surrogate for glioblastoma (GBM), there are also non-enhancing regions of infiltrative tumor within the T 2 -weighted lesion, which elude radiologic detection. Because non-enhancing GBM (Enh-) challenges clinical patient management as latent disease, this study sought to characterize ex vivo metabolic profiles from Enh- and CE GBM (Enh+) samples, alongside histological and in vivo MR parameters, to assist in defining criteria for estimating total tumor burden. Fifty-six patients with newly diagnosed GBM received a multi-parametric pre-surgical MR examination. Targets for obtaining image-guided tissue samples were defined based on in vivo parameters that were suspicious for tumor. The actual location from where tissue samples were obtained was recorded, and half of each sample was analyzed for histopathology while the other half was scanned using HR-MAS spectroscopy. The Enh+ and Enh- tumor samples demonstrated comparable mitotic activity, but also significant heterogeneity in microvascular morphology. Ex vivo spectroscopic parameters indicated similar levels of total choline and N-acetylaspartate between these contrast-based radiographic subtypes of GBM, and characteristic differences in the levels of myo-inositol, creatine/phosphocreatine, and phosphoethanolamine. Analysis of in vivo parameters at the sample locations were consistent with histological and ex vivo metabolic data. The similarity between ex vivo levels of choline and NAA, and between in vivo levels of choline, NAA and nADC in Enh+ and Enh- tumor, indicate that these parameters can be used in defining non-invasive metrics of total tumor burden for patients with GBM. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Characterization of Metabolic, Diffusion, and Perfusion Properties in GBM: Contrast-Enhancing versus Non-Enhancing Tumor

    Directory of Open Access Journals (Sweden)

    Adam Autry

    2017-12-01

    Full Text Available BACKGROUND: Although the contrast-enhancing (CE lesion on T1-weighted MR images is widely used as a surrogate for glioblastoma (GBM, there are also non-enhancing regions of infiltrative tumor within the T2-weighted lesion, which elude radiologic detection. Because non-enhancing GBM (Enh− challenges clinical patient management as latent disease, this study sought to characterize ex vivo metabolic profiles from Enh− and CE GBM (Enh+ samples, alongside histological and in vivo MR parameters, to assist in defining criteria for estimating total tumor burden. Methods: Fifty-six patients with newly diagnosed GBM received a multi-parametric pre-surgical MR examination. Targets for obtaining image-guided tissue samples were defined based on in vivo parameters that were suspicious for tumor. The actual location from where tissue samples were obtained was recorded, and half of each sample was analyzed for histopathology while the other half was scanned using HR-MAS spectroscopy. Results: The Enh+ and Enh− tumor samples demonstrated comparable mitotic activity, but also significant heterogeneity in microvascular morphology. Ex vivo spectroscopic parameters indicated similar levels of total choline and N-acetylaspartate between these contrast-based radiographic subtypes of GBM, and characteristic differences in the levels of myo-inositol, creatine/phosphocreatine, and phosphoethanolamine. Analysis of in vivo parameters at the sample locations were consistent with histological and ex vivo metabolic data. CONCLUSIONS: The similarity between ex vivo levels of choline and NAA, and between in vivo levels of choline, NAA and nADC in Enh+ and Enh− tumor, indicate that these parameters can be used in defining non-invasive metrics of total tumor burden for patients with GBM.

  7. DESIGN AND PERFORMANCE OF A XENOBIOTIC METABOLISM DATABASE MANAGER FOR METABOLIC SIMULATOR ENHANCEMENT AND CHEMICAL RISK ANALYSIS

    Science.gov (United States)

    A major uncertainty that has long been recognized in evaluating chemical toxicity is accounting for metabolic activation of chemicals resulting in increased toxicity. In silico approaches to predict chemical metabolism and to subsequently screen and prioritize chemicals for risk ...

  8. Enhanced 2,4-D Metabolism in Two Resistant Papaver rhoeas Populations from Spain

    Directory of Open Access Journals (Sweden)

    Joel Torra

    2017-09-01

    Full Text Available Corn poppy (Papaver rhoeas, the most problematic broadleaf weed in winter cereals in Southern Europe, has developed resistance to the widely-used herbicide, 2,4-D. The first reported resistance mechanism in this species to 2,4-D was reduced translocation from treated leaves to the rest of the plant. However, the presence of other non-target site resistance (NTSR mechanisms has not been investigated up to date. Therefore, the main objective of this research was to reveal if enhanced 2,4-D metabolism is also present in two Spanish resistant (R populations to synthetic auxins. With this aim, HPLC experiments at two 2,4-D rates (600 and 2,400 g ai ha−1 were conducted to identify and quantify the metabolites produced and evaluate possible differences in 2,4-D degradation between resistant (R and susceptible (S plants. Secondarily, to determine the role of cytochrome P450 in the resistance response, dose-response experiments were performed using malathion as its inhibitor. Three populations were used: S, only 2,4-D R (R-703 and multiple R to 2,4-D and ALS inhibitors (R-213. HPLC studies indicated the presence of two hydroxy metabolites in these R populations in shoots and roots, which were not detected in S plants, at both rates. Therefore, enhanced metabolism becomes a new NTSR mechanism in these two P. rhoeas populations from Spain. Results from the dose-response experiments also showed that pre-treatment of R plants with the cytochrome P450 (P450 inhibitor malathion reversed the phenotype to 2,4-D from resistant to susceptible in both R populations. Therefore, it could be hypothesized that a malathion inhibited P450 is responsible of the formation of the hydroxy metabolites detected in the metabolism studies. This and previous research indicate that two resistant mechanisms to 2,4-D could be present in populations R-703 and R-213: reduced translocation and enhanced metabolism. Future experiments are required to confirm these hypotheses

  9. Thioredoxin and thioredoxin reductase influence estrogen receptor α-mediated gene expression in human breast cancer cells

    OpenAIRE

    Rao, Abhi K; Ziegler, Yvonne S; McLeod, Ian X; Yates, John R; Nardulli, Ann M

    2009-01-01

    Accumulation of reactive oxygen species (ROS) in cells damages resident proteins, lipids, and DNA. In order to overcome the oxidative stress that occurs with ROS accumulation, cells must balance free radical production with an increase in the level of antioxidant enzymes that convert free radicals to less harmful species. We identified two antioxidant enzymes, thioredoxin (Trx) and Trx reductase (TrxR), in a complex associated with the DNA-bound estrogen receptor α (ERα). Western analysis and...

  10. Methanol-enhanced removal and metabolic conversion of formaldehyde by a black soybean from formaldehyde solutions.

    Science.gov (United States)

    Tan, Hao; Xiong, Yun; Li, Kun-Zhi; Chen, Li-Mei

    2017-02-01

    Methanol regulation of some biochemical and physiological characteristics in plants has been documented in several references. This study showed that the pretreatment of methanol with an appropriate concentration could stimulate the HCHO uptake by black soybean (BS) plants. The process of methanol-stimulated HCHO uptake by BS plants was optimized using the Central Composite Design and response surface methodology for the three variables, methanol concentration, HCHO concentration, and treatment time. Under optimized conditions, the best stimulation effect of methanol on HCHO uptake was obtained. 13 C-NMR analysis indicated that the H 13 CHO metabolism produced H 13 COOH, [2- 13 C]Gly, and [3- 13 C]Ser in BS plant roots. Methanol pretreatment enhanced the metabolic conversion of H 13 CHO in BS plant roots, which consequently increased HCHO uptake by BS plants. Therefore, methanol pretreatment might be used to increase HCHO uptake by plants in the phytoremediation of HCHO-polluted solutions.

  11. 2-deoxy-D-glucose-induced metabolic stress enhances resistance to Listeria monocytogenes infection in mice

    Science.gov (United States)

    Miller, E. S.; Bates, R. A.; Koebel, D. A.; Fuchs, B. B.; Sonnenfeld, G.

    1998-01-01

    Exposure to different forms of psychological and physiological stress can elicit a host stress response, which alters normal parameters of neuroendocrine homeostasis. The present study evaluated the influence of the metabolic stressor 2-deoxy-D-glucose (2-DG; a glucose analog, which when administered to rodents, induces acute periods of metabolic stress) on the capacity of mice to resist infection with the facultative intracellular bacterial pathogen Listeria monocytogenes. Female BDF1 mice were injected with 2-DG (500 mg/kg b. wt.) once every 48 h prior to, concurrent with, or after the onset of a sublethal dose of virulent L. monocytogenes. Kinetics of bacterial growth in mice were not altered if 2-DG was applied concurrently or after the start of the infection. In contrast, mice exposed to 2-DG prior to infection demonstrated an enhanced resistance to the listeria challenge. The enhanced bacterial clearance in vivo could not be explained by 2-DG exerting a toxic effect on the listeria, based on the results of two experiments. First, 2-DG did not inhibit listeria replication in trypticase soy broth. Second, replication of L. monocytogenes was not inhibited in bone marrow-derived macrophage cultures exposed to 2-DG. Production of neopterin and lysozyme, indicators of macrophage activation, were enhanced following exposure to 2-DG, which correlated with the increased resistance to L. monocytogenes. These results support the contention that the host response to 2-DG-induced metabolic stress can influence the capacity of the immune system to resist infection by certain classes of microbial pathogens.

  12. Identification of thioredoxin target disulfides in proteins released from barley aleurone layers

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, J.; Yang, Fen

    2010-01-01

    Thioredoxins are ubiquitous disulfide reductases involved in a wide range of cellular processes including DNA synthesis, oxidative stress response and apoptosis. In cereal seeds thioredoxins are proposed to facilitate the germination process by reducing disulfide bonds in storage proteins and other...

  13. Responsive eLearning exercises to enhance student interaction with metabolic pathways.

    Science.gov (United States)

    Roesler, William J; Dreaver-Charles, Kristine

    2018-05-01

    Successful learning of biochemistry requires students to engage with the material. In the past this often involved students writing out pathways by hand, and more recently directing students to online resources such as videos, songs, and animated slide presentations. However, even these latter resources do not really provide students an opportunity to engage with the material in an active fashion. As part of an online introductory metabolism course that was developed at our university, we created a series of twelve online interactive activities using Adobe Captivate 9. These activities targeted glycolysis, gluconeogenesis, the pentose phosphate pathway, glycogen metabolism, the citric acid cycle, and fatty acid oxidation. The interactive exercises consisted of two types. One involved dragging objects such as names of enzymes or allosteric modifiers to their correct drop locations such as a particular point in a metabolic pathway, a specific enzyme, and so forth. A second type involved clicking on objects, locations within a pathway, and so forth, in response to a particular question. In both types of exercises, students received feedback on their decisions in order to enhance learning. The student feedback received on these activities was very positive, and indicated that they found them to increase their confidence in the material and that they had learned the key principles of each pathway. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):223-229, 2018. © 2018 The International Union of Biochemistry and Molecular Biology.

  14. Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1

    Science.gov (United States)

    Lafferty Doty, Sharon; Shang, Tanya Q.; Wilson, Angela M.; Tangen, Jeff; Westergreen, Aram D.; Newman, Lee A.; Strand, Stuart E.; Gordon, Milton P.

    2000-06-01

    Chlorinated solvents, especially trichloroethylene (TCE), are the most widespread groundwater contaminants in the United States. Existing methods of pumping and treating are expensive and laborious. Phytoremediation, the use of plants for remediation of soil and groundwater pollution, is less expensive and has low maintenance; however, it requires large land areas and there are a limited number of suitable plants that are known to combine adaptation to a particular environment with efficient metabolism of the contaminant. In this work, we have engineered plants with a profound increase in metabolism of the most common contaminant, TCE, by introducing the mammalian cytochrome P450 2E1. This enzyme oxidizes a wide range of important pollutants, including TCE, ethylene dibromide, carbon tetrachloride, chloroform, and vinyl chloride. The transgenic plants had a dramatic enhancement in metabolism of TCE of up to 640-fold as compared with null vector control plants. The transgenic plants also showed an increased uptake and debromination of ethylene dibromide. Therefore, transgenic plants with this enzyme could be used for more efficient remediation of many sites contaminated with halogenated hydrocarbons.

  15. Identification of thioredoxin h-reducible disulphides in proteornes by differential labelling of cysteines: Insight into recognition and regulation of proteins in barley seeds by thioredoxin h

    DEFF Research Database (Denmark)

    Maeda, Kenji; Finnie, Christine; Svensson, Birte

    2005-01-01

    alpha-amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two-dimensional electrophoresis for convenient thioredoxin h-reducible disulphide identification in barley seed extracts without the need for protein purification...... or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four alpha-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located...... structurally close to the alpha-amylase binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects...

  16. Neuron-astrocyte interaction enhance GABAergic synaptic transmission in a manner dependent on key metabolic enzymes.

    Directory of Open Access Journals (Sweden)

    Przemysław eKaczor

    2015-04-01

    Full Text Available GABA is the major inhibitory neurotransmitter in the adult brain and mechanisms of GABAergic inhibition have been intensely investigated in the past decades. Recent studies provided evidence for an important role of astrocytes in shaping GABAergic currents. One of the most obvious, but yet poorly understood, mechanisms of the cross-talk between GABAergic currents and astrocytes is metabolism including neurotransmitter homeostasis. In particular, how modulation of GABAergic currents by astrocytes depends on key enzymes involved in cellular metabolism remains largely unknown. To address this issue, we have considered two simple models of neuronal cultures: nominally astrocyte-free neuronal culture (NC and neuronal-astrocytic co-cultures (ANCC and miniature Inhibitory Postsynaptic Currents (mIPSCs were recorded in control conditions and in the presence of respective enzyme blockers. We report that enrichment of neuronal culture with astrocytes results in a marked increase in mIPSC frequency. This enhancement of GABAergic activity was accompanied by increased number of GAD65 and vGAT puncta, indicating that at least a part of the frequency enhancement was due to increased number of synaptic contacts. Inhibition of glutamine synthetase (with MSO strongly reduced mIPSC frequency in ANCC but had no effect in NC. Moreover, treatment of ANCC with inhibitor of glycogen phosphorylase (BAYU6751 or with selective inhibitor of astrocytic Krebs cycle,fluoroacetate, resulted in a marked reduction of mIPSC frequency in ANCC having no effect in NC. We conclude that GABAergic synaptic transmission strongly depends on neuron-astrocyte interaction in a manner dependent on key metabolic enzymes as well as on the Krebs cycle.

  17. Metabolic Pathways Involved in Carbon Dioxide Enhanced Heat Tolerance in Bermudagrass

    Directory of Open Access Journals (Sweden)

    Jingjin Yu

    2017-09-01

    Full Text Available Global climate changes involve elevated temperature and CO2 concentration, imposing significant impact on plant growth of various plant species. Elevated temperature exacerbates heat damages, but elevated CO2 has positive effects on promoting plant growth and heat tolerance. The objective of this study was to identify metabolic pathways affected by elevated CO2 conferring the improvement of heat tolerance in a C4 perennial grass species, bermudagrass (Cynodon dactylon Pers.. Plants were planted under either ambient CO2 concentration (400 μmol⋅mol-1 or elevated CO2 concentration (800 μmol⋅mol-1 and subjected to ambient temperature (30/25°C, day/night or heat stress (45/40°C, day/night. Elevated CO2 concentration suppressed heat-induced damages and improved heat tolerance in bermudagrass. The enhanced heat tolerance under elevated CO2 was attributed to some important metabolic pathways during which proteins and metabolites were up-regulated, including light reaction (ATP synthase subunit and photosystem I reaction center subunit and carbon fixation [(glyceraldehyde-3-phosphate dehydrogenase, GAPDH, fructose-bisphosphate aldolase, phosphoglycerate kinase, sedoheptulose-1,7-bisphosphatase and sugars of photosynthesis, glycolysis (GAPDH, glucose, fructose, and galactose and TCA cycle (pyruvic acid, malic acid and malate dehydrogenase of respiration, amino acid metabolism (aspartic acid, methionine, threonine, isoleucine, lysine, valine, alanine, and isoleucine as well as the GABA shunt (GABA, glutamic acid, alanine, proline and 5-oxoproline. The up-regulation of those metabolic processes by elevated CO2 could at least partially contribute to the improvement of heat tolerance in perennial grass species.

  18. A thioredoxin-dependent peroxiredoxin Q from Corynebacterium glutamicum plays an important role in defense against oxidative stress.

    Directory of Open Access Journals (Sweden)

    Tao Su

    Full Text Available Peroxiredoxin Q (PrxQ that belonged to the cysteine-based peroxidases has long been identified in numerous bacteria, but the information on the physiological and biochemical functions of PrxQ remain largely lacking in Corynebacterium glutamicum. To better systematically understand PrxQ, we reported that PrxQ from model and important industrial organism C. glutamicum, encoded by the gene ncgl2403 annotated as a putative PrxQ, played important roles in adverse stress resistance. The lack of C. glutamicum prxQ gene resulted in enhanced cell sensitivity, increased ROS accumulation, and elevated protein carbonylation levels under adverse stress conditions. Accordingly, PrxQ-mediated resistance to adverse stresses mainly relied on the degradation of ROS. The physiological roles of PrxQ in resistance to adverse stresses were corroborated by its induced expression under adverse stresses, regulated directly by the stress-responsive ECF-sigma factor SigH. Through catalytical kinetic activity, heterodimer formation, and bacterial two-hybrid analysis, we proved that C. glutamicum PrxQ catalytically eliminated peroxides by exclusively receiving electrons from thioredoxin (Trx/thioredoxin reductase (TrxR system and had a broad range of oxidizing substrates, but a better efficiency for peroxynitrite and cumene hydroperoxide (CHP. Site-directed mutagenesis confirmed that the conserved Cys49 and Cys54 are the peroxide oxidation site and the resolving Cys residue, respectively. It was also discovered that C. glutamicum PrxQ mainly existed in monomer whether under its native state or functional state. Based on these results, a catalytic model of PrxQ is being proposed. Moreover, our result that C. glutamicum PrxQ can prevent the damaging effects of adverse stresses by acting as thioredoxin-dependent monomeric peroxidase could be further applied to improve the survival ability and robustness of the important bacterium during fermentation process.

  19. Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1

    Directory of Open Access Journals (Sweden)

    Teusink Bas

    2007-08-01

    Full Text Available Abstract Background Thioredoxin (TRX is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1. Results We have identified the trxB1-encoded thioredoxin reductase (TR as a key enzyme in the oxidative stress response of Lactobacillus plantarum WCFS1. Overexpression of the trxB1 gene resulted in a 3-fold higher TR activity in comparison to the wild-type strain. Subsequently, higher TR activity was associated with an increased resistance towards oxidative stress. We further determined the global transcriptional response to hydrogen peroxide stress in the trxB1-overexpression and wild-type strains grown in continuous cultures. Hydrogen peroxide stress and overproduction of TR collectively resulted in the up-regulation of 267 genes. Additionally, gene expression profiling showed significant differential expression of 27 genes in the trxB1-overexpression strain. Over expression of trxB1 was found to activate genes associated with DNA repair and stress mechanisms as well as genes associated with the activity of biosynthetic pathways for purine and sulfur-containing amino acids. A total of 16 genes showed a response to both TR overproduction and hydrogen peroxide stress. These genes are involved in the purine metabolism, energy metabolism (gapB as well as in stress-response (groEL, npr2, and manganese transport (mntH2. Conclusion Based on our findings we propose that overproduction of the trxB1-encoded TR in L. plantarum improves tolerance towards oxidative stress. This response coincides with simultaneous induction of a group of 16 transcripts of genes. Within this group of genes, most are associated with oxidative stress response. The obtained crossover between datasets may explain the phenotype of the trxB1-overexpression strain, which appears to be prepared for encountering

  20. Metabolic approaches to enhance transdermal drug delivery. 1. Effect of lipid synthesis inhibitors.

    Science.gov (United States)

    Tsai, J C; Guy, R H; Thornfeldt, C R; Gao, W N; Feingold, K R; Elias, P M

    1996-06-01

    The intercellular domains of the stratum corneum, which contain a mixture of cholesterol, free fatty acids, and ceramides, mediate both the epidermal permeability barrier and the transdermal delivery of both lipophilic and hydrophilic molecules. Prior studies have shown that each of the three key lipid classes is required for normal barrier function. For example, selective inhibition of either cholesterol, fatty acid, or ceramide synthesis in the epidermis delays barrier recovery rates after barrier perturbation of hairless mouse skin in vivo. In this study, we investigated the potential of certain inhibitors of lipid synthesis to enhance the transdermal delivery of lidocaine or caffeine as a result of their capacity to perturb barrier homeostasis. After acetone disruption of the barrier, the extent of lidocaine delivery and the degree of altered barrier function paralleled each other. Moreover, the further alteration in barrier function produced by either the fatty acid synthesis inhibitor 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), the cholesterol synthesis inhibitor fluvastatin (FLU), or cholesterol sulfate (CS) resulted in a further increase in lidocaine absorption. Furthermore, coapplications of TOFA and CS together caused an additive increase in lidocaine uptake. Finally, a comparable increase in drug delivery occurred when the barrier was disrupted initially with DMSO instead of acetone; coapplications of TOFA and FLU together again delayed barrier recovery and increased drug delivery by about 8-fold vs delivery from a standard enhancing vehicle. Whereas these metabolic inhibitors also variably increased the octanol/water partitioning of the drugs studied (perhaps via complexion or pH alterations), physicochemical effects of the inhibitors alone did not alter drug uptake in intact skin; i.e., passive mechanisms alone cannot account for the net increase in drug delivery. Our results show that modulations of epidermal lipid biosynthesis, following

  1. Ebselen: A substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidant

    OpenAIRE

    Zhao, Rong; Masayasu, Hiroyuki; Holmgren, Arne

    2002-01-01

    Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a seleno-organic compound with glutathione peroxidase-like activity is used in clinical trials against stroke. Human and bovine TrxR catalyzed the reduction of ebselen to ebselen selenol by NADPH with an apparent KM-value of 2.5 μM and a kcat of 588 min−1. The addition of thioredoxin (Trx) stimulated the TrxR-catalyzed reduction of ebselen several-fold. This result was caused by a very fast oxidation of reduced Trx by ebselen with a rate cons...

  2. Hibiscus chlorotic ringspot virus coat protein upregulates sulfur metabolism genes for enhanced pathogen defense.

    Science.gov (United States)

    Gao, Ruimin; Ng, Florence Kai Lin; Liu, Peng; Wong, Sek-Man

    2012-12-01

    In both Hibiscus chlorotic ringspot virus (HCRSV)-infected and HCRSV coat protein (CP) agroinfiltrated plant leaves, we showed that sulfur metabolism pathway related genes-namely, sulfite oxidase (SO), sulfite reductase, and adenosine 5'-phosphosulfate kinase-were upregulated. It led us to examine a plausible relationship between sulfur-enhanced resistance (SED) and HCRSV infection. We broadened an established method to include different concentrations of sulfur (0S, 1S, 2S, and 3S) to correlate them to symptom development of HCRSV-infected plants. We treated plants with glutathione and its inhibitor to verify the SED effect. Disease resistance was induced through elevated glutathione contents during HCRSV infection. The upregulation of SO was related to suppression of symptom development induced by sulfur treatment. In this study, we established that HCRSV-CP interacts with SO which, in turn, triggers SED and leads to enhanced plant resistance. Thus, we have discovered a new function of SO in the SED pathway. This is the first report to demonstrate that the interaction of a viral protein and host protein trigger SED in plants. It will be interesting if such interaction applies generally to other host-pathogen interactions that will lead to enhanced pathogen defense.

  3. Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin.

    Science.gov (United States)

    Shi, M; Zhao, S; Wang, Z-H; Stanley, D; Chen, X-X

    2016-12-01

    Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species. In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full-length cDNA sequence is composed of 959 bp containing a 321 bp open reading frame that encodes a predicted protein of 106 amino acids, a predicted molecular weight of 11.7 kDa and an isoelectric point of 5.03. PxTrx was mainly expressed in larval Malpighian tubules and the fat body. An enriched recombinant PxTrx had insulin disulphide reductase activity and stimulated Human Embryonic Kidney 293 (HEK293) cell proliferation. It also protected supercoiled DNA and living HEK293 cells from H 2 O 2 -induced damage. Parasitization by Cotesia vestalis and injections of 0.05 and 0.01 equivalents of C. vestalis Bracovirus (CvBv), the symbiotic virus carried by the parasitoid, led to down-regulation of PxTrx expression in host fat body. Taken together, our results indicate that PxTrx contributes to the maintenance of P. xylostella cellular haemostasis. Host fat body expression of PxTrx is strongly attenuated by parasitization and by injections of CvBv. © 2016 The Royal Entomological Society.

  4. Baicalein induces cell death in murine T cell lymphoma via inhibition of thioredoxin system.

    Science.gov (United States)

    Patwardhan, Raghavendra S; Pal, Debojyoti; Checker, Rahul; Sharma, Deepak; Sandur, Santosh K

    2017-10-01

    We have earlier demonstrated the radioprotective potential of baicalein using murine splenic lymphocytes. Here, we have studied the effect of baicalein on murine T cell lymphoma EL4 cells and investigated the underlying mechanism of action. We observed that baicalein induced a dose dependent cell death in EL4 cells in vitro and significantly reduced the frequency of cancer stem cells. Previously, we have reported that murine and human T cell lymphoma cells have increased oxidative stress tolerance capacity due to active thioredoxin system. Hence, we monitored the effect of baicalein on thioredoxin system in EL4 cells. Docking studies revealed that baicalein could bind to the active site of thioredoxin reductase. Baicalein treatment led to significant reduction in the activity of thioredoxin reductase and nuclear levels of thioredoxin-1 thereby increasing ASK1 levels and caspase-3 activity. Interestingly, CRISPR-Cas9 based knock-out of ASK1 or over-expression of thioredoxin-1 abolished anti-tumor effects of baicalein in EL4 cells. Further, baicalein administration significantly reduced intra-peritoneal tumor burden of EL4 cells in C57BL/6 mice. Thus, our study describes anti-tumor effects of baicalein in EL4 cells via inhibition of thioredoxin system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Enhanced Neuroplasticity by the Metabolic Enhancer Piracetam Associated with Improved Mitochondrial Dynamics and Altered Permeability Transition Pore Function

    Directory of Open Access Journals (Sweden)

    Carola Stockburger

    2016-01-01

    Full Text Available The mitochondrial cascade hypothesis of dementia assumes mitochondrial dysfunction leading to reduced energy supply, impaired neuroplasticity, and finally cell death as one major pathomechanism underlying the continuum from brain aging over mild cognitive impairment to initial and advanced late onset Alzheimer’s disease. Accordingly, improving mitochondrial function has become an important strategy to treat the early stages of this continuum. The metabolic enhancer piracetam has been proposed as possible prototype for those compounds by increasing impaired mitochondrial function and related aspects like mechanisms of neuroplasticity. We here report that piracetam at therapeutically relevant concentrations improves neuritogenesis in the human cell line SH-SY5Y over conditions mirroring the whole spectrum of age-associated cognitive decline. These effects go parallel with improvement of impaired mitochondrial dynamics shifting back fission and fusion balance to the energetically more favorable fusion site. Impaired fission and fusion balance can also be induced by a reduction of the mitochondrial permeability transition pore (mPTP function as atractyloside which indicates the mPTP has similar effects on mitochondrial dynamics. These changes are also reduced by piracetam. These findings suggest the mPTP as an important target for the beneficial effects of piracetam on mitochondrial function.

  6. Enhanced Neuroplasticity by the Metabolic Enhancer Piracetam Associated with Improved Mitochondrial Dynamics and Altered Permeability Transition Pore Function.

    Science.gov (United States)

    Stockburger, Carola; Miano, Davide; Pallas, Thea; Friedland, Kristina; Müller, Walter E

    2016-01-01

    The mitochondrial cascade hypothesis of dementia assumes mitochondrial dysfunction leading to reduced energy supply, impaired neuroplasticity, and finally cell death as one major pathomechanism underlying the continuum from brain aging over mild cognitive impairment to initial and advanced late onset Alzheimer's disease. Accordingly, improving mitochondrial function has become an important strategy to treat the early stages of this continuum. The metabolic enhancer piracetam has been proposed as possible prototype for those compounds by increasing impaired mitochondrial function and related aspects like mechanisms of neuroplasticity. We here report that piracetam at therapeutically relevant concentrations improves neuritogenesis in the human cell line SH-SY5Y over conditions mirroring the whole spectrum of age-associated cognitive decline. These effects go parallel with improvement of impaired mitochondrial dynamics shifting back fission and fusion balance to the energetically more favorable fusion site. Impaired fission and fusion balance can also be induced by a reduction of the mitochondrial permeability transition pore (mPTP) function as atractyloside which indicates the mPTP has similar effects on mitochondrial dynamics. These changes are also reduced by piracetam. These findings suggest the mPTP as an important target for the beneficial effects of piracetam on mitochondrial function.

  7. Metabolic engineering of the phenylpropanoid pathway enhances the antioxidant capacity of Saussurea involucrata.

    Directory of Open Access Journals (Sweden)

    Jian Qiu

    Full Text Available The rare wild species of snow lotus Saussurea involucrata is a commonly used medicinal herb with great pharmacological value for human health, resulting from its uniquely high level of phenylpropanoid compound production. To gain information on the phenylpropanid biosynthetic pathway genes in this critically important medicinal plant, global transcriptome sequencing was performed. It revealed that the phenylpropanoid pathway genes were well represented in S. involucrata. In addition, we introduced two key phenylpropanoid pathway inducing transcription factors (PAP1 and Lc into this medicinal plant. Transgenic S. involucrata co-expressing PAP1 and Lc exhibited purple pigments due to a massive accumulation of anthocyanins. The over-expression of PAP1 and Lc largely activated most of the phenylpropanoid pathway genes, and increased accumulation of several phenylpropanoid compounds significantly, including chlorogenic acid, syringin, cyanrine and rutin. Both ABTS (2,2'-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid and FRAP (ferric reducing anti-oxidant power assays revealed that the antioxidant capacity of transgenic S. involucrata lines was greatly enhanced over controls. In addition to providing a deeper understanding of the molecular basis of phenylpropanoid metabolism, our results potentially enable an alternation of bioactive compound production in S. involucrata through metabolic engineering.

  8. Glucocorticoids enhance muscle endurance and ameliorate Duchenne muscular dystrophy through a defined metabolic program.

    Science.gov (United States)

    Morrison-Nozik, Alexander; Anand, Priti; Zhu, Han; Duan, Qiming; Sabeh, Mohamad; Prosdocimo, Domenick A; Lemieux, Madeleine E; Nordsborg, Nikolai; Russell, Aaron P; MacRae, Calum A; Gerber, Anthony N; Jain, Mukesh K; Haldar, Saptarsi M

    2015-12-08

    Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC-KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although glucocorticoid receptor (GR)-mediated transactivation is often associated with muscle atrophy and other adverse effects of pharmacologic GC administration, our data define a distinct GR-induced gene regulatory pathway that contributes to therapeutic effects of GCs in DMD through proergogenic metabolic programming.

  9. Metabolic syndrome enhances endoplasmic reticulum, oxidative stress and leukocyte-endothelium interactions in PCOS.

    Science.gov (United States)

    Bañuls, Celia; Rovira-Llopis, Susana; Martinez de Marañon, Aranzazu; Veses, Silvia; Jover, Ana; Gomez, Marcelino; Rocha, Milagros; Hernandez-Mijares, Antonio; Victor, Victor M

    2017-06-01

    Polycystic ovary syndrome (PCOS) is associated with insulin resistance, which can lead to metabolic syndrome (MetS). Oxidative stress and leukocyte-endothelium interactions are related to PCOS. Our aim was to evaluate whether the presence of MetS in PCOS patients can influence endoplasmic reticulum (ER) and oxidative stress and leukocyte-endothelium interactions. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 148 PCOS women (116 without/32 with MetS) and 112 control subjects (87 without / 25 with MetS). Metabolic parameters, reactive oxygen species (ROS) production, ER stress markers (GRP78, sXBP1, ATF6), leukocyte-endothelium interactions, adhesion molecules (VCAM-1, ICAM-1, E-Selectin), TNF-α and IL-6 were determined. Total ROS, inflammatory parameters and adhesion molecules were enhanced in the presence of MetS (pPCOS+MetS group showed higher levels of IL-6 and ICAM-1 than controls (pPCOS and PCOS+MetS groups vs their respective controls (pPCOS groups (pPCOS+MetS patients exhibited higher GRP78 and ATF6 levels than controls and PCOS patients without MetS (pPCOS women, HOMA-IR was positively correlated with ICAM-1 (r=0.501; pPCOS, all of which are related to vascular complications. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Cocaine abstinence following chronic treatment alters cerebral metabolism in dopaminergic reward regions. Bromocriptine enhances recovery

    International Nuclear Information System (INIS)

    Clow, D.W.; Hammer, R.P. Jr.

    1991-01-01

    2-[14C]deoxyglucose autoradiography was used to determine local cerebral glucose utilization (lCGU) in rats following chronic cocaine treatment and subsequent abstinence. lCGU was examined in 43 discrete brain regions in animals which had received daily injections of cocaine for 14 days (10 mg/kg) followed by 3 days of saline or bromocriptine (10 mg/kg) treatment. Cocaine abstinence following chronic treatment significantly reduced lCGU in several regions including mesocorticolimbic structures such as ventral tegmental area, medial prefrontal cortex, and nucleus accumbens (NAc). Within the NAc, however, only the rostral pole showed significant reduction. In contrast, when bromocriptine treatment accompanied abstinence, lCGU was no longer reduced in mesocorticolimbic and most other regions, implying that metabolic recovery was enhanced by bromocriptine treatment during early abstinence following chronic cocaine treatment. These data suggest that cerebral metabolism is decreased during cocaine abstinence following chronic treatment in critical brain regions, and that this alteration can be prevented by treatment with direct-acting dopamine agonists such as bromocriptine

  11. Cocaine abstinence following chronic treatment alters cerebral metabolism in dopaminergic reward regions. Bromocriptine enhances recovery

    Energy Technology Data Exchange (ETDEWEB)

    Clow, D.W.; Hammer, R.P. Jr. (Univ. of Hawaii School of Medicine, Honolulu (USA))

    1991-01-01

    2-(14C)deoxyglucose autoradiography was used to determine local cerebral glucose utilization (lCGU) in rats following chronic cocaine treatment and subsequent abstinence. lCGU was examined in 43 discrete brain regions in animals which had received daily injections of cocaine for 14 days (10 mg/kg) followed by 3 days of saline or bromocriptine (10 mg/kg) treatment. Cocaine abstinence following chronic treatment significantly reduced lCGU in several regions including mesocorticolimbic structures such as ventral tegmental area, medial prefrontal cortex, and nucleus accumbens (NAc). Within the NAc, however, only the rostral pole showed significant reduction. In contrast, when bromocriptine treatment accompanied abstinence, lCGU was no longer reduced in mesocorticolimbic and most other regions, implying that metabolic recovery was enhanced by bromocriptine treatment during early abstinence following chronic cocaine treatment. These data suggest that cerebral metabolism is decreased during cocaine abstinence following chronic treatment in critical brain regions, and that this alteration can be prevented by treatment with direct-acting dopamine agonists such as bromocriptine.

  12. Combination of traditional mutation and metabolic engineering to enhance ansamitocin P-3 production in Actinosynnema pretiosum.

    Science.gov (United States)

    Du, Zhi-Qiang; Zhang, Yuan; Qian, Zhi-Gang; Xiao, Han; Zhong, Jian-Jiang

    2017-12-01

    Ansamitocin P-3 (AP-3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP-3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP-3 biosynthesis in Actinosynnema pretiosum. First, a mutant strain M was isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutation, which could produce AP-3 almost threefold that of wild type (WT) in 48 deep-well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13-17 in the M strain, a further 60% increase of AP-3 production in 250-ml shake flasks was achieved in the engineered strain M-asmUdpg:asm13-17 compared to the M strain, and its maximum AP-3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP-3 biosynthesis pathway were significantly increased in the M and M-asmUdpg:asm13-17 during fermentation compared to the WT. The good fermentation performance of the engineered strain was also confirmed in a lab-scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP-3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites. © 2017 Wiley Periodicals, Inc.

  13. Hormonal enhancement of insecticide efficacy in Tribolium castaneum: oxidative stress and metabolic aspects.

    Science.gov (United States)

    Plavšin, Ivana; Stašková, Tereza; Šerý, Michal; Smýkal, Vlastimil; Hackenberger, Branimir K; Kodrík, Dalibor

    2015-04-01

    Insect anti-stress responses, including those induced by insecticides, are controlled by adipokinetic hormones (AKHs). We examined the physiological consequences of Pyrap-AKH application on Tribolium castaneum adults (AKH-normal and AKH-deficient prepared by the RNAi technique) treated by two insecticides, pirimiphos-methyl and deltamethrin. Co-application of pirimiphos-methyl and/or deltamethrin with AKH significantly increased beetle mortality compared with application of the insecticides alone. This co-treatment was accompanied by substantial stimulation of general metabolism, as monitored by carbon dioxide production. Further, the insecticide treatment alone affected some basic markers of oxidative stress: it lowered total antioxidative capacity as well as the activity of superoxide dismutase in the beetle body; in addition, it enhanced the activity of catalase and glutathione-S-transferase. However, these discrepancies in oxidative stress markers were eliminated/reduced by co-application with Pyrap-AKH. We suggest that the elevation of metabolism, which is probably accompanied with faster turnover of toxins, might be responsible for the higher mortality that results after AKH and insecticide co-application. Changes in oxidative stress markers are probably not included in the mechanisms responsible for increased mortality. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Glucocorticoids enhance muscle endurance and ameliorate Duchenne muscular dystrophy through a defined metabolic program

    DEFF Research Database (Denmark)

    Morrison-Nozik, Alexander; Anand, Priti; Zhu, Han

    2015-01-01

    in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription......Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy...... factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC-KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although...

  15. Tilting Plant Metabolism for Improved Metabolite Biosynthesis and Enhanced Human Benefit

    Directory of Open Access Journals (Sweden)

    Bhekumthetho Ncube

    2015-07-01

    Full Text Available The immense chemical diversity of plant-derived secondary metabolites coupled with their vast array of biological functions has seen this group of compounds attract considerable research interest across a range of research disciplines. Medicinal and aromatic plants, in particular, have been exploited for this biogenic pool of phytochemicals for products such as pharmaceuticals, fragrances, dyes, and insecticides, among others. With consumers showing increasing interests in these products, innovative biotechnological techniques are being developed and employed to alter plant secondary metabolism in efforts to improve on the quality and quantity of specific metabolites of interest. This review provides an overview of the biosynthesis for phytochemical compounds with medicinal and other related properties and their associated biological activities. It also provides an insight into how their biosynthesis/biosynthetic pathways have been modified/altered to enhance production.

  16. Enhancing NAD+ salvage metabolism is neuroprotective in a PINK1 model of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Susann Lehmann

    2017-02-01

    Full Text Available Familial forms of Parkinson's disease (PD caused by mutations in PINK1 are linked to mitochondrial impairment. Defective mitochondria are also found in Drosophila models of PD with pink1 mutations. The co-enzyme nicotinamide adenine dinucleotide (NAD+ is essential for both generating energy in mitochondria and nuclear DNA repair through NAD+-consuming poly(ADP-ribose polymerases (PARPs. We found alterations in NAD+ salvage metabolism in Drosophila pink1 mutants and showed that a diet supplemented with the NAD+ precursor nicotinamide rescued mitochondrial defects and protected neurons from degeneration. Additionally, a mutation of Parp improved mitochondrial function and was neuroprotective in the pink1 mutants. We conclude that enhancing the availability of NAD+ by either the use of a diet supplemented with NAD+ precursors or the inhibition of NAD+-dependent enzymes, such as PARPs, which compete with mitochondria for NAD+, is a viable approach to preventing neurotoxicity associated with mitochondrial defects.

  17. Thioredoxin reductase 1 upregulates MCP-1 release in human endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhen-Bo [Institute of Biophysics, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Beijing (China); Shen, Xun, E-mail: shenxun@sun5.ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, and Graduate School of the Chinese Academy of Sciences, Beijing (China)

    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.

  18. Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Justin R. Prigge

    2017-06-01

    Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

  19. Oligo-Carrageenan Kappa-Induced Reducing Redox Status and Increase in TRR/TRX Activities Promote Activation and Reprogramming of Terpenoid Metabolism in Eucalyptus Trees

    Directory of Open Access Journals (Sweden)

    Alberto González

    2014-06-01

    Full Text Available In order to analyze whether the reducing redox status and activation of thioredoxin reductase (TRR/thioredoxin(TRX system induced by oligo-carrageenan (OC kappa in Eucalyptus globulus activate secondary metabolism increasing terpenoid synthesis, trees were sprayed on the leaves with water, with OC kappa, or with inhibitors of NAD(PH, ascorbate (ASC and (GSH synthesis and TRR activity, CHS-828, lycorine, buthionine sulfoximine (BSO and auranofine, respectively, and with OC kappa and cultivated for four months. The main terpenoids in control Eucalyptus trees were eucalyptol (76%, α-pinene (7.4%, aromadendrene (3.6%, silvestrene (2.8%, sabinene (2% and α-terpineol (0.9%. Treated trees showed a 22% increase in total essential oils as well as a decrease in eucalyptol (65% and sabinene (0.8% and an increase in aromadendrene (5%, silvestrene (7.8% and other ten terpenoids. In addition, treated Eucalyptus showed seven de novo synthesized terpenoids corresponding to carene, α-terpinene, α-fenchene, γ-maaliene, spathulenol and α-camphenolic aldehyde. Most increased and de novo synthesized terpenoids have potential insecticidal and antimicrobial activities. Trees treated with CHS-828, lycorine, BSO and auranofine and with OC kappa showed an inhibition of increased and de novo synthesized terpenoids. Thus, OC kappa-induced reducing redox status and activation of TRR/TRX system enhance secondary metabolism increasing the synthesis of terpenoids and reprogramming of terpenoid metabolism in Eucalyptus trees.

  20. Oligo-carrageenan kappa-induced reducing redox status and increase in TRR/TRX activities promote activation and reprogramming of terpenoid metabolism in Eucalyptus trees.

    Science.gov (United States)

    González, Alberto; Gutiérrez-Cutiño, Marlen; Moenne, Alejandra

    2014-06-05

    In order to analyze whether the reducing redox status and activation of thioredoxin reductase (TRR)/thioredoxin(TRX) system induced by oligo-carrageenan (OC) kappa in Eucalyptus globulus activate secondary metabolism increasing terpenoid synthesis, trees were sprayed on the leaves with water, with OC kappa, or with inhibitors of NAD(P)H, ascorbate (ASC) and (GSH) synthesis and TRR activity, CHS-828, lycorine, buthionine sulfoximine (BSO) and auranofine, respectively, and with OC kappa and cultivated for four months. The main terpenoids in control Eucalyptus trees were eucalyptol (76%), α-pinene (7.4%), aromadendrene (3.6%), silvestrene (2.8%), sabinene (2%) and α-terpineol (0.9%). Treated trees showed a 22% increase in total essential oils as well as a decrease in eucalyptol (65%) and sabinene (0.8%) and an increase in aromadendrene (5%), silvestrene (7.8%) and other ten terpenoids. In addition, treated Eucalyptus showed seven de novo synthesized terpenoids corresponding to carene, α-terpinene, α-fenchene, γ-maaliene, spathulenol and α-camphenolic aldehyde. Most increased and de novo synthesized terpenoids have potential insecticidal and antimicrobial activities. Trees treated with CHS-828, lycorine, BSO and auranofine and with OC kappa showed an inhibition of increased and de novo synthesized terpenoids. Thus, OC kappa-induced reducing redox status and activation of TRR/TRX system enhance secondary metabolism increasing the synthesis of terpenoids and reprogramming of terpenoid metabolism in Eucalyptus trees.

  1. Improvement of mitochondrial function and dynamics by the metabolic enhancer piracetam.

    Science.gov (United States)

    Stockburger, Carola; Kurz, Christopher; Koch, Konrad A; Eckert, Schamim H; Leuner, Kristina; Müller, Walter E

    2013-10-01

    The metabolic enhancer piracetam is used in many countries to treat cognitive impairment in aging, brain injuries, as well as dementia such as AD (Alzheimer's disease). As a specific feature of piracetam, beneficial effects are usually associated with mitochondrial dysfunction. In previous studies we were able to show that piracetam enhanced ATP production, mitochondrial membrane potential as well as neurite outgrowth in cell and animal models for aging and AD. To investigate further the effects of piracetam on mitochondrial function, especially mitochondrial fission and fusion events, we decided to assess mitochondrial morphology. Human neuroblastoma cells were treated with the drug under normal conditions and under conditions imitating aging and the occurrence of ROS (reactive oxygen species) as well as in stably transfected cells with the human wild-type APP (amyloid precursor protein) gene. This AD model is characterized by expressing only 2-fold more human Aβ (amyloid β-peptide) compared with control cells and therefore representing very early stages of AD when Aβ levels gradually increase over decades. Interestingly, these cells exhibit an impaired mitochondrial function and morphology under baseline conditions. Piracetam is able to restore this impairment and shifts mitochondrial morphology back to elongated forms, whereas there is no effect in control cells. After addition of a complex I inhibitor, mitochondrial morphology is distinctly shifted to punctate forms in both cell lines. Under these conditions piracetam is able to ameliorate morphology in cells suffering from the mild Aβ load, as well as mitochondrial dynamics in control cells.

  2. Mitochondrial isocitrate dehydrogenase is inactivated upon oxidation and reactivated by thioredoxin-dependent reduction in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Keisuke eYoshida

    2014-09-01

    Full Text Available Regulation of mitochondrial metabolism is essential for ensuring cellular growth and maintenance in plants. Based on redox-proteomics analysis, several proteins involved in diverse mitochondrial reactions have been identified as potential redox-regulated proteins. NAD+-dependent isocitrate dehydrogenase (IDH, a key enzyme in the tricarboxylic acid cycle, is one such candidate. In this study, we investigated the redox regulation mechanisms of IDH by biochemical procedures. In contrast to mammalian and yeast counterparts reported to date, recombinant IDH in Arabidopsis mitochondria did not show adenylate-dependent changes in enzymatic activity. Instead, IDH was inactivated by oxidation treatment and partially reactivated by subsequent reduction. Functional IDH forms a heterodimer comprising regulatory (IDH-r and catalytic (IDH-c subunits. IDH-r was determined to be the target of oxidative modifications forming an oligomer via intermolecular disulfide bonds. Mass spectrometric analysis combined with tryptic digestion of IDH-r indicated that Cys128 and Cys216 are involved in intermolecular disulfide bond formation. Furthermore, we showed that mitochondria-localized o-type thioredoxin (Trx-o promotes the reduction of oxidized IDH-r. These results suggest that IDH-r is susceptible to oxidative stress, and Trx-o serves to convert oxidized IDH-r to the reduced form that is necessary for active IDH complex.

  3. Enzymes or redox couples? The kinetics of thioredoxin and glutaredoxin reactions in a systems biology context

    NARCIS (Netherlands)

    Pillay, Ché S.; Hofmeyr, Jan Hendrik S; Olivier, Brett G.; Snoep, Jacky L.; Rohwer, Johann M.

    2009-01-01

    Systems biology approaches, such as kinetic modelling, could provide valuable insights into how thioredoxins, glutaredoxins and peroxiredoxins (here collectively called redoxins), and the systems that reduce these molecules are regulated. However, it is not clear whether redoxins should be described

  4. Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

    DEFF Research Database (Denmark)

    Björnberg, Olof; Viennet, Thibault; Skjoldager, Nicklas

    2015-01-01

    Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two...... enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s-1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli Trx......-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass...

  5. RECOGNITION DYNAMICS OF ESCHERICHIA COLI THIOREDOXIN PROBED USING MOLECULAR DYNAMICS AND BINDING FREE ENERGY CALCULATIONS

    Directory of Open Access Journals (Sweden)

    M. S. Shahul Hameed

    2016-03-01

    Full Text Available E. coli thioredoxin has been regarded as a hub protein as it interacts with, and regulates, numerous target proteins involved in a wide variety of cellular processes. Thioredoxin can form complexes with a variety of target proteins with a wide range of affinity, using a consensus binding surface. In this study an attempt to deduce the molecular basis for the observed multispecificity of E. coli thioredoxin has been made. In this manuscript it has been shown that structural plasticity, adaptable and exposed hydrophobic binding surface, surface electrostatics, closely clustered multiple hot spot residues and conformational changes brought about by the redox status of the protein have been shown to account for the observed multispecificity and molecular recognition of thioredoxin. Dynamical differences between the two redox forms of the enzyme have also been studied to account for their differing interactions with some target proteins.

  6. Sulforaphane-induced transcription of thioredoxin reductase in lens: possible significance against cataract formation

    Directory of Open Access Journals (Sweden)

    Varma SD

    2013-10-01

    Full Text Available Shambhu D Varma, Krish Chandrasekaran, Svitlana Kovtun Department of Ophthalmology and Visual Sciences, University of Maryland, Baltimore, MD, USA Purpose: Sulforaphane is a phytochemically derived organic isothiocyanate 1-isothiocyanato-4-methylsulfinyl-butane present naturally in crucifers, including broccoli and cauliflower. Biochemically, it has been reported to induce the transcription of several antioxidant enzymes. Since such enzymes have been implicated in preventing cataract formation triggered by the intraocular generation of oxy-radical species, the purpose of this investigation was to examine whether it could induce the formation of antioxidant enzymes in the eye lens. Thioredoxin reductase (TrxR was used as the target of such induction. Methods: Mice lenses were cultured for an overnight period of 17 hours in medium 199 fortified with 10% fetal calf serum. Incubation was conducted in the absence and presence of sulforaphane (5 µM. Subsequently, the lenses were homogenized in phosphate-buffered saline (PBS, followed by centrifugation. TrxR activity was determined in the supernatant by measuring the nicotinamide adenine dinucleotide phosphate (reduced (NADPH-dependent reduction of 5,5´-dithiobis-2-nitrobenzoic acid (DTNB. Non-specific reduction of DTNB was corrected for by conducting parallel determinations in the presence of aurothiomalate. The reduction of DTNB was followed spectrophotometrically at 410 nm. Results: The activity of TrxR in the lenses incubated with sulforaphane was found to be elevated to 18 times of that observed in lenses incubated without sulforaphane. It was also noticeably higher in the lenses incubated without sulforaphane than in the un-incubated fresh lenses. However, this increase was much lower than that observed for lenses incubated with sulforaphane. Conclusion: Sulforaphane has been found to enhance TrxR activity in the mouse lens in culture. In view of the protective effect of the antioxidant enzymes

  7. Enhanced glucose metabolism in cultured human skeletal muscle after Roux-en-Y gastric bypass surgery.

    Science.gov (United States)

    Nascimento, Emmani B M; Riedl, Isabelle; Jiang, Lake Qunfeng; Kulkarni, Sameer S; Näslund, Erik; Krook, Anna

    2015-01-01

    Roux-en-Y gastric bypass (RYGB) surgery rapidly increases whole body insulin sensitivity, with changes in several organs including skeletal muscle. Objectives were to determine whether improvements in insulin action in skeletal muscle may occur directly at the level of the myocyte or secondarily from changes in systemic factors associated with weight loss. Myotubes were derived before and after RYGB surgery. The setting was Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden. Eight patients (body mass index (BMI) 41.8 kg/m(2); age 41 yr) underwent RYGB surgery. Before and 6 months after RYGB surgery, skeletal muscle biopsies were collected from vastus lateralis muscle. Satellite cells derived from skeletal muscle biopsies were propagated in vitro as myoblasts and differentiated into myotubes. Expression of myogenic markers is increased in myoblasts derived from biopsies taken 6 months after bypass surgery, compared with their respective presurgery condition. Furthermore, glycogen synthesis, tyrosine phosphorylation of insulin receptor (IRS)-1-Tyr612 and Interleukin (IL)-8 secretion were increased, while fatty acid oxidation and circulating IL8 levels remain unaltered. Myotubes derived from muscle biopsies obtained after RYGB surgery displayed increased insulin-stimulated phosphorylation of protein kinase B (PKB)-Thr308 and proline-rich Akt substrate of 40 kDa (PRAS40)-Thr246. RYGB surgery is accompanied by enhanced glucose metabolism and insulin signaling, altered IL8 secretion and changes in mRNA levels and myogenic markers in cultured skeletal muscle cells. Thus, RYGB surgery involves intrinsic reprogramming of skeletal muscle to increase peripheral insulin sensitivity and glucose metabolism. Copyright © 2015 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.

  8. A Copolymer Scaffold Functionalized with Nanodiamond Particles Enhances Osteogenic Metabolic Activity and Bone Regeneration.

    Science.gov (United States)

    Yassin, Mohammed A; Mustafa, Kamal; Xing, Zhe; Sun, Yang; Fasmer, Kristine Eldevik; Waag, Thilo; Krueger, Anke; Steinmüller-Nethl, Doris; Finne-Wistrand, Anna; Leknes, Knut N

    2017-06-01

    Functionalizing polymer scaffolds with nanodiamond particles (nDPs) has pronounced effect on the surface properties, such as improved wettability, an increased active area and binding sites for cellular attachment and adhesion, and increased ability to immobilize biomolecules by physical adsorption. This study aims to evaluate the effect of poly(l-lactide-co-ε-caprolactone) (poly(LLA-co-CL)) scaffolds, functionalized with nDPs, on bone regeneration in a rat calvarial critical size defect. Poly(LLA-co-CL) scaffolds functionalized with nDPs are also compared with pristine scaffolds with reference to albumin adsorption and seeding efficiency of bone marrow stromal cells (BMSCs). Compared with pristine scaffolds, the experimental scaffolds exhibit a reduction in albumin adsorption and a significant increase in the seeding efficiency of BMSCs (p = 0.027). In the calvarial defects implanted with BMSC-seeded poly(LLA-co-CL)/nDPs scaffolds, live imaging at 12 weeks discloses a significant increase in osteogenic metabolic activity (p = 0.016). Microcomputed tomography, confirmed by histological data, reveals a substantial increase in bone volume (p = 0.021). The results show that compared with conventional poly(LLA-co-CL) scaffolds those functionalized with nDPs promote osteogenic metabolic activity and mineralization capacity. It is concluded that poly(LLA-co-CL) composite matrices functionalized with nDPs enhance osteoconductivity and therefore warrant further study as potential scaffolding material for bone tissue engineering. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

    Science.gov (United States)

    Fan, Yichong; Makar, Merna; Wang, Michael X; Ai, Hui-Wang

    2017-09-01

    Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

  10. Metabolic clues regarding the enhanced performance of elite endurance athletes from orchiectomy-induced hormonal changes.

    Science.gov (United States)

    Atwood, Craig S; Bowen, Richard L

    2007-01-01

    , thereby increasing power to weight ratio. Taken together, these hormonal changes act to limit glycogen utilization, delay fatigue and enhance recovery thereby allowing for optimal performances on a day-to-day basis. These insights provide the foundation for future studies on the endocrinology of exercise metabolism, and suggest that Lance Armstrong's athletic advantage was not due to drug use.

  11. Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths

    Directory of Open Access Journals (Sweden)

    Salinas Gustavo

    2010-04-01

    Full Text Available Abstract Background The thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with the selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum. Results We examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites. Conclusions Our results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and

  12. Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths

    Science.gov (United States)

    2010-01-01

    Background The thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm) parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with the selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR) as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum. Results We examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda) conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria) possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites. Conclusions Our results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and that canonical enzymes

  13. Thioredoxin from the Indianmeal moth Plodia interpunctella: cloning and test of the allergenic potential in mice.

    Directory of Open Access Journals (Sweden)

    Elisabeth Hoflehner

    Full Text Available BACKGROUND/OBJECTIVE: The Indianmeal moth Plodia interpunctella is a highly prevalent food pest in human dwellings, and has been shown to contain a number of allergens. So far, only one of these, the arginine kinase (Plo i 1 has been identified. OBJECTIVE: The aim of this study was to identify further allergens and characterise these in comparison to Plo i 1. METHOD: A cDNA library from whole adult P. interpunctella was screened with the serum of a patient with indoor allergy and IgE to moths, and thioredoxin was identified as an IgE-binding protein. Recombinant thioredoxin was generated in E. coli, and tested together with Plo i 1 and whole moth extracts in IgE immunoblots against a large panel of indoor allergic patients' sera. BALB/c mice were immunised with recombinant thioredoxin and Plo i 1, and antibody production, mediator release from RBL cells, T-cell proliferation and cytokine production were measured. RESULT: For the first time a thioredoxin from an animal species was identified as allergen. About 8% of the sera from patients with IgE against moth extracts reacted with recombinant P. interpunctella thioredoxin, compared to 25% reacting with recombinant Plo i 1. In immunised BALB/c mice, the recombinant allergens both induced classical Th2-biased immune responses such as induction IgE and IgG1 antibodies, upregulation of IL-5 and IL-4 and basophil degranulation. CONCLUSION: Thioredoxin from moths like Plo i 1 acts like a classical Type I allergen as do the thioredoxins from wheat or corn. This clearly supports the pan-allergen nature of thioredoxin. The designation Plo i 2 is suggested for the new P. interpunctella allergen.

  14. Is Oxidized Thioredoxin a Major Trigger for Cysteine Oxidation? Clues from a Redox Proteomics Approach

    OpenAIRE

    García-Santamarina, Sarela; Boronat, Susanna; Calvo, Isabel A.; Rodríguez-Gabriel, Miguel; Ayté, José; Molina, Henrik; Hidalgo, Elena

    2013-01-01

    This is a copy of an article published in the Antioxidants & Redox Signaling © Mary Ann Liebert, Inc. Antioxidants & Redox Signaling is available online at http://online.liebertpub.com Cysteine oxidation mediates oxidative stress toxicity and signaling. It has been long proposed that the thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (Trr), is not only involved in recycling classical Trx substrates, such as ribonucleotide reductase, but it also regulates g...

  15. Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism.

    Directory of Open Access Journals (Sweden)

    Azza Al-Mahrouki

    Full Text Available Ultrasound (US stimulated microbubbles (MB is a new treatment approach that sensitizes cancer cells to radiation (XRT. The molecular pathways in this response remain unelucidated, however, previous data has supported a role for cell membrane-metabolism related pathways including an up regulation of UDP glycosyltransferase 8 (UGT8, which catalyzes the transfer of galactose to ceramide, a lipid that is associated with the induction of apoptotic signalling. In this study, the role of UGT8 in responses of prostate tumours to ultrasound-stimulated microbubble radiation enhancement therapy is investigated. Experiments were carried out with cells in vitro and tumours in vivo in which UGT8 levels had been up regulated or down regulated. Genetically modified PC3 cells were treated with XRT, US+MB, or a combination of XRT+US+MB. An increase in the immunolabelling of ceramide was observed in cells where UGT8 was down-regulated as opposed to cells where UGT8 was either not regulated or was up-regulated. Clonogenic assays have revealed a decreased level of cellular survival with the down-regulation of UGT8. Xenograft tumours generated from stably transfected PC3 cells were also treated with US+MB, XRT or US+MB+XRT. Histology demonstrated more cellular damage in tumours with down-regulated UGT8 in comparison with control tumours. In contrast, tumours with up-regulated UGT8 had less damage than control tumours. Power Doppler imaging indicated a reduction in the vascular index with UGT8 down-regulation and photoacoustic imaging revealed a reduction in oxygen saturation. This was contrary to when UGT8 was up regulated. The down regulation of UGT8 led to the accumulation of ceramide resulting in more cell death signalling and therefore, a greater enhancement of radiation effect when vascular disruption takes place through the use of ultrasound-stimulated microbubbles.

  16. Nitrosative/oxidative stress conditions regulate thioredoxin-interacting protein (TXNIP) expression and thioredoxin-1 (TRX-1) nuclear localization.

    Science.gov (United States)

    Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

    2013-01-01

    Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras-ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H₂O₂, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.

  17. Nitrosative/oxidative stress conditions regulate thioredoxin-interacting protein (TXNIP expression and thioredoxin-1 (TRX-1 nuclear localization.

    Directory of Open Access Journals (Sweden)

    Fernando Toshio Ogata

    Full Text Available Thioredoxin (TRX-1 is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras-ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP, TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP. Once activated by the oxidants, SNAP and H₂O₂, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126, or in cells transfected with the Protein Enriched in Astrocytes (PEA-15, a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.

  18. Silencing the Girdin gene enhances radio-sensitivity of hepatocellular carcinoma via suppression of glycolytic metabolism.

    Science.gov (United States)

    Yu, Li; Sun, Yifan; Li, Jingjing; Wang, Yan; Zhu, Yuxing; Shi, Yong; Fan, Xiaojun; Zhou, Jianda; Bao, Ying; Xiao, Jie; Cao, Ke; Cao, Peiguo

    2017-08-15

    Radiotherapy has been used increasingly to treat primary hepatocellular carcinoma. Clinically, the main cause of radiotherapy failure is cellular radioresistance, conferred via glycolytic metabolism. Our previous study demonstrated that Girdin is upregulated in primary hepatocellular carcinoma and promotes the invasion and metastasis of tumor cells. However, whether Girdin underlies the radio-sensitivity of hepatocellular carcinoma remains unclear. A short hairpin RNA (shRNA) was used to silence CCDC88A (encoding Girdin), and real-time PCR was performed to determine CCDC88A mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of Girdin silencing on cellular radiosensitivity. Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the signaling pathway downstream of Girdin. Finally, animal experiments were performed to demonstrate the effect of CCDC88A silencing on the radiosensitivity of hepatoma in vivo. shRNA-induced Girdin silencing suppressed glycolysis and enhanced the radio-sensitivity of hepatic cell lines, HepG2 and Huh-7. Furthermore, silencing of Girdin inhibited the PI3K/AKT/HIF-1α signaling pathway, which is a central regulator of glycolysis. Girdin can regulate glycolysis in hepatocellular carcinoma cells through the PI3K/AKT/HIF-1α signaling pathway, which decreases the sensitivity of tumor cells to radiotherapy.

  19. A remediation performance model for enhanced metabolic reductive dechlorination of chloroethenes in fractured clay till

    Science.gov (United States)

    Manoli, Gabriele; Chambon, Julie C.; Bjerg, Poul L.; Scheutz, Charlotte; Binning, Philip J.; Broholm, Mette M.

    2012-04-01

    A numerical model of metabolic reductive dechlorination is used to describe the performance of enhanced bioremediation in fractured clay till. The model is developed to simulate field observations of a full scale bioremediation scheme in a fractured clay till and thereby to assess remediation efficiency and timeframe. A relatively simple approach is used to link the fermentation of the electron donor soybean oil to the sequential dechlorination of trichloroethene (TCE) while considering redox conditions and the heterogeneous clay till system (clay till matrix, fractures and sand stringers). The model is tested on lab batch experiments and applied to describe sediment core samples from a TCE-contaminated site. Model simulations compare favorably to field observations and demonstrate that dechlorination may be limited to narrow bioactive zones in the clay matrix around fractures and sand stringers. Field scale simulations show that the injected donor is expected to be depleted after 5 years, and that without donor re-injection contaminant rebound will occur in the high permeability zones and the mass removal will stall at 18%. Long remediation timeframes, if dechlorination is limited to narrow bioactive zones, and the need for additional donor injections to maintain dechlorination activity may limit the efficiency of ERD in low-permeability media. Future work should address the dynamics of the bioactive zones, which is essential to understand for predictions of long term mass removal.

  20. Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering.

    Science.gov (United States)

    Zhai, Yafei; Han, Donglei; Pan, Ying; Wang, Shuaishuai; Fang, Junqiang; Wang, Peng; Liu, Xian-wei

    2015-02-01

    Guanosine 5'-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5'-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase.

    OpenAIRE

    Caldas , Thérèse; Malki , Abderrahim; Kern , Renée; Abdallah , Jad; Richarme , Gilbert

    2006-01-01

    Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN func...

  2. Thioredoxin-1 promotes colorectal cancer invasion and metastasis through crosstalk with S100P.

    Science.gov (United States)

    Lin, Feiyan; Zhang, Peili; Zuo, Zhigui; Wang, Fule; Bi, Ruichun; Shang, Wenjing; Wu, Aihua; Ye, Ju; Li, Shaotang; Sun, Xuecheng; Wu, Jianbo; Jiang, Lei

    2017-08-10

    Thioredoxin-1 (Trx-1) is a small redox-regulating protein, which plays an important role in several cellular functions. Despite recent advances in understanding the biology of Trx-1, the role of Trx-1 and its underlying signaling mechanism in colorectal cancer (CRC) metastasis have not been extensively studied. In this study, we observed that Trx-1 expression is increased in CRC tissues compared to the paired non-cancerous tissues and is significantly correlated with clinical staging, lymph node metastasis and poor survival. Overexpression of Trx-1 enhanced CRC cell invasion and metastasis in vitro and in vivo. Conversely, suppression of Trx-1 expression decreased cell invasion and metastasis in vitro and in vivo. Moreover, Trx-1 activates S100P gene transcription. S100P, in turn, promotes Trx-1 expression and nuclear localization by upregulating p-ERK1/2 and downregulating TXNIP expression. Our finding provides new insight into the mechanism of Trx-1/S100P axis in the promotion of CRC metastasis, and suggests that the Trx-1/S100P axis and their related signaling pathways could be novel targets for the treatment of metastatic CRC. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Targeting of the Glutathione, Thioredoxin, and Nrf2 Antioxidant Systems in Head and Neck Cancer.

    Science.gov (United States)

    Roh, Jong-Lyel; Jang, Hyejin; Kim, Eun Hye; Shin, Daiha

    2017-07-10

    The glutathione (GSH), thioredoxin (Trx), and Nrf2 systems represent a major defense against reactive oxygen species (ROS), the cellular imbalance of which in cancer promotes growth and therapeutic resistance. This study investigated whether targeting the GSH, Trx, and Nrf2 antioxidant systems effectively eliminated head and neck cancer (HNC). At high concentrations, auranofin, but not buthionine sulfoximine (BSO) alone, decreased the viability of HNC, whereas even at low concentrations, auranofin plus BSO synergized to kill HNC cells. Dual silencing of the genes for GCLM and TrxR1 induced GSH depletion, Trx activity inhibition, and ROS accumulation, synergistically killing HNC cells. Inhibition of the GSH and Trx systems resulted in activation of the Nrf2-antioxidant response element (ARE) pathway, which may result in suboptimal GSH and Trx inhibition where HNC is resistant. Genetic inhibition of Nrf2 and/or HO-1 or trigonelline enhanced growth suppression, ROS accumulation, and cell death from GSH and Trx inhibition. The in vivo effects of GSH, Trx, and Nrf2 system inhibition were confirmed in a mouse HNC xenograft model by achieving growth inhibition >60% compared with those of control. Innovations: This study is the first to show that triple inhibition of GSH, Trx, and Nrf2 pathways could be an effective method to overcome the resistance of HNC. Inhibition of the Nrf2-ARE pathway in addition to dual inhibition of the GSH and Trx antioxidant systems can effectively eliminate resistant HNC. Antioxid. Redox Signal. 27, 106-114.

  4. Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin-related protein 14-mediated autophagy.

    Science.gov (United States)

    Zhen, Zijun; Yang, Kaibin; Ye, Litong; You, Zhiyao; Chen, Rirong; Liu, Ying; He, Youjian

    2017-07-01

    Paclitaxel is not as effective for neuroblastoma as most of the front-line chemotherapeutics due to drug resistance. This study explored the regulatory mechanism of paclitaxel-associated autophagy and potential solutions to paclitaxel resistance in neuroblastoma. The formation of autophagic vesicles was detected by scanning transmission electron microscopy and flow cytometry. The autophagy-associated proteins were assessed by western blot. Autophagy was induced and the autophagy-associated proteins LC3-I, LC3-II, Beclin 1, and thioredoxin-related protein 14 (TRP14), were found to be upregulated in neuroblastoma cells that were exposed to paclitaxel. The inhibition of Beclin 1 or TRP14 by siRNA increased the sensitivity of the tumor cells to paclitaxel. In addition, Beclin 1-mediated autophagy was regulated by TRP14. Furthermore, the TRP14 inhibitor suberoylanilide hydroxamic acid (SAHA) downregulated paclitaxel-induced autophagy and enhanced the anticancer effects of paclitaxel in normal control cancer cells but not in cells with upregulated Beclin 1 and TRP14 expression. Our findings showed that paclitaxel-induced autophagy in neuroblastoma cells was regulated by TRP14 and that SAHA could sensitize neuroblastoma cells to paclitaxel by specifically inhibiting TRP14. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  5. Enhanced volatile fatty acids production from anaerobic fermentation of food waste: A mini-review focusing on acidogenic metabolic pathways.

    Science.gov (United States)

    Zhou, Miaomiao; Yan, Binghua; Wong, Jonathan W C; Zhang, Yang

    2018-01-01

    Recently, efficient disposal of food waste (FW) with potential resource recovery has attracted great attentions. Due to its easily biodegradable nature, rich nutrient availability and high moisture content, FW is regarded as favorable substrate for anaerobic digestion (AD). Both waste disposal and energy recovery can be fulfilled during AD of FW. Volatile fatty acids (VFAs) which are the products of the first-two stages of AD, are widely applied in chemical industry as platform chemicals recently. Concentration and distribution of VFAs is the result of acidogenic metabolic pathways, which can be affected by the micro-environment (e.g. pH) in the digester. Hence, the clear elucidation of the acidogenic metabolic pathways is essential for optimization of acidogenic process for efficient product recovery. This review summarizes major acidogenic metabolic pathways and regulating strategies for enhancing VFAs recovery during acidogenic fermentation of FW. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Biophysical and Structural Characterization of the Thioredoxin-binding Domain of Protein Kinase ASK1 and Its Interaction with Reduced Thioredoxin

    Czech Academy of Sciences Publication Activity Database

    Košek, Dalibor; Kylarová, Salome; Pšenáková, Katarína; Řežábková, L.; Herman, P.; Večeř, J.; Obšilová, Veronika; Obšil, T.

    2014-01-01

    Roč. 289, č. 35 (2014), s. 24463-24474 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA14-10061S Institutional support: RVO:67985823 Keywords : ASK1 * thioredoxin * AUC * SAXS * coiled-coiled domain Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  7. Effect of acrolein and glutathione depleting agents on thioredoxin

    International Nuclear Information System (INIS)

    Yang Xianmei; Wu Xuli; Choi, Young Eun; Kern, Julie C.; Kehrer, James P.

    2004-01-01

    Acrolein is a widespread environmental pollutant that reacts rapidly with nucleophiles, especially cellular thiols. In addition to glutathione (GSH), thioredoxin (Trx) and thioredoxin reductase (TR) contain thiol groups and may react with electrophiles. In the present study, A549 cells treated with 5-25 μM acrolein for 30 min lost cellular Trx activity in a dose-dependent fashion. Over 90% of Trx activity was lost at concentrations of 25 μM or greater. In contrast, Trx protein content, as assessed by western blotting, was not altered immediately after the 30 min acrolein treatment. Both Trx activity and protein levels increased 4 h after the acrolein treatment. However, Trx activity remained below control levels at 24 h. A similar dose-response relationship was seen with TR in A549 cells exposed to acrolein. There was, however, a rapid recovery of TR activity such that it attained normal levels by 4 h after doses ≤75 μM acrolein. Diethyl maleate (DEM), a common but not highly specific, agent used to deplete GSH, also inactivated Trx. A 2 h exposure of A549 cells to 1 mM DEM depleted cellular GSH by ∼50% and diminished Trx activity by over 67%. Lower DEM doses (0.125 mM and 0.25 mM) for 1 h had no significant effect on GSH but significantly decreased Trx activity 12 and 23%, respectively. Similar to immediately after acrolein exposure, DEM did not affect Trx protein levels. A Trx-1-GFP fusion protein was transfected into A549 cells. While the fusion protein was expressed, the Trx component was inactive by the insulin reducing assay. In summary, Trx and TR are inactivated by acrolein. In addition, the GSH depleting agent DEM inactivates Trx somewhat more effectively than it depletes GSH. The Trx-1-GFP fusion protein, while readily expressed, appears to have little or no activity, perhaps because the small size of Trx-1 (12 kDa) is affected by the larger GFP

  8. Resolution of oxidative stress by thioredoxin reductase: Cysteine versus selenocysteine

    Directory of Open Access Journals (Sweden)

    Brian Cunniff

    2014-01-01

    Full Text Available Thioredoxin reductase (TR catalyzes the reduction of thioredoxin (TRX, which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1–4, thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1 and mitochondrial TR2 (Sec-TR2 that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2. In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP, but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic

  9. Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

    Science.gov (United States)

    Besson, Marie Thérèse; Alegría, Karin; Garrido-Gerter, Pamela; Barros, Luis Felipe; Liévens, Jean-Charles

    2015-01-01

    Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93). We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP) impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK) which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to mediate the hGluT3

  10. Enhanced neuronal glucose transporter expression reveals metabolic choice in a HD Drosophila model.

    Directory of Open Access Journals (Sweden)

    Marie Thérèse Besson

    Full Text Available Huntington's disease is a neurodegenerative disorder caused by toxic insertions of polyglutamine residues in the Huntingtin protein and characterized by progressive deterioration of cognitive and motor functions. Altered brain glucose metabolism has long been suggested and a possible link has been proposed in HD. However, the precise function of glucose transporters was not yet determined. Here, we report the effects of the specifically-neuronal human glucose transporter expression in neurons of a Drosophila model carrying the exon 1 of the human huntingtin gene with 93 glutamine repeats (HQ93. We demonstrated that overexpression of the human glucose transporter in neurons ameliorated significantly the status of HD flies by increasing their lifespan, reducing their locomotor deficits and rescuing eye neurodegeneration. Then, we investigated whether increasing the major pathways of glucose catabolism, glycolysis and pentose-phosphate pathway (PPP impacts HD. To mimic increased glycolytic flux, we overexpressed phosphofructokinase (PFK which catalyzes an irreversible step in glycolysis. Overexpression of PFK did not affect HQ93 fly survival, but protected from photoreceptor loss. Overexpression of glucose-6-phosphate dehydrogenase (G6PD, the key enzyme of the PPP, extended significantly the lifespan of HD flies and rescued eye neurodegeneration. Since G6PD is able to synthesize NADPH involved in cell survival by maintenance of the redox state, we showed that tolerance to experimental oxidative stress was enhanced in flies co-expressing HQ93 and G6PD. Additionally overexpressions of hGluT3, G6PD or PFK were able to circumvent mitochondrial deficits induced by specific silencing of genes necessary for mitochondrial homeostasis. Our study confirms the involvement of bioenergetic deficits in HD course; they can be rescued by specific expression of a glucose transporter in neurons. Finally, the PPP and, to a lesser extent, the glycolysis seem to

  11. Decreased in vitro mitochondrial function is associated with enhanced brain metabolism, blood flow, and memory in Surf1-deficient mice

    Science.gov (United States)

    Lin, Ai-Ling; Pulliam, Daniel A; Deepa, Sathyaseelan S; Halloran, Jonathan J; Hussong, Stacy A; Burbank, Raquel R; Bresnen, Andrew; Liu, Yuhong; Podlutskaya, Natalia; Soundararajan, Anuradha; Muir, Eric; Duong, Timothy Q; Bokov, Alex F; Viscomi, Carlo; Zeviani, Massimo; Richardson, Arlan G; Van Remmen, Holly; Fox, Peter T; Galvan, Veronica

    2013-01-01

    Recent studies have challenged the prevailing view that reduced mitochondrial function and increased oxidative stress are correlated with reduced longevity. Mice carrying a homozygous knockout (KO) of the Surf1 gene showed a significant decrease in mitochondrial electron transport chain Complex IV activity, yet displayed increased lifespan and reduced brain damage after excitotoxic insults. In the present study, we examined brain metabolism, brain hemodynamics, and memory of Surf1 KO mice using in vitro measures of mitochondrial function, in vivo neuroimaging, and behavioral testing. We show that decreased respiration and increased generation of hydrogen peroxide in isolated Surf1 KO brain mitochondria are associated with increased brain glucose metabolism, cerebral blood flow, and lactate levels, and with enhanced memory in Surf1 KO mice. These metabolic and functional changes in Surf1 KO brains were accompanied by higher levels of hypoxia-inducible factor 1 alpha, and by increases in the activated form of cyclic AMP response element-binding factor, which is integral to memory formation. These findings suggest that Surf1 deficiency-induced metabolic alterations may have positive effects on brain function. Exploring the relationship between mitochondrial activity, oxidative stress, and brain function will enhance our understanding of cognitive aging and of age-related neurologic disorders. PMID:23838831

  12. In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates

    NARCIS (Netherlands)

    Poblete-Castro, I.; Binger, D.; Rodrigues, A.; Becker, J.; Martins Dos Santos, V.A.P.; Wittmann, C.

    2013-01-01

    Here, we present systems metabolic engineering driven by in-silico modeling to tailor Pseudomonas putida for synthesis of medium chain length PHAs on glucose. Using physiological properties of the parent wild type as constraints, elementary flux mode analysis of a large-scale model of the metabolism

  13. Respiration and nitrogen assimilation: targeting mitochondria-associated metabolism as a means to enhance nitrogen use efficiency.

    Science.gov (United States)

    Foyer, Christine H; Noctor, Graham; Hodges, Michael

    2011-02-01

    Considerable advances in our understanding of the control of mitochondrial metabolism and its interactions with nitrogen metabolism and associated carbon/nitrogen interactions have occurred in recent years, particularly highlighting important roles in cellular redox homeostasis. The tricarboxylic acid (TCA) cycle is a central metabolic hub for the interacting pathways of respiration, nitrogen assimilation, and photorespiration, with components that show considerable flexibility in relation to adaptations to the different functions of mitochondria in photosynthetic and non-photosynthetic cells. By comparison, the operation of the oxidative pentose phosphate pathway appears to represent a significant limitation to nitrogen assimilation in non-photosynthetic tissues. Valuable new insights have been gained concerning the roles of the different enzymes involved in the production of 2-oxoglutarate (2-OG) for ammonia assimilation, yielding an improved understanding of the crucial role of cellular energy balance as a broker of co-ordinate regulation. Taken together with new information on the mechanisms that co-ordinate the expression of genes involved in organellar functions, including energy metabolism, and the potential for exploiting the existing flexibility for NAD(P)H utilization in the respiratory electron transport chain to drive nitrogen assimilation, the evidence that mitochondrial metabolism and machinery are potential novel targets for the enhancement of nitrogen use efficiency (NUE) is explored.

  14. Ebselen: A thioredoxin reductase-dependent catalyst for α-tocopherol quinone reduction

    International Nuclear Information System (INIS)

    Fang Jianguo; Zhong Liangwei; Zhao Rong; Holmgren, Arne

    2005-01-01

    The thioredoxin system, composed of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH, is a powerful protein disulfide reductase system with a broad substrate specificity. Recently the selenazol drug ebselen was shown to be a substrate for both mammalian TrxR and Trx. We examined if α-tocopherol quinone (TQ), a product of α-tocopherol oxidation, is reduced by ebselen in the presence of TrxR, since TQ was not a substrate for the enzyme itself. Ebselen reduction of TQ in the presence of TrxR was caused by ebselen selenol, generated from fast reduction of ebselen by the enzyme. TQ has no intrinsic antioxidant activity, while the product of reduction of TQ, α-tocopherolhydroquinone (TQH 2 ), is a potent antioxidant. The thioredoxin system dependence of ebselen to catalyze reduction of other oxidized species, such as hydrogen peroxide, dehydroascorbate, and peroxynitrite, is discussed. The ability of ebselen to reduce TQ via the thioredoxin system is a novel mechanism to explain the effects of the drug as an antioxidant in vivo

  15. Dissecting molecular interactions involved in recognition of target disulfides by the barley thioredoxin system

    DEFF Research Database (Denmark)

    Björnberg, Olof; Maeda, Kenji; Svensson, Birte

    2012-01-01

    Thioredoxin reduces disulfide bonds, thus regulating activities of target proteins in various biological systems, e.g., inactivation of inhibitors of starch hydrolases and proteases in germinating plant seeds. In the three-dimensional structure of a complex with barley α-amylase/subtilisin inhibi......Thioredoxin reduces disulfide bonds, thus regulating activities of target proteins in various biological systems, e.g., inactivation of inhibitors of starch hydrolases and proteases in germinating plant seeds. In the three-dimensional structure of a complex with barley α...... thioredoxin reductase. HvTrxh2 M88G and M88A adjacent to the invariant cis-proline lost efficiency in both BASI disulfide reduction and recycling by thioredoxin reductase. These effects were further pronounced in M88P lacking a backbone NH group. Remarkably, HvTrxh2 E86R in the same loop displayed overall...... retained catalytic properties, with the exception of a 3-fold increased activity toward BASI. From the 104VGA106 loop, a backbone hydrogen bond donated by A106 appears to be important for target disulfide recognition as A106P lost 90% activity toward BASI but was efficiently recycled by thioredoxin...

  16. Thioredoxin interacting protein and its association with clinical outcome in gastro-oesophageal adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Caroline M. Woolston

    2013-01-01

    Full Text Available The overall prognosis for operable gastro-oesophageal adenocarcinoma remains poor and therefore neoadjuvant chemotherapy has become the standard of care, in addition to radical surgery. Certain anticancer agents (e.g. anthracyclines and cisplatin generate damaging reactive oxygen species as by-products of their mechanism of action. Drug effectiveness can therefore depend upon the presence of cellular redox buffering systems that are often deregulated in cancer. The expression of the redox protein, thioredoxin interacting protein, was assessed in gastro-oesophageal adenocarcinomas. Thioredoxin interacting protein expression was assessed using conventional immunohistochemistry on a tissue microarray of 140 adenocarcinoma patients treated by primary surgery alone and 88 operable cases treated with neoadjuvant chemotherapy. In the primary surgery cases, high thioredoxin interacting protein expression associated with a lack of lymph node involvement (p=0.005, no perineural invasion (p=0.030 and well/moderate tumour differentiation (p=0.033. In the neoadjuvant tumours, high thioredoxin interacting protein expression was an independent marker for improved disease specific survival (p=0.002 especially in cases with anthracycline-based regimes (p=0.008. This study highlights the potential of thioredoxin interacting protein as a biomarker for response in neoadjuvant treated gastro-oesophageal adenocarcinoma and may represent a useful therapeutic target due to its association with tumour progression.

  17. Physical Activity Enhances Metabolic Fitness Independently of Cardiorespiratory Fitness in Marathon Runners

    Directory of Open Access Journals (Sweden)

    M. J. Laye

    2015-01-01

    Full Text Available High levels of cardiovascular fitness (CRF and physical activity (PA are associated with decreased mortality and risk to develop metabolic diseases. The independent contributions of CRF and PA to metabolic disease risk factors are unknown. We tested the hypothesis that runners who run consistently >50 km/wk and/or >2 marathons/yr for the last 5 years have superior metabolic fitness compared to matched sedentary subjects (CRF, age, gender, and BMI. Case-control recruitment of 31 pairs of runner-sedentary subjects identified 10 matched pairs with similar VO2max (mL/min/kg (similar-VO2max. The similar-VO2max group was compared with a group of age, gender, and BMI matched pairs who had the largest difference in VO2max (different-VO2max. Primary outcomes that defined metabolic fitness including insulin response to an oral glucose tolerance test, fasting lipids, and fasting insulin were superior in runners versus sedentary controls despite similar VO2max. Furthermore, performance (velocity at VO2max, running economy, improved exercise metabolism (lactate threshold, and skeletal muscle levels of mitochondrial proteins were superior in runners versus sedentary controls with similar VO2max. In conclusion subjects with a high amount of PA have more positive metabolic health parameters independent of CRF. PA is thus a good marker against metabolic diseases.

  18. NADPH-Thioredoxin Reductase C Mediates the Response to Oxidative Stress and Thermotolerance in the Cyanobacterium Anabaena sp PCC7120

    NARCIS (Netherlands)

    Sanchez-Riego, Ana M.; Mata-Cabana, Alejandro; Galmozzi, CarlaV.; Florencio, Francisco J.

    2016-01-01

    NADPH-thioredoxin reductase C (NTRC) is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thiioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of

  19. Inhibition of Sphingolipid Metabolism Enhances Resveratrol Chemotherapy in Human Gastric Cancer Cells

    OpenAIRE

    Shin, Kyong-Oh; Park, Nam-Young; Seo, Cho-Hee; Hong, Seon-Pyo; Oh, Ki-Wan; Hong, Jin-Tae; Han, Sang-Kil; Lee, Yong-Moon

    2012-01-01

    Resveratrol, a chemopreventive agent, is rapidly metabolized in the intestine and liver via glucuronidation. Thus, the pharmacokinetics of resveratrol limits its efficacy. To improve efficacy, the activity of resveratrol was investigated in the context of sphingolipid metabolism in human gastric cancer cells. Diverse sphingolipid metabolites, including dihydroceramides (DHCer), were tested for their ability to induce resveratrol cytotoxicity. Exposure to resveratrol (100 ?M) for 24 hr induced...

  20. Metabolic Engineering of the Actinomycete Amycolatopsis sp. Strain ATCC 39116 towards Enhanced Production of Natural Vanillin.

    Science.gov (United States)

    Fleige, Christian; Meyer, Florian; Steinbüchel, Alexander

    2016-06-01

    The Gram-positive bacterium Amycolatopsis sp. ATCC 39116 is used for the fermentative production of natural vanillin from ferulic acid on an industrial scale. The strain is known for its outstanding tolerance to this toxic product. In order to improve the productivity of the fermentation process, the strain's metabolism was engineered for higher final concentrations and molar yields. Degradation of vanillin could be decreased by more than 90% through deletion of the vdh gene, which codes for the central vanillin catabolism enzyme, vanillin dehydrogenase. This mutation resulted in improvement of the final concentration of vanillin by more than 2.2 g/liter, with a molar yield of 80.9%. Further improvement was achieved with constitutive expression of the vanillin anabolism genes ech and fcs, coding for the enzymes feruloyl-coenzyme A (CoA) synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech). The transcription of both genes was shown to be induced by ferulic acid, which explains the unwanted adaptation phase in the fermentation process before vanillin was efficiently produced by the wild-type cells. Through the constitutive and enhanced expression of the two genes, the adaptation phase was eliminated and a final vanillin concentration of 19.3 g/liter, with a molar yield of 94.9%, was obtained. Moreover, an even higher final vanillin concentration of 22.3 g/liter was achieved, at the expense of a lower molar yield, by using an improved feeding strategy. This is the highest reported vanillin concentration reached in microbial fermentation processes without extraction of the product. Furthermore, the vanillin was produced almost without by-products, with a molar yield that nearly approached the theoretical maximum. Much effort has been put into optimization of the biotechnological production of natural vanillin. The demand for this compound is growing due to increased consumer concerns regarding chemically produced food additives. Since this compound is toxic to most

  1. Lifespan decrease in a Caenorhabditis elegans mutant lacking TRX-1, a thioredoxin expressed in ASJ sensory neurons.

    Science.gov (United States)

    Miranda-Vizuete, Antonio; Fierro González, Juan Carlos; Gahmon, Gabriele; Burghoorn, Jan; Navas, Plácido; Swoboda, Peter

    2006-01-23

    Thioredoxins are a class of small proteins that play a key role in regulating many cellular redox processes. We report here the characterization of the first member of the thioredoxin family in metazoans that is mainly associated with neurons. The Caenorhabditis elegans gene B0228.5 encodes a thioredoxin (TRX-1) that is expressed in ASJ ciliated sensory neurons, and to some extent also in the posterior-most intestinal cells. TRX-1 is active at reducing protein disulfides in the presence of a heterologous thioredoxin reductase. A mutant worm strain carrying a null allele of the trx-1 gene displays a reproducible decrease in both mean and maximum lifespan when compared to wild-type. The identification and characterization of TRX-1 paves the way to use C. elegans as an in vivo model to study the role of thioredoxins in lifespan and nervous system physiology and pathology.

  2. CCAAT/Enhancer Binding Protein β in relation to ER Stress, Inflammation, and Metabolic Disturbances

    Directory of Open Access Journals (Sweden)

    Sophie E. van der Krieken

    2015-01-01

    Full Text Available The prevalence of the metabolic syndrome and underlying metabolic disturbances increase rapidly in developed countries. Various molecular targets are currently under investigation to unravel the molecular mechanisms that cause these disturbances. This is done in attempt to counter or prevent the negative health consequences of the metabolic disturbances. Here, we reviewed the current knowledge on the role of C/EBP-β in these metabolic disturbances. C/EBP-β deletion in mice resulted in downregulation of hepatic lipogenic genes and increased expression of β-oxidation genes in brown adipose tissue. Furthermore, C/EBP-β is important in the differentiation and maturation of adipocytes and is increased during ER stress and proinflammatory conditions. So far, studies were only conducted in animals and in cell systems. The results found that C/EBP-β is an important transcription factor within the metabolic disturbances of the metabolic system. Therefore, it is interesting to examine the potential role of C/EBP-β at molecular and physiological level in humans.

  3. Physical activity enhances metabolic fitness independently of cardiorespiratory fitness in marathon runners

    DEFF Research Database (Denmark)

    Laye, M J; Nielsen, M B; Hansen, L S

    2015-01-01

    High levels of cardiovascular fitness (CRF) and physical activity (PA) are associated with decreased mortality and risk to develop metabolic diseases. The independent contributions of CRF and PA to metabolic disease risk factors are unknown. We tested the hypothesis that runners who run consisten......High levels of cardiovascular fitness (CRF) and physical activity (PA) are associated with decreased mortality and risk to develop metabolic diseases. The independent contributions of CRF and PA to metabolic disease risk factors are unknown. We tested the hypothesis that runners who run...... consistently >50 km/wk and/or >2 marathons/yr for the last 5 years have superior metabolic fitness compared to matched sedentary subjects (CRF, age, gender, and BMI). Case-control recruitment of 31 pairs of runner-sedentary subjects identified 10 matched pairs with similar VO2max (mL/min/kg) (similar-VO2max......). The similar-VO2max group was compared with a group of age, gender, and BMI matched pairs who had the largest difference in VO2max (different-VO2max). Primary outcomes that defined metabolic fitness including insulin response to an oral glucose tolerance test, fasting lipids, and fasting insulin were superior...

  4. The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

    DEFF Research Database (Denmark)

    Skjoldager, Nicklas; Bang, Maria Blanner; Rykær, Martin

    2017-01-01

    The NADPH-dependent homodimeric flavoenzyme thioredoxin reductase (TrxR) provides reducing equivalents to thioredoxin, a key regulator of various cellular redox processes. Crystal structures of photo-inactivated thioredoxin reductase (TrxR) from the Gram-positive bacterium Lactococcus lactis have...... been determined. These structures reveal novel molecular features that provide further insight into the mechanisms behind the sensitivity of this enzyme toward visible light. We propose that a pocket on the si-face of the isoalloxazine ring accommodates oxygen that reacts with photo-excited FAD...... thus be a widespread feature among bacterial TrxR with the described characteristics, which affords applications in clinical photo-therapy of drug-resistant bacteria....

  5. Glutathione transferases are structural and functional outliers in the thioredoxin fold.

    Science.gov (United States)

    Atkinson, Holly J; Babbitt, Patricia C

    2009-11-24

    Glutathione transferases (GSTs) are ubiquitous scavengers of toxic compounds that fall, structurally and functionally, within the thioredoxin fold suprafamily. The fundamental catalytic capability of GSTs is catalysis of the nucleophilic addition or substitution of glutathione at electrophilic centers in a wide range of small electrophilic compounds. While specific GSTs have been studied in detail, little else is known about the structural and functional relationships between different groupings of GSTs. Through a global analysis of sequence and structural similarity, it was determined that variation in the binding of glutathione between the two major subgroups of cytosolic (soluble) GSTs results in a different mode of glutathione activation. Additionally, the convergent features of glutathione binding between cytosolic GSTs and mitochondrial GST kappa are described. The identification of these structural and functional themes helps to illuminate some of the fundamental contributions of the thioredoxin fold to catalysis in the GSTs and clarify how the thioredoxin fold can be modified to enable new functions.

  6. Enhancements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism and carcinogenic risk via NNK/arsenic interaction

    International Nuclear Information System (INIS)

    Lee, H.-L.; Chang, Louis W.; Wu, J.-P.; Ueng, Y.-F.; Tsai, M.-H.; Hsieh, Dennis Paul Hsientang; Lin Pinpin

    2008-01-01

    Epidemiological studies indicated an enhancement of cigarette smoke-induced carcinogenicity, including hepatocellular carcinoma, by arsenic. We believe that arsenic will enhance the expression of hepatic CYP2A enzyme and NNK metabolism (a cigarette smoke component), thus its metabolites, and carcinogenic DNA adducts. Male ICR mice were exposed to NNK (0.5 mg/mouse) and sodium arsenite (0, 10, or 20 mg/kg) daily via gavaging for 10 days and their urine was collected at day 10 for NNK metabolite analysis. Liver samples were also obtained for CYP2A enzyme and DNA adducts evaluations. Both the cyp2a4/5 mRNA levels and the CYP2A enzyme activity were significantly elevated in arsenic-treated mice liver. Furthermore, urinary NNK metabolites in NNK/arsenic co-treated mice also increased compared to those treated with NNK alone. Concomitantly, DNA adducts (N 7 -methylguanine and O 6 -methylguanine) were significantly elevated in the livers of mice co-treated with NNK and arsenic. Our findings provide clear evidence that arsenic increased NNK metabolism by up-regulation of CYP2A expression and activity leading to an increased NNK metabolism and DNA adducts (N 7 -methylguanine and O 6 -methylguanine). These findings suggest that in the presence of arsenic, NNK could induce greater DNA adducts formation in hepatic tissues resulting in higher carcinogenic potential

  7. Plateau hypoxia attenuates the metabolic activity of intestinal flora to enhance the bioavailability of nifedipine.

    Science.gov (United States)

    Zhang, Juanhong; Chen, Yuyan; Sun, Yuemei; Wang, Rong; Zhang, Junmin; Jia, Zhengping

    2018-11-01

    Nifedipine is completely absorbed by the gastrointestinal tract and its pharmacokinetics and metabolism may be influenced by microorganisms. If gut microbes are involved in the metabolism of nifedipine, plateau hypoxia may regulate the bioavailability and the therapeutic effect of nifedipine by altering the metabolic activity of the gut microbiota. We herein demonstrated for the first time that gut flora is involved in the metabolism of nifedipine by in vitro experiments. In addition, based on the results of 16S rRNA analysis of feces in rats after acute plateau, we first confirmed that the plateau environment could cause changes in the number and composition of intestinal microbes. More importantly, these changes in flora could lead to a slower metabolic activity of nifedipine in the body after an acute plateau, resulting in increased bioavailability and therapeutic efficacy of nifedipine. Our research will provide basis and new ideas for changes in the fecal flora of human acutely entering the plateau, and contribute to rational drug use of nifedipine.

  8. Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

    Science.gov (United States)

    Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

    2013-07-01

    Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

  9. Unifying mechanical and thermodynamic descriptions across the thioredoxin protein family.

    Science.gov (United States)

    Mottonen, James M; Xu, Minli; Jacobs, Donald J; Livesay, Dennis R

    2009-05-15

    We compare various predicted mechanical and thermodynamic properties of nine oxidized thioredoxins (TRX) using a Distance Constraint Model (DCM). The DCM is based on a nonadditive free energy decomposition scheme, where entropic contributions are determined from rigidity and flexibility of structure based on distance constraints. We perform averages over an ensemble of constraint topologies to calculate several thermodynamic and mechanical response functions that together yield quantitative stability/flexibility relationships (QSFR). Applied to the TRX protein family, QSFR metrics display a rich variety of similarities and differences. In particular, backbone flexibility is well conserved across the family, whereas cooperativity correlation describing mechanical and thermodynamic couplings between the residue pairs exhibit distinctive features that readily standout. The diversity in predicted QSFR metrics that describe cooperativity correlation between pairs of residues is largely explained by a global flexibility order parameter describing the amount of intrinsic flexibility within the protein. A free energy landscape is calculated as a function of the flexibility order parameter, and key values are determined where the native-state, transition-state, and unfolded-state are located. Another key value identifies a mechanical transition where the global nature of the protein changes from flexible to rigid. The key values of the flexibility order parameter help characterize how mechanical and thermodynamic response is linked. Variation in QSFR metrics and key characteristics of global flexibility are related to the native state X-ray crystal structure primarily through the hydrogen bond network. Furthermore, comparison of three TRX redox pairs reveals differences in thermodynamic response (i.e., relative melting point) and mechanical properties (i.e., backbone flexibility and cooperativity correlation) that are consistent with experimental data on thermal stabilities

  10. Prognostic value of serum thioredoxin levels in ischemic stroke.

    Science.gov (United States)

    Yu, Tieer; Zhang, Wanli; Lin, Yuanshao; Li, Qian; Xue, Jie; Cai, Zhengyi; Cheng, Yifan; Shao, Bei

    2017-11-01

    Thioredoxin (Trx) is one of significant antioxidative molecules to diminish oxidative stress. Current evidence suggests that Trx is a potent antioxidant with cytoprotective functions. The aim of our study was to investigate specifically the association between serum Trx levels and acute ischemic stroke (AIS) patients. 198 AIS patients and 75 controls were enrolled to the study. Serum Trx levels were measured using an enzyme-linked immunosorbent assay (ELISA). Stroke severity was assessed with the National Institutes of Health Stroke Scale (NIHSS) score on admission. Clinical endpoint was functional outcome measured by Barthel Index (BI) 3 months after admission. Multivariate binary logistic regression analyses were performed to identify predictors. We found that serum Trx levels were significantly increased in patients as compared to controls. Serum Trx was an independent biomarker to predict ischemic stroke (OR, 1.264; 95% CI, 1.04-1.537; P = 0.019). In addition, there was a negative correlation between NIHSS score at admission and serum Trx levels in cardioembolic stroke patients (r = -0.422; P = 0.013). Furthermore, higher serum Trx levels in AIS patients were associated with favorable functional outcome. Serum Trx was an independent predictor for the functional outcome (OR, 0.862; 95% CI, 0.75-0.991; P = 0.037). Serum Trx might be as a biomarker of cardioembolic stroke severity. Increased serum Trx levels could be a useful tool to predict good prognosis in patients with AIS.

  11. Thioredoxin priming prolongs lung allograft survival by promoting immune tolerance.

    Directory of Open Access Journals (Sweden)

    Hanbo Hu

    Full Text Available Tolerance to allograft antigen is the major challenge and final goal of transplant medicine. Our previous study demonstrated that thioredoxin-1 (Trx priming of donor lung significantly protected allogeneic lung graft. To determine whether Trx priming of donor lung inhibits allograft rejection, extends allograft survival and induces immune tolerance, orthotopic left lung transplantation was performed from Lewis to Sprague-Dawley rats without immunosuppression. Donor lungs were primed with Trx at 4°C for 4 hr prior to transplantation. After up to 37 days post-transplantation, allograft lung morphology, recipient T cell and humoral alloantigen-specific immune responses were examined. We found that Trx-primed lungs exhibited much reduced acute rejection and associated lung injuries resulting in loss of graft functional area at 5-37 days post-transplant in contrast to the control groups. CD4+ T cells from the recipients with Trx-primed grafts responded to the stimulation of dendritic cells (DCs of donor origin, in contrast to DCs from the third party, with significantly reduced proliferation. Consistent with above findings, we observed that CD4+Foxp3+ regulatory T cells in spleen cells from the recipients with Trx-primed grafts were significantly increased compared to controls, and CD4+ T cells from the recipients with Trx-primed grafts produced much higher levels of immunosuppressive cytokine, IL-10 when stimulated with allogeneic donor DCs. In addition, humoral immune tolerance was also induced as there was no significant increase levels of serum antibodies against donor antigens in Trx-lung recipients when re-challenged with allogeneic donor antigens. Our results demonstrate that one-time Trx-priming of donor lung grafts prior to transplantation significantly prolongs the survival of the grafts through inducing or promoting cellular and humoral alloantigen-specific immune tolerance, which might be associated with the induction of

  12. Enhancement of cadmium bioremediation by endophytic bacterium Bacillus sp. L14 using industrially used metabolic inhibitors (DCC or DNP)

    International Nuclear Information System (INIS)

    Luo Shenglian; Xiao Xiao; Xi Qiang; Wan Yong; Chen Liang; Zeng Guangming; Liu Chengbin; Guo Hanjun; Chen Jueliang

    2011-01-01

    Bioremediations of cadmium by endophytic bacterium (EB) L14 (Bacillus sp.) in the presence of industrially used metabolic inhibitors (DCC or DNP) were investigated. In the presence of DCC or DNP, the biomass population of EB L14 was greatly inhibited. However, the cadmium removal of EB L14 increased from 73.6% (in the absence of DCC or DNP) to 93.7% and 80.8%, respectively. The analysis of total and intracellular cadmium concentrations during 24 h of incubation indicated that this enhanced cadmium removal was the inhibition effect of DCC or DNP on the cations export resistance system of EB L14. This unique property strongly indicated the superiority of this endophyte for practical application in cadmium bioremediation in the presence of industrially used metabolic inhibitors.

  13. Thioredoxin induces Tregs to generate an immunotolerant tumor microenvironment in metastatic melanoma

    Science.gov (United States)

    Wang, Xiaogang; Dong, Haisheng; Li, Qi; Li, Yingxian; Hong, An

    2015-01-01

    Metastatic melanoma is a highly aggressive cancer that is very difficult to treat. Additionally, the antitumor immune reaction of melanoma is still unclear. Here we demonstrate an association between the expression and secretion of the antioxidant protein thioredoxin (TRX) and increasing tumor stage and metastasis in melanoma. To elucidate the role of TRX in melanoma, we assessed the correlation of TRX expression with different disease parameters in melanoma. We also examined the in vitro and in vivo effects of modulating TRX levels in melanoma cells using various methods of TRX depletion and augmentation. We further explored the effects of TRX on the cytokine milieu and the ability of TRX to regulate the proportion and specific activities of T-cell populations. We demonstrate that TRX expression correlates with Treg representation in clinical samples and, that modulation of TRX influences the induction of Tregs and the generation of an immunotolerant cytokine profile in mouse serum. Using a murine metastatic melanoma model, we identified a tumor immunoevasion mechanism whereby melanoma cell-secreted TRX enhances Treg infiltration. TRX displays chemotactic effects in recruiting Tregs, stimulates the conversion of conventional T cells to Tregs, and confers survival advantage to Tregs in the tumor microenvironment. In turn, this increase of Tregs generates immunotolerance in tissues and therefore decreases antitumor immune reactions. These results elucidate a mechanism by which TRX promotes metastatic melanoma in part through Treg recruitment to inhibit T-cell antitumor effects and suggest that TRX antibody may be useful in the clinic as a therapy against melanoma. PMID:26405597

  14. Chromium supplementation enhances the metabolic response of steers to lipopolysaccharide (LPS) challenge

    Science.gov (United States)

    The effect of chromium (Cr; KemTRACE®brandChromiumProprionate 0.04%, Kemin Industries) supplementation on the metabolic response to LPS challenge was examined. Steers (n=20; 235±4 kg body weight (BW)) received a premix that added 0 (Con) or 0.2 mg/kg Cr to the total diet (DM (dry matter) basis) for ...

  15. Galanin enhances systemic glucose metabolism through enteric Nitric Oxide Synthase-expressed neurons

    Directory of Open Access Journals (Sweden)

    Anne Abot

    2018-04-01

    Full Text Available Objective: Decreasing duodenal contraction is now considered as a major focus for the treatment of type 2 diabetes. Therefore, identifying bioactive molecules able to target the enteric nervous system, which controls the motility of intestinal smooth muscle cells, represents a new therapeutic avenue. For this reason, we chose to study the impact of oral galanin on this system in diabetic mice. Methods: Enteric neurotransmission, duodenal contraction, glucose absorption, modification of gut–brain axis, and glucose metabolism (glucose tolerance, insulinemia, glucose entry in tissue, hepatic glucose metabolism were assessed. Results: We show that galanin, a neuropeptide expressed in the small intestine, decreases duodenal contraction by stimulating nitric oxide release from enteric neurons. This is associated with modification of hypothalamic nitric oxide release that favors glucose uptake in metabolic tissues such as skeletal muscle, liver, and adipose tissue. Oral chronic gavage with galanin in diabetic mice increases insulin sensitivity, which is associated with an improvement of several metabolic parameters such as glucose tolerance, fasting blood glucose, and insulin. Conclusion: Here, we demonstrate that oral galanin administration improves glucose homeostasis via the enteric nervous system and could be considered a therapeutic potential for the treatment of T2D. Keywords: Galanin, Enteric nervous system, Diabetes

  16. Programming Saposin-Mediated Compensatory Metabolic Sinks for Enhanced Ubiquinone Production.

    Science.gov (United States)

    Xu, Wen; Yuan, Jifeng; Yang, Shuiyun; Ching, Chi-Bun; Liu, Jiankang

    2016-12-16

    Microbial synthesis of ubiquinone by fermentation processes has been emerging in recent years. However, as ubiquinone is a primary metabolite that is tightly regulated by the host central metabolism, tweaking the individual pathway components could only result in a marginal improvement on the ubiquinone production. Given that ubiquinone is stored in the lipid bilayer, we hypothesized that introducing additional metabolic sink for storing ubiquinone might improve the CoQ 10 production. As human lipid binding/transfer protein saposin B (hSapB) was reported to extract ubiquinone from the lipid bilayer and form the water-soluble complex, hSapB was chosen to build a compensatory metabolic sink for the ubiquinone storage. As a proof-of-concept, hSapB-mediated metabolic sink systems were devised and systematically investigated in the model organism of Escherichia coli. The hSapB-mediated periplasmic sink resulted in more than 200% improvement of CoQ 8 over the wild type strain. Further investigation revealed that hSapB-mediated sink systems could also improve the CoQ 10 production in a CoQ 10 -hyperproducing E. coli strain obtained by a modular pathway rewiring approach. As the design principles and the engineering strategies reported here are generalizable to other microbes, compensatory sink systems will be a method of significant interest to the synthetic biology community.

  17. A new nano-enhanced technology proposed to quantify intracellular detection of radiation-induced metabolic processes.

    Science.gov (United States)

    Malak, Henryk; Richmond, Robert; Dicello, J F

    2011-02-01

    A new approach to intracellular detection and imaging of metabolic processes and pathways is presented that uses surface plasmon resonance to enhance interactions between photon-absorbing metabolites and metal nanoparticles in contact with cells in vitro or in vivo. Photon absorption in the nanoparticles creates plasmon fields, enhancing intrinsic metabolite fluorescence, thereby increasing absorption and emission rates, creating new spectral emission bands, shortening fluorescence lifetimes, becoming more photo-stable and increasing fluorescent resonance energy transfer efficiency. Because the cells remain viable, it is proposed that the method may be used to interrogate cells prior to and after irradiation, with the potential for automated analyses of intracellular interactive pathways associated with radiation exposures at lower doses than existing technologies. The design and concepts of the instrument are presented along with data for unexposed cells.

  18. The Reducing Capacity of Thioredoxin on Oxidized Thiols in Boiled Wort

    DEFF Research Database (Denmark)

    Murmann, Anne N.; Hägglund, Per; Svensson, Birte

    2017-01-01

    system was also capable of increasing the free thiol concentration, although with lower efficiency to 187 and 170 μM, respectively. The presence of sulfite, an important antioxidant in beer secreted by the yeast during fermentation, was found to inactivate thioredoxin by sulfitolysis. Reduction......Free thiol-containing proteins are suggested to work as antioxidants in beer, but the majority of thiols in wort are present in their oxidized form as disulfides and are therefore not active as antioxidants. Thioredoxin, a disulfide-reducing protein, is released into the wort from some yeast...... and fluorescence detection of thiol-derivatives. When boiled wort was incubated with all components of the thioredoxin system at pH 7.0 and 25 °C for 60 min under anaerobic conditions, the free thiol concentration increased from 25 to 224 μM. At pH values similar to wort (pH 5.7) and beer (pH 4.5), the thioredoxin...

  19. Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1

    NARCIS (Netherlands)

    Serrano, L.M.; Molenaar, D.; Wels, M.W.W.; Teusink, B.; Bron, P.A.; Vos, de W.M.; Smid, E.J.

    2007-01-01

    Background - Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1. Results - We have identified the

  20. Thioredoxin reductase is a key factor in the oxidative stress response of Lactobacillus plantarum WCFS1

    NARCIS (Netherlands)

    Serrano, L.M.; Molenaar, D; Sanders, M.W.W.; Teusink, B.; Bron, P.A.; Vos, W.M. de; Smid, E.J.

    2007-01-01

    ABSTRACT: BACKGROUND: Thioredoxin (TRX) is a powerful disulfide oxido-reductase that catalyzes a wide spectrum of redox reactions in the cell. The aim of this study is to elucidate the role of the TRX system in the oxidative stress response in Lactobacillus plantarum WCFS1. RESULTS: We have

  1. Metabolic Engineering of the Actinomycete Amycolatopsis sp. Strain ATCC 39116 towards Enhanced Production of Natural Vanillin

    OpenAIRE

    Fleige, Christian; Meyer, Florian; Steinbüchel, Alexander

    2016-01-01

    The Gram-positive bacterium Amycolatopsis sp. ATCC 39116 is used for the fermentative production of natural vanillin from ferulic acid on an industrial scale. The strain is known for its outstanding tolerance to this toxic product. In order to improve the productivity of the fermentation process, the strain's metabolism was engineered for higher final concentrations and molar yields. Degradation of vanillin could be decreased by more than 90% through deletion of the vdh gene, which codes for ...

  2. Differential stimulation of luminol-enhanced chemiluminescence (CL) and arachidonic acid metabolism in rat peritoneal neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Sturm, R.J.; Adams, L.M.; Cullinan, C.A.; Berkenkopf, J.W.; Weichman, B.M.

    1986-03-05

    Phorbol 12-myristate, 13-acetate (PMA) induced the production of radical oxygen species (ROS) from rat peritoneal neutrophils as assessed by CL. ROS generation occurred in a time- (maximum at 13.5 min) and dose- (concentration range of 1.7-498 nM) related fashion. However, 166 nM PMA did not induce either cyclooxygenase (CO) or lipoxygenase (LPO) product formation by 20 min post-stimulation. Conversely, A23187, at concentrations between 0.1 and 10 ..mu..M, stimulated both pathways of arachidonic acid metabolism, but had little or no effect upon ROS production. When suboptimal concentrations of PMA (5.5 nM) and A23187 (0.1-1 ..mu..M) were coincubated with the neutrophils, a synergistic ROS response was elicited. However, arachidonic acid metabolism in the presence of PMA was unchanged relative to A12187 alone. Nordihydroguaiaretic acid (NDGA) inhibited both PMA-induced CL (IC/sub 50/ = 0.9 ..mu..M) and A23187-induced arachidonic acid metabolism (IC/sub 50/ = 1.7 ..mu..M and 6.0 ..mu..M for LPO and CO, respectively). The mixed LPO-CO inhibitor, BW755C, behaved in a qualitatively similar manner to NDGA, whereas the CO inhibitors, indomethacin, piroxicam and naproxen had no inhibitory effect on ROS generation at concentrations as high as 100 ..mu..M. These results suggest that NDGA and BW755C may inhibit CL and arachidonic acid metabolism by distinct mechanisms in rat neutrophils.

  3. Neuron-astrocyte interaction enhance GABAergic synaptic transmission in a manner dependent on key metabolic enzymes.

    OpenAIRE

    Przemysław eKaczor; Dariusz eRakus; Jerzy Władysław Mozrzymas; Jerzy Władysław Mozrzymas

    2015-01-01

    GABA is the major inhibitory neurotransmitter in the adult brain and mechanisms of GABAergic inhibition have been intensely investigated in the past decades. Recent studies provided evidence for an important role of astrocytes in shaping GABAergic currents. One of the most obvious, but yet poorly understood, mechanisms of the cross-talk between GABAergic currents and astrocytes is metabolism including neurotransmitter homeostasis. In particular, how modulation of GABAergic currents by astrocy...

  4. Kinetic and thermodynamic properties of two barley thioredoxin h isozymes, HvTrxh1 and HvTrxh2

    DEFF Research Database (Denmark)

    Maeda, Kenji; Hägglund, Per; Björnberg, Olof

    2010-01-01

    -dependent fluorescence, and the barley isozymes, reaction kinetics and thermodynamic properties were readily determined. The reaction constants were 60% higher for HvTrxh1 than HvTrxh2, while their redox potentials were very similar. The primary nucleophile, Cys(N), of the active site Trp-Cys(N)-Gly-Pro-Cys......Barley thioredoxin h isozymes 1 (HvTrxh1) and barley thioredoxin h isozymes 2 (HvTrxh2) show distinct spatiotemporal distribution in germinating seeds. Using a novel approach involving measurement of bidirectional electron transfer rates between Escherichia coli thioredoxin, which exhibits redox...

  5. Phytochemical Composition and Metabolic Performance Enhancing Activity of Dietary Berries Traditionally Used by Native North Americans

    Science.gov (United States)

    Burns Kraft, Tristan F.; Dey, Moul; Rogers, Randy B.; Ribnicky, David M.; Gipp, David M.; Cefalu, William T.; Raskin, Ilya; Lila, Mary Ann

    2009-01-01

    Four wild berry species, Amelanchier alnifolia, Viburnum trilobum, Prunus virginiana, and Shepherdia argentea, all integral to the traditional subsistence diet of Native American tribal communities, were evaluated to elucidate phytochemical composition and bioactive properties related to performance and human health. Biological activity was screened using a range of bioassays that assessed the potential for these little-known dietary berries to affect diabetic microvascular complications, hyperglycemia, pro-inflammatory gene expression, and metabolic syndrome symptoms. Non-polar constituents from berries, including carotenoids, were potent inhibitors of aldose reductase (an enzyme involved in the etiology of diabetic microvascular complications) whereas the polar constituents, mainly phenolic acids, anthocyanins, and proanthocyanidins, were hypoglycemic agents and strong inhibitors of IL-1β and COX-2 gene expression. Berry samples also showed the ability to modulate lipid metabolism and energy expenditure in a manner consistent with improving metabolic syndrome. The results demonstrate that these berries traditionally consumed by tribal cultures contain a rich array of phytochemicals that have the capacity to promote health and protect against chronic diseases, such as diabetes. PMID:18211018

  6. Enhanced photosynthetic capacity increases nitrogen metabolism through the coordinated regulation of carbon and nitrogen assimilation in Arabidopsis thaliana.

    Science.gov (United States)

    Otori, Kumi; Tanabe, Noriaki; Maruyama, Toshiki; Sato, Shigeru; Yanagisawa, Shuichi; Tamoi, Masahiro; Shigeoka, Shigeru

    2017-09-01

    Plant growth and productivity depend on interactions between the metabolism of carbon and nitrogen. The sensing ability of internal carbon and nitrogen metabolites (the C/N balance) enables plants to regulate metabolism and development. In order to investigate the effects of an enhanced photosynthetic capacity on the metabolism of carbon and nitrogen in photosynthetically active tissus (source leaves), we herein generated transgenic Arabidopsis thaliana plants (ApFS) that expressed cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in their chloroplasts. The phenotype of ApFS plants was indistinguishable from that of wild-type plants at the immature stage. However, as plants matured, the growth of ApFS plants was superior to that of wild-type plants. Starch levels were higher in ApFS plants than in wild-type plants at 2 and 5 weeks. Sucrose levels were also higher in ApFS plants than in wild-type plants, but only at 5 weeks. On the other hand, the contents of various free amino acids were lower in ApFS plants than in wild-type plants at 2 weeks, but were similar at 5 weeks. The total C/N ratio was the same in ApFS plants and wild-type plants, whereas nitrite levels increased in parallel with elevations in nitrate reductase activity at 5 weeks in ApFS plants. These results suggest that increases in the contents of photosynthetic intermediates at the early growth stage caused a temporary imbalance in the free-C/free-N ratio and, thus, the feedback inhibition of the expression of genes involved in the Calvin cycle and induction of the expression of those involved in nitrogen metabolism due to supply deficient free amino acids for maintenance of the C/N balance in source leaves of ApFS plants.

  7. Enhancing Carbon Fixation by Metabolic Engineering: A Model System of Complex Network Modulation

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Gregory Stephanopoulos

    2008-04-10

    In the first two years of this research we focused on the development of a DNA microarray for transcriptional studies in the photosynthetic organism Synechocystis and the elucidation of the metabolic pathway for biopolymer synthesis in this organism. In addition we also advanced the molecular biological tools for metabolic engineering of biopolymer synthesis in Synechocystis and initiated a series of physiological studies for the elucidation of the carbon fixing pathways and basic central carbon metabolism of these organisms. During the last two-year period we focused our attention on the continuation and completion of the last task, namely, the development of tools for basic investigations of the physiology of these cells through, primarily, the determination of their metabolic fluxes. The reason for this decision lies in the importance of fluxes as key indicators of physiology and the high level of information content they carry in terms of identifying rate limiting steps in a metabolic pathway. While flux determination is a well-advanced subject for heterotrophic organisms, for the case of autotrophic bacteria, like Synechocystis, some special challenges had to be overcome. These challenges stem mostly from the fact that if one uses {sup 13}C labeled CO{sub 2} for flux determination, the {sup 13}C label will mark, at steady state, all carbon atoms of all cellular metabolites, thus eliminating the necessary differentiation required for flux determination. This peculiarity of autotrophic organisms makes it imperative to carry out flux determination under transient conditions, something that had not been accomplished before. We are pleased to report that we have solved this problem and we are now able to determine fluxes in photosynthetic organisms from stable isotope labeling experiments followed by measurements of label enrichment in cellular metabolites using Gas Chromatography-Mass Spectrometry. We have conducted extensive simulations to test the method and

  8. Overexpression of plastidial thioredoxins f and m differentially alters photosynthetic activity and response to oxidative stress in tobacco plants

    Directory of Open Access Journals (Sweden)

    Pascal eREY

    2013-10-01

    Full Text Available Plants display a remarkable diversity of thioredoxins (Trxs, reductases controlling the thiol redox status of proteins. The physiological function of many of them remains elusive, particularly for plastidial Trxs f and m, which are presumed based on biochemical data to regulate photosynthetic reactions and carbon metabolism. Recent reports revealed that Trxs f and m participate in vivo in the control of starch metabolism and cyclic photosynthetic electron transfer around photosystem I, respectively. To further delineate their in planta function, we compared the photosynthetic characteristics, the level and/or activity of various Trx targets and the responses to oxidative stress in transplastomic tobacco plants overexpressing either Trx f or Trx m. We found that plants overexpressing Trx m specifically exhibit altered growth, reduced chlorophyll content, impaired photosynthetic linear electron transfer and decreased pools of glutathione and ascorbate. In both transplastomic lines, activities of two enzymes involved in carbon metabolism, NADP-malate dehydrogenase and NADP-glyceraldehyde-3-phosphate dehydrogenase are markedly and similarly altered. In contrast, plants overexpressing Trx m specifically display increased capacity for methionine sulfoxide reductases, enzymes repairing damaged proteins by regenerating methionine from oxidized methionine. Finally, we also observed that transplastomic plants exhibit distinct responses when exposed to oxidative stress conditions generated by methyl viologen or exposure to high light combined with low temperature, the plants overexpressing Trx m being notably more tolerant than Wt and those overexpressing Trx f. Altogether, these data indicate that Trxs f and m fulfill distinct physiological functions. They prompt us to propose that the m type is involved in key processes linking photosynthetic activity, redox homeostasis and antioxidant mechanisms in the chloroplast.

  9. THE THIOREDOXIN SYSTEM IN REGULATING MCF-7 CELL PROLIFERATION UNDER REDOX STATUS MODULATION

    Directory of Open Access Journals (Sweden)

    E. A. Stepovaya

    2016-01-01

    Full Text Available Introduction. Despite the available data on tumor cell functioning under the conditions of free radical-mediated oxidation, the mechanisms of redox regulation, cell proliferation management and apoptosis avoidance remain understudied.The objective of the study was to identify the role of the thioredoxin system in regulating MCF-7 breast cancer cell proliferation under redox status modulation with 1.4-dithioerythritol.Material and methods. The studies were conducted on the MCF-7 breast cancer cell line, grown in adherent cell culture. Cell redox status was modulated with5 mM N-ethylmaleimide – an SH group and peptide inhibitor and5 mM 1.4-dithioerythritol – a thiol group protector. The cell cycle was evaluated by flow cytometry, the same technique was used to measure the reactive oxygen species concentration. The levels of reduced and oxidized glutathione and the activity of thioredoxin reductase were identified by spectrophotometry. The intracellular concentrations of thioredoxin, cyclin E and cyclin-dependent kinase 2 were determined by Western blot analysis.Results and discussion. The essential role of the thioredoxin system in regulating MCF-7 breast cancer cell proliferation was exhibited. S-phase arrest under the effect of N-ethylmaleimide and G0/G1-phase arrest under the effect of 1.4-dithioerythritol are associated with the changes in the activity of redox-sensitive protein complexes (cyclins and cyclin-dependent kinases that regulate cell proliferation.Conclusion. Redoxdependent modulation of proliferation regulating intracellular protein activity occurs due to the thioredoxin system. This is a promising research area for seeking molecular targets of breast cell malignization. 

  10. Comprehensively Characterizing the Thioredoxin Interactome In Vivo Highlights the Central Role Played by This Ubiquitous Oxidoreductase in Redox Control*

    Science.gov (United States)

    Arts, Isabelle S.; Vertommen, Didier; Baldin, Francesca; Laloux, Géraldine; Collet, Jean-François

    2016-01-01

    Thioredoxin (Trx) is a ubiquitous oxidoreductase maintaining protein-bound cysteine residues in the reduced thiol state. Here, we combined a well-established method to trap Trx substrates with the power of bacterial genetics to comprehensively characterize the in vivo Trx redox interactome in the model bacterium Escherichia coli. Using strains engineered to optimize trapping, we report the identification of a total 268 Trx substrates, including 201 that had never been reported to depend on Trx for reduction. The newly identified Trx substrates are involved in a variety of cellular processes, ranging from energy metabolism to amino acid synthesis and transcription. The interaction between Trx and two of its newly identified substrates, a protein required for the import of most carbohydrates, PtsI, and the bacterial actin homolog MreB was studied in detail. We provide direct evidence that PtsI and MreB contain cysteine residues that are susceptible to oxidation and that participate in the formation of an intermolecular disulfide with Trx. By considerably expanding the number of Trx targets, our work highlights the role played by this major oxidoreductase in a variety of cellular processes. Moreover, as the dependence on Trx for reduction is often conserved across species, it also provides insightful information on the interactome of Trx in organisms other than E. coli. PMID:27081212

  11. Thioredoxins, Glutaredoxins, and Peroxiredoxins—Molecular Mechanisms and Health Significance: from Cofactors to Antioxidants to Redox Signaling

    Science.gov (United States)

    Hanschmann, Eva-Maria; Godoy, José Rodrigo; Berndt, Carsten; Hudemann, Christoph

    2013-01-01

    Abstract Thioredoxins (Trxs), glutaredoxins (Grxs), and peroxiredoxins (Prxs) have been characterized as electron donors, guards of the intracellular redox state, and “antioxidants”. Today, these redox catalysts are increasingly recognized for their specific role in redox signaling. The number of publications published on the functions of these proteins continues to increase exponentially. The field is experiencing an exciting transformation, from looking at a general redox homeostasis and the pathological oxidative stress model to realizing redox changes as a part of localized, rapid, specific, and reversible redox-regulated signaling events. This review summarizes the almost 50 years of research on these proteins, focusing primarily on data from vertebrates and mammals. The role of Trx fold proteins in redox signaling is discussed by looking at reaction mechanisms, reversible oxidative post-translational modifications of proteins, and characterized interaction partners. On the basis of this analysis, the specific regulatory functions are exemplified for the cellular processes of apoptosis, proliferation, and iron metabolism. The importance of Trxs, Grxs, and Prxs for human health is addressed in the second part of this review, that is, their potential impact and functions in different cell types, tissues, and various pathological conditions. Antioxid. Redox Signal. 19, 1539–1605. PMID:23397885

  12. Lack of mitochondrial thioredoxin o1 is compensated by antioxidant components under salinity in Arabidopsis thaliana plants.

    Science.gov (United States)

    Calderón, Aingeru; Sánchez-Guerrero, Antonio; Ortiz-Espín, Ana; Martínez-Alcalá, Isabel; Camejo, Daymi; Jiménez, Ana; Sevilla, Francisca

    2018-02-15

    In a changing environment, plants are able to acclimate to the new conditions by regulating their metabolism through the antioxidant and redox systems involved in the stress response. Here we studied a mitochondrial thioredoxin in wild type (WT) Arabidopis thaliana and two Attrxo1 mutant lines grown in the absence or presence of 100 mM NaCl. Compared to WT plants, no evident phenotype was observed in the mutant plants in control condition, although they had higher number of stomata, loss of water, nitric oxide and carbonyl protein contents as well as higher activity of superoxide dismutase (SOD) and catalase enzymes than WT plants. Under salinity, the mutants presented lower water loss and higher stomatal closure, H 2 O 2 and lipid peroxidation levels accompanied by higher enzymatic activity of catalase and the different SOD isoenzymes compared to WT plants. These inductions may collaborate in the maintenance of plant integrity and growth observed under saline conditions, possibly as a way to compensate the lack of TRXo1. We discuss the potential of TRXo1 to influence the development of the whole plant under saline conditions, which have great value for the agronomy of plants growing under unfavourable environment. This article is protected by copyright. All rights reserved.

  13. Low molecular weight thiols and thioredoxins are important players in Hg(II) resistance in Thermus thermophilus HB27.

    Science.gov (United States)

    Norambuena, J; Wang, Y; Hanson, T; Boyd, J M; Barkay, T

    2017-11-17

    Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low molecular weight and protein thiols may be important cell components used in Hg resistance. To date, the role of low molecular weight thiols in Hg-detoxification remains understudied. The mercury resistance ( mer ) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low molecular weight thiol metabolism. This mer operon encodes for an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low molecular weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in presence of either a thiol oxidizing agent or a thiol alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 μM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus spp. mer operons. The presence of a thiol related gene was also detected in some alphaprotobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II) buffering agents while Hg is reduced by MerA. Importance The survival of microorganisms in presence of toxic metals is central to life's sustainability. The affinity of thiol groups to toxic heavy metals drives microbe-metal interactions and modulate metal toxicity. Mercury detoxification ( mer ) genes likely originated early in microbial evolution among geothermal environments. Little is

  14. Thioredoxin mitigates radiation-induced hematopoietic stem cell injury in mice

    Directory of Open Access Journals (Sweden)

    Pasupathi Sundaramoorthy

    2017-11-01

    Full Text Available Abstract Background Radiation exposure poses a significant threat to public health. Hematopoietic injury is one of the major manifestations of acute radiation sickness. Protection and/or mitigation of hematopoietic stem cells (HSCs from radiation injury is an important goal in the development of medical countermeasure agents (MCM. We recently identified thioredoxin (TXN as a novel molecule that has marked protective and proliferative effects on HSCs. In the current study, we investigated the effectiveness of TXN in rescuing mice from a lethal dose of total body radiation (TBI and in enhancing hematopoietic reconstitution following a lethal dose of irradiation. Methods We used in-vivo and in-vitro methods to understand the biological and molecular mechanisms of TXN on radiation mitigation. BABL/c mice were used for the survival study and a flow cytometer was used to quantify the HSC population and cell senescence. A hematology analyzer was used for the peripheral blood cell count, including white blood cells (WBCs, red blood cells (RBCs, hemoglobin, and platelets. Colony forming unit (CFU assay was used to study the colongenic function of HSCs. Hematoxylin and eosin staining was used to determine the bone marrow cellularity. Senescence-associated β-galactosidase assay was used for cell senescence. Western blot analysis was used to evaluate the DNA damage and senescence protein expression. Immunofluorescence staining was used to measure the expression of γ-H2AX foci for DNA damage. Results We found that administration of TXN 24 h following irradiation significantly mitigates BALB/c mice from TBI-induced death: 70% of TXN-treated mice survived, whereas only 25% of saline-treated mice survived. TXN administration led to enhanced recovery of peripheral blood cell counts, bone marrow cellularity, and HSC population as measured by c-Kit+Sca-1+Lin– (KSL cells, SLAM + KSL cells and CFUs. TXN treatment reduced cell senescence and radiation

  15. Towards the understanding of microbial metabolism in relation to microbial enhanced oil recovery

    DEFF Research Database (Denmark)

    Halim, Amalia Yunita; Nielsen, Sidsel Marie; Nielsen, Kristian Fog

    2017-01-01

    In this study, Bacillus licheniformis 421 was used as a model organism to understand the effects of microbial cell growth and metabolite production under anaerobic conditions in relation to microbial enhanced oil recovery. The bacterium was able to grow anaerobically on different carbon compounds...

  16. Enhancement of Naringenin Biosynthesis from Tyrosine by Metabolic Engineering of Saccharomyces cerevisiae.

    Science.gov (United States)

    Lyu, Xiaomei; Ng, Kuan Rei; Lee, Jie Lin; Mark, Rita; Chen, Wei Ning

    2017-08-09

    Flavonoids are an important class of plant polyphenols that possess a variety of health benefits. In this work, S. cerevisiae was metabolically engineered to produce the flavonoid naringenin, using tyrosine as the precursor. Our strategy to improve naringenin production comprised three modules. In module 1, we employed a modified GAL system to overexpress the genes of the naringenin biosynthesis pathway and investigated their synergistic action. In module 2, we simultaneously up-regulated acetyl-CoA production and down-regulated fatty acid biosynthesis in order to increase the precursor supply, malonyl-CoA. In module 3, we engineered the tyrosine biosynthetic pathway to eliminate the feedback inhibition of tyrosine and also down-regulated competing pathways. It was found that modules 1 and 3 played important roles in improving naringenin production. We succeeded in producing up to ∼90 mg/L of naringenin in our final strain, which is a 20-fold increase as compared to the parental strain.

  17. Hypoxia Induces a Metabolic Shift and Enhances the Stemness and Expansion of Cochlear Spiral Ganglion Stem/Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Hsin-Chien Chen

    2015-01-01

    Full Text Available Previously, we demonstrated that hypoxia (1% O2 enhances stemness markers and expands the cell numbers of cochlear stem/progenitor cells (SPCs. In this study, we further investigated the long-term effect of hypoxia on stemness and the bioenergetic status of cochlear spiral ganglion SPCs cultured at low oxygen tensions. Spiral ganglion SPCs were obtained from postnatal day 1 CBA/CaJ mouse pups. The measurement of oxygen consumption rate, extracellular acidification rate (ECAR, and intracellular adenosine triphosphate levels corresponding to 20% and 5% oxygen concentrations was determined using a Seahorse XF extracellular flux analyzer. After low oxygen tension cultivation for 21 days, the mean size of the hypoxia-expanded neurospheres was significantly increased at 5% O2; this correlated with high-level expression of hypoxia-inducible factor-1 alpha (Hif-1α, proliferating cell nuclear antigen (PCNA, cyclin D1, Abcg2, nestin, and Nanog proteins but downregulated expression of p27 compared to that in a normoxic condition. Low oxygen tension cultivation tended to increase the side population fraction, with a significant difference found at 5% O2 compared to that at 20% O2. In addition, hypoxia induced a metabolic energy shift of SPCs toward higher basal ECARs and higher maximum mitochondrial respiratory capacity but lower proton leak than under normoxia, where the SPC metabolism was switched toward glycolysis in long-term hypoxic cultivation.

  18. The progestin etonogestrel enhances the respiratory response to metabolic acidosis in newborn rats. Evidence for a mechanism involving supramedullary structures.

    Science.gov (United States)

    Loiseau, Camille; Osinski, Diane; Joubert, Fanny; Straus, Christian; Similowski, Thomas; Bodineau, Laurence

    2014-05-01

    Central congenital hypoventilation syndrome is a neuro-respiratory disease characterized by the dysfunction of the CO2/H(+) chemosensitive neurons of the retrotrapezoid nucleus/parafacial respiratory group. A recovery of CO2/H(+) chemosensitivity has been observed in some central congenital hypoventilation syndrome patients coincidental with contraceptive treatment by a potent progestin, desogestrel (Straus et al., 2010). The mechanisms of this progestin effect remain unknown, although structures of medulla oblongata, midbrain or diencephalon are known to be targets for progesterone. In the present study, on ex vivo preparations of central nervous system of newborn rats, we show that acute exposure to etonogestrel (active metabolite of desogestrel) enhanced the increased respiratory frequency induced by metabolic acidosis via a mechanism involving supramedullary structures located in pontine, mesencephalic or diencephalic regions. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. Limited uptake, translocation and enhanced metabolic degradation contribute to glyphosate tolerance in Mucuna pruriens var. utilis plants.

    Science.gov (United States)

    Rojano-Delgado, Antonia María; Cruz-Hipolito, Hugo; De Prado, Rafael; Luque de Castro, María Dolores; Franco, Antonio Rodríguez

    2012-01-01

    Velvet bean (Mucuna pruriens, Fabaceae) plants exhibits an innate, very high resistance (i.e., tolerance) to glyphosate similar to that of plants which have acquired resistance to this herbicide as a trait. We analyzed the uptake of [(14)C]-glyphosate by leaves and its translocation to meristematic tissues, and used scanning electron micrographs to further analyze the cuticle and 3D capillary electrophoresis to investigate a putative metabolism capable of degrading the herbicide. Velvet bean exhibited limited uptake of glyphosate and impaired translocation of the compound to meristematic tissues. Also, for the first time in a higher plant, two concurrent pathways capable of degrading glyphosate to AMPA, Pi, glyoxylate, sarcosine and formaldehyde as end products were identified. Based on the results, the innate tolerance of velvet bean to glyphosate is possibly a result of the combined action of the previous three traits, namely: limited uptake, impaired translocation and enhanced degradation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal

    DEFF Research Database (Denmark)

    Kristiansen, Rikke; Nguyen, Hien Thi Thu; Saunders, Aaron Marc

    2013-01-01

    Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to ‘Candidatus Accumuliba......Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to ‘Candidatus....... japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaerarelated PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate...... by ‘Candidatus Accumulibacter phosphatis’, and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation....

  1. Immunoproteomics based identification of thioredoxin reductase GliT and novel Aspergillus fumigatus antigens for serologic diagnosis of invasive aspergillosis.

    Science.gov (United States)

    Shi, Li-ning; Li, Fang-qiu; Huang, Mei; Lu, Jing-fen; Kong, Xiao-xiang; Wang, Shi-qin; Shao, Hai-feng

    2012-01-18

    There has been a rising incidence of invasive aspergillosis (IA) in critically ill patients, even in the absence of an apparent predisposing immunodeficiency. The diagnosis of IA is difficult because clinical signs are not sensitive and specific, and serum galactomannan has relatively low sensitivity in this group of patients. Therefore, more prompt and accurate disease markers for early diagnosis are needed. To establish disease markers demands a thorough knowledge of fungal antigens which may be detected in the serum or other body fluids of patients. Herein we report novel immunodominant antigens identified from extracellular proteins of Aspergillus fumigatus. Extracellular proteins of A. fumigatus were separated by two-dimensional electrophoresis (2-DE) and probed with the sera from critically ill patients with proven IA. The immunoreactive protein spots were identified by MALDI-TOF mass spectrometry (MALDI-TOF -MS). Forty spots from 2DE gels were detected and 17 different proteins were identified as immunogenic in humans. Function annotation revealed that most of these proteins were metabolic enzymes involved in carbohydrate, fatty acid, amino acid, and energy metabolism. One of the proteins, thioredoxin reductase GliT (TR), which showed the best immunoactivity, was analyzed further for secretory signals, protein localization, and homology. The results indicated that TR is a secretory protein with a signal sequence exhibiting a high probability for secretion. Furthermore, TR did not match any human proteins, and had low homology with most other fungi. The recombinant TR was recognized by the sera of all proven IA patients with different underlying diseases in this study. The immunoreactive proteins identified in this study may be helpful for the diagnosis of IA in critically ill patients. Our results indicate that TR and other immunodominant antigens have potential as biomarkers for the serologic diagnosis of invasive aspergillosis.

  2. Immunoproteomics based identification of thioredoxin reductase GliT and novel Aspergillus fumigatus antigens for serologic diagnosis of invasive aspergillosis

    Directory of Open Access Journals (Sweden)

    Shi Li-ning

    2012-01-01

    Full Text Available Abstract Background There has been a rising incidence of invasive aspergillosis (IA in critically ill patients, even in the absence of an apparent predisposing immunodeficiency. The diagnosis of IA is difficult because clinical signs are not sensitive and specific, and serum galactomannan has relatively low sensitivity in this group of patients. Therefore, more prompt and accurate disease markers for early diagnosis are needed. To establish disease markers demands a thorough knowledge of fungal antigens which may be detected in the serum or other body fluids of patients. Herein we report novel immunodominant antigens identified from extracellular proteins of Aspergillus fumigatus. Results Extracellular proteins of A. fumigatus were separated by two-dimensional electrophoresis (2-DE and probed with the sera from critically ill patients with proven IA. The immunoreactive protein spots were identified by MALDI-TOF mass spectrometry (MALDI-TOF -MS. Forty spots from 2DE gels were detected and 17 different proteins were identified as immunogenic in humans. Function annotation revealed that most of these proteins were metabolic enzymes involved in carbohydrate, fatty acid, amino acid, and energy metabolism. One of the proteins, thioredoxin reductase GliT (TR, which showed the best immunoactivity, was analyzed further for secretory signals, protein localization, and homology. The results indicated that TR is a secretory protein with a signal sequence exhibiting a high probability for secretion. Furthermore, TR did not match any human proteins, and had low homology with most other fungi. The recombinant TR was recognized by the sera of all proven IA patients with different underlying diseases in this study. Conclusions The immunoreactive proteins identified in this study may be helpful for the diagnosis of IA in critically ill patients. Our results indicate that TR and other immunodominant antigens have potential as biomarkers for the serologic diagnosis

  3. Molecular cloning and characteristic analysis of a thioredoxin from ...

    African Journals Online (AJOL)

    user

    2012-04-12

    Apr 12, 2012 ... which is conserved in bacteria, plants, and animals. (Spyrou et al. .... 1 h with HRP-conjugated secondary antibodies. Proteins .... metabolism. They are .... Neobenedenia melleni (Monogenea: Capsalidae) from marine fishes in Fujian ... (BbTrx) from the entomopathogenic fungus Beauveria bassiana and.

  4. Mechanisms relevant to the enhanced virulence of a dihydroxynaphthalene-melanin metabolically engineered entomopathogen.

    Directory of Open Access Journals (Sweden)

    Min-Nan Tseng

    Full Text Available The entomopathogenic fungus Metarhizium anisopliae MA05-169 is a transformant strain that has been metabolically engineered to express dihydroxynaphthalene-melanin biosynthesis genes. In contrast to the wild type strain, the transformant displays a greater resistance to environmental stress and a higher virulence toward target insect host. However, the underlying mechanisms for these characteristics remain unclear; hence experiments were initiated to explore the possible mechanism(s through physiological and molecular approaches. Although both transformant and wild type strains could infect and share the same insect host range, the former germinated faster and produced more appressoria than the latter, both in vivo and in vitro. The transformant showed a significantly shorter median lethal time (LT50 when infecting the diamondback moth (Plutella xylostella and the striped flea beetle (Phyllotreta striolata, than the wild type. Additionally, the transformant was more tolerant to reactive oxygen species (ROS, produced 40-fold more orthosporin and notably overexpressed the transcripts of the pathogenicity-relevant hydrolytic enzymes (chitinase, protease, and phospholipase genes in vivo. In contrast, appressorium turgor pressure and destruxin A content were slightly decreased compared to the wild type. The transformant's high anti-stress tolerance, its high virulence against five important insect pests (cowpea aphid Aphis craccivora, diamondback moth Pl. xylostella, striped flea beetle Ph. striolata, and silverleaf whitefly Bemisia argentifolii and its capacity to colonize the root system are key properties for its potential bio-control field application.

  5. Metabolic engineering of Escherichia coli to enhance shikimic acid production from sorbitol.

    Science.gov (United States)

    Liu, Xianglei; Lin, Jun; Hu, Haifeng; Zhou, Bin; Zhu, Baoquan

    2014-09-01

    Shikimic acid (SA) is the key synthetic material of Oseltamivir, which is an effective drug for the prevention and treatment of influenza. In this study, to block the downstream metabolic pathway of SA, the shikimate kinase isoenzyme genes aroK and aroL were deleted by Red recombination. Moreover, the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed by constructing the recombinant vector pETDuet-GBAE. As a result, SA production of E. coli BW25113 (∆aroL/aroK, DE3)/pETDuet-GBAE reached 1,077.6 mg/l when low amounts of sorbitol (5 g/l) were fed in shake flasks. The yield was 3.7 times that when glucose was used (P sorbitol was an optimized carbon source for the high efficient accumulation of SA for the first time, which was applicable to use in the industry for high yields and low consumption.

  6. Engineering functional artificial hybrid proteins between poplar peroxiredoxin II and glutaredoxin or thioredoxin

    International Nuclear Information System (INIS)

    Rouhier, Nicolas; Gama, Filipe; Wingsle, Gunnar; Gelhaye, Eric; Gans, Pierre; Jacquot, Jean-Pierre

    2006-01-01

    The existence of natural peroxiredoxin-glutaredoxin hybrid enzymes in several bacteria is in line with previous findings indicating that poplar peroxiredoxin II can use glutaredoxin as an electron donor. This peroxiredoxin remains however unique since it also uses thioredoxin with a quite good efficiency. Based on the existing fusions, we have created artificial enzymes containing a poplar peroxiredoxin module linked to glutaredoxin or thioredoxin modules. The recombinant fusion enzymes folded properly into non-covalently bound homodimers or homotetramers. Two of the three protein constructs exhibit peroxidase activity, a reaction where the two modules need to function together, but they also display enzymatic activities specific of each module. In addition, mass spectrometry analyses indicate that the Prx module can be both glutathiolated or overoxidized in vitro. This is discussed in the light of the Prx reactivity

  7. Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERα in complex with synthetic ligands

    International Nuclear Information System (INIS)

    Cura, Vincent; Gangloff, Monique; Eiler, Sylvia; Moras, Dino; Ruff, Marc

    2007-01-01

    A new crystallization strategy: the presence of cleaved thioredoxin fusion is critical for crystallization of the estrogen nuclear receptor ligand binding domain in complex with synthetic ligands. This novel technique should be regarded as an interesting alternative for crystallization of difficult proteins. The ligand-binding domain (LBD) of human oestrogen receptor α was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD–ligand–coactivator peptide complexes at 2.3 Å resolution. This technique is likely to be applicable to other low-solubility proteins

  8. Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis.

    Science.gov (United States)

    Tanaka, Toru; Hosoi, Fumihito; Yamaguchi-Iwai, Yuko; Nakamura, Hajime; Masutani, Hiroshi; Ueda, Shugo; Nishiyama, Akira; Takeda, Shunichi; Wada, Hiromi; Spyrou, Giannis; Yodoi, Junji

    2002-04-02

    Thioredoxin-2 (Trx-2) is a mitochondria-specific member of the thioredoxin superfamily. Mitochondria have a crucial role in the signal transduction for apoptosis. To investigate the biological significance of Trx-2, we cloned chicken TRX-2 cDNA and generated clones of the conditional Trx-2-deficient cells using chicken B-cell line, DT40. Here we show that TRX-2 is an essential gene and that Trx-2-deficient cells undergo apoptosis upon repression of the TRX-2 transgene, showing an accumulation of intracellular reactive oxygen species (ROS). Cytochrome c is released from mitochondria, while caspase-9 and caspase-3, but not caspase-8, are activated upon inhibition of the TRX-2 transgene. In addition, Trx-2 and cytochrome c are co-immunoprecipitated in an in vitro assay. These results suggest that mitochondrial Trx-2 is essential for cell viability, playing a crucial role in the scavenging ROS in mitochondria and regulating the mitochondrial apoptosis signaling pathway.

  9. Repurposing Auranofin, Ebselen, and PX-12 as Antimicrobial Agents Targeting the Thioredoxin System

    Directory of Open Access Journals (Sweden)

    Holly C. May

    2018-03-01

    Full Text Available As microbial resistance to drugs continues to rise at an alarming rate, finding new ways to combat pathogens is an issue of utmost importance. Development of novel and specific antimicrobial drugs is a time-consuming and expensive process. However, the re-purposing of previously tested and/or approved drugs could be a feasible way to circumvent this long and costly process. In this review, we evaluate the U.S. Food and Drug Administration tested drugs auranofin, ebselen, and PX-12 as antimicrobial agents targeting the thioredoxin system. These drugs have been shown to act on bacterial, fungal, protozoan, and helminth pathogens without significant toxicity to the host. We propose that the thioredoxin system could serve as a useful therapeutic target with broad spectrum antimicrobial activity.

  10. Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    LENUS (Irish Health Repository)

    Douillard, Francois P

    2011-08-09

    Abstract Background The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and\\/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis. Results Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU\\/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment. Conclusions This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.

  11. Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase.

    Science.gov (United States)

    Gandin, Valentina; Fernandes, Aristi Potamitou; Rigobello, Maria Pia; Dani, Barbara; Sorrentino, Francesca; Tisato, Francesco; Björnstedt, Mikael; Bindoli, Alberto; Sturaro, Alberto; Rella, Rocco; Marzano, Cristina

    2010-01-15

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH (nicotinamide adenine dinucleotide phosphate), plays a central role in regulating cellular redox homeostasis and signaling pathways. TrxR, overexpressed in many tumor cells and contributing to drug resistance, has emerged as a new target for anticancer drugs. Gold complexes have been validated as potent TrxR inhibitors in vitro in the nanomolar range. In order to obtain potent and selective TrxR inhibitors, we have synthesized a series of linear, 'auranofin-like' gold(I) complexes all containing the [Au(PEt(3))](+) synthon and the ligands: Cl(-), Br(-), cyanate, thiocyanate, ethylxanthate, diethyldithiocarbamate and thiourea. Phosphine gold(I) complexes efficiently inhibited cytosolic and mitochondrial TrxR at concentrations that did not affect the two related oxidoreductases glutathione reductase (GR) and glutathione peroxidase (GPx). The inhibitory effect of the redox proteins was also observed intracellularly in cancer cells pretreated with gold(I) complexes. Gold(I) compounds were found to induce antiproliferative effects towards several human cancer cells some of which endowed with cisplatin or multidrug resistance. In addition, they were able to activate caspase-3 and induce apoptosis observed as nucleosome formation and sub-G1 cell accumulation. The complexes with thiocyanate and xanthate ligands were particularly effective in inhibiting thioredoxin reductase and inducing apoptosis. Pharmacodynamic studies in human ovarian cancer cells allowed for the correlation of intracellular drug accumulation with TrxR inhibition that leads to the induction of apoptosis via the mitochondrial pathway.

  12. Protective effects of the thioredoxin and glutaredoxin systems in dopamine-induced cell death

    OpenAIRE

    Arodin, Lisa; Miranda-Vizuete, Antonio; Swoboda, Peter; Fernandes, Aristi P.

    2014-01-01

    Although the etiology of sporadic Parkinson disease (PD) is unknown, it is well established that oxidative stress plays an important role in the pathogenic mechanism. The thioredoxin (Trx) and glutaredoxin (Grx) systems are two central systems upholding the sulfhydryl homeostasis by reducing disulfides and mixed disulfides within the cell and thereby protecting against oxidative stress. By examining the expression of redox proteins in human postmortem PD brains, we found the levels of Trx1 an...

  13. Cell death by SecTRAPs: thioredoxin reductase as a prooxidant killer of cells.

    Directory of Open Access Journals (Sweden)

    Karin Anestål

    Full Text Available BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins can be formed from the selenoprotein thioredoxin reductase (TrxR by targeting of its selenocysteine (Sec residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity.

  14. Enhanced thyroid iodine metabolism in patients with triiodothyronine-predominant Graves' disease

    International Nuclear Information System (INIS)

    Takamatsu, J.; Hosoya, T.; Naito, N.

    1988-01-01

    Some patients with hyperthyroid Graves' disease have increased serum T3 and normal or even low serum T4 levels during treatment with antithyroid drugs. These patients with elevated serum T3 to T4 ratios rarely have a remission of their hyperthyroidism. The aim of this study was to investigate thyroid iodine metabolism in such patients, whom we termed T3-predominant Graves' disease. Mean thyroid radioactive iodine uptake was 51.0 +/- 18.1% ( +/- SD) at 3 h, and it decreased to 38.9 +/- 20.1% at 24 h in 31 patients with T3-predominant Graves' disease during treatment. It was 20.0 +/- 11.4% at 3 h and increased to 31.9 +/- 16.0% at 24 h in 17 other patients with hyperthyroid Graves' disease who had normal serum T3 and T4 levels and a normal serum T3 to T4 ratio during treatment (control Graves' disease). The activity of serum TSH receptor antibodies was significantly higher in the patients with T3-predominant Graves' disease than in control Graves' disease patients. From in vitro studies of thyroid tissue obtained at surgery, both thyroglobulin content and iodine content in thyroglobulin were significantly lower in patients with T3-predominant Graves' disease than in the control Graves' disease patients. Thyroid peroxidase (TPO) activity determined by a guaiacol assay was 0.411 +/- 0.212 g.u./mg protein in the T3-predominant Graves' disease patients, significantly higher than that in the control Graves' disease patients. Serum TPO autoantibody levels determined by immunoprecipitation also were greater in T3-predominant Graves' disease patients than in control Graves' disease patients. Binding of this antibody to TPO slightly inhibited the enzyme activity of TPO, but this effect of the antibody was similar in the two groups of patients

  15. Effect of signal peptide on stability and folding of Escherichia coli thioredoxin.

    Directory of Open Access Journals (Sweden)

    Pranveer Singh

    Full Text Available The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

  16. Effect of signal peptide on stability and folding of Escherichia coli thioredoxin.

    Science.gov (United States)

    Singh, Pranveer; Sharma, Likhesh; Kulothungan, S Rajendra; Adkar, Bharat V; Prajapati, Ravindra Singh; Ali, P Shaik Syed; Krishnan, Beena; Varadarajan, Raghavan

    2013-01-01

    The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

  17. Nicotinamide nucleotide transhydrogenase (Nnt) links the substrate requirement in brain mitochondria for hydrogen peroxide removal to the thioredoxin/peroxiredoxin (Trx/Prx) system.

    Science.gov (United States)

    Lopert, Pamela; Patel, Manisha

    2014-05-30

    Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H2O2) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H2O2 catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP(+), provides the link between mitochondrial respiration and H2O2 detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H2O2 in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H2O2, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H2O2 catabolism, (b) decreased NADPH and increased NADP(+) levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H2O2 and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H2O2 antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Whey Protein Supplementation Enhances Whole Body Protein Metabolism and Performance Recovery after Resistance Exercise: A Double-Blind Crossover Study.

    Science.gov (United States)

    West, Daniel W D; Abou Sawan, Sidney; Mazzulla, Michael; Williamson, Eric; Moore, Daniel R

    2017-07-11

    No study has concurrently measured changes in free-living whole body protein metabolism and exercise performance during recovery from an acute bout of resistance exercise. We aimed to determine if whey protein ingestion enhances whole body net protein balance and recovery of exercise performance during overnight (10 h) and 24 h recovery after whole body resistance exercise in trained men. In a double-blind crossover design, 12 trained men (76 ± 8 kg, 24 ± 4 years old, 14% ± 5% body fat; means ± standard deviation (SD)) performed resistance exercise in the evening prior to consuming either 25 g of whey protein (PRO; MuscleTech 100% Whey) or an energy-matched placebo (CHO) immediately post-exercise (0 h), and again the following morning (~10 h of recovery). A third randomized trial, completed by the same participants, involving no exercise and no supplement served as a rested control trial (Rest). Participants ingested [ 15 N]glycine to determine whole body protein kinetics and net protein balance over 10 and 24 h of recovery. Performance was assessed pre-exercise and at 0, 10, and 24 h of recovery using a battery of tests. Net protein balance tended to improve in PRO ( P = 0.064; effect size (ES) = 0.61, PRO vs. CHO) during overnight recovery. Over 24 h, net balance was enhanced in PRO ( P = 0.036) but not in CHO ( P = 0.84; ES = 0.69, PRO vs. CHO), which was mediated primarily by a reduction in protein breakdown (PRO protein supplementation improved MVC (ES = 0.76), REP (ES = 0.44), and peak power (ES = 0.55). In conclusion, whey protein supplementation enhances whole body anabolism, and may improve acute recovery of exercise performance after a strenuous bout of resistance exercise.

  19. Microbial metabolism and community structure in response to bioelectrochemically enhanced remediation of petroleum hydrocarbon-contaminated soil.

    Science.gov (United States)

    Lu, Lu; Huggins, Tyler; Jin, Song; Zuo, Yi; Ren, Zhiyong Jason

    2014-04-01

    This study demonstrates that electrodes in a bioelectrochemical system (BES) can potentially serve as a nonexhaustible electron acceptor for in situ bioremediation of hydrocarbon contaminated soil. The deployment of BES not only eliminates aeration or supplement of electron acceptors as in contemporary bioremediation but also significantly shortens the remediation period and produces sustainable electricity. More interestingly, the study reveals that microbial metabolism and community structure distinctively respond to the bioelectrochemically enhanced remediation. Tubular BESs with carbon cloth anode (CCA) or biochar anode (BCA) were inserted into raw water saturated soils containing petroleum hydrocarbons for enhancing in situ remediation. Results show that total petroleum hydrocarbon (TPH) removal rate almost doubled in soils close to the anode (63.5-78.7%) than that in the open circuit positive controls (37.6-43.4%) during a period of 64 days. The maximum current density from the BESs ranged from 73 to 86 mA/m(2). Comprehensive microbial and chemical characterizations and statistical analyses show that the residual TPH has a strongly positive correlation with hydrocarbon-degrading microorganisms (HDM) numbers, dehydrogenase activity, and lipase activity and a negative correlation with soil pH, conductivity, and catalase activity. Distinctive microbial communities were identified at the anode, in soil with electrodes, and soil without electrodes. Uncommon electrochemically active bacteria capable of hydrocarbon degradation such as Comamonas testosteroni, Pseudomonas putida, and Ochrobactrum anthropi were selectively enriched on the anode, while hydrocarbon oxidizing bacteria were dominant in soil samples. Results from genus or phylum level characterizations well agree with the data from cluster analysis. Data from this study suggests that a unique constitution of microbial communities may play a key role in BES enhancement of petroleum hydrocarbons

  20. Enhanced thyroid iodine metabolism in patients with triiodothyronine-predominant Graves' disease

    Energy Technology Data Exchange (ETDEWEB)

    Takamatsu, J.; Hosoya, T.; Naito, N.; Yoshimura, H.; Kohno, Y.; Tarutani, O.; Kuma, K.; Sakane, S.; Takeda, K.; Mozai, T.

    1988-01-01

    Some patients with hyperthyroid Graves' disease have increased serum T3 and normal or even low serum T4 levels during treatment with antithyroid drugs. These patients with elevated serum T3 to T4 ratios rarely have a remission of their hyperthyroidism. The aim of this study was to investigate thyroid iodine metabolism in such patients, whom we termed T3-predominant Graves' disease. Mean thyroid radioactive iodine uptake was 51.0 +/- 18.1% ( +/- SD) at 3 h, and it decreased to 38.9 +/- 20.1% at 24 h in 31 patients with T3-predominant Graves' disease during treatment. It was 20.0 +/- 11.4% at 3 h and increased to 31.9 +/- 16.0% at 24 h in 17 other patients with hyperthyroid Graves' disease who had normal serum T3 and T4 levels and a normal serum T3 to T4 ratio during treatment (control Graves' disease). The activity of serum TSH receptor antibodies was significantly higher in the patients with T3-predominant Graves' disease than in control Graves' disease patients. From in vitro studies of thyroid tissue obtained at surgery, both thyroglobulin content and iodine content in thyroglobulin were significantly lower in patients with T3-predominant Graves' disease than in the control Graves' disease patients. Thyroid peroxidase (TPO) activity determined by a guaiacol assay was 0.411 +/- 0.212 g.u./mg protein in the T3-predominant Graves' disease patients, significantly higher than that in the control Graves' disease patients. Serum TPO autoantibody levels determined by immunoprecipitation also were greater in T3-predominant Graves' disease patients than in control Graves' disease patients. Binding of this antibody to TPO slightly inhibited the enzyme activity of TPO, but this effect of the antibody was similar in the two groups of patients.

  1. Macranthoidin B Modulates Key Metabolic Pathways to Enhance ROS Generation and Induce Cytotoxicity and Apoptosis in Colorectal Cancer

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    Xing Fan

    2018-04-01

    Full Text Available Background/Aims: Induction of oxidative stress and reactive oxygen species (ROS mediated-apoptosis have been utilized as effective strategies in anticancer therapy. Macranthoidin B (MB is a potent inducer of ROS-mediated apoptosis in cancer, but its mechanism of action is poorly understood. Method: Superoxide production with MB exposure in colorectal cancer (CRC cells was measured using lucigenin chemiluminescence and real-time PCR. MB’s inhibitory effect on proliferation and viability of CRC cells was determined by proliferation assays. MB’s effect on apoptosis of CRC cells was determined by Western blotting and annexin V-FITC/PI staining. MB’s effect on the growth of CRC xenografts in mice was assessed. An established metabolomics profiling platform combining ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS with gas chromatography-mass spectrometry (GC-MS was performed to determine MB’s effect on total metabolite variation in CRC cells. Results: We found that MB increases ROS generation via modulating key metabolic pathways. Using metabolomics profiling platform combining LC-MS with GC-MS, a total of 236 metabolites were identified in HCT-116 cells in which 31 metabolites were determined to be significantly regulated (p ≤ 0.05 after MB exposure. A number of key metabolites revealed by metabolomics analysis include glucose, fructose, citrate, arginine, phenylalanine, and S-adenosylhomocysteine (SAH, suggesting specific modulation of metabolism on carbohydrates, amino acids and peptides, lipids, nucleotide, cofactors and vitamins in HCT-116 CRC cells with MB treatment highly associated with apoptosis triggered by enhanced ROS and activated caspase-3. Conclusion: Our results demonstrate that MB represses CRC cell proliferation by inducing ROS-mediated apoptosis.

  2. Macranthoidin B Modulates Key Metabolic Pathways to Enhance ROS Generation and Induce Cytotoxicity and Apoptosis in Colorectal Cancer.

    Science.gov (United States)

    Fan, Xing; Rao, Jun; Zhang, Ziwei; Li, Dengfeng; Cui, Wenhao; Zhang, Jun; Wang, Hua; Tou, Fangfang; Zheng, Zhi; Shen, Qiang

    2018-01-01

    Induction of oxidative stress and reactive oxygen species (ROS) mediated-apoptosis have been utilized as effective strategies in anticancer therapy. Macranthoidin B (MB) is a potent inducer of ROS-mediated apoptosis in cancer, but its mechanism of action is poorly understood. Superoxide production with MB exposure in colorectal cancer (CRC) cells was measured using lucigenin chemiluminescence and real-time PCR. MB's inhibitory effect on proliferation and viability of CRC cells was determined by proliferation assays. MB's effect on apoptosis of CRC cells was determined by Western blotting and annexin V-FITC/PI staining. MB's effect on the growth of CRC xenografts in mice was assessed. An established metabolomics profiling platform combining ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS) with gas chromatography-mass spectrometry (GC-MS) was performed to determine MB's effect on total metabolite variation in CRC cells. We found that MB increases ROS generation via modulating key metabolic pathways. Using metabolomics profiling platform combining LC-MS with GC-MS, a total of 236 metabolites were identified in HCT-116 cells in which 31 metabolites were determined to be significantly regulated (p ≤ 0.05) after MB exposure. A number of key metabolites revealed by metabolomics analysis include glucose, fructose, citrate, arginine, phenylalanine, and S-adenosylhomocysteine (SAH), suggesting specific modulation of metabolism on carbohydrates, amino acids and peptides, lipids, nucleotide, cofactors and vitamins in HCT-116 CRC cells with MB treatment highly associated with apoptosis triggered by enhanced ROS and activated caspase-3. Our results demonstrate that MB represses CRC cell proliferation by inducing ROS-mediated apoptosis. © 2018 The Author(s). Published by S. Karger AG, Basel.

  3. Alleviation of reactive oxygen species enhances PUFA accumulation in Schizochytrium sp. through regulating genes involved in lipid metabolism

    Directory of Open Access Journals (Sweden)

    Sai Zhang

    2018-06-01

    Full Text Available The unicellular heterotrophic thraustochytrids are attractive candidates for commercial polyunsaturated fatty acids (PUFA production. However, the reactive oxygen species (ROS generated in their aerobic fermentation process often limits their PUFA titer. Yet, the specific mechanisms of ROS involvement in the crosstalk between oxidative stress and intracellular lipid synthesis remain poorly described. Metabolic engineering to improve the PUFA yield in thraustochytrids without compromising growth is an important aspect of economic feasibility. To fill this gap, we overexpressed the antioxidative gene superoxide dismutase (SOD1 by integrating it into the genome of thraustochytrid Schizochytrium sp. PKU#Mn4 using a novel genetic transformation system. This study reports the ROS alleviation, enhanced PUFA production and transcriptome changes resulting from the SOD1 overexpression. SOD1 activity in the recombinant improved by 5.2–71.6% along with 7.8–38.5% decline in ROS during the fermentation process. Interestingly, the total antioxidant capacity in the recombinant remained higher than wild-type and above zero in the entire process. Although lipid profile was similar to that of wild-type, the concentrations of major fatty acids in the recombinant were significantly (p ≤ 0.05 higher. The PUFA titer increased up to 1232 ± 41 mg/L, which was 32.9% higher (p ≤ 0.001 than the wild type. Transcriptome analysis revealed strong downregulation of genes potentially involved in β-oxidation of fatty acids in peroxisome and upregulation of genes catalyzing lipid biosynthesis. Our results enrich the knowledge on stress-induced PUFA biosynthesis and the putative role of ROS in the regulation of lipid metabolism in oleaginous thraustochytrids. This study provides a new and alternate strategy for cost-effective industrial fermentation of PUFA. Keywords: Polyunsaturated fatty acids, Schizochytrium sp., Superoxide dismutase, Transgene

  4. Overexpression of Genes Encoding Glycolytic Enzymes in Corynebacterium glutamicum Enhances Glucose Metabolism and Alanine Production under Oxygen Deprivation Conditions

    Science.gov (United States)

    Yamamoto, Shogo; Gunji, Wataru; Suzuki, Hiroaki; Toda, Hiroshi; Suda, Masako; Jojima, Toru; Inui, Masayuki

    2012-01-01

    We previously reported that Corynebacterium glutamicum strain ΔldhAΔppc+alaD+gapA, overexpressing glyceraldehyde-3-phosphate dehydrogenase-encoding gapA, shows significantly improved glucose consumption and alanine formation under oxygen deprivation conditions (T. Jojima, M. Fujii, E. Mori, M. Inui, and H. Yukawa, Appl. Microbiol. Biotechnol. 87:159–165, 2010). In this study, we employ stepwise overexpression and chromosomal integration of a total of four genes encoding glycolytic enzymes (herein referred to as glycolytic genes) to demonstrate further successive improvements in C. glutamicum glucose metabolism under oxygen deprivation. In addition to gapA, overexpressing pyruvate kinase-encoding pyk and phosphofructokinase-encoding pfk enabled strain GLY2/pCRD500 to realize respective 13% and 20% improved rates of glucose consumption and alanine formation compared to GLY1/pCRD500. Subsequent overexpression of glucose-6-phosphate isomerase-encoding gpi in strain GLY3/pCRD500 further improved its glucose metabolism. Notably, both alanine productivity and yield increased after each overexpression step. After 48 h of incubation, GLY3/pCRD500 produced 2,430 mM alanine at a yield of 91.8%. This was 6.4-fold higher productivity than that of the wild-type strain. Intracellular metabolite analysis showed that gapA overexpression led to a decreased concentration of metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, suggesting that the overexpression resolved a bottleneck in glycolysis. Changing ratios of the extracellular metabolites by overexpression of glycolytic genes resulted in reduction of the intracellular NADH/NAD+ ratio, which also plays an important role on the improvement of glucose consumption. Enhanced alanine dehydrogenase activity using a high-copy-number plasmid further accelerated the overall alanine productivity. Increase in glycolytic enzyme activities is a promising approach to make drastic progress in growth-arrested bioprocesses. PMID

  5. Enhanced sulfamethoxazole degradation through ammonia oxidizing bacteria co-metabolism and fate of transformation products.

    Science.gov (United States)

    Kassotaki, Elissavet; Buttiglieri, Gianluigi; Ferrando-Climent, Laura; Rodriguez-Roda, Ignasi; Pijuan, Maite

    2016-05-01

    The occurrence of the widely-used antibiotic sulfamethoxazole (SFX) in wastewaters and surface waters has been reported in a large number of studies. However, the results obtained up-to-date have pointed out disparities in its removal. This manuscript explores the enhanced biodegradation potential of an enriched culture of Ammonia Oxidizing Bacteria (AOB) towards SFX. Several sets of batch tests were conducted to establish a link between SFX degradation and specific ammonia oxidation rate. The occurrence, degradation and generation of SFX and some of its transformation products (4-Nitro SFX, Desamino-SFX and N(4)-Acetyl-SFX) was also monitored. A clear link between the degradation of SFX and the nitrification rate was found, resulting in an increased SFX removal at higher specific ammonia oxidation rates. Moreover, experiments conducted under the presence of allylthiourea (ATU) did not present any removal of SFX, suggesting a connection between the AMO enzyme and SFX degradation. Long term experiments (up to 10 weeks) were also conducted adding two different concentrations (10 and 100 μg/L) of SFX in the influent of a partial nitrification sequencing batch reactor, resulting in up to 98% removal. Finally, the formation of transformation products during SFX degradation represented up to 32%, being 4-Nitro-SFX the most abundant. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation.

    Science.gov (United States)

    Gupta, Sonam; Verma, Dinesh Kumar; Biswas, Joyshree; Rama Raju, K Siva; Joshi, Neeraj; Wahajuddin; Singh, Sarika

    2014-08-01

    This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

    Science.gov (United States)

    Plancarte, Agustin; Nava, Gabriela

    2015-02-01

    Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase

  8. Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Wenqing; Zhang, Baoxin; Duan, Dongzhu [State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000 (China); Wu, Jincai [College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000 (China); Fang, Jianguo, E-mail: fangjg@lzu.edu.cn [State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000 (China); College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000 (China)

    2012-08-01

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100 mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo. -- Highlights: ► Curcumin induces oxidative stress by targeting the thioredoxin system. ► Curcumin-modified TrxR quantitatively oxidizes NADPH to generate ROS. ► Knockdown of TrxR1 augments curcumin's cytotoxicity in HeLa cells.

  9. Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells

    International Nuclear Information System (INIS)

    Cai, Wenqing; Zhang, Baoxin; Duan, Dongzhu; Wu, Jincai; Fang, Jianguo

    2012-01-01

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100 mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo. -- Highlights: ► Curcumin induces oxidative stress by targeting the thioredoxin system. ► Curcumin-modified TrxR quantitatively oxidizes NADPH to generate ROS. ► Knockdown of TrxR1 augments curcumin's cytotoxicity in HeLa cells. ► Curcumin

  10. Whey Protein Supplementation Enhances Whole Body Protein Metabolism and Performance Recovery after Resistance Exercise: A Double-Blind Crossover Study

    Directory of Open Access Journals (Sweden)

    Daniel W. D. West

    2017-07-01

    Full Text Available No study has concurrently measured changes in free-living whole body protein metabolism and exercise performance during recovery from an acute bout of resistance exercise. We aimed to determine if whey protein ingestion enhances whole body net protein balance and recovery of exercise performance during overnight (10 h and 24 h recovery after whole body resistance exercise in trained men. In a double-blind crossover design, 12 trained men (76 ± 8 kg, 24 ± 4 years old, 14% ± 5% body fat; means ± standard deviation (SD performed resistance exercise in the evening prior to consuming either 25 g of whey protein (PRO; MuscleTech 100% Whey or an energy-matched placebo (CHO immediately post-exercise (0 h, and again the following morning (~10 h of recovery. A third randomized trial, completed by the same participants, involving no exercise and no supplement served as a rested control trial (Rest. Participants ingested [15N]glycine to determine whole body protein kinetics and net protein balance over 10 and 24 h of recovery. Performance was assessed pre-exercise and at 0, 10, and 24 h of recovery using a battery of tests. Net protein balance tended to improve in PRO (P = 0.064; effect size (ES = 0.61, PRO vs. CHO during overnight recovery. Over 24 h, net balance was enhanced in PRO (P = 0.036 but not in CHO (P = 0.84; ES = 0.69, PRO vs. CHO, which was mediated primarily by a reduction in protein breakdown (PRO < CHO; P < 0.01. Exercise decreased repetitions to failure (REP, maximal strength (MVC, peak and mean power, and countermovement jump performance (CMJ at 0 h (all P < 0.05 vs. Pre. At 10 h, there were small-to-moderate effects for enhanced recovery of the MVC (ES = 0.56, mean power (ES = 0.49, and CMJ variables (ES: 0.27–0.49 in PRO. At 24 h, protein supplementation improved MVC (ES = 0.76, REP (ES = 0.44, and peak power (ES = 0.55. In conclusion, whey protein supplementation enhances whole body anabolism, and may improve acute recovery of

  11. Metabolic enhancer piracetam attenuates rotenone induced oxidative stress: a study in different rat brain regions.

    Science.gov (United States)

    Verma, Dinesh Kumar; Joshi, Neeraj; Raju, Kunumuri Sivarama; Wahajuddin, Muhammad; Singh, Rama Kant; Singh, Sarika

    2015-01-01

    Piracetam is clinically being used nootropic drug but the details of its neuroprotective mechanism are not well studied. The present study was conducted to assess the effects of piracetam on rotenone induced oxidative stress by using both ex vivo and in vivo test systems. Rats were treated with piracetam (600 mg/kg b.w. oral) for seven constitutive days prior to rotenone administration (intracerebroventricular, 12 µg) in rat brain. Rotenone induced oxidative stress was assessed after 1 h and 24 h of rotenone administration. Ex vivo estimations were performed by using two experimental designs. In one experimental design the rat brain homogenate was treated with rotenone (1 mM, 2 mM and 4 mM) and rotenone+piracetam (10 mM) for 1 h. While in second experimental design the rats were pretreated with piracetam for seven consecutive days. On eighth day the rats were sacrificed, brain homogenate was prepared and treated with rotenone (1 mM, 2 mM and 4mM) for 1h. After treatment the glutathione (GSH) and malondialdehyde (MDA) levels were estimated in brain homogenate. In vivo study showed that pretreatment of piracetam offered significant protection against rotenone induced decreased GSH and increased MDA level though the protection was region specific. But the co-treatment of piracetam with rotenone did not offer significant protection against rotenone induced oxidative stress in ex vivo study. Whereas ex vivo experiments in rat brain homogenate of piracetam pretreated rats, showed the significant protection against rotenone induced oxidative stress. Findings indicated that pretreatment of piracetam significantly attenuated the rotenone induced oxidative stress though the protection was region specific. Piracetam treatment to rats led to its absorption and accumulation in different brain regions as assessed by liquid chromatography mass spectrometry/mass spectrometry. In conclusion, study indicates the piracetam is able to enhance the antioxidant capacity in brain cells

  12. Impaired cross-talk between the thioredoxin and glutathione systems is related to ASK-1 mediated apoptosis in neuronal cells exposed to mercury.

    Science.gov (United States)

    Branco, Vasco; Coppo, Lucia; Solá, Susana; Lu, Jun; Rodrigues, Cecília M P; Holmgren, Arne; Carvalho, Cristina

    2017-10-01

    Mercury (Hg) compounds target both cysteine (Cys) and selenocysteine (Sec) residues in peptides and proteins. Thus, the components of the two major cellular antioxidant systems - glutathione (GSH) and thioredoxin (Trx) systems - are likely targets for mercurials. Hg exposure results in GSH depletion and Trx and thioredoxin reductase (TrxR) are prime targets for mercury. These systems have a wide-range of common functions and interaction between their components has been reported. However, toxic effects over both systems are normally treated as isolated events. To study how the interaction between the glutathione and thioredoxin systems is affected by Hg, human neuroblastoma (SH-SY5Y) cells were exposed to 1 and 5μM of inorganic mercury (Hg 2+ ), methylmercury (MeHg) or ethylmercury (EtHg) and examined for TrxR, GSH and Grx levels and activities, as well as for Trx redox state. Phosphorylation of apoptosis signalling kinase 1 (ASK1), caspase-3 activity and the number of apoptotic cells were evaluated to investigate the induction of Trx-mediated apoptotic cell death. Additionally, primary cerebellar neurons from mice depleted of mitochondrial Grx2 (mGrx2D) were used to examine the link between Grx activity and Trx function. Results showed that Trx was affected at higher exposure levels than TrxR, especially for EtHg. GSH levels were only significantly affected by exposure to a high concentration of EtHg. Depletion of GSH with buthionine sulfoximine (BSO) severely increased Trx oxidation by Hg. Notably, EtHg-induced oxidation of Trx was significantly enhanced in primary neurons of mGrx2D mice. Our results suggest that GSH/Grx acts as backups for TrxR in neuronal cells to maintain Trx turnover during Hg exposure, thus linking different mechanisms of molecular and cellular toxicity. Finally, Trx oxidation by Hg compounds was associated to apoptotic hallmarks, including increased ASK-1 phosphorylation, caspase-3 activation and increased number of apoptotic cells

  13. The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase.

    Science.gov (United States)

    Caldas, Thérèse; Malki, Abderrahim; Kern, Renée; Abdallah, Jad; Richarme, Gilbert

    2006-05-12

    Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.

  14. Crystallization and preliminary X-ray analysis of the N-terminal domain of human thioredoxin-interacting protein

    International Nuclear Information System (INIS)

    Polekhina, Galina; Ascher, David Benjamin; Kok, Shie Foong; Waltham, Mark

    2011-01-01

    The N-terminal domain of thioredoxin-interacting protein has been expressed, purified and crystallized. The crystals belonged to a monoclinic space group and diffracted to 3 Å resolution using synchrotron radiation. Thioredoxin-interacting protein (TXNIP) is a negative regulator of thioredoxin and its roles in the pathologies of diabetes and cardiovascular diseases have marked it out as a potential drug target. Expression of TXNIP is robustly induced under various stress conditions such as high glucose, heat shock, UV, H 2 O 2 and mechanical stress amongst others. Elevated levels of TXNIP result in the sequestration and inactivation of thioredoxin, leading to cellular oxidative stress. For some time, this was the only known function of TXNIP; however, more recently the protein has been shown to play a role in regulation of glucose uptake and activation of the inflammasome. Based on the primary sequence, TXNIP is remotely related to β-arrestins, which include the visual arrestins. TXNIP has thus been classified as a member of the α-arrestin family, which to date includes five other members. None of the other α-arrestins are known to interact with thioredoxin, although curiously one has been implicated in glucose uptake. In order to gain insight into the structure–function relationships of the α-arrestin protein family, and particularly that of TXNIP, the N-terminal domain of TXNIP has been crystallized. The crystals belonged to a monoclinic space group and diffracted to 3 Å resolution using synchrotron radiation

  15. Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway.

    Science.gov (United States)

    Jeong, Yu Jeong; An, Chul Han; Woo, Su Gyeong; Park, Ji Hye; Lee, Ki-Won; Lee, Sang-Hoon; Rim, Yeonggil; Jeong, Hyung Jae; Ryu, Young Bae; Kim, Cha Young

    2016-09-01

    The biosynthesis of flavonoids such as anthocyanin and stilbenes has attracted increasing attention because of their potential health benefits. Anthocyanins and stilbenes share common phenylpropanoid precursor pathways. We previously reported that the overexpression of sweetpotato IbMYB1a induced anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum) plants. In the present study, transgenic tobacco (Nicotiana tabacum SR1) plants (STS-OX and ROST-OX) expressing the RpSTS gene encoding stilbene synthase from rhubarb (Rheum palmatum L. cv. Jangyeop) and the RpSTS and VrROMT genes encoding resveratrol O-methyltransferase from frost grape (Vitis riparia) were generated under the control of 35S promoter. Phenotypic alterations in floral organs, such as a reduction in floral pigments and male sterility, were observed in STS-OX transgenic tobacco plants. However, we failed to obtain STS-OX and ROST-OX plants with high levels of resveratrol compounds. Therefore, to improve the production of resveratrol derivatives in plants, we cross-pollinated flowers of STS-OX or ROST-OX and IbMYB1a-OX transgenic lines (SM and RSM). Phenotypic changes in vegetative and reproductive development of SM and RSM plants were observed. Furthermore, by HPLC and LC-MS analyses, we found enhanced production of resveratrol derivatives such as piceid, piceid methyl ether, resveratrol methyl ether O-hexoside, and 5-methyl resveratrol-3,4'-O-β-D-diglucopyranoside in SM and RSM cross-pollinated lines. Here, total contents of trans- and cis-piceids ranged from approximately 104-240 µg/g fresh weight in SM (F2). Collectively, we suggest that coexpression of RpSTS and IbMYB1a via cross-pollination can induce enhanced production of resveratrol compounds in plants by increasing metabolic flux into stilbenoid biosynthesis.

  16. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

    Directory of Open Access Journals (Sweden)

    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  17. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

    KAUST Repository

    Aouida, Mustapha; Li, Lixin; Mahjoub, Ali; Alshareef, Sahar; Ali, Zahir; Piatek, Agnieszka Anna; Mahfouz, Magdy M.

    2015-01-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

  18. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

    KAUST Repository

    Aouida, Mustapha

    2015-04-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

  19. The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by beta-amyloid peptide.

    Science.gov (United States)

    Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, W E

    2010-05-01

    beta-Amyloid peptide (Abeta) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Abeta-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Abeta and on neurite outgrowth in PC12 cells were investigated. Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Abeta(1-42). Similar protective effects against Abeta(1-42) were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Abeta load was markedly diminished in the brain of those animals after treatment with piracetam. Abeta production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Abeta-induced mitochondrial dysfunction and Abeta-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Abeta on brain function.

  20. Metabolic engineering of the pentose phosphate pathway for enhanced limonene production in the cyanobacterium Synechocysti s sp. PCC 6803.

    Science.gov (United States)

    Lin, Po-Cheng; Saha, Rajib; Zhang, Fuzhong; Pakrasi, Himadri B

    2017-12-13

    Isoprenoids are diverse natural compounds, which have various applications as pharmaceuticals, fragrances, and solvents. The low yield of isoprenoids in plants makes them difficult for cost-effective production, and chemical synthesis of complex isoprenoids is impractical. Microbial production of isoprenoids has been considered as a promising approach to increase the yield. In this study, we engineered the model cyanobacterium Synechocystis sp. PCC 6803 for sustainable production of a commercially valuable isoprenoid, limonene. Limonene synthases from the plants Mentha spicata and Citrus limon were expressed in cyanobacteria for limonene production. Production of limonene was two-fold higher with limonene synthase from M. spicata than that from C. limon. To enhance isoprenoid production, computational strain design was conducted by applying the OptForce strain design algorithm on Synechocystis 6803. Based on the metabolic interventions suggested by this algorithm, genes (ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase) in the pentose phosphate pathway were overexpressed, and a geranyl diphosphate synthase from the plant Abies grandis was expressed to optimize the limonene biosynthetic pathway. The optimized strain produced 6.7 mg/L of limonene, a 2.3-fold improvement in productivity. Thus, this study presents a feasible strategy to engineer cyanobacteria for photosynthetic production of isoprenoids.

  1. The NADPH thioredoxin reductase C functions as an electron donor to 2-Cys peroxiredoxin in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

    International Nuclear Information System (INIS)

    Sueoka, Keigo; Yamazaki, Teruaki; Hiyama, Tetsuo; Nakamoto, Hitoshi

    2009-01-01

    An NADPH thioredoxin reductase C was co-purified with a 2-Cys peroxiredoxin by the combination of anion exchange chromatography and electroelution from gel slices after native PAGE from a thermophilic cyanobacterium Thermosynechococcus elongatus as an NAD(P)H oxidase complex induced by oxidative stress. The result provided a strong evidence that the NADPH thioredoxin reductase C interacts with the 2-Cys peroxiredoxin in vivo. An in vitro reconstitution assay with purified recombinant proteins revealed that both proteins were essential for an NADPH-dependent reduction of H 2 O 2 . These results suggest that the reductase transfers the reducing power from NADPH to the peroxiredoxin, which reduces peroxides in the cyanobacterium under oxidative stress. In contrast with other NADPH thioredoxin reductases, the NADPH thioredoxin reductase C contains a thioredoxin-like domain in addition to an NADPH thioredoxin reductase domain in the same polypeptide. Each domain contains a conserved CXYC motif. A point mutation at the CXYC motif in the NADPH thioredoxin reductase domain resulted in loss of the NADPH oxidation activity, while a mutation at the CXYC motif in the thioredoxin-like domain did not affect the electron transfer, indicating that this motif is not essential in the electron transport from NADPH to the 2-Cys peroxiredoxin.

  2. Protein chaperones Q8ZP25_SALTY from Salmonella typhimurium and HYAE_ECOLI from Escherichia coli exhibit thioredoxin-like structures despite lack of canonical thioredoxin active site sequence motive

    Science.gov (United States)

    Parish, David; Benach, Jordi; Liu, Goahua; Singarapu, Kiran Kumar; Xiao, Rong; Acton, Thomas; Su, Min; Bansal, Sonal; Prestegard, James H.; Hunt, John; Montelione, Gaetano T.

    2010-01-01

    The structure of the 142-residue protein Q8ZP25_SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE_ECOLI encoded in the genome of Eschericia coli was determined by NMR. The two proteins belong to Pfam [1] PF07449, which currently comprises 50 members, and belongs itself to the ‘thioredoxin-like clan’. However, protein HYAE_ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE_ECOLI was previously classified as a [NiFe] hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides. PMID:19039680

  3. Resveratrol protects the ovary against chromium-toxicity by enhancing endogenous antioxidant enzymes and inhibiting metabolic clearance of estradiol

    International Nuclear Information System (INIS)

    Banu, Sakhila K.; Stanley, Jone A.; Sivakumar, Kirthiram K.; Arosh, Joe A.; Burghardt, Robert C.

    2016-01-01

    Resveratrol (RVT), a polyphenolic component in grapes and red wine, has been known for its cytoprotective actions against several diseases. However, beneficial effects of RVT against early exposure to endocrine disrupting chemicals (EDCs) have not been understood. EDCs are linked to several ovarian diseases such as premature ovarian failure, polycystic ovary syndrome, early menopause and infertility in women. Hexavalent chromium (CrVI) is a heavy metal EDC, and widely used in > 50 industries. Environmental contamination with CrVI in the US is rapidly increasing, predisposing the human to several illnesses including cancers and still birth. Our lab has been involved in determining the molecular mechanism of CrVI-induced female infertility and intervention strategies to mitigate CrVI effects. Lactating mother rats were exposed to CrVI (50 ppm potassium dichromate) from postpartum days 1–21 through drinking water with or without RVT (10 mg/kg body wt., through oral gavage daily). During this time, F1 females received respective treatments through mother's milk. On postnatal day (PND) 25, blood and the ovary, kidney and liver were collected from the F1 females for analyses. CrVI increased atresia of follicles by increasing cytochrome C and cleaved caspase-3; decreasing antiapoptotic proteins; decreasing estradiol (E 2 ) biosynthesis and enhancing metabolic clearance of E 2 , increasing oxidative stress and decreasing endogenous antioxidants. RVT mitigated the effects of CrVI by upregulating cell survival proteins and AOXs; and restored E 2 levels by inhibiting hydroxylation, glucuronidation and sulphation of E 2 . This is the first study to report the protective effects of RVT against any toxicant in the ovary. - Highlights: • Resveratrol (RVT) protects the ovary against CrVI-toxicity. • RVT mitigated CrVI-induced apoptosis and follicle atresia. • RVT restored estradiol level against CrVI-toxicity. • RVT inhibited metabolic clearance of estradiol in the

  4. Resveratrol protects the ovary against chromium-toxicity by enhancing endogenous antioxidant enzymes and inhibiting metabolic clearance of estradiol

    Energy Technology Data Exchange (ETDEWEB)

    Banu, Sakhila K., E-mail: skbanu@cvm.tamu.edu; Stanley, Jone A.; Sivakumar, Kirthiram K.; Arosh, Joe A.; Burghardt, Robert C.

    2016-07-15

    Resveratrol (RVT), a polyphenolic component in grapes and red wine, has been known for its cytoprotective actions against several diseases. However, beneficial effects of RVT against early exposure to endocrine disrupting chemicals (EDCs) have not been understood. EDCs are linked to several ovarian diseases such as premature ovarian failure, polycystic ovary syndrome, early menopause and infertility in women. Hexavalent chromium (CrVI) is a heavy metal EDC, and widely used in > 50 industries. Environmental contamination with CrVI in the US is rapidly increasing, predisposing the human to several illnesses including cancers and still birth. Our lab has been involved in determining the molecular mechanism of CrVI-induced female infertility and intervention strategies to mitigate CrVI effects. Lactating mother rats were exposed to CrVI (50 ppm potassium dichromate) from postpartum days 1–21 through drinking water with or without RVT (10 mg/kg body wt., through oral gavage daily). During this time, F1 females received respective treatments through mother's milk. On postnatal day (PND) 25, blood and the ovary, kidney and liver were collected from the F1 females for analyses. CrVI increased atresia of follicles by increasing cytochrome C and cleaved caspase-3; decreasing antiapoptotic proteins; decreasing estradiol (E{sub 2}) biosynthesis and enhancing metabolic clearance of E{sub 2}, increasing oxidative stress and decreasing endogenous antioxidants. RVT mitigated the effects of CrVI by upregulating cell survival proteins and AOXs; and restored E{sub 2} levels by inhibiting hydroxylation, glucuronidation and sulphation of E{sub 2}. This is the first study to report the protective effects of RVT against any toxicant in the ovary. - Highlights: • Resveratrol (RVT) protects the ovary against CrVI-toxicity. • RVT mitigated CrVI-induced apoptosis and follicle atresia. • RVT restored estradiol level against CrVI-toxicity. • RVT inhibited metabolic clearance of

  5. Thioredoxin 1 modulates apoptosis induced by bioactive compounds in prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Aida Rodriguez-Garcia

    2017-08-01

    Full Text Available Accumulating evidence suggests that natural bioactive compounds, alone or in combination with traditional chemotherapeutic agents, could be used as potential therapies to fight cancer. In this study, we employed four natural bioactive compounds (curcumin, resveratrol, melatonin, and silibinin and studied their role in redox control and ability to promote apoptosis in androgen sensitive and insensitive prostate cancer cells. Here is shown that curcumin and resveratrol promote ROS production and induce apoptosis in LNCaP and PC-3. An increase in reactive species is a trigger event in curcumin-induced apoptosis and a consequence of resveratrol effects on other pathways within these cells. Moreover, here we demonstrated that these four compounds affect differently one of the main intracellular redox regulator, the thioredoxin system. Exposure to curcumin and resveratrol promoted TRX1 oxidation and altered its subcellular location. Furthermore, resveratrol diminished TRX1 levels in PC-3 cells and increased the expression of its inhibitor TXNIP. Conversly, melatonin and silibinin only worked as cytostatic agents, reducing ROS levels and showing preventive effects against TRX oxidation. All together, this work explores the effect of compounds currently tested as chemo-preventive agents in prostate cancer therapy, on the TRX1 redox state and function. Our work shows the importance that the TRX system might have within the differences found in their mechanisms of action. These bioactive compounds trigger different responses and affect ROS production and redox systems in prostate cancer cells, suggesting the key role that redox-related pathways might play in processes like differentiation or survival in prostate cancer. Keywords: Thioredoxin, Thioredoxin reductase, TXNIP, Prostate cancer, Redox signaling, Apoptosis

  6. Epalrestat increases glutathione, thioredoxin, and heme oxygenase-1 by stimulating Nrf2 pathway in endothelial cells

    Directory of Open Access Journals (Sweden)

    Kaori Yama

    2015-04-01

    Full Text Available Epalrestat (EPS is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH in rat Schwann cells. GSH plays a crucial role in protecting endothelial cells from oxidative stress, thereby preventing vascular diseases. Here we show that EPS increases GSH levels in not only Schwann cells but also endothelial cells. Treatment of bovine aortic endothelial cells (BAECs, an in vitro model of the vascular endothelium, with EPS caused a dramatic increase in intracellular GSH levels. This was concomitant with the up-regulation of glutamate cysteine ligase, an enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Moreover, EPS stimulated the expression of thioredoxin and heme oxygenase-1, which have important redox regulatory functions in endothelial cells. Nuclear factor erythroid 2-related factor 2 (Nrf2 is a key transcription factor that regulates the expression of antioxidant genes. EPS increased nuclear Nrf2 levels in BAECs. Nrf2 knockdown by siRNA suppressed the EPS-induced glutamate cysteine ligase, thioredoxin-1, and heme oxygenase-1 expression. Interestingly, LY294002, an inhibitor of phosphatidylinositol 3-kinase, abolished the EPS-stimulated GSH synthesis, suggesting that the kinase is associated with Nrf2 activation induced by EPS. Furthermore, EPS reduced the cytotoxicity induced by H2O2 and tert-butylhydroperoxide, indicating that EPS plays a role in protecting cells from oxidative stress. Taken together, the results provide evidence that EPS exerts new beneficial effects on endothelial cells by increasing GSH, thioredoxin, and heme oxygenase-1 levels through the activation of Nrf2. We suggest that EPS has the potential to prevent several vascular diseases caused by oxidative stress.

  7. Thioredoxin-dependent Redox Regulation of Chloroplastic Phosphoglycerate Kinase from Chlamydomonas reinhardtii*

    Science.gov (United States)

    Morisse, Samuel; Michelet, Laure; Bedhomme, Mariette; Marchand, Christophe H.; Calvaresi, Matteo; Trost, Paolo; Fermani, Simona; Zaffagnini, Mirko; Lemaire, Stéphane D.

    2014-01-01

    In photosynthetic organisms, thioredoxin-dependent redox regulation is a well established mechanism involved in the control of a large number of cellular processes, including the Calvin-Benson cycle. Indeed, 4 of 11 enzymes of this cycle are activated in the light through dithiol/disulfide interchanges controlled by chloroplastic thioredoxin. Recently, several proteomics-based approaches suggested that not only four but all enzymes of the Calvin-Benson cycle may withstand redox regulation. Here, we characterized the redox features of the Calvin-Benson enzyme phosphoglycerate kinase (PGK1) from the eukaryotic green alga Chlamydomonas reinhardtii, and we show that C. reinhardtii PGK1 (CrPGK1) activity is inhibited by the formation of a single regulatory disulfide bond with a low midpoint redox potential (−335 mV at pH 7.9). CrPGK1 oxidation was found to affect the turnover number without altering the affinity for substrates, whereas the enzyme activation appeared to be specifically controlled by f-type thioredoxin. Using a combination of site-directed mutagenesis, thiol titration, mass spectrometry analyses, and three-dimensional modeling, the regulatory disulfide bond was shown to involve the not strictly conserved Cys227 and Cys361. Based on molecular mechanics calculation, the formation of the disulfide is proposed to impose structural constraints in the C-terminal domain of the enzyme that may lower its catalytic efficiency. It is therefore concluded that CrPGK1 might constitute an additional light-modulated Calvin-Benson cycle enzyme with a low activity in the dark and a TRX-dependent activation in the light. These results are also discussed from an evolutionary point of view. PMID:25202015

  8. Comparative molecular modeling study of Arabidopsis NADPH-dependent thioredoxin reductase and its hybrid protein.

    Directory of Open Access Journals (Sweden)

    Yuno Lee

    Full Text Available 2-Cys peroxiredoxins (Prxs play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C of thioredoxin reductase (TrxR in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D, which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD simulations on AtNTRC and AtNTRA-(Trx-D proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D because of following reasons: i unstable and unfavorable interaction of the linker region, ii shifted Trx domain, and iii different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.

  9. Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Klausen, Louise Kjær; Prag, Søren

    2009-01-01

    in the cytoplasm and nucleus. Txnl1 has thioredoxin activity with a redox potential of about -250 mV. Mutant Txnl1 with one active site cysteine replaced by serine formed disulfide bonds to eEF1A1, a substrate-recruiting factor of the 26S proteasome. eEF1A1 is therefore a likely physiological substrate....... In response to knock-down of Txnl1, ubiquitin-protein conjugates were moderately stabilised. Hence, Txnl1 is the first example of a direct connection between protein reduction and proteolysis, two major intracellular protein quality control mechanisms....

  10. Inactivation of barley limit dextrinase inhibitor by thioredoxin-catalysed disulfide reduction

    DEFF Research Database (Denmark)

    Jensen, Johanne Mørch; Hägglund, Per; Christensen, Hans Erik Mølager

    2012-01-01

    and one glutathionylated cysteine. Here, thioredoxin is shown to progressively reduce disulfide bonds in LDI accompanied by loss of activity. A preferential reduction of the glutathionylated cysteine, as indicated by thiol quantification and molecular mass analysis using electrospray ionisation mass......Barley limit dextrinase (LD) that catalyses hydrolysis of α-1,6 glucosidic linkages in starch-derived dextrins is inhibited by limit dextrinase inhibitor (LDI) found in mature seeds. LDI belongs to the chloroform/methanol soluble protein family (CM-protein family) and has four disulfide bridges...... spectrometry, was not related to LDI inactivation. LDI reduction is proposed to cause conformational destabilisation leading to loss of function....

  11. Hexavalent chromium causes the oxidation of thioredoxin in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Myers, Judith M.; Antholine, William E.; Myers, Charles R.

    2008-01-01

    Hexavalent chromium [Cr(VI)] species such as chromates are cytotoxic. Inhalational exposure is a primary concern in many Cr-related industries and their immediate environments, and bronchial epithelial cells are directly exposed to inhaled Cr(VI). Chromates are readily taken up by cells and are reduced to reactive Cr species which may also result in the generation of reactive oxygen species (ROS). The thioredoxin (Trx) system has a key role in the maintenance of cellular thiol redox balance and is essential for cell survival. Cells normally maintain the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins largely in the reduced state. Redox Western blots were used to assess the redox status of the thioredoxins in normal human bronchial epithelial cells (BEAS-2B) incubated with soluble Na 2 CrO 4 or insoluble ZnCrO 4 for different periods of time. Both chromates caused a dose- and time-dependent oxidation of Trx2 and Trx1. Trx2 was more susceptible in that it could all be converted to the oxidized form, whereas a small amount of reduced Trx1 remained even after prolonged treatment with higher Cr concentrations. Only one of the dithiols, presumably the active site, of Trx1 was oxidized by Cr(VI). Cr(VI) did not cause significant GSH depletion or oxidation indicating that Trx oxidation does not result from a general oxidation of cellular thiols. With purified Trx and thioredoxin reductase (TrxR) in vitro, Cr(VI) also resulted in Trx oxidation. It was determined that purified TrxR has pronounced Cr(VI) reducing activity, so competition for electron flow from TrxR might impair its ability to reduce Trx. The in vitro data also suggested some direct redox interaction between Cr(VI) and Trx. The ability of Cr(VI) to cause Trx oxidation in cells could contribute to its cytotoxic effects, and could have important implications for cell survival, redox-sensitive cell signaling, and the cells' tolerance of other oxidant insults

  12. Thioredoxins in Redox Maintenance and Survival during Oxidative Stress of Bacteroides fragilis▿ †

    OpenAIRE

    Reott, Michael A.; Parker, Anita C.; Rocha, Edson R.; Smith, C. Jeffrey

    2009-01-01

    The anaerobe Bacteroides fragilis is a gram-negative, opportunistic pathogen that is highly aerotolerant and can persist in aerobic environments for extended periods. In this study, the six B. fragilis thioredoxins (Trxs) were investigated to determine their role during oxidative stress. Phylogenetic analyses of Trx protein sequences indicated that four of the six Trxs (TrxA, TrxC, TrxD, and TrxF) belong to the M-type Trx class but were associated with two different M-type lineages. TrxE and ...

  13. Human truncated thioredoxin (Trx80) as a novel mitogenic cytokine for white blood cells

    OpenAIRE

    Pekkari, Klas

    2001-01-01

    Thioredoxin (Trx) is a 12 kDa protein present in all species with a well-conserved active site sequence comprising -Cys-Gly-Pro-Cys-, which catalyzes oxido-reductase reactions. Trx regulates the activity of transcription factors and intracellular signalling pathways, and secreted Trx is a co-cytokine with several interleukins. In addition to full-length Trx a 10 kDa C-terminally truncated form of the protein is produced mainly by monocytes. This protein has unique eosinophil...

  14. Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

    OpenAIRE

    Roberts, Irma N.; Lam, Xuan Tam; Miranda, Helder; Kieselbach, Thomas; Funk, Christiane

    2012-01-01

    The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (...

  15. Acute Metabolic Alkalosis Enhances Response of C3H Mouse Mammary Tumors to the Weak Base Mitoxantrone

    Directory of Open Access Journals (Sweden)

    Natarajan Raghunand

    2001-01-01

    Full Text Available Uptake of weak acid and weak base chemotherapeutic drugs by tumors is greatly influenced by the tumor extracellular/interstitial pH (pHe, the intracellular pH (pHi maintained by the tumor cells, and by the ionization properties of the drug itself. The acid-outside plasmalemmal pH gradient in tumors acts to exclude weak base drugs like the anthracyclines, anthraquinones, and vinca alkaloids from the cells, leading to a substantial degree of “physiological drug resistance” in tumors. We have induced acute metabolic alkalosis in C3H tumor-bearing C3H/hen mice, by gavage and by intraperitoneal (i.p. administration of NaHCO3. 31P magnetic resonance spectroscopic measurements of 3-aminopropylphosphonate show increases of up to 0.6 pH units in tumor pHe, and 0.2 to 0.3 pH units in hind leg tissue pHe, within 2 hours of i.p. administration of NaHCO3. Theoretical calculations of mitoxantrone uptake into tumor and normal (hind leg tissue at the measured pH, and pHI values indicate that a gain in therapeutic index of up to 3.3-fold is possible with NaHCO3 pretreatment. Treatment of C3H tumor-bearing mice with 12 mg/kg mitoxantrone resulted in a tumor growth delay of 9 days, whereas combined NaHCO3mitoxantrone therapy resulted in an enhancement of the TGD to 16 days.

  16. Metabolic effect of TAp63α : enhanced glycolysis and pentose phosphate pathway, resulting in increased antioxidant defense

    NARCIS (Netherlands)

    D'Alessandro, Angelo; Amelio, Ivano; Berkers, Celia R; Antonov, Alexey; Vousden, Karen H; Melino, Gerry; Zolla, Lello

    2014-01-01

    TAp63α is a member of the p53 family, which plays a central role in epithelial cancers. Recently, a role has emerged for p53 family members in cancer metabolic modulation. In order to assess whether TAp63α plays a role in cancer metabolism, we exploited p53-null osteosarcoma Tet-On Saos-2 cells, in

  17. Specific SIRT1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation

    NARCIS (Netherlands)

    Feige, Jérôme N.; Lagouge, Marie; Canto, Carles; Strehle, Axelle; Houten, Sander M.; Milne, Jill C.; Lambert, Philip D.; Mataki, Chikage; Elliott, Peter J.; Auwerx, Johan

    2008-01-01

    The NAD(+)-dependent deacetylase SIRT1 controls metabolic processes in response to low nutrient availability. We report the metabolic phenotype of mice treated with SRT1720, a specific and potent synthetic activator of SIRT1 that is devoid of direct action on AMPK. SRT1720 administration robustly

  18. Royal Jelly Reduces Cholesterol Levels, Ameliorates Aβ Pathology and Enhances Neuronal Metabolic Activities in a Rabbit Model of Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Yongming Pan

    2018-03-01

    Full Text Available Alzheimer’s disease (AD is the most common form of dementia characterized by aggregation of amyloid β (Aβ and neuronal loss. One of the risk factors for AD is high cholesterol levels, which are known to promote Aβ deposition. Previous studies have shown that royal jelly (RJ, a product of worker bees, has potential neuroprotective effects and can attenuate Aβ toxicity. However, little is known about how RJ regulates Aβ formation and its effects on cholesterol levels and neuronal metabolic activities. Here, we investigated whether RJ can reduce cholesterol levels, regulate Aβ levels and enhance neuronal metabolic activities in an AD rabbit model induced by 2% cholesterol diet plus copper drinking water. Our results suggest that RJ significantly reduced the levels of plasma total cholesterol (TC and low density lipoprotein-cholesterol (LDL-C, and decreased the level of Aβ in rabbit brains. RJ was also shown to markedly ameliorate amyloid deposition in AD rabbits from Aβ immunohistochemistry and thioflavin-T staining. Furthermore, our study suggests that RJ can reduce the expression levels of β-site APP cleaving enzyme-1 (BACE1 and receptor for advanced glycation end products (RAGE, and increase the expression levels of low density lipoprotein receptor-related protein 1 (LRP-1 and insulin degrading enzyme (IDE. In addition, we found that RJ remarkably increased the number of neurons, enhanced antioxidant capacities, inhibited activated-capase-3 protein expression, and enhanced neuronal metabolic activities by increasing N-acetyl aspartate (NAA and glutamate and by reducing choline and myo-inositol in AD rabbits. Taken together, our data demonstrated that RJ could reduce cholesterol levels, regulate Aβ levels and enhance neuronal metabolic activities in AD rabbits, providing preclinical evidence that RJ treatment has the potential to protect neurons and prevent AD.

  19. Metabolic Agents that Enhance ATP can Improve Cognitive Functioning: A Review of the Evidence for Glucose, Oxygen, Pyruvate, Creatine, and l-Carnitine

    Directory of Open Access Journals (Sweden)

    Lauren Owen

    2011-08-01

    Full Text Available Over the past four or five decades, there has been increasing interest in the neurochemical regulation of cognition. This field received considerable attention in the 1980s, with the identification of possible cognition enhancing agents or “smart drugs”. Even though many of the optimistic claims for some agents have proven premature, evidence suggests that several metabolic agents may prove to be effective in improving and preserving cognitive performance and may lead to better cognitive aging through the lifespan. Aging is characterized by a progressive deterioration in physiological functions and metabolic processes. There are a number of agents with the potential to improve metabolic activity. Research is now beginning to identify these various agents and delineate their potential usefulness for improving cognition in health and disease. This review provides a brief overview of the metabolic agents glucose, oxygen, pyruvate, creatine, and l-carnitine and their beneficial effects on cognitive function. These agents are directly responsible for generating ATP (adenosine triphosphate the main cellular currency of energy. The brain is the most metabolically active organ in the body and as such is particularly vulnerable to disruption of energy resources. Therefore interventions that sustain adenosine triphosphate (ATP levels may have importance for improving neuronal dysfunction and loss. Moreover, recently, it has been observed that environmental conditions and diet can affect transgenerational gene expression via epigenetic mechanisms. Metabolic agents might play a role in regulation of nutritional epigenetic effects. In summary, the reviewed metabolic agents represent a promising strategy for improving cognitive function and possibly slowing or preventing cognitive decline.

  20. Overexpression of thioredoxin in islets transduced by a lentiviral vector prolongs graft survival in autoimmune diabetic NOD mice

    Directory of Open Access Journals (Sweden)

    Sytwu Huey-Kang

    2009-08-01

    Full Text Available Abstract Pancreatic islet transplantation is considered an appropriate treatment to achieve insulin independence in type I diabetic patients. However, islet isolation and transplantation-induced oxidative stress and autoimmune-mediated destruction are still the major obstacles to the long-term survival of graft islets in this potential therapy. To protect islet grafts from inflammatory damage and prolong their survival, we transduced islets with an antioxidative gene thioredoxin (TRX using a lentiviral vector before transplantation. We hypothesized that the overexpression of TRX in islets would prolong islet graft survival when transplanted into diabetic non-obese diabetic (NOD mice. Methods Islets were isolated from NOD mice and transduced with lentivirus carrying TRX (Lt-TRX or enhanced green fluorescence protein (Lt-eGFP, respectively. Transduced islets were transplanted under the left kidney capsule of female diabetic NOD mice, and blood glucose concentration was monitored daily after transplantation. The histology of the islet graft was assessed at the end of the study. The protective effect of TRX on islets was investigated. Results The lentiviral vector effectively transduced islets without altering the glucose-stimulating insulin-secretory function of islets. Overexpression of TRX in islets reduced hydrogen peroxide-induced cytotoxicity in vitro. After transplantation into diabetic NOD mice, euglycemia was maintained for significantly longer in Lt-TRX-transduced islets than in Lt-eGFP-transduced islets; the mean graft survival was 18 vs. 6.5 days (n = 9 and 10, respectively, p Conclusion We successfully transduced the TRX gene into islets and demonstrated that these genetically modified grafts are resistant to inflammatory insult and survived longer in diabetic recipients. Our results further support the concept that the reactive oxygen species (ROS scavenger and antiapoptotic functions of TRX are critical to islet survival after

  1. An atlas of the thioredoxin fold class reveals the complexity of function-enabling adaptations.

    Science.gov (United States)

    Atkinson, Holly J; Babbitt, Patricia C

    2009-10-01

    The group of proteins that contain a thioredoxin (Trx) fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features-including variations on a dithiol CxxC active site motif-that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif-only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.

  2. Serum and urinary thioredoxin concentrations are associated with severity of children hydronephrosis.

    Science.gov (United States)

    Xu, Zhe-Ming; Li, Min-Ju; Tao, Chang

    2017-03-01

    Ureteropelvic junction obstruction (UPJO) is the most common cause of hydronephrosis in children. This study was to assess the relationship between serum thioredoxin (S-Trx) and urinary thioredoxin (U-Trx) concentrations and severity of children hydronephrosis caused by UPJO. This study included 156 hydronephrosis children with unilateral UPJO and 80 healthy children. S-Trx and U-Trx concentrations were measured using enzyme-linked immunosorbent assay. U-Trx/creatinine (cr) ratio was calculated. S-Trx and U-Trx concentrations and U-Trx/cr ratio were significantly higher in hydronephrosis children than in healthy children. They were significantly correlated with split renal function, anterior-posterior diameter and Society for Fetal Urology classification, as well as were independently related to the split renal function 30mm and Society for Fetal Urology grade IV. Under receiver operating characteristic curves, U-Trx/cr ratio showed the higher predictive value compared to S-Trx and U-Trx concentrations. Increased S-Trx and U-Trx concentrations, especially U-Trx/cr ratio, are closely associated with the severity of children hydronephrosis, substantializing Trx as a promising biomarker for the progression of children hydronephrosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Regulation of the activity of the tumor suppressor PTEN by thioredoxin in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Song, Zuohe; Saghafi, Negin; Gokhale, Vijay; Brabant, Marc; Meuillet, Emmanuelle J.

    2007-01-01

    Human Thioredoxin-1 (hTrx-1) is a small redox protein with a molecular weight of 12 kDa that contains two cysteine residues found in its catalytic site. HTrx-1 plays an important role in cell growth, apoptosis, and cancer patient prognosis. Recently, we have demonstrated that hTrx-1 binds to the C2 domain of the human tumor suppressor, PTEN, in a redox dependent manner. This binding leads to the inhibition of PTEN lipid phosphatase activity in mammalian tissue culture systems. In this study, we show that over-expression of hTrx-1 in Drosophila melanogaster promotes cell growth and proliferation during eye development as measured by eye size and ommatidia size. Furthermore, hTrx-1 rescues the small eye phenotype induced by the over-expression of PTEN. We demonstrate that this rescue of the PTEN-induced eye size phenotype requires cysteine-218 in the C2 domain of PTEN. We also show that hTrx-1 over-expression results in increased Akt phosphorylation in fly head extracts supporting our observations that the hTrx-1-induced eye size increase results from the inhibition of PTEN activity. Our study confirms the redox regulation of PTEN through disulfide bond formation with the hTrx-1 in Drosophila and suggests conserved mechanisms for thioredoxins and their interactions with the phosphatidylinositol-3-kinase signaling pathway in humans and fruit flies

  4. The Effects of Acrolein on the Thioredoxin System: Implications for Redox-Sensitive Signaling

    Science.gov (United States)

    Myers, Charles R.; Myers, Judith M.; Kufahl, Timothy D.; Forbes, Rachel; Szadkowski, Adam

    2012-01-01

    The reactive aldehyde acrolein is a ubiquitous environmental pollutant and is also generated endogenously. It is a strong electrophile and reacts rapidly with nucleophiles including thiolates. This review focuses on the effects of acrolein on thioredoxin reductase (TrxR) and thioredoxin (Trx), which are major regulators of intracellular protein thiol redox balance. Acrolein causes irreversible effects on TrxR and Trx, which are consistent with the formation of covalent adducts to selenocysteine and cysteine residues that are key to their activity. TrxR and Trx are more sensitive than some other redox-sensitive proteins, and their prolonged inhibition could disrupt a number of redox-sensitive functions in cells. Among these effects are the oxidation of peroxiredoxins and the activation of apoptosis signal regulating kinase (ASK1). ASK1 promotes MAP kinase activation, and p38 activation contributes to apoptosis and a number of other acrolein-induced stress responses. Overall, the disruption of the TrxR/Trx system by acrolein could be significant early and prolonged events that affects many aspects of redox-sensitive signaling and oxidant stress. PMID:21812108

  5. Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.

    Energy Technology Data Exchange (ETDEWEB)

    Scholle, M. D.; Banach, B. S.; Hamdan, S. M.; Richardson, C. C.; Kay, B. K.; Biosciences Division; Amunix, Inc.; Univ. of Illinois at Chicago; Harvard Medical School

    2008-11-01

    Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5{Delta}22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5{Delta}22 with Trx, under oxidizing conditions, with an IC50 of {approx} 10 {micro}M.

  6. Novel Insights in Mammalian Catalase Heme Maturation: Effect of NO and Thioredoxin-1

    Science.gov (United States)

    Chakravarti, Ritu; Gupta, Karishma; Majors, Alana; Ruple, Lisa; Aronica, Mark; Stuehr, Dennis J.

    2016-01-01

    Catalase is a tetrameric heme-containing enzyme with essential antioxidant functions in biology. Multiple factors including nitric oxide (NO) have been shown to attenuate its activity. However, the possible impact of NO in relation to the maturation of active catalase, including its heme acquisition and tetramer formation, has not been investigated. We found that NO attenuates heme insertion into catalase in both short-term and long-term incubations. The NO inhibition in catalase heme incorporation was associated with defective oligomerization of catalase, such that inactive catalase monomers and dimers accumulated in place of the mature tetrameric enzyme. We also found that GAPDH plays a key role in mediating these NO effects on the structure and activity of catalase. Moreover, the NO sensitivity of catalase maturation could be altered up or down by manipulating the cellular expression level or activity of thioredoxin-1, a known protein-SNO denitrosylase enzyme. In a mouse model of allergic inflammatory asthma, we found that lungs from allergen-challenged mice contained a greater percentage of dimeric catalase relative to tetrameric catalase in the unchallenged control, suggesting that the mechanisms described here are in play in the allergic asthma model. Together, our study shows how maturation of active catalase can be influenced by NO, S-nitrosylated GAPDH, and thioredoxin-1, and how maturation may become compromised in inflammatory conditions such as asthma. PMID:25659933

  7. Novel insights in mammalian catalase heme maturation: effect of NO and thioredoxin-1.

    Science.gov (United States)

    Chakravarti, Ritu; Gupta, Karishma; Majors, Alana; Ruple, Lisa; Aronica, Mark; Stuehr, Dennis J

    2015-05-01

    Catalase is a tetrameric heme-containing enzyme with essential antioxidant functions in biology. Multiple factors including nitric oxide (NO) have been shown to attenuate its activity. However, the possible impact of NO in relation to the maturation of active catalase, including its heme acquisition and tetramer formation, has not been investigated. We found that NO attenuates heme insertion into catalase in both short-term and long-term incubations. The NO inhibition in catalase heme incorporation was associated with defective oligomerization of catalase, such that inactive catalase monomers and dimers accumulated in place of the mature tetrameric enzyme. We also found that GAPDH plays a key role in mediating these NO effects on the structure and activity of catalase. Moreover, the NO sensitivity of catalase maturation could be altered up or down by manipulating the cellular expression level or activity of thioredoxin-1, a known protein-SNO denitrosylase enzyme. In a mouse model of allergic inflammatory asthma, we found that lungs from allergen-challenged mice contained a greater percentage of dimeric catalase relative to tetrameric catalase in the unchallenged control, suggesting that the mechanisms described here are in play in the allergic asthma model. Together, our study shows how maturation of active catalase can be influenced by NO, S-nitrosylated GAPDH, and thioredoxin-1, and how maturation may become compromised in inflammatory conditions such as asthma. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Brucella abortus Induces a Warburg Shift in Host Metabolism That Is Linked to Enhanced Intracellular Survival of the Pathogen.

    Science.gov (United States)

    Czyż, Daniel M; Willett, Jonathan W; Crosson, Sean

    2017-08-01

    Intracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected with Brucella abortus undergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well described in cancer cells and also occurs in activated inflammatory cells. B. abortus efficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affect in vitro growth of B. abortus in axenic culture but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, and B. abortus uses this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens. IMPORTANCE Brucella spp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected with Brucella abortus undergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism

  9. Polyamine metabolism in ripening tomato fruit. II. Polyamine metabolism and synthesis in relation to enhanced putrescine content and storage life of alc tomato fruit

    International Nuclear Information System (INIS)

    Rastogi, R.; Davies, P.J.

    1991-01-01

    The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele alc) and contain three times as much putrescine as the standard Rutgers variety (Alc) at the ripe stage. Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-alc-a near isogenic line possessing the allele alc, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in alc pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and alc fruit showed a decrease in the metabolism of [1,4- 14 C]putrescine and [terminal labeled- 3 H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-alc fruit, and as a result it was significantly higher in alc fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in alc fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase

  10. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin–cadmium induced diabetic nephrotoxic rats

    International Nuclear Information System (INIS)

    Kandasamy, Neelamegam; Ashokkumar, Natarajan

    2014-01-01

    Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)–cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine, blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ–Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ–Cd induced diabetic nephrotoxic rats. - Highlights: • Diabetic rats are more susceptible to cadmium nephrotoxicity. • Cadmium plays as a cumulative

  11. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin–cadmium induced diabetic nephrotoxic rats

    Energy Technology Data Exchange (ETDEWEB)

    Kandasamy, Neelamegam; Ashokkumar, Natarajan, E-mail: npashokkumar1@gmail.com

    2014-09-01

    Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)–cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine, blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ–Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ–Cd induced diabetic nephrotoxic rats. - Highlights: • Diabetic rats are more susceptible to cadmium nephrotoxicity. • Cadmium plays as a cumulative

  12. Brevetoxin-2, is a unique inhibitor of the C-terminal redox center of mammalian thioredoxin reductase-1.

    Science.gov (United States)

    Chen, Wei; Tuladhar, Anupama; Rolle, Shantelle; Lai, Yanhao; Rodriguez Del Rey, Freddy; Zavala, Cristian E; Liu, Yuan; Rein, Kathleen S

    2017-08-15

    Karenia brevis, the Florida red tide dinoflagellate produces a suite of neurotoxins known as the brevetoxins. The most abundant of the brevetoxins PbTx-2, was found to inhibit the thioredoxin-thioredoxin reductase system, whereas the PbTx-3 has no effect on this system. On the other hand, PbTx-2 activates the reduction of small disulfides such as 5,5'-dithio-bis-(2-nitrobenzoic acid) by thioredoxin reductase. PbTx-2 has an α, β-unsaturated aldehyde moiety which functions as an efficient electrophile and selenocysteine conjugates are readily formed. PbTx-2 blocks the inhibition of TrxR by the inhibitor curcumin, whereas curcumin blocks PbTx-2 activation of TrxR. It is proposed that the mechanism of inhibition of thioredoxin reduction is via the formation of a Michael adduct between selenocysteine and the α, β-unsaturated aldehyde moiety of PbTx-2. PbTx-2 had no effect on the rates of reactions catalyzed by related enzymes such as glutathione reductase, glutathione peroxidase or glutaredoxin. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Investigation of Epidermal Growth Factor, Tumor Necrosis Factor-alpha and Thioredoxin System in Rats Exposed to Cerebral Ischemia

    Directory of Open Access Journals (Sweden)

    Erol-Demirbilek Melike

    2016-09-01

    Full Text Available Background: Thioredoxin reductase (TrxR, epidermal growth factor (EGF and tumor necrosis factor-α (TNF-α have neuroprotective/neurotoxic effects in cerebral ischemia. We aimed to investigate the TrxR activity, EGF and TNF-α levels in cerebral ischemic, sham-operated and non-ischemic rat brains.

  14. Time course of hydrogen peroxide-thioredoxin balance and its influence on the intracellular signalling in myocardial infarction.

    Science.gov (United States)

    Schenkel, Paulo Cavalheiro; Tavares, Angela Maria Vicente; Fernandes, Rafael Oliveira; Diniz, Gabriela Placoná; Ludke, Ana Raquel Lehenbauer; Ribeiro, Maria Flavia Marques; Araujo, Alex Sander da Rosa; Barreto-Chaves, Maria Luiza; Belló-Klein, Adriane

    2012-06-01

    We investigated the myocardial thioredoxin-1 and hydrogen peroxide concentrations and their association with some prosurvival and pro-apoptotic proteins, during the transition from myocardial infarction (MI) to heart failure in rats. Male Wistar rats were divided into the following six groups: three sham-operated groups and three MI groups, each at at 2, 7 and 28 days postsurgery. Cardiac function was analysed by echocardiography; the concentration of H(2)O(2) and the ratio of reduced to oxidized glutathione were measured spectrophotometrically, while the myocardial immunocontent of thioredoxin-1, angiotensin II, angiotensin II type 1 and type 2 receptors, p-JNK/JNK, p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK3β/GSK3β was evaluated by Western blot. Our results show that thioredoxin-1 appears to make an important contribution to the reduced H(2)O(2) concentration. It was associated with lower JNK expression in the early period post-MI (2 days). However, thioredoxin-1 decreased, while renin-angiotensin system markers and levels of H(2)O(2) increased, over 28 days post-MI, in parallel with some signalling proteins involved in maladaptative cardiac remodelling and ventricular dysfunction. These findings provide insight into the time course profile of endogenous antioxidant adaptation to ischaemic injury, which may be useful for the design of therapeutical strategies targeting oxidative stress post-MI.

  15. Increased levels of thioredoxin in patients with abdominal aortic aneurysms (AAAs). A potential link of oxidative stress with AAA evolution

    DEFF Research Database (Denmark)

    Martinez-Pinna, R; Lindholt, Jes S.; Blanco-Colio, L M

    2010-01-01

    Oxidative stress is a main mechanism involved in vascular pathologies. Increased thioredoxin (TRX) levels have been observed in several oxidative stress-associated cardiovascular diseases. We aim to test the potential role of TRX as a biomarker of oxidative stress in abdominal aortic aneurysm (AAA)....

  16. Genetic variation in thioredoxin interacting protein (TXNIP) is associated with hypertriglyceridaemia and blood pressure in diabetes mellitus

    NARCIS (Netherlands)

    Greevenbroek, van M.M.J.; Vermeulen, V.; Feskens, E.J.M.; Evelo, V.T.; Kruijshoop, M.; Hoebee, B.; Kallen, van der C.J.H.; Bruin, de T.W.A.

    2007-01-01

    Aims Thioredoxin interacting protein (TXNIP) is an attractive candidate gene for diabetes or diabetic dyslipidaemia, since TXNIP is the strongest glucose-responsive gene in pancreatic B-cells, TXNIP deficiency in a mouse model is associated with hyperlipidaemia and TXNIP is located in the 1q21-1q23

  17. Effect of the acquisition enhancing drug piracetam on rat cerebral energy metabolism. Comparison with naftidrofuryl and methamphetamine

    NARCIS (Netherlands)

    Nickolson, V.J.; Wolthuis, O.L.

    1976-01-01

    The effects of Piracetam, Naftidrofuryl and methamphetamine on several parameters of cerebral energy metabolism have been studied. At variance with some reports in the literature neither Piracetam nor Naftidrofuryl affected the cerebral contents of adenine nucleotides and, accordingly, both

  18. Immunosuppressive activity enhances central carbon metabolism and bioenergetics in myeloid-derived suppressor cells in vitro models

    Directory of Open Access Journals (Sweden)

    Hammami Ines

    2012-07-01

    Full Text Available Abstract Background The tumor microenvironment contains a vast array of pro- and anti-inflammatory cytokines that alter myelopoiesis and lead to the maturation of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs. Incubating bone marrow (BM precursors with a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF and interleukin-6 (IL-6 generated a tumor-infiltrating MDSC-like population that impaired anti-tumor specific T-cell functions. This in vitro experimental approach was used to simulate MDSC maturation, and the cellular metabolic response was then monitored. A complementary experimental model that inhibited L-arginine (L-Arg metabolizing enzymes in MSC-1 cells, an immortalized cell line derived from primary MDSCs, was used to study the metabolic events related to immunosuppression. Results Exposure of BM cells to GM-CSF and IL-6 activated, within 24 h, L-Arg metabolizing enzymes which are responsible for the MDSCs immunosuppressive potential. This was accompanied by an increased uptake of L-glutamine (L-Gln and glucose, the latter being metabolized by anaerobic glycolysis. The up-regulation of nutrient uptake lead to the accumulation of TCA cycle intermediates and lactate as well as the endogenous synthesis of L-Arg and the production of energy-rich nucleotides. Moreover, inhibition of L-Arg metabolism in MSC-1 cells down-regulated central carbon metabolism activity, including glycolysis, glutaminolysis and TCA cycle activity, and led to a deterioration of cell bioenergetic status. The simultaneous increase of cell specific concentrations of ATP and a decrease in ATP-to-ADP ratio in BM-derived MDSCs suggested cells were metabolically active during maturation. Moreover, AMP-activated protein kinase (AMPK was activated during MDSC maturation in GM-CSF and IL-6–treated cultures, as revealed by the continuous increase of AMP-to-ATP ratios and the phosphorylation of AMPK. Likewise, AMPK activity was

  19. Adrenergic pathway activation enhances brown adipose tissue metabolism: A [18 F]FDG PET/CT study in mice

    International Nuclear Information System (INIS)

    Mirbolooki, M. Reza; Upadhyay, Sanjeev Kumar; Constantinescu, Cristian C.; Pan, Min-Liang; Mukherjee, Jogeshwar

    2014-01-01

    Objective: Pharmacologic approaches to study brown adipocyte activation in vivo with a potential of being translational to humans are desired. The aim of this study was to examine pre- and postsynaptic targeting of adrenergic system for enhancing brown adipose tissue (BAT) metabolism quantifiable by [ 18 F]fluoro-2-deoxyglucose ([ 18 F]FDG) positron emission tomography (PET)/computed tomography (CT) in mice. Methods: A β 3 -adrenoreceptor selective agonist (CL 316243), an adenylyl cyclase enzyme activator (forskolin) and a potent blocker of presynaptic norepinephrine transporter (atomoxetine), were injected through the tail vein of Swiss Webster mice 30 minutes before intravenous (iv) administration of [ 18 F]FDG. The mice were placed on the PET/CT bed for 30 min PET acquisition followed by 10 min CT acquisition for attenuation correction and anatomical delineation of PET images. Results: Activated interscapular (IBAT), cervical, periaortic and intercostal BAT were observed in 3-dimentional analysis of [ 18 F]FDG PET images. CL 316243 increased the total [ 18 F]FDG standard uptake value (SUV) of IBAT 5-fold greater compared to that in placebo-treated mice. It also increased the [ 18 F]FDG SUV of white adipose tissue (2.4-fold), and muscle (2.7-fold), as compared to the control. There was no significant difference in heart, brain, spleen and liver uptakes between groups. Forskolin increased [ 18 F]FDG SUV of IBAT 1.9-fold greater than that in placebo-treated mice. It also increased the [ 18 F]FDG SUV of white adipose tissue (2.2-fold) and heart (5.4-fold) compared to control. There was no significant difference in muscle, brain, spleen, and liver uptakes between groups. Atomoxetine increased [ 18 F]FDG SUV of IBAT 1.7-fold greater than that in placebo-treated mice. There were no significant differences in all other organs compared to placebo-treated mice except liver (1.6 fold increase). A positive correlation between SUV levels of IBAT and CT Hounsfield unit (HU

  20. The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by ß-amyloid peptide

    Science.gov (United States)

    Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, WE

    2010-01-01

    Background and purpose: β-Amyloid peptide (Aβ) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. Experimental approach: We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Aβ-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Aβ and on neurite outgrowth in PC12 cells were investigated. Key results: Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Aβ1-42. Similar protective effects against Aβ1-42 were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Aβ load was markedly diminished in the brain of those animals after treatment with piracetam. Aβ production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Aβ-induced mitochondrial dysfunction and Aβ-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Conclusion and implications: Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Aβ on brain function. This article is commented on by Moncada, pp. 217–219 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00706.x and to view related papers by Pravdic et al. and Puerta et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00698.x and http://dx.doi.org/10.1111/j

  1. Hyperglycemia regulates thioredoxin-ROS activity through induction of thioredoxin-interacting protein (TXNIP) in metastatic breast cancer-derived cells MDA-MB-231

    International Nuclear Information System (INIS)

    Turturro, Francesco; Friday, Ellen; Welbourne, Tomas

    2007-01-01

    We studied the RNA expression of the genes in response to glucose from 5 mM (condition of normoglycemia) to 20 mM (condition of hyperglycemia/diabetes) by microarray analysis in breast cancer derived cell line MDA-MB-231. We identified the thioredoxin-interacting protein (TXNIP), whose RNA level increased as a gene product particularly sensitive to the variation of the level of glucose in culture media. We investigated the kinesis of the TXNIP RNA and protein in response to glucose and the relationship between this protein and the related thioredoxin (TRX) in regulating the level of reactive oxygen species (ROS) in MDA-MB-231 cells. MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating. For glucose shift (5/20), cells were plated in 5 mM glucose and shifted to 20 mM at time 0. Cells were analyzed with Affymetrix Human U133A microarray chip and gene expression profile was obtained. Semi-quantitative RT-PCR and Western blot was used to validate the expression of TXNIP RNA and protein in response to glucose, respectively. ROS were detected by CM-H2DCFDA (5–6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate) and measured for mean fluorescence intensity with flow cytometry. TRX activity was assayed by the insulin disulfide reducing assay. We found that the regulation of TXNIP gene expression by glucose in MDA-MB-231 cells occurs rapidly within 6 h of its increased level (20 mM glucose) and persists through the duration of the conditions of hyperglycemia. The increased level of TXNIP RNA is followed by increased level of protein that is associated with increasing levels of ROS and reduced TRX activity. The inhibition of the glucose transporter GLUT1 by phloretin notably reduces TXNIP RNA level and the inhibition of the p38 MAP kinase activity by SB203580 reverses the effects of TXNIP on ROS-TRX activity. In this study we show that TXNIP is an oxidative stress responsive gene and its expression is exquisitely regulated by

  2. Hyperglycemia regulates thioredoxin-ROS activity through induction of thioredoxin-interacting protein (TXNIP in metastatic breast cancer-derived cells MDA-MB-231

    Directory of Open Access Journals (Sweden)

    Friday Ellen

    2007-06-01

    Full Text Available Abstract Background We studied the RNA expression of the genes in response to glucose from 5 mM (condition of normoglycemia to 20 mM (condition of hyperglycemia/diabetes by microarray analysis in breast cancer derived cell line MDA-MB-231. We identified the thioredoxin-interacting protein (TXNIP, whose RNA level increased as a gene product particularly sensitive to the variation of the level of glucose in culture media. We investigated the kinesis of the TXNIP RNA and protein in response to glucose and the relationship between this protein and the related thioredoxin (TRX in regulating the level of reactive oxygen species (ROS in MDA-MB-231 cells. Methods MDA-MB-231 cells were grown either in 5 or 20 mM glucose chronically prior to plating. For glucose shift (5/20, cells were plated in 5 mM glucose and shifted to 20 mM at time 0. Cells were analyzed with Affymetrix Human U133A microarray chip and gene expression profile was obtained. Semi-quantitative RT-PCR and Western blot was used to validate the expression of TXNIP RNA and protein in response to glucose, respectively. ROS were detected by CM-H2DCFDA (5–6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and measured for mean fluorescence intensity with flow cytometry. TRX activity was assayed by the insulin disulfide reducing assay. Results We found that the regulation of TXNIP gene expression by glucose in MDA-MB-231 cells occurs rapidly within 6 h of its increased level (20 mM glucose and persists through the duration of the conditions of hyperglycemia. The increased level of TXNIP RNA is followed by increased level of protein that is associated with increasing levels of ROS and reduced TRX activity. The inhibition of the glucose transporter GLUT1 by phloretin notably reduces TXNIP RNA level and the inhibition of the p38 MAP kinase activity by SB203580 reverses the effects of TXNIP on ROS-TRX activity. Conclusion In this study we show that TXNIP is an oxidative stress responsive

  3. PaTrx1 and PaTrx3, Two Cytosolic Thioredoxins of the Filamentous Ascomycete Podospora anserina Involved in Sexual Development and Cell Degeneration▿ †

    OpenAIRE

    Malagnac, Fabienne; Klapholz, Benjamin; Silar, Philippe

    2007-01-01

    In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2, and PaTrx3 shows that PaTrx1 is the major thioredoxin inv...

  4. Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

    International Nuclear Information System (INIS)

    Wang, Yijun; Lu, Hongjuan; Wang, Dongxu; Li, Shengrong; Sun, Kang; Wan, Xiaochun; Taylor, Ethan Will; Zhang, Jinsong

    2012-01-01

    Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. A high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does not

  5. Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yijun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Lu, Hongjuan [Productivity Center of Jiangsu Province, Nanjing 210042, Jiangsu (China); Wang, Dongxu; Li, Shengrong; Sun, Kang; Wan, Xiaochun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Taylor, Ethan Will [Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, Greensboro, NC 27402 (United States); Zhang, Jinsong, E-mail: zjs@ahau.edu.cn [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China)

    2012-12-15

    Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. A high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does not

  6. Increased iron availability resulting from increased CO2 enhances carbon and nitrogen metabolism in the economical marine red macroalga Pyropia haitanensis (Rhodophyta).

    Science.gov (United States)

    Chen, Binbin; Zou, Dinghui; Yang, Yufeng

    2017-04-01

    Ocean acidification caused by rising CO 2 is predicted to increase the concentrations of dissolved species of Fe(II) and Fe(III), leading to the enhanced photosynthetic carbon sequestration in some algal species. In this study, the carbon and nitrogen metabolism in responses to increased iron availability under two CO 2 levels (390 μL L -1 and 1000 μL L -1 ), were investigated in the maricultivated macroalga Pyropia haitanensis (Rhodophyta). The results showed that, elevated CO 2 increased soluble carbonhydrate (SC) contents, resulting from enhanced photosynthesis and photosynthetic pigment synthesis in this algae, but declined its soluble protein (SP) contents, resulting in increased ratio of SC/SP. This enhanced photosynthesis performance and carbon accumulation was more significant under iron enrichment condition in seawater, with higher iron uptake rate at high CO 2 level. As a key essential biogenic element for algae, Fe-replete functionally contributed to P. haitanensis photosynthesis. Increased SC fundamentally provided carbon skeletons for nitrogen assimilation. The significant increase of carbon and nitrogen assimilation finally contributed to enhanced growth in this alga. This was also intuitively reflected by respiration that provided energy for cellular metabolism and algal growth. We propose that, in the predicted scenario of rising atmospheric CO 2 , P. haitanensis is capable to adjust its physiology by increasing its carbon and nitrogen metabolism to acclimate the acidified seawater, at the background of global climate change and simultaneously increased iron concentration due to decreased pH levels. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Bee's honey attenuates non-alcoholic steatohepatitis-induced hepatic injury through the regulation of thioredoxin-interacting protein-NLRP3 inflammasome pathway.

    Science.gov (United States)

    Xiao, Jia; Liu, Yingxia; Xing, Feiyue; Leung, Tung Ming; Liong, Emily C; Tipoe, George L

    2016-06-01

    We aim to examine whether honey ameliorates hepatic injury in non-alcoholic steatohepatitis (NASH) animal and cell line steatosis models. NASH was induced in female Sprague-Dawley rat by 8-week feeding with a high-fat diet. During the experiment, 5 g/kg honey was intragastrically fed daily. Rat normal hepatocyte BRL-3A cell was treated with sodium palmitate (SP) to induce steatosis in the absence or presence of honey pre-treatment or specific siRNA/overexpress plasmid of thioredoxin-interacting protein (TXNIP) or antagonist/agonist of Nod-like receptor protein 3 (NLRP3). Honey significantly improved the high-fat-diet-induced hepatic injury, steatosis, fibrosis, oxidative stress, and inflammation in rats. Honey also inhibited the overexpression of TXNIP and the activation of NLRP3 inflammasome. These effects were replicated in BRL-3A cell line which showed that the down-regulation of TXNIP or inhibition of NLRP3 contributed to the suppression of NLRP3 inflammasome activation, inflammation, and re-balanced lipid metabolism. In contrast, overexpression of TXNIP or agonism of NLRP3 exacerbated the cellular damage induced by SP. Suppression of the TXNIP-NLRP3 inflammasome pathway may partly contribute to the amelioration of hepatic injury during the progression of NASH by honey. Targeting hepatic TXNIP-NLRP3 inflammasome pathway is a potential therapeutic way for the prevention and treatment of NASH.

  8. The thioredoxin reductase--Thioredoxin redox system cleaves the interchain disulphide bond of botulinum neurotoxins on the cytosolic surface of synaptic vesicles.

    Science.gov (United States)

    Pirazzini, Marco; Azarnia Tehran, Domenico; Zanetti, Giulia; Lista, Florigio; Binz, Thomas; Shone, Clifford C; Rossetto, Ornella; Montecucco, Cesare

    2015-12-01

    Botulinum neurotoxins (BoNTs) are Janus toxins, as they are at the same time the most deadly substances known and one of the safest drugs used in human therapy. They specifically block neurotransmission at peripheral nerves through the proteolysis of SNARE proteins, i.e. the essential proteins which are the core of the neuroexocytosis machinery. Even if BoNTs are traditionally known as seven main serotypes, their actual number is much higher as each serotype exists in many different subtypes, with individual biological properties and little antigenic relations. Since BoNTs can be used as biological weapons, and the only currently available therapy is based on immunological approaches, the existence of so many different subtypes is a major safety problem. Nevertheless, all BoNT isoforms are structurally similar and intoxicate peripheral nerve endings via a conserved mechanism. They consist of two chains linked by a unique disulphide bond which must be reduced to enable their toxicity. We found that thioredoxin 1 and its reductase compose the cell redox system responsible for this reduction, and its inhibition via specific chemicals significantly reduces BoNTs activity, in vitro as well as in vivo. Such molecules can be considered as lead compounds for the development of pan-inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. The thioredoxin TRX-1 regulates adult lifespan extension induced by dietary restriction in Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Fierro-Gonzalez, Juan Carlos; Gonzalez-Barrios, Maria; Miranda-Vizuete, Antonio; Swoboda, Peter

    2011-01-01

    Highlights: → First in vivo data for thioredoxin in dietary-restriction-(DR)-induced longevity. → Thioredoxin (trx-1) loss suppresses longevity of eat-2 mutant, a genetic DR model. → trx-1 overexpression extends wild-type longevity, but not that of eat-2 mutant. → Longevity by dietary deprivation (DD), a non-genetic DR model, requires trx-1. → trx-1 expression in ASJ neurons of aging adults is increased in response to DD. -- Abstract: Dietary restriction (DR) is the only environmental intervention known to extend adult lifespan in a wide variety of animal models. However, the genetic and cellular events that mediate the anti-aging programs induced by DR remain elusive. Here, we used the nematode Caenorhabditis elegans to provide the first in vivo evidence that a thioredoxin (TRX-1) regulates adult lifespan extension induced by DR. We found that deletion of the gene trx-1 completely suppressed the lifespan extension caused by mutation of eat-2, a genetic surrogate of DR in the worm. However, trx-1 deletion only partially suppressed the long lifespan caused by mutation of the insulin-like receptor gene daf-2 or by mutation of the sensory cilia gene osm-5. A trx-1::GFP translational fusion expressed from its own promoter in ASJ neurons (Ptrx-1::trx-1::GFP) rescued the trx-1 deletion-mediated suppression of the lifespan extension caused by mutation of eat-2. This rescue was not observed when trx-1::GFP was expressed from the ges-1 promoter in the intestine. In addition, overexpression of Ptrx-1::trx-1::GFP extended lifespan in wild type, but not in eat-2 mutants. trx-1 deletion almost completely suppressed the lifespan extension induced by dietary deprivation (DD), a non-genetic, nutrient-based model of DR in the worm. Moreover, DD upregulated the expression of a trx-1 promoter-driven GFP reporter gene (Ptrx-1::GFP) in ASJ neurons of aging adults, but not that of control Pgpa-9::GFP (which is also expressed in ASJ neurons). We propose that DR activates TRX-1

  10. The thioredoxin TRX-1 regulates adult lifespan extension induced by dietary restriction in Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Fierro-Gonzalez, Juan Carlos [Karolinska Institute, Center for Biosciences at NOVUM, Department of Biosciences and Nutrition, S-141 83 Huddinge (Sweden); Gonzalez-Barrios, Maria [Centro Andaluz de Biologia del Desarrollo (CABD-CSIC), Departamento de Fisiologia, Anatomia y Biologia Celular, Universidad Pablo de Olavide, E-41013 Sevilla (Spain); Miranda-Vizuete, Antonio, E-mail: amirviz@upo.es [Centro Andaluz de Biologia del Desarrollo (CABD-CSIC), Departamento de Fisiologia, Anatomia y Biologia Celular, Universidad Pablo de Olavide, E-41013 Sevilla (Spain); Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, E-41013 Sevilla (Spain); Swoboda, Peter, E-mail: peter.swoboda@ki.se [Karolinska Institute, Center for Biosciences at NOVUM, Department of Biosciences and Nutrition, S-141 83 Huddinge (Sweden)

    2011-03-18

    Highlights: {yields} First in vivo data for thioredoxin in dietary-restriction-(DR)-induced longevity. {yields} Thioredoxin (trx-1) loss suppresses longevity of eat-2 mutant, a genetic DR model. {yields} trx-1 overexpression extends wild-type longevity, but not that of eat-2 mutant. {yields} Longevity by dietary deprivation (DD), a non-genetic DR model, requires trx-1. {yields} trx-1 expression in ASJ neurons of aging adults is increased in response to DD. -- Abstract: Dietary restriction (DR) is the only environmental intervention known to extend adult lifespan in a wide variety of animal models. However, the genetic and cellular events that mediate the anti-aging programs induced by DR remain elusive. Here, we used the nematode Caenorhabditis elegans to provide the first in vivo evidence that a thioredoxin (TRX-1) regulates adult lifespan extension induced by DR. We found that deletion of the gene trx-1 completely suppressed the lifespan extension caused by mutation of eat-2, a genetic surrogate of DR in the worm. However, trx-1 deletion only partially suppressed the long lifespan caused by mutation of the insulin-like receptor gene daf-2 or by mutation of the sensory cilia gene osm-5. A trx-1::GFP translational fusion expressed from its own promoter in ASJ neurons (Ptrx-1::trx-1::GFP) rescued the trx-1 deletion-mediated suppression of the lifespan extension caused by mutation of eat-2. This rescue was not observed when trx-1::GFP was expressed from the ges-1 promoter in the intestine. In addition, overexpression of Ptrx-1::trx-1::GFP extended lifespan in wild type, but not in eat-2 mutants. trx-1 deletion almost completely suppressed the lifespan extension induced by dietary deprivation (DD), a non-genetic, nutrient-based model of DR in the worm. Moreover, DD upregulated the expression of a trx-1 promoter-driven GFP reporter gene (Ptrx-1::GFP) in ASJ neurons of aging adults, but not that of control Pgpa-9::GFP (which is also expressed in ASJ neurons). We propose

  11. Antisense Suppression of 2-Cysteine Peroxiredoxin in Arabidopsis Specifically Enhances the Activities and Expression of Enzymes Associated with Ascorbate Metabolism But Not Glutathione Metabolism1

    Science.gov (United States)

    Baier, Margarete; Noctor, Graham; Foyer, Christine H.; Dietz, Karl-Josef

    2000-01-01

    The aim of this study was to characterize the effect of decreased 2-cysteine peroxiredoxin (2-CP) on the leaf anti-oxidative system in Arabidopsis. At three stages of leaf development, two lines of transgenic Arabidopsis mutants with decreased contents of chloroplast 2-CP were compared with wild type and a control line transformed with an empty vector. Glutathione contents and redox state were similar in all plants, and no changes in transcript levels for enzymes involved in glutathione metabolism were observed. Transcript levels for chloroplastic glutathione peroxidase were much lower than those for 2-CP, and both cytosolic and chloroplastic glutathione peroxidase were not increased in the mutants. In contrast, the foliar ascorbate pool was more oxidized in the mutants, although the difference decreased with plant age. The activities of thylakoid and stromal ascorbate peroxidase and particularly monodehydroascorbate reductase were increased as were transcripts for these enzymes. No change in dehydroascorbate reductase activity was observed, and effects on transcript abundance for glutathione reductase, catalase, and superoxide dismutase were slight or absent. The results demonstrate that 2-CP forms an integral part of the anti-oxidant network of chloroplasts and is functionally interconnected with other defense systems. Suppression of 2-CP leads to increased expression of other anti-oxidative genes possibly mediated by increased oxidation state of the leaf ascorbate pool. PMID:11027730

  12. Mechanisms of metabonomic for a gateway drug: nicotine priming enhances behavioral response to cocaine with modification in energy metabolism and neurotransmitter level.

    Directory of Open Access Journals (Sweden)

    Hongyu Li

    Full Text Available Nicotine, one of the most commonly used drugs, has become a major concern because tobacco serves as a gateway drug and is linked to illicit drug abuse, such as cocaine and marijuana. However, previous studies mainly focused on certain genes or neurotransmitters which have already been known to participate in drug addiction, lacking endogenous metabolic profiling in a global view. To further explore the mechanism by which nicotine modifies the response to cocaine, we developed two conditioned place preference (CPP models in mice. In threshold dose model, mice were pretreated with nicotine, followed by cocaine treatment at the dose of 2 mg/kg, a threshold dose of cocaine to induce CPP in mice. In high-dose model, mice were only treated with 20 mg/kg cocaine, which induced a significant CPP. (1H nuclear magnetic resonance based on metabonomics was used to investigate metabolic profiles of the nucleus accumbens (NAc and striatum. We found that nicotine pretreatment dramatically increased CPP induced by 2 mg/kg cocaine, which was similar to 20 mg/kg cocaine-induced CPP. Interestingly, metabolic profiles showed considerable overlap between these two models. These overlapped metabolites mainly included neurotransmitters as well as the molecules participating in energy homeostasis and cellular metabolism. Our results show that the reinforcing effect of nicotine on behavioral response to cocaine may attribute to the modification of some specific metabolites in NAc and striatum, thus creating a favorable metabolic environment for enhancing conditioned rewarding effect of cocaine. Our findings provide an insight into the effect of cigarette smoking on cocaine dependence and the underlying mechanism.

  13. Metabolically stable bradykinin B2 receptor agonists enhance transvascular drug delivery into malignant brain tumors by increasing drug half-life

    Directory of Open Access Journals (Sweden)

    Glen Daniel

    2009-05-01

    Full Text Available Abstract Background The intravenous co-infusion of labradimil, a metabolically stable bradykinin B2 receptor agonist, has been shown to temporarily enhance the transvascular delivery of small chemotherapy drugs, such as carboplatin, across the blood-brain tumor barrier. It has been thought that the primary mechanism by which labradimil does so is by acting selectively on tumor microvasculature to increase the local transvascular flow rate across the blood-brain tumor barrier. This mechanism of action does not explain why, in the clinical setting, carboplatin dosing based on patient renal function over-estimates the carboplatin dose required for target carboplatin exposure. In this study we investigated the systemic actions of labradimil, as well as other bradykinin B2 receptor agonists with a range of metabolic stabilities, in context of the local actions of the respective B2 receptor agonists on the blood-brain tumor barrier of rodent malignant gliomas. Methods Using dynamic contrast-enhanced MRI, the pharmacokinetics of gadolinium-diethyltriaminepentaacetic acid (Gd-DTPA, a small MRI contrast agent, were imaged in rodents bearing orthotopic RG-2 malignant gliomas. Baseline blood and brain tumor tissue pharmacokinetics were imaged with the 1st bolus of Gd-DTPA over the first hour, and then re-imaged with a 2nd bolus of Gd-DTPA over the second hour, during which normal saline or a bradykinin B2 receptor agonist was infused intravenously for 15 minutes. Changes in mean arterial blood pressure were recorded. Imaging data was analyzed using both qualitative and quantitative methods. Results The decrease in systemic blood pressure correlated with the known metabolic stability of the bradykinin B2 receptor agonist infused. Metabolically stable bradykinin B2 agonists, methionine-lysine-bradykinin and labradimil, had differential effects on the transvascular flow rate of Gd-DTPA across the blood-brain tumor barrier. Both methionine

  14. Analysis of metabolic networks of Streptomyces leeuwenhoekii C34 by means of a genome scale model: Prediction of modifications that enhance the production of specialized metabolites.

    Science.gov (United States)

    Razmilic, Valeria; Castro, Jean F; Andrews, Barbara; Asenjo, Juan A

    2018-07-01

    The first genome scale model (GSM) for Streptomyces leeuwenhoekii C34 was developed to study the biosynthesis pathways of specialized metabolites and to find metabolic engineering targets for enhancing their production. The model, iVR1007, consists of 1,722 reactions, 1,463 metabolites, and 1,007 genes, it includes the biosynthesis pathways of chaxamycins, chaxalactins, desferrioxamines, ectoine, and other specialized metabolites. iVR1007 was validated using experimental information of growth on 166 different sources of carbon, nitrogen and phosphorous, showing an 83.7% accuracy. The model was used to predict metabolic engineering targets for enhancing the biosynthesis of chaxamycins and chaxalactins. Gene knockouts, such as sle03600 (L-homoserine O-acetyltransferase), and sle39090 (trehalose-phosphate synthase), that enhance the production of the specialized metabolites by increasing the pool of precursors were identified. Using the algorithm of flux scanning based on enforced objective flux (FSEOF) implemented in python, 35 and 25 over-expression targets for increasing the production of chaxamycin A and chaxalactin A, respectively, that were not directly associated with their biosynthesis routes were identified. Nineteen over-expression targets that were common to the two specialized metabolites studied, like the over-expression of the acetyl carboxylase complex (sle47660 (accA) and any of the following genes: sle44630 (accA_1) or sle39830 (accA_2) or sle27560 (bccA) or sle59710) were identified. The predicted knockouts and over-expression targets will be used to perform metabolic engineering of S. leeuwenhoekii C34 and obtain overproducer strains. © 2018 Wiley Periodicals, Inc.

  15. Identification of Thioredoxin Target Disulfides Using Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2014-01-01

    Thioredoxins (Trx) are small redox proteins that reduce disulfide bonds in various target proteins and maintain cellular thiol redox control. Here, a thiol-specific labeling and affinity enrichment approach for identification and relative quantification of Trx target disulfides in complex protein...... reduction is determined by LC-MS/MS-based quantification of tryptic peptides labeled with "light" (12C) and "heavy" (13C) ICAT reagents. The methodology can be adapted to monitor the effect of different reductants or oxidants on the redox status of thiol/disulfide proteomes in biological systems....... extracts is described. The procedure utilizes the isotope-coded affinity tag (ICAT) reagents containing a thiol reactive iodoacetamide group and a biotin affinity tag to target peptides containing reduced cysteine residues. The identification of substrates for Trx and the extent of target disulfide...

  16. Two Lactococcus lactis thioredoxin paralogues play different roles in responses to arsenate and oxidative stress

    DEFF Research Database (Denmark)

    Efler, Petr; Kilstrup, Mogens; Johnsen, Stig

    2015-01-01

    Thioredoxin (Trx) maintains intracellular thiol groups in a reduced state and is involved in a wide range of cellular processes, including ribonucleotide reduction, sulphur assimilation, oxidative stress responses and arsenate detoxification. The industrially important lactic acid bacterium...... Lactococcus lactis contains two Trxs. TrxA is similar to the well-characterized Trx homologue from Escherichia coli and contains the common WCGPC active site motif, while TrxD is atypical and contains an aspartate residue in the active site (WCGDC). To elucidate the physiological roles of the two Trx...... to the wild-type. The lack of TrxA also appears to impair methionine sulphoxide reduction. Both ΔtrxA and ΔtrxD strains displayed growth inhibition after treatment with sodium arsenate and tellurite as compared with the wild-type, suggesting partially overlapping functions of TrxA and TrxD. Overall...

  17. Structure of the thioredoxin-fold domain of human phosducin-like protein 2

    International Nuclear Information System (INIS)

    Lou, Xiaochu; Bao, Rui; Zhou, Cong-Zhao; Chen, Yuxing

    2009-01-01

    The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Human phosducin-like protein 2 (hPDCL2) has been identified as belonging to subgroup II of the phosducin (Pdc) family. The members of this family share an N-terminal helix domain and a C-terminal thioredoxin-fold (Trx-fold) domain. The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 Å resolution and resembled the Trx-fold domain of rat phosducin. Comparative structural analysis revealed the structural basis of their putative functional divergence

  18. Thioredoxin 80-Activated-Monocytes (TAMs) Inhibit the Replication of Intracellular Pathogens

    DEFF Research Database (Denmark)

    Cortes-Bratti, Ximena; Brasseres, Eugenie; Herrera-Rodriquez, Fabiola

    2011-01-01

    Background: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs). Principal Findings: In this investigation we present evidence...... for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L...... in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome. Significance: Our results show that Trx80 potentiates the bactericidal activities...

  19. The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity

    NARCIS (Netherlands)

    Cantó, Carles; Houtkooper, Riekelt H.; Pirinen, Eija; Youn, Dou Y.; Oosterveer, Maaike H.; Cen, Yana; Fernandez-Marcos, Pablo J.; Yamamoto, Hiroyasu; Andreux, Pénélope A.; Cettour-Rose, Philippe; Gademann, Karl; Rinsch, Chris; Schoonjans, Kristina; Sauve, Anthony A.; Auwerx, Johan

    2012-01-01

    As NAD(+) is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38-both NAD(+) consumers-increases NAD(+)

  20. Protein metabolism in the rat cerebral cortex in vivo and in vitro as affected by the acquisition enhancing drug piracetam

    NARCIS (Netherlands)

    Nickolson, V.J.; Wolthuis, O.L.

    1976-01-01

    The effect of Piracetam on rat cerebral protein metabolism in vivo and in vitro was studied. It was found that the drug stimulates the uptake of labelled leucine by cerebral cortex slices, has no effect on the incorporation of leucine into cerebral protein, neither in slices nor in vivo, but

  1. Enhanced Fructose Utilization Mediated by SLC2A5 Is a Unique Metabolic Feature of Acute Myeloid Leukemia with Therapeutic Potential.

    Science.gov (United States)

    Chen, Wen-Lian; Wang, Yue-Ying; Zhao, Aihua; Xia, Li; Xie, Guoxiang; Su, Mingming; Zhao, Linjing; Liu, Jiajian; Qu, Chun; Wei, Runmin; Rajani, Cynthia; Ni, Yan; Cheng, Zhen; Chen, Zhu; Chen, Sai-Juan; Jia, Wei

    2016-11-14

    Rapidly proliferating leukemic progenitor cells consume substantial glucose, which may lead to glucose insufficiency in bone marrow. We show that acute myeloid leukemia (AML) cells are prone to fructose utilization with an upregulated fructose transporter GLUT5, which compensates for glucose deficiency. Notably, AML patients with upregulated transcription of the GLUT5-encoding gene SLC2A5 or increased fructose utilization have poor outcomes. Pharmacological blockage of fructose uptake ameliorates leukemic phenotypes and potentiates the cytotoxicity of the antileukemic agent, Ara-C. In conclusion, this study highlights enhanced fructose utilization as a metabolic feature of AML and a potential therapeutic target. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Characterization of a Thioredoxin-1 Gene from Taenia solium and Its Encoding Product

    Science.gov (United States)

    Jiménez, Lucía; Rodríguez-Lima, Oscar; Ochoa-Sánchez, Alicia; Landa, Abraham

    2015-01-01

    Taenia solium thioredoxin-1 gene (TsTrx-1) has a length of 771 bp with three exons and two introns. The core promoter gene presents two putative stress transcription factor binding sites, one putative TATA box, and a transcription start site (TSS). TsTrx-1 mRNA is expressed higher in larvae than in adult. This gene encodes a protein of 107 amino acids that presents the Trx active site (CGPC), the classical secondary structure of the thioredoxin fold, and the highest degree of identity with the Echinococcus granulosus Trx. A recombinant TsTrx-1 (rTsTrx-1) was produced in Escherichia coli with redox activity. Optimal activity for rTsTrx-1 was at pH 6.5 in the range of 15 to 25°C. The enzyme conserved activity for 3 h and lost it in 24 h at 37°C. rTsTrx-1 lost 50% activity after 1 h and lost activity completely in 24 h at temperatures higher than 55°C. Best storage temperature for rTsTrx-1 was at −70°C. It was inhibited by high concentrations of H2O2 and methylglyoxal (MG), but it was inhibited neither by NaCl nor by anti-rTsTrx-1 rabbit antibodies that strongly recognized a ~12 kDa band in extracts from several parasites. These TsTrx-1 properties open the opportunity to study its role in relationship T. solium-hosts. PMID:26090410

  3. The effects of chromium(VI) on the thioredoxin system: Implications for redox regulation

    Science.gov (United States)

    Myers, Charles R.

    2014-01-01

    Hexavalent chromium [Cr(VI)] compounds are highly redox active and have long been recognized as potent cytotoxins and carcinogens. The intracellular reduction of Cr(VI) generates reactive Cr intermediates, which are themselves strong oxidants, as well as superoxide, hydrogen peroxide, and hydroxyl radical. These probably contribute to the oxidative damage and effects on redox-sensitive transcription factors that have been reported. However, the identification of events that initiate these signaling changes has been elusive. More recent studies show that Cr(VI) causes irreversible inhibition of thioredoxin reductase (TrxR) and oxidation of thioredoxin (Trx) and peroxiredoxin (Prx). Mitochondrial Trx2/Prx3 are more sensitive to Cr(VI) treatment than cytosolic Trx1/Prx1, although both compartments show thiol oxidation with higher doses or longer treatments. Thiol redox proteomics demonstrate that Trx2, Prx3, and Trx1 are among the most sensitive proteins in cells to Cr(VI) treatment. Their oxidation could therefore represent initiating events that have widespread implications for protein thiol redox control and for multiple aspects of redox signaling. This review summarizes the effects of Cr(VI) on the TrxR/Trx system and how these events could influence a number of downstream redox signaling systems that are influenced by Cr(VI) exposure. Some of the signaling events discussed include the activation of apoptosis signal regulating kinase and MAP kinases (p38 and JNK) and the modulation of a number of redox-sensitive transcription factors including AP-1, NF-κB, p53, and Nrf2. PMID:22542445

  4. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Kirkensgaard, Kristine G. [Carlsberg Laboratory (Denmark); Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark (Denmark); Hägglund, Per; Finnie, Christine; Svensson, Birte [Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark (Denmark); Henriksen, Anette, E-mail: anette@crc.dk [Carlsberg Laboratory (Denmark)

    2009-09-01

    The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R{sub cryst} of 19.0% and an R{sub free} of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation.

  5. Thioredoxin-1 attenuates sepsis-induced cardiomyopathy after cecal ligation and puncture in mice.

    Science.gov (United States)

    Wilson, Rickesha L; Selvaraju, Vaithinathan; Lakshmanan, Rajesh; Thirunavukkarasu, Mahesh; Campbell, Jacob; McFadden, David W; Maulik, Nilanjana

    2017-12-01

    Sepsis is a leading cause of mortality among patients in intensive care units across the USA. Thioredoxin-1 (Trx-1) is an essential 12 kDa cytosolic protein that, apart from maintaining the cellular redox state, possesses multifunctional properties. In this study, we explored the possibility of controlling adverse myocardial depression by overexpression of Trx-1 in a mouse model of severe sepsis. Adult C57BL/6J and Trx-1 Tg/+ mice were divided into wild-type sham (WTS), wild-type cecal ligation and puncture (WTCLP), Trx-1 Tg/+ sham (Trx-1 Tg/+ S), and Trx-1 Tg/+ CLP groups. Cardiac function was evaluated before surgery, 6 and 24 hours after CLP surgery. Immunohistochemical and Western blot analysis were performed after 24 hours in heart tissue sections. Echocardiography analysis showed preserved cardiac function in the Trx-1 Tg/+ CLP group compared with the WTCLP group. Similarly, Western blot analysis revealed increased expression of Trx-1, heme oxygenase-1 (HO-1), survivin (an inhibitor of apoptosis [IAP] protein family), and decreased expression of thioredoxin-interacting protein (TXNIP), caspase-3, and 3- nitrotyrosine in the Trx-1 Tg/+ CLP group compared with the WTCLP group. Immunohistochemical analysis showed reduced 4-hydroxynonenal, apoptosis, and vascular leakage in the cardiac tissue of Trx-1 Tg/+ CLP mice compared with mice in the WTCLP group. Our results indicate that overexpression of Trx-1 attenuates cardiac dysfunction during CLP. The mechanism of action may involve reduction of oxidative stress, apoptosis, and vascular permeability through activation of Trx-1/HO-1 and anti-apoptotic protein survivin. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

    International Nuclear Information System (INIS)

    Kirkensgaard, Kristine G.; Hägglund, Per; Finnie, Christine; Svensson, Birte; Henriksen, Anette

    2009-01-01

    The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R cryst of 19.0% and an R free of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation

  7. Analysis of the Interactions Between Thioredoxin and 20 Selenoproteins in Chicken.

    Science.gov (United States)

    Liu, Qi; Yang, Jie; Cai, Jingzeng; Luan, Yilin; Sattar, Hamid; Liu, Man; Xu, Shiwen; Zhang, Ziwei

    2017-10-01

    Thioredoxin (Trx) is a small molecular protein with complicated functions in a number of processes, including inflammation, apoptosis, embryogenesis, cardiovascular disease, and redox regulation. Some selenoproteins, such as glutathione peroxidase (Gpx), iodothyronine deiodinase (Dio), and thioredoxin reductase (TR), are involved in redox regulation. However, whether there are interactions between Trx and selenoproteins is still not known. In the present paper, we used a Modeller, Hex 8.0.0, and the KFC2 Server to predict the interactions between Trx and selenoproteins. We used the Modeller to predict the target protein in objective format and assess the accuracy of the results. Molecular interaction studies with Trx and selenoproteins were performed using the molecular docking tools in Hex 8.0.0. Next, we used the KFC2 Server to further test the protein binding sites. In addition to the selenoprotein physiological functions, we also explored potential relationships between Trx and selenoproteins beyond all the results we got. The results demonstrate that Trx has the potential to interact with 19 selenoproteins, including iodothyronine deiodinase 1 (Dio1), iodothyronine deiodinase 3 (Dio3), glutathione peroxidase 1 (Gpx1), glutathione peroxidase 2 (Gpx2), glutathione peroxidase 3 (Gpx3), glutathione peroxidase 4 (Gpx4), selenoprotein H (SelH), selenoprotein I (SelI), selenoprotein M (SelM), selenoprotein N (SelN), selenoprotein T (SelT), selenoprotein U (SelU), selenoprotein W (SelW), selenoprotein 15 (Sep15), methionine sulfoxide reductase B (Sepx1), selenophosphate synthetase 1 (SPS1), TR1, TR2, and TR3, among which TR1, TR2, TR3, SPS1, Sep15, SelN, SelM, SelI, Gpx2, Gpx3, Gpx4, and Dio3 exhibited intense correlations with Trx. However, additional experiments are needed to verify them.

  8. Thioredoxin reductase deficiency potentiates oxidative stress, mitochondrial dysfunction and cell death in dopaminergic cells.

    Directory of Open Access Journals (Sweden)

    Pamela Lopert

    Full Text Available Mitochondria are considered major generators of cellular reactive oxygen species (ROS which are implicated in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD. We have recently shown that isolated mitochondria consume hydrogen peroxide (H₂O₂ in a substrate- and respiration-dependent manner predominantly via the thioredoxin/peroxiredoxin (Trx/Prx system. The goal of this study was to determine the role of Trx/Prx system in dopaminergic cell death. We asked if pharmacological and lentiviral inhibition of the Trx/Prx system sensitized dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂ levels and death in response to toxicants implicated in PD. Incubation of N27 dopaminergic cells or primary rat mesencephalic cultures with the Trx reductase (TrxR inhibitor auranofin in the presence of sub-toxic concentrations of parkinsonian toxicants paraquat; PQ or 6-hydroxydopamine; 6OHDA (for N27 cells resulted in a synergistic increase in H₂O₂ levels and subsequent cell death. shRNA targeting the mitochondrial thioredoxin reductase (TrxR2 in N27 cells confirmed the effects of pharmacological inhibition. A synergistic decrease in maximal and reserve respiratory capacity was observed in auranofin treated cells and TrxR2 deficient cells following incubation with PQ or 6OHDA. Additionally, TrxR2 deficient cells showed decreased basal mitochondrial oxygen consumption rates. These data demonstrate that inhibition of the mitochondrial Trx/Prx system sensitizes dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂, and cell death. Therefore, in addition to their role in the production of cellular H₂O₂ the mitochondrial Trx/Prx system serve as a major sink for cellular H₂O₂ and its disruption may contribute to dopaminergic pathology associated with PD.

  9. An atlas of the thioredoxin fold class reveals the complexity of function-enabling adaptations.

    Directory of Open Access Journals (Sweden)

    Holly J Atkinson

    2009-10-01

    Full Text Available The group of proteins that contain a thioredoxin (Trx fold is huge and diverse. Assessment of the variation in catalytic machinery of Trx fold proteins is essential in providing a foundation for understanding their functional diversity and predicting the function of the many uncharacterized members of the class. The proteins of the Trx fold class retain common features-including variations on a dithiol CxxC active site motif-that lead to delivery of function. We use protein similarity networks to guide an analysis of how structural and sequence motifs track with catalytic function and taxonomic categories for 4,082 representative sequences spanning the known superfamilies of the Trx fold. Domain structure in the fold class is varied and modular, with 2.8% of sequences containing more than one Trx fold domain. Most member proteins are bacterial. The fold class exhibits many modifications to the CxxC active site motif-only 56.8% of proteins have both cysteines, and no functional groupings have absolute conservation of the expected catalytic motif. Only a small fraction of Trx fold sequences have been functionally characterized. This work provides a global view of the complex distribution of domains and catalytic machinery throughout the fold class, showing that each superfamily contains remnants of the CxxC active site. The unifying context provided by this work can guide the comparison of members of different Trx fold superfamilies to gain insight about their structure-function relationships, illustrated here with the thioredoxins and peroxiredoxins.

  10. Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1.

    Science.gov (United States)

    Hannemann, Liya; Suppanz, Ida; Ba, Qiaorui; MacInnes, Katherine; Drepper, Friedel; Warscheid, Bettina; Koch, Hans-Georg

    2016-01-20

    YchF/Ola1 are unconventional members of the universally conserved GTPase family because they preferentially hydrolyze ATP rather than GTP. These ATPases have been associated with various cellular processes and pathologies, including DNA repair, tumorigenesis, and apoptosis. In particular, a possible role in regulating the oxidative stress response has been suggested for both bacterial and human YchF/Ola1. In this study, we analyzed how YchF responds to oxidative stress and how it potentially regulates the antioxidant response. Our data identify a redox-regulated monomer-dimer equilibrium of YchF as a key event in the functional cycle of YchF. Upon oxidative stress, the oxidation of a conserved and surface-exposed cysteine residue promotes YchF dimerization, which is accompanied by inhibition of the ATPase activity. No dimers were observed in a YchF mutant lacking this cysteine. In vitro, the YchF dimer is dissociated by thioredoxin 1 (TrxA) and this stimulates the ATPase activity. The physiological significance of the YchF-thioredoxin 1 interaction was demonstrated by in vivo cross-linking, which validated this interaction in living cells. This approach also revealed that both the ATPase domain and the helical domain of YchF are in contact with TrxA. YchF/Ola1 are the first redox-regulated members of the universally conserved GTPase family and are inactivated by oxidation of a conserved cysteine residue within the nucleotide-binding motif. Our data provide novel insights into the regulation of the so far ill-defined YchF/Ola1 family of proteins and stipulate their role as negative regulators of the oxidative stress response.

  11. Cyclophosphamide as a potent inhibitor of tumor thioredoxin reductase in vivo

    International Nuclear Information System (INIS)

    Wang Xufang; Zhang Jinsong; Xu Tongwen

    2007-01-01

    Cyclophosphamide (CTX) is in the nitrogen mustard group of alkylating antineoplastic chemotherapeutic agents. It is one of the most frequently used antitumor agents for the treatment of a broad spectrum of human cancers. Thioredoxin reductase (TrxR) catalyze the NADPH-dependent reduction of thioredoxin and play an important role in multiple cellular events related to carcinogenesis including cell proliferation, apoptosis, and cell signaling. This enzyme represents a promising target for the development of cytostatic agents. The purpose of this study is to determine whether CTX could target TrxR in vivo. Lewis lung carcinoma and solid H22 hepatoma treated with 50-250 mg/kg CTX for 3 h lost TrxR activity in a dose-dependent fashion. Over 75% and 95% of TrxR activity was lost at the dose of 250 mg/kg. There was, however, a recovery of TrxR activity such that it attained normal levels by 120 h after a dose of 250 mg/kg. In addition, we found that CTX caused a preferential TrxR inhibition over other antioxidant enzymes, such as glutathione peroxidase, catalase, and superoxide dismutase. We also used ascites H22 cells to investigate cancer cells response after TrxR was inhibited by CTX in vivo since CTX is needed to be activated by liver cytochrome P450 enzymes. The time course and dose-dependent changes of cellular TrxR activity were similar with those in tumor tissue. CTX caused a dose-dependent cellular proliferation inhibition which was positively correlated with TrxR inhibition at 3 h. Furthermore, when 3 h CTX-treated cells with various TrxR backgrounds, harvested from ascites-bearing mice, were implanted into mice, the proliferations of these cells were again proportionally dependent on TrxR activity. The TrxR inhibition could thereby be considered as a crucial mechanism contributing to anticancer effect seen upon clinical use of CTX

  12. Differential labelling of cysteines for simultaneous identification of thioredoxin h-reducible disulphides in native protein extracts: insight into recognition and regulation of proteins in barley seeds by thioredoxin h

    DEFF Research Database (Denmark)

    Maeda, Kenji; Finnie, Christine; Svensson, Birte

    2005-01-01

    . Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four a-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located structurally close to the alpha-amylase...... binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects of the recognition mechanism......) to be distinguished from those inaccessible or disulphide bound form (pyridylethylated) according to the mass difference in the peptide mass maps obtained by matrixassistend laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in vitro reduction of disulphides in recombinant barley a-amylase...

  13. Pioglitazone enhances expression of genes involved in mitochondrial oxidative metabolism in skeletal muscle of women with polycystic ovary syndrome (PCOS)

    DEFF Research Database (Denmark)

    Skov, Vibe

    Aims                Polycystic ovary syndrome (PCOS) is a common endocrine disorder in premenopausal women and is associated with insulin resistance increasing the risk for developing type 2 diabetes mellitus. Studies have shown that thiazolidinediones (TZD) improve metabolic disturbances in PCOS...... patients. We hypothesized that the effect of TZD in PCOS is in part mediated by changes in the transcriptional profile of muscle favoring insulin sensitivity. Methods Using the HG-U133 2.0 Plus expression array from Affymetrix, we examined the effect of pioglitazone (30 mg/day for 16 weeks) on gene...... expression in skeletal muscle of 10 obese women with PCOS (dataset 1). Furthermore, evaluation of gene expression changes between PCOS patients before treatment and control subjects were performed (dataset 2). All subjects were metabolically characterised by a euglycemic-hyperinsulinemic clamp combined...

  14. Atypical Thioredoxins in Poplar: The Glutathione-Dependent Thioredoxin-Like 2.1 Supports the Activity of Target Enzymes Possessing a Single Redox Active Cysteine1[W

    Science.gov (United States)

    Chibani, Kamel; Tarrago, Lionel; Gualberto, José Manuel; Wingsle, Gunnar; Rey, Pascal; Jacquot, Jean-Pierre; Rouhier, Nicolas

    2012-01-01

    Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways. PMID:22523226

  15. Phytochemical composition and metabolic performance-enhancing activity of dietary berries traditionally used by Native North Americans.

    Science.gov (United States)

    Burns Kraft, Tristan F; Dey, Moul; Rogers, Randy B; Ribnicky, David M; Gipp, David M; Cefalu, William T; Raskin, Ilya; Lila, Mary Ann

    2008-02-13

    Four wild berry species, Amelanchier alnifolia, Viburnum trilobum, Prunus virginiana, and Shepherdia argentea, all integral to the traditional subsistence diet of Native American tribal communities, were evaluated to elucidate phytochemical composition and bioactive properties related to performance and human health. Biological activity was screened using a range of bioassays that assessed the potential for these little-known dietary berries to affect diabetic microvascular complications, hyperglycemia, pro-inflammatory gene expression, and metabolic syndrome symptoms. Nonpolar constituents from berries, including carotenoids, were potent inhibitors of aldose reductase (an enzyme involved in the etiology of diabetic microvascular complications), whereas the polar constituents, mainly phenolic acids, anthocyanins, and proanthocyanidins, were hypoglycemic agents and strong inhibitors of IL-1beta and COX-2 gene expression. Berry samples also showed the ability to modulate lipid metabolism and energy expenditure in a manner consistent with improving metabolic syndrome. The results demonstrate that these berries traditionally consumed by tribal cultures contain a rich array of phytochemicals that have the capacity to promote health and protect against chronic diseases, such as diabetes.

  16. An enhanced in vivo stable isotope labeling by amino acids in cell culture (SILAC) model for quantification of drug metabolism enzymes.

    Science.gov (United States)

    MacLeod, A Kenneth; Fallon, Padraic G; Sharp, Sheila; Henderson, Colin J; Wolf, C Roland; Huang, Jeffrey T-J

    2015-03-01

    Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of

  17. Nanoscale zero-valent iron/persulfate enhanced upflow anaerobic sludge blanket reactor for dye removal: Insight into microbial metabolism and microbial community

    Science.gov (United States)

    Pan, Fei; Zhong, Xiaohan; Xia, Dongsheng; Yin, Xianze; Li, Fan; Zhao, Dongye; Ji, Haodong; Liu, Wen

    2017-03-01

    This study investigated the efficiency of nanoscale zero-valent iron combined with persulfate (NZVI/PS) for enhanced degradation of brilliant red X-3B in an upflow anaerobic sludge blanket (UASB) reactor, and examined the effects of NZVI/PS on anaerobic microbial communities during the treatment process. The addition of NZVI (0.5 g/L) greatly enhanced the decolourization rate of X-3B from 63.8% to 98.4%. The Biolog EcoPlateTM technique was utilized to examine microbial metabolism in the reactor, and the Illumina MiSeq high-throughput sequencing revealed 22 phyla and 88 genera of the bacteria. The largest genera (Lactococcus) decreased from 33.03% to 7.94%, while the Akkermansia genera increased from 1.69% to 20.23% according to the abundance in the presence of 0.2 g/L NZVI during the biological treatment process. Meanwhile, three strains were isolated from the sludge in the UASB reactors and identified by 16 S rRNA analysis. The distribution of three strains was consistent with the results from the Illumina MiSeq high throughput sequencing. The X-ray photoelectron spectroscopy results indicated that Fe(0) was transformed into Fe(II)/Fe(III) during the treatment process, which are beneficial for the microorganism growth, and thus promoting their metabolic processes and microbial community.

  18. Nanoscale zero-valent iron/persulfate enhanced upflow anaerobic sludge blanket reactor for dye removal: Insight into microbial metabolism and microbial community

    Science.gov (United States)

    Pan, Fei; Zhong, Xiaohan; Xia, Dongsheng; Yin, Xianze; Li, Fan; Zhao, Dongye; Ji, Haodong; Liu, Wen

    2017-01-01

    This study investigated the efficiency of nanoscale zero-valent iron combined with persulfate (NZVI/PS) for enhanced degradation of brilliant red X-3B in an upflow anaerobic sludge blanket (UASB) reactor, and examined the effects of NZVI/PS on anaerobic microbial communities during the treatment process. The addition of NZVI (0.5 g/L) greatly enhanced the decolourization rate of X-3B from 63.8% to 98.4%. The Biolog EcoPlateTM technique was utilized to examine microbial metabolism in the reactor, and the Illumina MiSeq high-throughput sequencing revealed 22 phyla and 88 genera of the bacteria. The largest genera (Lactococcus) decreased from 33.03% to 7.94%, while the Akkermansia genera increased from 1.69% to 20.23% according to the abundance in the presence of 0.2 g/L NZVI during the biological treatment process. Meanwhile, three strains were isolated from the sludge in the UASB reactors and identified by 16 S rRNA analysis. The distribution of three strains was consistent with the results from the Illumina MiSeq high throughput sequencing. The X-ray photoelectron spectroscopy results indicated that Fe(0) was transformed into Fe(II)/Fe(III) during the treatment process, which are beneficial for the microorganism growth, and thus promoting their metabolic processes and microbial community. PMID:28300176

  19. Protein Chaperones Q8ZP25_SALTY from Salmonella Typhimurium and HYAE_ECOLI from Escherichia coli Exhibit Thioredoxin-like Structures Despite Lack of Canonical Thioredoxin Active Site Sequence Motif

    Energy Technology Data Exchange (ETDEWEB)

    Parish, D.; Benach, J; Liu, G; Singarapu, K; Xiao, R; Acton, T; Hunt, J; Montelione, G; Szyperski, T; et. al.

    2008-01-01

    The structure of the 142-residue protein Q8ZP25 SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE ECOLI was previously classified as a (NiFe) hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.

  20. Protein chaperones Q8ZP25_SALTY from Salmonella typhimurium and HYAE_ECOLI from Escherichia coli exhibit thioredoxin-like structures despite lack of canonical thioredoxin active site sequence motif.

    Science.gov (United States)

    Parish, David; Benach, Jordi; Liu, Goahua; Singarapu, Kiran Kumar; Xiao, Rong; Acton, Thomas; Su, Min; Bansal, Sonal; Prestegard, James H; Hunt, John; Montelione, Gaetano T; Szyperski, Thomas

    2008-12-01

    The structure of the 142-residue protein Q8ZP25_SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE_ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247-D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the 'thioredoxin-like clan'. However, protein HYAE_ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE_ECOLI was previously classified as a [NiFe] hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.

  1. High Temperature During Rice Grain Filling Enhances Aspartate Metabolism in Grains and Results in Accumulation of Aspartate-Family Amino Acids and Protein Components

    Directory of Open Access Journals (Sweden)

    Cheng-gang LIANG

    2013-09-01

    Full Text Available Global warming causes the exacerbation of rice growing environment, which seriously affects rice growth and reproduction, and finally results in the decrease of rice yield and quality. We investigated the activities of aspartate metabolism enzymes in grains, and the contents of Aspartate-family amino acids and protein components to further understand the effects of high temperature (HT on rice nutritional quality during rice grain filling. Under HT, the average activities of aspartate aminotransferase (AAT and aspartokinase (AK in grains significantly increased, the amino acid contents of aspartate (Asp, lysine (Lys, threonine (Thr, methionine (Met and isoleucine (Ile and the protein contents of albumin, globulin, prolamin and glutelin also significantly increased. The results indicated that HT enhanced Asp metabolism during rice grain filling and the enhancement of Asp metabolism might play an important role in the increase of Asp-family amino acids and protein components in grains. In case of the partial appraisal of the change of Asp-family amino acids and protein components under HT, we introduced eight indicators (amino acid or protein content, ratio of amino acid or protein, amino acid or protein content per grain and amino acid or protein content per panicle to estimate the effects of HT. It is suggested that HT during rice grain filling was benefit for the accumulation of Asp-family amino acids and protein components. Combined with the improvement of Asp-family amino acid ratio in grains under HT, it is suggested that HT during grain filling may improve the rice nutritional quality. However, the yields of parts of Asp-family amino acids and protein components were decreased under HT during rice grain filling.

  2. RNA-Seq Reveals Enhanced Sugar Metabolism in Streptococcus mutans Co-cultured with Candida albicans within Mixed-Species Biofilms

    Science.gov (United States)

    He, Jinzhi; Kim, Dongyeop; Zhou, Xuedong; Ahn, Sang-Joon; Burne, Robert A.; Richards, Vincent P.; Koo, Hyun

    2017-01-01

    Early childhood caries (ECC), which can lead to rampant tooth-decay that is painful and costly to treat, is one of the most prevalent infectious diseases affecting children worldwide. Previous studies support that interactions between Streptococcus mutans and Candida albicans are associated with the pathogenesis of ECC. The presence of Candida enhances S. mutans growth, fitness and accumulation within biofilms in vitro, although the molecular basis for these behaviors is undefined. Using an established co-cultivation biofilm model and RNA-Seq, we investigated how C. albicans influences the transcriptome of S. mutans. The presence of C. albicans dramatically altered gene expression in S. mutans in the dual-species biofilm, resulting in 393 genes differentially expressed, compared to mono-species biofilms of S. mutans. By Gene Ontology analysis, the majority of up-regulated genes were related to carbohydrate transport and metabolic/catabolic processes. KEGG pathway impact analysis showed elevated pyruvate and galactose metabolism, suggesting that co-cultivation with C. albicans influences carbohydrate utilization by S. mutans. Analysis of metabolites confirmed the increases in carbohydrate metabolism, with elevated amounts of formate in the culture medium of co-cultured biofilms. Moreover, co-cultivation with C. albicans altered transcription of S. mutans signal transduction (comC and ciaRH) genes associated with fitness and virulence. Interestingly, the expression of genes for mutacins (bacteriocins) and CRISPR were down-regulated. Collectively, the data provide a comprehensive insight into S. mutans transcriptomic changes induced by C. albicans, and offer novel insights into how bacterial–fungal interactions may enhance the severity of dental caries. PMID:28642749

  3. RNA-Seq Reveals Enhanced Sugar Metabolism in Streptococcus mutans Co-cultured with Candida albicans within Mixed-Species Biofilms

    Directory of Open Access Journals (Sweden)

    Jinzhi He

    2017-06-01

    Full Text Available Early childhood caries (ECC, which can lead to rampant tooth-decay that is painful and costly to treat, is one of the most prevalent infectious diseases affecting children worldwide. Previous studies support that interactions between Streptococcus mutans and Candida albicans are associated with the pathogenesis of ECC. The presence of Candida enhances S. mutans growth, fitness and accumulation within biofilms in vitro, although the molecular basis for these behaviors is undefined. Using an established co-cultivation biofilm model and RNA-Seq, we investigated how C. albicans influences the transcriptome of S. mutans. The presence of C. albicans dramatically altered gene expression in S. mutans in the dual-species biofilm, resulting in 393 genes differentially expressed, compared to mono-species biofilms of S. mutans. By Gene Ontology analysis, the majority of up-regulated genes were related to carbohydrate transport and metabolic/catabolic processes. KEGG pathway impact analysis showed elevated pyruvate and galactose metabolism, suggesting that co-cultivation with C. albicans influences carbohydrate utilization by S. mutans. Analysis of metabolites confirmed the increases in carbohydrate metabolism, with elevated amounts of formate in the culture medium of co-cultured biofilms. Moreover, co-cultivation with C. albicans altered transcription of S. mutans signal transduction (comC and ciaRH genes associated with fitness and virulence. Interestingly, the expression of genes for mutacins (bacteriocins and CRISPR were down-regulated. Collectively, the data provide a comprehensive insight into S. mutans transcriptomic changes induced by C. albicans, and offer novel insights into how bacterial–fungal interactions may enhance the severity of dental caries.

  4. Protective effect of bioflavonoid myricetin enhances carbohydrate metabolic enzymes and insulin signaling molecules in streptozotocin-cadmium induced diabetic nephrotoxic rats.

    Science.gov (United States)

    Kandasamy, Neelamegam; Ashokkumar, Natarajan

    2014-09-01

    Diabetic nephropathy is the kidney disease that occurs as a result of diabetes. The present study was aimed to evaluate the therapeutic potential of myricetin by assaying the activities of key enzymes of carbohydrate metabolism, insulin signaling molecules and renal function markers in streptozotocin (STZ)-cadmium (Cd) induced diabetic nephrotoxic rats. After myricetin treatment schedule, blood and tissue samples were collected to determine plasma glucose, insulin, hemoglobin, glycosylated hemoglobin and renal function markers, carbohydrate metabolic enzymes in the liver and insulin signaling molecules in the pancreas and skeletal muscle. A significant increase of plasma glucose, glycosylated hemoglobin, urea, uric acid, creatinine, blood urea nitrogen (BUN), urinary albumin, glycogen phosphorylase, glucose-6-phosphatase, and fructose-1,6-bisphosphatase and a significant decrease of plasma insulin, hemoglobin, hexokinase, glucose-6-phosphate dehydrogenase, glycogen and glycogen synthase with insulin signaling molecule expression were found in the STZ-Cd induced diabetic nephrotoxic rats. The administration of myricetin significantly normalizes the carbohydrate metabolic products like glucose, glycated hemoglobin, glycogen phosphorylase and gluconeogenic enzymes and renal function markers with increase insulin, glycogen, glycogen synthase and insulin signaling molecule expression like glucose transporter-2 (GLUT-2), glucose transporter-4 (GLUT-4), insulin receptor-1 (IRS-1), insulin receptor-2 (IRS-2) and protein kinase B (PKB). Based on the data, the protective effect of myricetin was confirmed by its histological annotation of the pancreas, liver and kidney tissues. These findings suggest that myricetin improved carbohydrate metabolism which subsequently enhances glucose utilization and renal function in STZ-Cd induced diabetic nephrotoxic rats. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. 1H, 13C and 15N chemical shift assignments of the thioredoxin from the obligate anaerobe Desulfovibrio vulgaris Hildenborough.

    Science.gov (United States)

    Garcin, Edwige B; Bornet, Olivier; Pieulle, Laetitia; Guerlesquin, Françoise; Sebban-Kreuzer, Corinne

    2011-10-01

    Thioredoxins are ubiquitous key antioxidant enzymes which play an essential role in cell defense against oxidative stress. They maintain the redox homeostasis owing to the regulation of thiol-disulfide exchange. In the present paper, we report the full resonance assignments of (1)H, (13)C and (15)N atoms for the reduced and oxidized forms of Desulfovibrio vulgaris Hildenborough thioredoxin 1 (Trx1). 2D and 3D heteronuclear NMR experiments were performed using uniformly (15)N-, (13)C-labelled Trx1. Chemical shifts of 97% of the backbone and 90% of the side chain atoms were obtained for the oxidized and reduced form (BMRB deposits with accession number 17299 and 17300, respectively).

  6. Improved mitochondrial function in brain aging and Alzheimer disease - the new mechanism of action of the old metabolic enhancer piracetam

    Directory of Open Access Journals (Sweden)

    Kristina Leuner

    2010-09-01

    Full Text Available Piracetam, the prototype of the so-called nootropic drugs’ is used since many years in different countries to treat cognitive impairment in aging and dementia. Findings that piracetam enhances fluidity of brain mitochondrial membranes led to the hypothesis that piracetam might improve mitochondrial function, e.g. might enhance ATP synthesis. This assumption has recently been supported by a number of observations showing enhanced mitochondrial membrane potential (MMP, enhanced ATP production, and reduced sensitivity for apoptosis in a variety of cell and animal models for aging and Alzheimer disease (AD. As a specific consequence, substantial evidence for elevated neuronal plasticity as a specific effect of piracetam has emerged. Taken together, these new findings can explain many of the therapeutic effects of piracetam on cognition in aging and dementia as well as different situations of brain dysfunctions.

  7. Inhibition of the thioredoxin system in the brain and liver of zebra-seabreams exposed to waterborne methylmercury

    International Nuclear Information System (INIS)

    Branco, Vasco; Canario, Joao; Holmgren, Arne; Carvalho, Cristina

    2011-01-01

    Mercury compounds were recently found to interact in vitro with the thioredoxin system, inhibiting both Thioredoxin (Trx) and Thioredoxin reductase (TrxR). In order to evaluate if Trx and TrxR are affected in vivo by methylmercury (MeHg), we exposed juvenile zebra-seabreams to different concentrations of this toxicant in water for 28 days followed by a 14-day depuration period. Methylmercury accumulated to a larger extent in the kidney and liver of fishes, but decreased significantly during the depuration. During the exposure, MeHg percentage in the liver reached levels above 90% of total mercury (HgT) decreasing to 60% of HgT by the end of the depuration period. In the kidney, MeHg accounted for 50-70% of HgT. In the brain and muscle, mercury accumulated throughout the exposure with all mercury being MeHg. The total mercury kept increasing in these organs during the depuration period. However, in the brain, this increase in HgT was accompanied by a decrease in the MeHg percentage (∼ 10%). In the liver, both Trx and TrxR activities were significantly reduced (TrxR - 40%; Trx - 70%) by the end of the exposure, but recovered to control levels (100%) during the depuration. In the brain, both enzymes where inhibited during the depuration period (TrxR - 75%; Trx - 70%) when some production of inorganic mercury was detected. Activity of glutathione reductase showed increased levels when TrxR activity was low, suggesting complementarity between both systems. These results indicate that in vivo the thioredoxin system is a toxicological target for MeHg with TrxR being particularly affected.

  8. Dynamic gene expression for metabolic engineering of mammalian cells in culture.

    Science.gov (United States)

    Le, Huong; Vishwanathan, Nandita; Kantardjieff, Anne; Doo, Inseok; Srienc, Michael; Zheng, Xiaolu; Somia, Nikunj; Hu, Wei-Shou

    2013-11-01

    Recombinant mammalian cells are the major hosts for the production of protein therapeutics. In addition to high expression of the product gene, a hyper-producer must also harbor superior phenotypic traits related to metabolism, protein secretion, and growth control. Introduction of genes endowing the relevant hyper-productivity traits is a strategy frequently used to enhance the productivity. Most of such cell engineering efforts have been performed using constitutive expression systems. However, cells respond to various environmental cues and cellular events dynamically according to cellular needs. The use of inducible systems allows for time dependent expression, but requires external manipulation. Ideally, a transgene's expression should be synchronous to the host cell's own rhythm, and at levels appropriate for the objective. To that end, we identified genes with different expression dynamics and intensity ranges using pooled transcriptome data. Their promoters may be used to drive the expression of the transgenes following the desired dynamics. We isolated the promoter of the Thioredoxin-interacting protein (Txnip) gene and demonstrated its capability to drive transgene expression in concert with cell growth. We further employed this Chinese hamster promoter to engineer dynamic expression of the mouse GLUT5 fructose transporter in Chinese hamster ovary (CHO) cells, enabling them to utilize sugar according to cellular needs rather than in excess as typically seen in culture. Thus, less lactate was produced, resulting in a better growth rate, prolonged culture duration, and higher product titer. This approach illustrates a novel concept in metabolic engineering which can potentially be used to achieve dynamic control of cellular behaviors for enhanced process characteristics. © 2013 Published by Elsevier Inc.

  9. NADPH-dependent thioredoxin reductase C plays a role in nonhost disease resistance against Pseudomonas syringae pathogens by regulating chloroplast-generated reactive oxygen species

    Directory of Open Access Journals (Sweden)

    Yasuhiro Ishiga

    2016-04-01

    Full Text Available Chloroplasts are cytoplasmic organelles for photosynthesis in eukaryotic cells. In addition, recent studies have shown that chloroplasts have a critical role in plant innate immunity against invading pathogens. Hydrogen peroxide is a toxic by-product from photosynthesis, which also functions as a signaling compound in plant innate immunity. Therefore, it is important to regulate the level of hydrogen peroxide in response to pathogens. Chloroplasts maintain components of the redox detoxification system including enzymes such as 2-Cys peroxiredoxins (2-Cys Prxs, and NADPH-dependent thioredoxin reductase C (NTRC. However, the significance of 2-Cys Prxs and NTRC in the molecular basis of nonhost disease resistance is largely unknown. We evaluated the roles of Prxs and NTRC using knock-out mutants of Arabidopsis in response to nonhost Pseudomonas syringae pathogens. Plants lacking functional NTRC showed localized cell death (LCD accompanied by the elevated accumulation of hydrogen peroxide in response to nonhost pathogens. Interestingly, the Arabidopsis ntrc mutant showed enhanced bacterial growth and disease susceptibility of nonhost pathogens. Furthermore, the expression profiles of the salicylic acid (SA and jasmonic acid (JA-mediated signaling pathways and phytohormone analyses including SA and JA revealed that the Arabidopsis ntrc mutant shows elevated JA-mediated signaling pathways in response to nonhost pathogen. These results suggest the critical role of NTRC in plant innate immunity against nonhost P. syringae pathogens.

  10. Direct association of thioredoxin-1 (TRX) with macrophage migration inhibitory factor (MIF): regulatory role of TRX on MIF internalization and signaling.

    Science.gov (United States)

    Son, Aoi; Kato, Noriko; Horibe, Tomohisa; Matsuo, Yoshiyuki; Mochizuki, Michika; Mitsui, Akira; Kawakami, Koji; Nakamura, Hajime; Yodoi, Junji

    2009-10-01

    Thioredoxin-1 (TRX) is a small (14 kDa) multifunctional protein with the redox-active site Cys-Gly-Pro-Cys. Macrophage migration inhibitory factor (MIF) is a 12 kDa cytokine belonging to the TRX family. Historically, when we purified TRX from the supernatant of ATL-2 cells, a 12 kDa protein was identified along with TRX, which was later proved to be MIF. Here, we show that TRX and MIF form a complex in the cell and the culture supernatant of ATL-2 cells. Using a BIAcore assay, we confirmed that TRX has a specific affinity with MIF. We also found that extracellular MIF was more effectively internalized into the ATL-2 cells expressing TRX on the cell surface, than the Jurkat T cells which do not express surface TRX. Moreover, anti-TRX antibody blocked the MIF internalization, suggesting that the cell surface TRX is involved in MIF internalization into the cells. Furthermore, anti-TRX antibody inhibited MIF-mediated enhancement of TNF-alpha production from macrophage RAW264.7 cells. These results suggest that the cell surface TRX serves as one of the MIF binding molecules or MIF receptor component and inhibits MIF-mediated inflammatory signals.

  11. Ocean Warming Enhances Malformations, Premature Hatching, Metabolic Suppression and Oxidative Stress in the Early Life Stages of a Keystone Squid

    Science.gov (United States)

    Rosa, Rui; Pimentel, Marta S.; Boavida-Portugal, Joana; Teixeira, Tatiana; Trübenbach, Katja; Diniz, Mário

    2012-01-01

    Background The knowledge about the capacity of organisms’ early life stages to adapt to elevated temperatures is very limited but crucial to understand how marine biota will respond to global warming. Here we provide a comprehensive and integrated view of biological responses to future warming during the early ontogeny of a keystone invertebrate, the squid Loligo vulgaris. Methodology/Principal Findings Recently-spawned egg masses were collected and reared until hatching at present day and projected near future (+2°C) temperatures, to investigate the ability of early stages to undergo thermal acclimation, namely phenotypic altering of morphological, behavioural, biochemical and physiological features. Our findings showed that under the projected near-future warming, the abiotic conditions inside the eggs promoted metabolic suppression, which was followed by premature hatching. Concomitantly, the less developed newborns showed greater incidence of malformations. After hatching, the metabolic burst associated with the transition from an encapsulated embryo to a planktonic stage increased linearly with temperature. However, the greater exposure to environmental stress by the hatchlings seemed to be compensated by physiological mechanisms that reduce the negative effects on fitness. Heat shock proteins (HSP70/HSC70) and antioxidant enzymes activities constituted an integrated stress response to ocean warming in hatchlings (but not in embryos). Conclusions/Significance The stressful abiotic conditions inside eggs are expected to be aggravated under the projected near-future ocean warming, with deleterious effects on embryo survival and growth. Greater feeding challenges and the lower thermal tolerance limits of the hatchlings are strictly connected to high metabolic demands associated with the planktonic life strategy. Yet, we found some evidence that, in the future, the early stages might support higher energy demands by adjusting some cellular functional properties

  12. Western diet enhances intestinal tumorigenesis in Min/+ mice, associating with mucosal metabolic and inflammatory stress and loss of Apc heterozygosity.

    Science.gov (United States)

    Niku, Mikael; Pajari, Anne-Maria; Sarantaus, Laura; Päivärinta, Essi; Storvik, Markus; Heiman-Lindh, Anu; Suokas, Santeri; Nyström, Minna; Mutanen, Marja

    2017-01-01

    Western-type diet (WD) is a risk factor for colorectal cancer, but the underlying mechanisms are poorly understood. We investigated the interaction of WD and heterozygous mutation in the Apc gene on adenoma formation and metabolic and immunological changes in the histologically normal intestinal mucosa of Apc Min/+ (Min/+) mice. The diet used was high in saturated fat and low in calcium, vitamin D, fiber and folate. The number of adenomas was twofold higher in the WD mice compared to controls, but adenoma size, proliferation or apoptosis did not differ. The ratio of the Min to wild-type allele was higher in the WD mice, indicating accelerated loss of Apc heterozygosity (LOH). Densities of intraepithelial CD3ε + T lymphocytes and of mucosal FoxP3 + regulatory T cells were higher in the WD mice, implying inflammatory changes. Western blot analyses from the mucosa of the WD mice showed suppressed activation of the ERK and AKT pathways and a tendency for reduced activation of the mTOR pathway as measured in phosphoS6/S6 levels. The expression of pyruvate dehydrogenase kinase 4 was up-regulated in both mRNA and protein levels. Gene expression analyses showed changes in oxidation/reduction, fatty acid and monosaccharide metabolic pathways, tissue organization, cell fate and regulation of apoptosis. Together, our results suggest that the high-risk Western diet primes the intestine to tumorigenesis through synergistic effects in energy metabolism, inflammation and oxidative stress, which culminate in the acceleration of LOH of the Apc gene. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. SPIN1, negatively regulated by miR-148/152, enhances Adriamycin resistance via upregulating drug metabolizing enzymes and transporter in breast cancer.

    Science.gov (United States)

    Chen, Xu; Wang, Ya-Wen; Gao, Peng

    2018-05-09

    Spindlin1 (SPIN1), a protein highly expressed in several human cancers, has been correlated with tumorigenesis and development. Alterations of drug metabolizing enzymes and drug transporters are major determinants of chemoresistance in tumor cells. However, whether the metabolizing enzymes and transporters are under the control of SPIN1 in breast cancer chemoresistance has not yet been defined. SPIN1 expression in breast cancer cells and tissues was detected by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Chemosensitivity assays in vitro and in vivo were performed to determine the effect of SPIN1 on Adriamycin resistance. Downstream effectors of SPIN1 were screened by microarray and confirmed by qRT-PCR and Western blot. Luciferase assay and Western blot were used to identify miRNAs regulating SPIN1. We showed that SPIN1 was significantly elevated in drug-resistant breast cancer cell lines and tissues, compared with the chemosensitive ones. SPIN1 enhanced Adriamycin resistance of breast cancer cells in vitro, and downregulation of SPIN1 by miRNA could decrease Adriamycin resistance in vivo. Mechanistically, drug metabolizing enzymes and transporter CYP2C8, UGT2B4, UGT2B17 and ABCB4 were proven to be downstream effectors of SPIN1. Notably, SPIN1 was identified as a direct target of the miR-148/152 family (miR-148a-3p, miR-148b-3p and miR-152-3p). As expected, miR-148a-3p, miR-148b-3p or miR-152-3p could increase Adriamycin sensitivity in breast cancer cells in vitro. Moreover, high expression of SPIN1 or low expression of the miR-148/152 family predicted poorer survival in breast cancer patients. Our results establish that SPIN1, negatively regulated by the miR-148/152 family, enhances Adriamycin resistance in breast cancer via upregulating the expression of drug metabolizing enzymes and drug transporter.

  14. Enhanced

    Directory of Open Access Journals (Sweden)

    Martin I. Bayala

    2014-06-01

    Full Text Available Land Surface Temperature (LST is a key parameter in the energy balance model. However, the spatial resolution of the retrieved LST from sensors with high temporal resolution is not accurate enough to be used in local-scale studies. To explore the LST–Normalised Difference Vegetation Index relationship potential and obtain thermal images with high spatial resolution, six enhanced image sharpening techniques were assessed: the disaggregation procedure for radiometric surface temperatures (TsHARP, the Dry Edge Quadratic Function, the Difference of Edges (Ts∗DL and three models supported by the relationship of surface temperature and water stress of vegetation (Normalised Difference Water Index, Normalised Difference Infrared Index and Soil wetness index. Energy Balance Station data and in situ measurements were used to validate the enhanced LST images over a mixed agricultural landscape in the sub-humid Pampean Region of Argentina (PRA, during 2006–2010. Landsat Thematic Mapper (TM and Moderate Resolution Imaging Spectroradiometer (EOS-MODIS thermal datasets were assessed for different spatial resolutions (e.g., 960, 720 and 240 m and the performances were compared with global and local TsHARP procedures. Results suggest that the Ts∗DL technique is the most adequate for simulating LST to high spatial resolution over the heterogeneous landscape of a sub-humid region, showing an average root mean square error of less than 1 K.

  15. Molecular cloning and characterization of Fasciola gigantica thioredoxin-glutathione reductase.

    Science.gov (United States)

    Changklungmoa, Narin; Kueakhai, Pornanan; Sangpairoj, Kant; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Riengrojpitak, Suda; Sobhon, Prasert; Chaithirayanon, Kulathida

    2015-06-01

    The Fasciola gigantica thioredoxin-glutathione reductase (FgTGR) gene is a fusion between thioredoxin reductase (TR) and a glutaredoxin (Grx) gene. FgTGR was cloned by polymerase chain reaction (PCR) from adult complementary DNA (cDNA), and its sequences showed two isoforms, i.e., the cytosolic and mitochondrial FgTGR. Cytosolic FgTGR (cytFgTGR) was composed of 2370 bp, and its peptide had no signal sequence and hence was not a secreted protein. Mitochondrial FgTGR (mitFgTGR) was composed of 2506 bp with a signal peptide of 43 amino acids; therefore, it was a secreted protein. The putative cytFgTGR and mitFgTGR peptides comprised of 598 and 641 amino acids, respectively, with a molecular weight of 65.8 kDa for cytFgTGR and mitFgTGR, with a conserved sequence (CPYC) of TR, and ACUG and CVNVGC of Grx domains. The recombinant FgTGR (rFgTGR) was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTGR). The FgTGR protein expression, estimated by indirect ELISA using the rabbit anti-rFgTGR as probe, showed high levels of expression in eggs, and 2- and 4-week-old juveniles and adults. The rFgTGR exhibited specific activities in the 5,5'-dithiobis (2-nitro-benzoic acid) (DTNB) reductase assay for TR activity and in β-hydroxyethul disulfide (HED) for Grx activity. When analyzed by immunoblotting and immunohistochemistry, rabbit anti-rFgTGR reacted with natural FgTGR at a molecular weight of 66 kDa from eggs, whole body fraction (WB) of metacercariae, NEJ, 2- and 4-week-old juveniles and adults, and the tegumental antigen (TA) of adult. The FgTGR protein was expressed at high levels in the tegument of 2- and 4-week-old juveniles. The FgTGR may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or a drug target.

  16. Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions.

    Science.gov (United States)

    Mancusso, Romina; Cruz, Eduardo; Cataldi, Marcela; Mendoza, Carla; Fuchs, James; Wang, Hsin; Yang, Xiaomin; Tasayco, María Luisa

    2004-04-06

    Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to

  17. Exogenous spermidine is enhancing tomato tolerance to salinity-alkalinity stress by regulating chloroplast antioxidant system and chlorophyll metabolism.

    Science.gov (United States)

    Li, Jianming; Hu, Lipan; Zhang, Li; Pan, Xiongbo; Hu, Xiaohui

    2015-12-29

    Salinity-alkalinity stress is known to adversely affect a variety of processes in plants, thus inhibiting growth and decreasing crop yield. Polyamines protect plants against a variety of environmental stresses. However, whether exogenous spermidine increases the tolerance of tomato seedlings via effects on chloroplast antioxidant enzymes and chlorophyll metabolism is unknown. In this study, we examined the effect of exogenous spermidine on chlorophyll synthesis and degradation pathway intermediates and related enzyme activities, as well as chloroplast ultrastructure, gene expression, and antioxidants in salinity-alkalinity-stressed tomato seedlings. Salinity-alkalinity stress disrupted chlorophyll metabolism and hindered uroorphyrinogen III conversion to protoporphyrin IX. These effects were more pronounced in seedlings of cultivar Zhongza No. 9 than cultivar Jinpengchaoguan. Under salinity-alkalinity stress, exogenous spermidine alleviated decreases in the contents of total chlorophyll and chlorophyll a and b in seedlings of both cultivars following 4 days of stress. With extended stress, exogenous spermidine reduced the accumulation of δ-aminolevulinic acid, porphobilinogen, and uroorphyrinogen III and increased the levels of protoporphyrin IX, Mg-protoporphyrin IX, and protochlorophyllide, suggesting that spermidine promotes the conversion of uroorphyrinogen III to protoporphyrin IX. The effect occurred earlier in cultivar Jinpengchaoguan than in cultivar Zhongza No. 9. Exogenous spermidine also alleviated the stress-induced increases in malondialdehyde content, superoxide radical generation rate, chlorophyllase activity, and expression of the chlorophyllase gene and the stress-induced decreases in the activities of antioxidant enzymes, antioxidants, and expression of the porphobilinogen deaminase gene. In addition, exogenous spermidine stabilized the chloroplast ultrastructure in stressed tomato seedlings. The tomato cultivars examined exhibited different

  18. Exercise Prevents Enhanced Postoperative Neuroinflammation and Cognitive Decline and Rectifies the Gut Microbiome in a Rat Model of Metabolic Syndrome.

    Science.gov (United States)

    Feng, Xiaomei; Uchida, Yosuke; Koch, Lauren; Britton, Steve; Hu, Jun; Lutrin, David; Maze, Mervyn

    2017-01-01

    Postoperative cognitive decline (PCD) can affect in excess of 10% of surgical patients and can be considerably higher with risk factors including advanced age, perioperative infection, and metabolic conditions such as obesity and insulin resistance. To define underlying pathophysiologic processes, we used animal models including a rat model of metabolic syndrome generated by breeding for a trait of low aerobic exercise tolerance. After 35 generations, the low capacity runner (LCR) rats differ 10-fold in their aerobic exercise capacity from high capacity runner (HCR) rats. The LCR rats respond to surgical procedure with an abnormal phenotype consisting of exaggerated and persistent PCD and failure to resolve neuroinflammation. We determined whether preoperative exercise can rectify the abnormal surgical phenotype. Following institutional approval of the protocol each of male LCR and male HCR rats were randomly assigned to four groups and subjected to isoflurane anesthesia and tibia fracture with internal fixation (surgery) or anesthesia alone (sham surgery) and to a preoperative exercise regimen that involved walking for 10 km on a treadmill over 6 weeks (exercise) or being placed on a stationary treadmill (no exercise). Feces were collected before and after exercise for assessment of gut microbiome. Three days following surgery or sham surgery the rats were tested for ability to recall a contextual aversive stimulus in a trace fear conditioning paradigm. Thereafter some rats were euthanized and the hippocampus harvested for analysis of inflammatory mediators. At 3 months, the remainder of the rats were tested for memory recall by the probe test in a Morris Water Maze. Postoperatively, LCR rats exhibited exaggerated cognitive decline both at 3 days and at 3 months that was prevented by preoperative exercise. Similarly, LCR rats had excessive postoperative neuroinflammation that was normalized by preoperative exercise. Diversity of the gut microbiome in the

  19. Fractal structure in the volumetric contrast enhancement of malignant gliomas as a marker of oxidative metabolic pathway gene expression

    NARCIS (Netherlands)

    Miller, Kai J.; Berendsen, Sharon; Seute, Tatjana; Yeom, Kristen; Gephardt, Melanie H.; Grant, Gerald A.; Robe, Pierre A.

    2017-01-01

    Background: Fractal structure is found throughout many processes in nature, and often arises from sets of simple rules. We examined MRI contrast enhancement patterns from glioblastoma patients for evidence of fractal structure and correlated these with gene expression patterns. Methods: For 39

  20. Crassulacean acid metabolism enhances underwater photosynthesis and diminishes photorespiration in the aquatic plant Isoetes australis

    DEFF Research Database (Denmark)

    Pedersen, Ole; Rich, S.M.; Pulido Pérez, Cristina

    2011-01-01

    Underwater photosynthesis by aquatic plants is often limited by low availability of CO2, and photorespiration can be high. Some aquatic plants utilize crassulacean acid metabolism (CAM) photosynthesis. The benefits of CAM for increased underwater photosynthesis and suppression of photorespiration...... photorespiration was evident at a range of O2 concentrations, including values below air equilibrium. At a high O2 concentration of 2.2-fold the atmospheric equilibrium concentration, net photosynthesis was reduced substantially and, although it remained positive in leaves containing high malate concentrations...... were evaluated for Isoetes australis, a submerged plant that inhabits shallow temporary rock pools. • Leaves high or low in malate were evaluated for underwater net photosynthesis and apparent photorespiration at a range of CO2 and O2 concentrations. • CAM activity was indicated by 9.7-fold higher leaf...

  1. Altered cortisol metabolism in polycystic ovary syndrome: insulin enhances 5alpha-reduction but not the elevated adrenal steroid production rates.

    Science.gov (United States)

    Tsilchorozidou, Tasoula; Honour, John W; Conway, Gerard S

    2003-12-01

    Androgen excess in women with polycystic ovary syndrome (PCOS) may be ovarian and/or adrenal in origin, and one proposed contributing mechanism is altered cortisol metabolism. Increased peripheral metabolism of cortisol may occur by enhanced inactivation of cortisol by 5alpha-reductase (5alpha-R) or impaired reactivation of cortisol from cortisone by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) resulting in decreased negative feedback suppression of ACTH secretion maintaining normal plasma cortisol concentrations at the expense of androgen excess. We have tested whether any enzyme dysregulation was related to circulating insulin or androgen concentrations in women with PCOS and have sought to clarify their relationship with obesity. First, to avoid obesity-related effects on cortisol metabolism, 18 lean women with PCOS were compared with 19 lean controls who were closely matched for body mass index (BMI). Second, the impact of obesity was studied in a cross-section of 42 PCOS women of a broad range of BMI. We measured 24-h urinary excretion of steroid metabolites by gas chromatography/mass spectrometry and fasting metabolic and hormone profiles. Urinary excretion of androgens [androsterone (P = 0.003), etiocholanolone (P = 0.02), and C19 steroid sulfates (P = 0.009)], cortisone metabolites [tetrahydrocortisone (THE) (P = 0.02), alpha-cortolone (P lean PCOS subjects when compared with controls. A significantly higher 5alpha-tetrahydrocortisol (5alpha-THF)/5beta-THF ratio (P = 0.04) and a significantly lower alpha-THF + THF + alpha-cortol/THE + cortolones ratio (P = 0.01) were found in lean PCOS women compared with lean controls, indicating both enhanced 5alpha-R and reduced 11beta-HSD1 activities. A decreased THE/cortolones ratio (P = 0.03) was also found in lean PCOS women compared with lean controls, indicating increased 20 alpha/beta-HSD activity. In the group of 42 PCOS subjects, measures of 5alpha/5beta reduction were positively correlated with the

  2. An extensive case study of hairy-root cultures for enhanced secondary-metabolite production through metabolic-pathway engineering.

    Science.gov (United States)

    Mehrotra, Shakti; Rahman, Laiq Ur; Kukreja, Arun Kumar

    2010-08-23

    An intrinsic improvement is taking place in the methodologies for the development of culture systems with first-rate production of plant-based molecules. The blending of HR (hairy root) cultures with ME (metabolic engineering) approaches offers new insights into, and possibilities for, improving the system productivity for known and/or novel high-value plant-derived active compounds. The introduction and expression of foreign genes in plants results in improvement of cellular activities by manipulating enzymatic, regulatory and transport function of the cell. The rational amendments in the rate-limiting steps of a biosynthetic pathway as well as inactivating the inefficient pathway(s) for by-product formation can be accomplished either through single-step engineering or through the multi-step engineering. The hierarchical control of any metabolic process can lead the engineer to apply the ME ideas and principles to any of the strata, including transcriptional, moving on to translational and enzymatic activity. The HR culture systems offer a remarkable potential for commercial production of a number of low-volume, but high-value, secondary metabolites. Taking HR as a model system, in the present review, we discuss engineering principles and perceptions to exploit secondary-metabolite pathways for the production of important bioactive compounds. We also talk about requisites and possible challenges that occur during ME, with emphasis on examples of various HR systems. Furthermore, it also highlights the utilization of global information obtained from '-omic' platforms in order to explore pathway architecture, structural and functional aspects of important enzymes and genes that can support the design of sets of engineering, resulting in the generation of wide-ranging views of DNA sequence-to-metabolite passageway networking and their control to obtain desired results.

  3. VLDL from Metabolic Syndrome Individuals Enhanced Lipid Accumulation in Atria with Association of Susceptibility to Atrial Fibrillation.

    Science.gov (United States)

    Lee, Hsiang-Chun; Lin, Hsin-Ting; Ke, Liang-Yin; Wei, Chi; Hsiao, Yi-Lin; Chu, Chih-Sheng; Lai, Wen-Ter; Shin, Shyi-Jang; Chen, Chu-Huang; Sheu, Sheng-Hsiung; Wu, Bin-Nan

    2016-01-20

    Metabolic syndrome (MetS) represents a cluster of metabolic derangements. Dyslipidemia is an important factor in MetS and is related to atrial fibrillation (AF). We hypothesized that very low density lipoproteins (VLDL) in MetS (MetS-VLDL) may induce atrial dilatation and vulnerability to AF. VLDL was therefore separated from normal (normal-VLDL) and MetS individuals. Wild type C57BL/6 male mice were divided into control, normal-VLDL (nVLDL), and MetS-VLDL (msVLDL) groups. VLDL (15 µg/g) and equivalent volumes of saline were injected via tail vein three times a week for six consecutive weeks. Cardiac chamber size and function were measured by echocardiography. MetS-VLDL significantly caused left atrial dilation (control, n = 10, 1.64 ± 0.23 mm; nVLDL, n = 7, 1.84 ± 0.13 mm; msVLDL, n = 10, 2.18 ± 0.24 mm; p < 0.0001) at week 6, associated with decreased ejection fraction (control, n = 10, 62.5% ± 7.7%, vs. msVLDL, n = 10, 52.9% ± 9.6%; p < 0.05). Isoproterenol-challenge experiment resulted in AF in young msVLDL mice. Unprovoked AF occurred only in elderly msVLDL mice. Immunohistochemistry showed excess lipid accumulation and apoptosis in msVLDL mice atria. These findings suggest a pivotal role of VLDL in AF pathogenesis for MetS individuals.

  4. Enhanced metabolic versatility of planktonic sulfur-oxidizing γ-proteobacteria in an oxygen-deficient coastal ecosystem

    Directory of Open Access Journals (Sweden)

    Alejandro A. Murillo

    2014-07-01

    Full Text Available Sulfur-oxidizing Gamma-proteobacteria are abundant in marine oxygen-deficient waters, and appear to play a key role in a previously unrecognized cryptic sulfur cycle. Metagenomic analyses of members of the uncultured SUP05 lineage in the Canadian seasonally anoxic fjord Saanich Inlet (SI, hydrothermal plumes in the Guaymas Basin (GB and single cell genomics analysis of two ARCTIC96BD-19 representatives from the South Atlantic Sub-Tropical Gyre (SASG have shown them to be metabolically versatile. However, SI and GB SUP05 bacteria seem to be obligate chemolithoautotrophs, whereas ARCTIC96BD-19 has the genetic potential for aerobic respiration. Here, we present results of a metagenomic analysis of sulfur-oxidizing Gamma-proteobacteria (GSO, closely related to the SUP05/ARCTIC96BD-19 clade, from a coastal ecosystem in the eastern South Pacific (ESP. This ecosystem experiences seasonal anoxia and accumulation of nitrite and ammonium at depth, with a corresponding increase in the abundance of GSO representatives. The ESP-GSOs appear to have a significantly different gene complement than those from Saanich Inlet, Guaymas Basin and SASG. Genomic analyses of de novo assembled contigs indicate the presence of a complete aerobic respiratory complex based on the cytochrome bc1 oxidase. Furthermore, they appear to encode a complete TCA cycle and several transporters for dissolved organic carbon species, suggesting a mixotrophic lifestyle. Thus, the success of sulfur-oxidizing Gamma-proteobacteria in oxygen-deficient marine ecosystems appears due not only to their previously recognized anaerobic metabolic versatility, but also to their capacity to function under aerobic conditions using different carbon sources. Finally, members of ESP-GSO cluster also have the genetic potential for reducing nitrate to ammonium based on the nirBD genes, and may therefore facilitate a tighter coupling of the nitrogen and sulfur cycles in oxygen-deficient waters.

  5. Detection of transketolase in bone marrow-derived insulin-producing cells: benfotiamine enhances insulin synthesis and glucose metabolism.

    Science.gov (United States)

    Oh, Seh-Hoon; Witek, Rafal P; Bae, Si-Hyun; Darwiche, Houda; Jung, Youngmi; Pi, Liya; Brown, Alicia; Petersen, Bryon E

    2009-01-01

    Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus.

  6. Detection of Transketolase in Bone Marrow—Derived Insulin-Producing Cells: Benfotiamine Enhances Insulin Synthesis and Glucose Metabolism

    Science.gov (United States)

    Witek, Rafal P.; Bae, Si-Hyun; Darwiche, Houda; Jung, Youngmi; Pi, Liya; Brown, Alicia; Petersen, Bryon E.

    2009-01-01

    Adult bone marrow (BM)-derived insulin-producing cells (IPCs) are capable of regulating blood glucose levels in chemically induced hyperglycemic mice. Using cell transplantation therapy, fully functional BM-derived IPCs help to mediate treatment of diabetes mellitus. Here, we demonstrate the detection of the pentose phosphate pathway enzyme, transketolase (TK), in BM-derived IPCs cultured under high-glucose conditions. Benfotiamine, a known activator of TK, was not shown to affect the proliferation of insulinoma cell line, INS-1; however, when INS-1 cells were cultured with oxythiamine, an inhibitor of TK, cell proliferation was suppressed. Treatment with benfotiamine activated glucose metabolism in INS-1 cells in high-glucose culture conditions, and appeared to maximize the BM-derived IPCs ability to synthesize insulin. Benfotiamine was not shown to induce the glucose receptor Glut-2, however it was shown to activate glucokinase, the enzyme responsible for conversion of glucose to glucose-6-phosphate. Furthermore, benfotiamine-treated groups showed upregulation of the downstream glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH). However, in cells where the pentose phosphate pathway was blocked by oxythiamine treatment, there was a clear downregulation of Glut-2, glucokinase, insulin, and GAPDH. When benfotiamine was used to treat mice transplanted with BM-derived IPCs transplanted, their glucose level was brought to a normal range. The glucose challenge of normal mice treated with benfotiamine lead to rapidly normalized blood glucose levels. These results indicate that benfotiamine activates glucose metabolism and insulin synthesis to prevent glucose toxicity caused by high concentrations of blood glucose in diabetes mellitus. PMID:18393672

  7. Modulation of Tryptophan and Serotonin Metabolism as a Biochemical Basis of the Behavioral Effects of Use and Withdrawal of Androgenic-Anabolic Steroids and Other Image- and Performance-Enhancing Agents

    Directory of Open Access Journals (Sweden)

    Abdulla A-B Badawy

    2018-02-01

    Full Text Available Modulation of tryptophan (Trp metabolism may underpin the behavioral effects of androgenic-anabolic steroids (AAS and associated image and performance enhancers. Euphoria, arousal, and decreased anxiety observed with moderate use and exercise may involve enhanced cerebral serotonin synthesis and function by increased release of albumin-bound Trp and estrogen-mediated liver Trp 2,3-dioxygenase (TDO inhibition and enhancement of serotonin function. Aggression, anxiety, depression, personality disorders, and psychosis, observed on withdrawal of AAS or with use of large doses, can be caused by decreased serotonin synthesis due to TDO induction on withdrawal, excess Trp inhibiting the 2 enzymes of serotonin synthesis, and increased cerebral levels of neuroactive kynurenines. Exercise and excessive protein and branched-chain amino acid intakes may aggravate the effects of large AAS dosage. The hypothesis is testable in humans and experimental animals by measuring parameters of Trp metabolism and disposition and related metabolic processes.

  8. Pathways of Lipid Metabolism in Marine Algae, Co-Expression Network, Bottlenecks and Candidate Genes for Enhanced Production of EPA and DHA in Species of Chromista

    Directory of Open Access Journals (Sweden)

    Alice Mühlroth

    2013-11-01

    Full Text Available The importance of n-3 long chain polyunsaturated fatty acids (LC-PUFAs for human health has received more focus the last decades, and the global consumption of n-3 LC-PUFA has increased. Seafood, the natural n-3 LC-PUFA source, is harvested beyond a sustainable capacity, and it is therefore imperative to develop alternative n-3 LC-PUFA sources for both eicosapentaenoic acid (EPA, 20:5n-3 and docosahexaenoic acid (DHA, 22:6n-3. Genera of algae such as Nannochloropsis, Schizochytrium, Isochrysis and Phaedactylum within the kingdom Chromista have received attention due to their ability to produce n-3 LC-PUFAs. Knowledge of LC-PUFA synthesis and its regulation in algae at the molecular level is fragmentary and represents a bottleneck for attempts to enhance the n-3 LC-PUFA levels for industrial production. In the present review, Phaeodactylum tricornutum has been used to exemplify the synthesis and compartmentalization of n-3 LC-PUFAs. Based on recent transcriptome data a co-expression network of 106 genes involved in lipid metabolism has been created. Together with recent molecular biological and metabolic studies, a model pathway for n-3 LC-PUFA synthesis in P. tricornutum has been proposed, and is compared to industrialized species of Chromista. Limitations of the n-3 LC-PUFA synthesis by enzymes such as thioesterases, elongases, acyl-CoA synthetases and acyltransferases are discussed and metabolic bottlenecks are hypothesized such as the supply of the acetyl-CoA and NADPH. A future industrialization will depend on optimization of chemical compositions and increased biomass production, which can be achieved by exploitation of the physiological potential, by selective breeding and by genetic engineering.

  9. The ACE-2/Ang1-7/Mas cascade enhances bone structure and metabolism following angiotensin-II type 1 receptor blockade.

    Science.gov (United States)

    Abuohashish, Hatem M; Ahmed, Mohammed M; Sabry, Dina; Khattab, Mahmoud M; Al-Rejaie, Salim S

    2017-07-15

    The renin angiotensin system (RAS) regulates numerous systemic functions and is expressed locally in skeletal tissues. Angiotensin1-7 (Ang1-7) is a beneficial member of the RAS, and the therapeutic effects of a large number of angiotensin receptors blockers (ARBs) are mediated by an Ang1-7-dependent cascade. This study examines whether the reported osteo-preservative effects of losartan are mediated through the angiotensin converting enzyme2 (ACE-2)/Ang1-7/Mas pathway in ovariectomized (OVX) rats. Sham and OVX animals received losartan (10mg/kg/d p.o.) for 6 weeks. A specific Mas receptor blocker (A-779) was delivered via mini-osmotic pumps during the losartan treatment period. Serum and urine bone metabolism biomarker levels were measured. Bone trabecular and cortical morphometry were quantified in distal femurs, whereas mineral contents were estimated in ashed bones, serum and urine. Finally, the expression of RAS components, the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) was determined. Losartan significantly improved the elevated bone metabolism marker levels and altered trabecular and cortical structures in OVX animals, and restored normal urinary and skeletal mineral levels. Mas receptor inhibition significantly abolished all osteo-protective effects of losartan and enhanced the deleterious effects of OVX. Losartan enhanced OVX-induced up-regulation of ACE-1, AngII, angiotensin type 1 (AT 1 ) receptor and RANKL expression, and increased ACE-2, Ang1-7, Mas and OPG expression in OVX animals. However, A-779 significantly eradicated the effects of losartan on RAS components and RANKL/OPG expression. Thus, Ang1-7 are involved in the osteo-preservative effects of losartan via Mas receptor, which may add therapeutic value to this well-known antihypertensive agent. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Manumycin A Is a Potent Inhibitor of Mammalian Thioredoxin Reductase-1 (TrxR-1).

    Science.gov (United States)

    Tuladhar, Anupama; Rein, Kathleen S

    2018-04-12

    The anticancer effect of manumycin A (Man A) has been attributed to the inhibition of farnesyl transferase (FTase), an enzyme that is responsible for post-translational modification of Ras proteins. However, we have discovered that Man A inhibits mammalian cytosolic thioredoxin reductase 1 (TrxR-1) in a time-dependent manner, with an IC 50 of 272 nM with preincubation and 1586 nM without preincubation. The inhibition of TrxR-1 by Man A is irreversible and is the result of a covalent interaction between Man A and TrxR-1. Evidence presented herein demonstrates that Man A forms a Michael adduct with the selenocysteine residue, which is located in the C-terminal redox center of TrxR-1. Inhibitors of TrxR-1, which act through this mechanism, convert TrxR-1 into a SecTRAP, which utilizes NADPH to reduce oxygen to superoxide radical anion (O 2 -• ).

  11. Chaperone-like properties of tobacco plastid thioredoxins f and m

    Science.gov (United States)

    Sanz-Barrio, Ruth; Fernández-San Millán, Alicia; Carballeda, Jon; Corral-Martínez, Patricia; Seguí-Simarro, José M.; Farran, Inmaculada

    2012-01-01

    Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein. PMID:21948853

  12. Nitric oxide induces thioredoxin-1 nuclear translocation: Possible association with the p21Ras survival pathway

    International Nuclear Information System (INIS)

    Arai, Roberto J.; Masutani, H.; Yodoi, J.; Debbas, V.; Laurindo, Francisco R.; Stern, A.; Monteiro, Hugo P.

    2006-01-01

    One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21 RasC118S ). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway

  13. Redox regulation of mast cell histamine release in thioredoxin-1 (TRX) transgenic mice.

    Science.gov (United States)

    Son, Aoi; Nakamura, Hajime; Kondo, Norihiko; Matsuo, Yoshiyuki; Liu, Wenrui; Oka, Shin-ichi; Ishii, Yasuyuki; Yodoi, Junji

    2006-02-01

    Thioredoxin-1 (TRX) is a stress-inducible redox-regulatory protein with antioxidative and anti-inflammatory effects. Here we show that the release of histamine from mast cells elicited by cross-linking of high-affinity receptor for IgE (FcepsilonRI) was significantly suppressed in TRX transgenic (TRX-tg) mice compared to wild type (WT) mice. Intracellular reactive oxygen species (ROS) of mast cells stimulated by IgE and antigen was also reduced in TRX-tg mice compared to WT mice. Whereas there was no difference in the production of cytokines (IL-6 and TNF-alpha) from mast cells in response to 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) stimulation in TRX-tg and WT mice. Immunological status of TRX-tg mice inclined to T helper (Th) 2 dominant in primary immune response, although there was no difference in the population of dendritic cells (DCs) and regulatory T cells. We conclude that the histamine release from mast cells in TRX-tg mice is suppressed by inhibition of ROS generation. As ROS are involved in mast cell activation and facilitate mediator release, TRX may be a key signaling molecule regulating the early events in the IgE signaling in mast cells and the allergic inflammation.

  14. Thioredoxin ameliorates cutaneous inflammation by regulating the epithelial production and release of pro-Inflammatory cytokines

    Directory of Open Access Journals (Sweden)

    Hai eTian

    2013-09-01

    Full Text Available Human thioredoxin-1 (TRX is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys-. It has been demonstrated that systemic administration and transgenic overexpression of TRX ameliorate inflammation in various animal models, but its anti-inflammatory mechanism is not well characterized. We investigated the anti-inflammatory effects of topically applied recombinant human TRX (rhTRX in a murine irritant contact dermatitis (ICD induced by croton oil. Topically applied rhTRX was distributed only in the skin tissues under both non-inflammatory and inflammatory conditions, and significantly suppressed the inflammatory response by inhibiting the production of cytokines and chemokines, such as TNF-α, Il-1β, IL-6, CXCL-1, and MCP-1. In an in vitro study, rhTRX also significantly inhibited the formation of cytokines and chemokines produced by keratinocytes after exposure to croton oil and phorbol 12-myristate 13-acetate. These results indicate that TRX prevents skin inflammation via the inhibition of local formation of inflammatory cytokines and chemokines. As a promising new approach, local application of TRX may be useful for the treatment of various skin and mucosal inflammatory disorders.

  15. Nuclear thioredoxin-1 is required to suppress cisplatin-mediated apoptosis of MCF-7 cells

    International Nuclear Information System (INIS)

    Chen, Xiao-Ping; Liu, Shou; Tang, Wen-Xin; Chen, Zheng-Wang

    2007-01-01

    Different cell line with increased thioredoxin-1 (Trx-1) showed a decreased or increased sensitivity to cell killing by cisplatin. Recently, several studies found that the subcellular localization of Trx-1 is closely associated with its functions. In this study, we explored the association of the nuclear Trx-1 with the cisplatin-mediated apoptosis of breast cancer cells MCF-7. Firstly, we found that higher total Trx-1 accompanied by no change of nuclear Trx-1 can not influence apoptosis induced by cisplatin in MCF-7 cells transferred with Trx-1 cDNA. Secondly, higher nuclear Trx-1 accompanied by no change of total Trx-1 can protect cells from apoptosis induced by cisplatin. Thirdly, high nuclear Trx-1 involves in the cisplatin-resistance in cisplatin-resistive cells. Meanwhile, we found that the mRNA level of p53 is closely correlated with the level of nuclear Trx-1. In summary, we concluded that the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53

  16. Interaction between M-like protein and macrophage thioredoxin facilitates antiphagocytosis for Streptococcus equi ssp. zooepidemicus.

    Directory of Open Access Journals (Sweden)

    Zhe Ma

    Full Text Available Streptococcus equi ssp. zooepidemicus (S. zooepidemicus, S.z is one of the common pathogens that can cause septicemia, meningitis, and mammitis in domesticated species. M-like protein (SzP is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of S. zooepidemicus and porcine thioredoxin (TRX was identified by the yeast two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and the oxidized forms of TRX without inhibiting TRX activity. Membrane anchored SzP was able to recruit TRX to the surface, which would facilitate the antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting C3 convertase activity and associating with factor H (FH. TRX alone inhibited C3 cleavage and C3a production, and the inhibitory effect was additive when FH was also present. TRX inhibited C3 deposition on the bacterial surface when it was recruited by SzP. These new findings indicated that S. zooepidemicus used SzP to recruit TRX and regulated the alternative complement pathways to evade the host immune phagocytosis.

  17. Interaction between M-Like Protein and Macrophage Thioredoxin Facilitates Antiphagocytosis for Streptococcus equi ssp. zooepidemicus

    Science.gov (United States)

    Ma, Zhe; Zhang, Hui; Zheng, Junxi; Li, Yue; Yi, Li; Fan, Hongjie; Lu, Chengping

    2012-01-01

    Streptococcus equi ssp. zooepidemicus (S. zooepidemicus, S.z) is one of the common pathogens that can cause septicemia, meningitis, and mammitis in domesticated species. M-like protein (SzP) is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of S. zooepidemicus and porcine thioredoxin (TRX) was identified by the yeast two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and the oxidized forms of TRX without inhibiting TRX activity. Membrane anchored SzP was able to recruit TRX to the surface, which would facilitate the antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting C3 convertase activity and associating with factor H (FH). TRX alone inhibited C3 cleavage and C3a production, and the inhibitory effect was additive when FH was also present. TRX inhibited C3 deposition on the bacterial surface when it was recruited by SzP. These new findings indicated that S. zooepidemicus used SzP to recruit TRX and regulated the alternative complement pathways to evade the host immune phagocytosis. PMID:22384152

  18. Changes in maternal serum thioredoxin (TRX) levels after delivery in preeclamptic and normotensive pregnant women.

    Science.gov (United States)

    Vitoratos, Nicolaos; Vlahos, Nikos F; Economou, Emanuel; Panoulis, Konstatninos; Creatsas, George

    2012-01-01

    To investigate changes of maternal plasma thioredoxin (TRX) levels after delivery in preeclamptic and normotensive pregnant women. Ten normotensive women (group A) were compared to 17 women with severe preeclampsia (group B). TRX levels were assessed in maternal plasma, immediately after delivery and 12-16 weeks postpartum. There were no differences in plasma TRX levels between the two groups immediately antepartum (p = 0.095). A significant reduction in plasma TRX levels was found immediately following delivery only in normotensive group (117.76 ± 37.19 ng/mL vs. 43.45 ± 21.11 ng/mL, p = 0.002), but not in women with preeclampsia (80.42 ± 59.95 ng/mL vs. 53.82 ± 44.34 ng/mL, p = 0.12). Plasma TRX levels remained unchanged in women with preeclampsia (80.42 ± 59.95 ng/mL vs. 55.37 ± 52.23 ng/mL, p = 0.2) at 12-14 weeks postpartum.

  19. Gold(III) bis(thiosemicarbazonate) compounds in breast cancer cells: Cytotoxicity and thioredoxin reductase targeting.

    Science.gov (United States)

    Rodríguez-Fanjul, Vanessa; López-Torres, Elena; Mendiola, M Antonia; Pizarro, Ana María

    2018-03-25

    Gold(III) compounds have received increasing attention in cancer research. Three gold complexes of general formula [Au III L]Cl, where L is benzil bis(thiosemicarbazonate), compound 1, benzil bis(4-methyl-3-thiosemicarbazonate), compound 2, or benzil bis(4-cyclohexyl-3-thiosemicarbazonate), compound 3, have been synthesized and fully characterized, including the X-ray crystal structure of compound 3, confirming square-planar geometry around the gold(III) centre. Compound 1 showed moderate cytotoxicity and accumulation in MCF7 breast cancer cells but did not inhibit thioredoxin reductase (TrxR) activity and did not induce reactive oxygen species (ROS) production. Compound 2, the least cytotoxic, was found to be capable of modestly inhibiting TrxR activity and produced low levels of ROS in the MCF7 cell line. The most cytotoxic compound, 3, had the highest cellular accumulation and its distribution pattern showed a clear preference for the cytosol and mitochondria of MCF7 cells. It readily hampered intracellular TrxR activity leading to a dramatic alteration of the cellular redox state and to the induction of cell death. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  20. Redox Signaling Mediated by Thioredoxin and Glutathione Systems in the Central Nervous System.

    Science.gov (United States)

    Ren, Xiaoyuan; Zou, Lili; Zhang, Xu; Branco, Vasco; Wang, Jun; Carvalho, Cristina; Holmgren, Arne; Lu, Jun

    2017-11-01

    The thioredoxin (Trx) and glutathione (GSH) systems play important roles in maintaining the redox balance in the brain, a tissue that is prone to oxidative stress due to its high-energy demand. These two disulfide reductase systems are active in various areas of the brain and are considered to be critical antioxidant systems in the central nervous system (CNS). Various neuronal disorders have been characterized to have imbalanced redox homeostasis. Recent Advances: In addition to their detrimental effects, recent studies have highlighted that reactive oxygen species/reactive nitrogen species (ROS/RNS) act as critical signaling molecules by modifying thiols in proteins. The Trx and GSH systems, which reversibly regulate thiol modifications, regulate redox signaling involved in various biological events in the CNS. In this review, we focus on the following: (i) how ROS/RNS are produced and mediate signaling in CNS; (ii) how Trx and GSH systems regulate redox signaling by catalyzing reversible thiol modifications; (iii) how dysfunction of the Trx and GSH systems causes alterations of cellular redox signaling in human neuronal diseases; and (iv) the effects of certain small molecules that target thiol-based signaling pathways in the CNS. Further study on the roles of thiol-dependent redox systems in the CNS will improve our understanding of the pathogenesis of many human neuronal disorders and also help to develop novel protective and therapeutic strategies against neuronal diseases. Antioxid. Redox Signal. 27, 989-1010.

  1. The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant.

    Science.gov (United States)

    Collet, Jean-Francois; Peisach, Daniel; Bardwell, James C A; Xu, Zhaohui

    2005-07-01

    Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron-sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essential for the assembly of the iron-sulfur was an additional cysteine that replaced the proline at position three of the CGPC motif. We solved the crystalline structure at 2.3 Angstroms for one of these variants, TrxA(CACA). The mutant protein crystallized as a dimer in which the iron-sulfur cluster is replaced by two intermolecular disulfide bonds. The catalytic site, which forms the dimer interface, crystallized in two different conformations. In one of them, the replacement of the CGPC motif by CACA has a dramatic effect on the structure and causes the unraveling of an extended alpha-helix. In both conformations, the second cysteine residue of the CACA motif is surface-exposed, which contrasts with wildtype thioredoxin where the second cysteine of the CXXC motif is buried. This exposure of a pair of vicinal cysteine residues apparently allows thioredoxin to acquire an iron-sulfur cofactor at its active site, and thus a new activity and mechanism of action.

  2. The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant

    Energy Technology Data Exchange (ETDEWEB)

    Collet, Jean-Francois; Peisach, Daniel; Bardwell, James C.A.; Xu, Zhaohui [Michigan

    2010-07-13

    Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron-sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essential for the assembly of the iron-sulfur was an additional cysteine that replaced the proline at position three of the CGPC motif. We solved the crystalline structure at 2.3 {angstrom} for one of these variants, TrxA(CACA). The mutant protein crystallized as a dimer in which the iron-sulfur cluster is replaced by two intermolecular disulfide bonds. The catalytic site, which forms the dimer interface, crystallized in two different conformations. In one of them, the replacement of the CGPC motif by CACA has a dramatic effect on the structure and causes the unraveling of an extended {alpha}-helix. In both conformations, the second cysteine residue of the CACA motif is surface-exposed, which contrasts with wildtype thioredoxin where the second cysteine of the CXXC motif is buried. This exposure of a pair of vicinal cysteine residues apparently allows thioredoxin to acquire an iron-sulfur cofactor at its active site, and thus a new activity and mechanism of action.

  3. The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron–sulfur cluster in an Escherichia coli thioredoxin mutant

    Science.gov (United States)

    Collet, Jean-Francois; Peisach, Daniel; Bardwell, James C.A.; Xu, Zhaohui

    2005-01-01

    Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron–sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essential for the assembly of the iron–sulfur was an additional cysteine that replaced the proline at position three of the CGPC motif. We solved the crystalline structure at 2.3 Å for one of these variants, TrxA(CACA). The mutant protein crystallized as a dimer in which the iron–sulfur cluster is replaced by two intermolecular disulfide bonds. The catalytic site, which forms the dimer interface, crystallized in two different conformations. In one of them, the replacement of the CGPC motif by CACA has a dramatic effect on the structure and causes the unraveling of an extended α-helix. In both conformations, the second cysteine residue of the CACA motif is surface-exposed, which contrasts with wildtype thioredoxin where the second cysteine of the CXXC motif is buried. This exposure of a pair of vicinal cysteine residues apparently allows thioredoxin to acquire an iron–sulfur cofactor at its active site, and thus a new activity and mechanism of action. PMID:15987909

  4. Evidence for a Role of Chloroplastic m-Type Thioredoxins in the Biogenesis of Photosystem II in Arabidopsis1[C][W][OPEN

    Science.gov (United States)

    Wang, Peng; Liu, Jun; Liu, Bing; Feng, Dongru; Da, Qingen; Wang, Peng; Shu, Shengying; Su, Jianbin; Zhang, Yang; Wang, Jinfa; Wang, Hong-Bin

    2013-01-01

    Chloroplastic m-type thioredoxins (TRX m) are essential redox regulators in the light regulation of photosynthetic metabolism. However, recent genetic studies have revealed novel functions for TRX m in meristem development, chloroplast morphology, cyclic electron flow, and tetrapyrrole synthesis. The focus of this study is on the putative role of TRX m1, TRX m2, and TRX m4 in the biogenesis of the photosynthetic apparatus in Arabidopsis (Arabidopsis thaliana). To that end, we investigated the impact of single, double, and triple TRX m deficiency on chloroplast development and the accumulation of thylakoid protein complexes. Intriguingly, only inactivation of three TRX m genes led to pale-green leaves and specifically reduced stability of the photosystem II (PSII) complex, implying functional redundancy between three TRX m isoforms. In addition, plants silenced for three TRX m genes displayed elevated levels of reactive oxygen species, which in turn interrupted the transcription of photosynthesis-related nuclear genes but not the expression of chloroplast-encoded PSII core proteins. To dissect the function of TRX m in PSII biogenesis, we showed that TRX m1, TRX m2, and TRX m4 interact physically with minor PSII assembly intermediates as well as with PSII core subunits D1, D2, and CP47. Furthermore, silencing three TRX m genes disrupted the redox status of intermolecular disulfide bonds in PSII core proteins, most notably resulting in elevated accumulation of oxidized CP47 oligomers. Taken together, our results suggest an important role for TRX m1, TRX m2, and TRX m4 proteins in the biogenesis of PSII, and they appear to assist the assembly of CP47 into PSII. PMID:24151299

  5. Enhanced metabolic effect of erythropoietin and keto acids in CRF patients on low-protein diet: Czech multicenter study.

    Science.gov (United States)

    Teplan, Vladimír; Schück, Otto; Knotek, Antonín; Hajný, Jan; Horácková, Miroslava; Kvapil, Milan

    2003-03-01

    Our study is designed to establish whether supplementation with erythropoietin (EPO) exerts additional beneficial metabolic effects in patients with chronic renal failure (CRF) treated with keto acids (KAs) on a low-protein diet (LPD). A long-term, prospective, randomized study was designed to use three therapeutic protocols: (A) EPO plus KAs plus LPD (group I), (B) EPO plus LPD (group II), and (C) LPD (group III). One hundred eighty-six randomly selected patients (90 men, 96 women; age, 22 to 78 years) with a creatinine clearance of 22 to 36 mL/min were monitored at the beginning and at every 6 months for 3 years. During the study period, glomerular filtration rate measured as inulin clearance decreased slightly (from 26.2 +/- 3.4 to 23.4 +/- 4.1 mL/min in group I), 27.4 +/- 4.8 to 20.2 +/- 4.4 mL/min in group II, and 26.8 +/- 3.6 to 17.4 +/- 4.1 mL/min in group III; P < 0.01). Serum urea levels also declined (P < 0.01), more pronouncedly in group I (P < 0.025). In group I, there was a significant increase in levels of leucine (P < 0.01) and albumin (P < 0.01) and a decrease in proteinuria (P < 0.01). Analysis of the lipid spectrum showed a mild, yet significant, decrease in total cholesterol and low-density lipoprotein cholesterol levels (P < 0.025), more pronounced in group I. In group I, there was a decrease in plasma triglyceride levels (from 362.85 +/- 115.05 mg/dL [4.1 +/- 1.3 mmol/L] to values as low as 203.55 +/- 70.80 mg/dL [2.3 +/- 0.8 mmol/L]; P < 0.01), whereas high-density lipoprotein cholesterol levels increased (from 34.75 +/- 7.72 mg/dL [0.9 +/- 0.2 mmol/L] to 46.33 +/- 7.72 mg/dL [1.2 +/- 0.2 mmol/L]; P < 0.025). Mean arterial blood pressure was stable. EPO supplementation in patients with CRF administered KAs potentiates the beneficial effects on metabolism of proteins, amino acids, and lipids. Long-term coadministration of EPO, KA, and LPD was associated with a delay in progression of renal failure and reduction in proteinuria.

  6. Engineering Cellular Metabolism

    DEFF Research Database (Denmark)

    Nielsen, Jens; Keasling, Jay

    2016-01-01

    Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds...... of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation....

  7. Development of a bread delivery vehicle for dietary prebiotics to enhance food functionality targeted at those with metabolic syndrome

    Science.gov (United States)

    Costabile, Adele; Walton, Gemma E; Tzortzis, George; Vulevic, Jelena; Charalampopoulos, Dimitris; Gibson, Glenn R

    2015-01-01

    Prebiotics are dietary carbohydrates that favourably modulate the gut microbiota. The aims of the present study were to develop a functional prebiotic bread using Bimuno®, (galactooligosaccharide (B-GOS) mixture), for modulation of the gut microbiota in vitro in individuals at risk of metabolic syndrome. A control bread, (no added prebiotic) and positive control bread (containing equivalent carbohydrate to B-GOS bread) were also developed. A 3-stage continuous in vitro colonic model was used to assess prebiotic functionality of the breads. Bacteria were quantified by fluorescence in situ hybridization and short chain fatty acids by gas chromatography. Ion-exchange chromatography was used to determine GOS concentration after bread production. Following B-GOS bread fermentation numbers of bifidobacteria and lactobacilli were significantly higher compared to controls. There was no significant degradation of B-GOS during bread manufacture, indicating GOS withstood the manufacturing process. Furthermore, based on previous research, increased bifidobacteria and butyrate levels could be of benefit to those with obesity related conditions. Our findings support utilization of prebiotic enriched bread for improving gastrointestinal health. PMID:26099034

  8. Dietary supplementation with omega-3 fatty acids and oleate enhances exercise training effects in patients with metabolic syndrome.

    Science.gov (United States)

    Ortega, Juan F; Morales-Palomo, Felix; Fernandez-Elias, Valentin; Hamouti, Nassim; Bernardo, Francisco J; Martin-Doimeadios, Rosa C; Nelson, Rachael K; Horowitz, Jeffrey F; Mora-Rodriguez, Ricardo

    2016-08-01

    We studied the effects of exercise training alone or combined with dietary supplementation of omega-3 polyunsaturated fatty acids (Ω-3PUFA) and oleate on metabolic syndrome (MSyn) components and other markers of cardiometabolic health. Thirty-six patients with MSyn underwent 24 weeks of high-intensity interval training. In a double-blind randomized design, half of the group ingested 500 mL/day of semi-skim milk (8 g of fat; placebo milk) whereas the other half ingested 500 mL/day of skim milk enriched with 275 mg of Ω-3PUFA and 7.5 g of oleate (Ω-3 + OLE). Ω-3 + OLE treatment elevated 30% plasma Ω-3PUFA but not significantly (P = 0.286). Improvements in VO2peak (12.8%), mean blood pressure (-7.1%), waist circumference (-1.8%), body fat mass (-2.9%), and trunk fat mass (-3.3%) were similar between groups. However, insulin sensitivity (measured by intravenous glucose tolerance test), serum concentration of C-reactive protein, and high-density lipoprotein improved only in the Ω-3 + OLE group by 31.5%, 32.1%, and 10.3%, respectively (all P exercise training in patients with MSyn. © 2016 The Obesity Society.

  9. Ultrasound enhances calcium absorption of jujube fruit by regulating the cellular calcium distribution and metabolism of cell wall polysaccharides.

    Science.gov (United States)

    Zhi, Huanhuan; Liu, Qiqi; Xu, Juan; Dong, Yu; Liu, Mengpei; Zong, Wei

    2017-12-01

    Ultrasound has been applied in fruit pre-washing processes. However, it is not sufficient to protect fruit from pathogenic infection throughout the entire storage period, and sometimes ultrasound causes tissue damage. The goal of this study was to investigate the effects of calcium chloride (CaCl 2 , 10 g L -1 ) and ultrasound (350 W at 40 kHz), separately and in combination, on jujube fruit quality, antioxidant status, tissue Ca 2+ content and distribution along with cell wall metabolism at 20 °C for 6 days. All three treatments significantly maintained fruit firmness and peel color, reduced respiration rate, decay incidence, superoxide anion, hydrogen peroxide and malondialdehyde and preserved higher enzymatic (superoxide dismutase, catalase and peroxidase) and non-enzymatic (ascorbic acid and glutathione) antioxidants compared with the control. Moreover, the combined treatment was more effective in increasing tissue Ca 2+ content and distribution, inhibiting the generation of water-soluble and CDTA-soluble pectin fractions, delaying the solubilization of Na 2 CO 3 -soluble pectin and having lower activities of cell wall-modifying enzymes (polygalacturonase and pectate lyase) during storage. These results demonstrated that the combination of CaCl 2 and ultrasound has potential commercial application to extend the shelf life of jujube fruit by facilitating Ca 2+ absorption and stabilizing the cell wall structure. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  10. Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves.

    Science.gov (United States)

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Navarro, Alicia; Arrillaga, Isabel; Segura, Juan

    2014-05-01

    Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  11. Enhancement of Farnesyl Diphosphate Pool as Direct Precursor of Sesquiterpenes Through Metabolic Engineering of the Mevalonate Pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Asadollahi, Mohammadali; Maury, Jerome; Schalk, M.

    2010-01-01

    , resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous overexpression of tHMG1 and repression of ERG9 did...... not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring the ergosterol biosynthesis were observed in the ERG9 repressed strains transformed with the plasmids...

  12. Redox Regulation Of Metabolic And Signaling Pathways By Thioredoxin And Glutaredoxin In Nitric Oxide Treated Hepatoblastoma Cells

    Directory of Open Access Journals (Sweden)

    C. Alicia Padilla Peña

    2015-08-01

    Conclusions: Trx1 and Grx1 exert contradictory influences on HepG2 cells. They are required for proliferation but they also contribute to antiproliferative effect of NO, associated to Akt1 redox changes.

  13. Enhanced activity of carbohydrate- and lipid-metabolizing enzymes in insecticide-resistant populations of the maize weevil, Sitophilus zeamais.

    Science.gov (United States)

    Araújo, R A; Guedes, R N C; Oliveira, M G A; Ferreira, G H

    2008-08-01

    Insecticide resistance is frequently associated with fitness disadvantages in the absence of insecticides. However, intense past selection with insecticides may allow the evolution of fitness modifier alleles that mitigate the cost of insecticide resistance and their consequent fitness disadvantages. Populations of Sitophilus zeamais with different levels of susceptibility to insecticides show differences in the accumulation and mobilization of energy reserves. These differences may allow S. zeamais to better withstand toxic compounds without reducing the beetles' reproductive fitness. Enzymatic assays with carbohydrate- and lipid-metabolizing enzymes were, therefore, carried out to test this hypothesis. Activity levels of trehalase, glycogen phosphorylase, lipase, glycosidase and amylase were determined in two insecticide-resistant populations showing (resistant cost) or not showing (resistant no-cost) associated fitness cost, and in an insecticide-susceptible population. Respirometry bioassays were also carried out with these weevil populations. The resistant no-cost population showed significantly higher body mass and respiration rate than the other two populations, which were similar. No significant differences in glycogen phosphorylase and glycosidase were observed among the populations. Among the enzymes studied, trehalase and lipase showed higher activity in the resistant cost population. The results obtained in the assays with amylase also indicate significant differences in activity among the populations, but with higher activity in the resistant no-cost population. The inverse activity trends of lipases and amylases in both resistant populations, one showing fitness disadvantage without insecticide exposure and the other not showing it, may underlay the mitigation of insecticide resistance physiological costs observed in the resistant no-cost population. The higher amylase activity observed in the resistant no-cost population may favor energy storage

  14. Chloroplastic thioredoxin-f and thioredoxin-m1/4 play important roles in brassinosteroids-induced changes in CO2 assimilation and cellular redox homeostasis in tomato

    OpenAIRE

    Cheng, Fei; Zhou, Yan-Hong; Xia, Xiao-Jian; Shi, Kai; Zhou, Jie; Yu, Jing-Quan

    2014-01-01

    Chloroplast thioredoxins (TRXs) and glutathione function as redox messengers in the regulation of photosynthesis. In this work, the roles of chloroplast TRXs in brassinosteroids (BRs)-induced changes in cellular redox homeostasis and CO2 assimilation were studied in the leaves of tomato plants. BRs-deficient d ^im plants showed decreased transcripts of TRX-f, TRX-m2, TRX-m1/4, and TRX-x, while exogenous BRs significantly induced CO2 assimilation and the expression of TRX-f, TRX-m2, TRX-m1/4, ...

  15. Severity of murine collagen-induced arthritis correlates with increased CYP7B activity: enhancement of dehydroepiandrosterone metabolism by interleukin-1beta.

    Science.gov (United States)

    Dulos, John; Verbraak, Evert; Bagchus, Wilma M; Boots, Annemieke M H; Kaptein, Allard

    2004-10-01

    The endogenous steroid dehydroepiandrosterone (DHEA) has been reported to play a role in rheumatoid arthritis (RA). DHEA is metabolized by the P450 enzyme CYP7B into 7alpha-OH-DHEA, which has immunostimulating properties. This study was undertaken to investigate the putative role of CYP7B in arthritis using murine collagen-induced arthritis (CIA), an interleukin-1beta (IL-1beta)-dependent model. DBA/1J mice were immunized and administered a booster with type II collagen. The presence of 7alpha-OH-DHEA was determined in both arthritic and nonarthritic joints and the serum of CIA mice by radioimmunoassay. CYP7B messenger RNA (mRNA) expression was analyzed in synovial biopsy samples, and in fibroblast-like synoviocytes (FLS) isolated from these synovial biopsy samples, by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, the regulatory role of IL-1beta on CYP7B activity in FLS was determined using RT-PCR, Western blotting, and high-performance liquid chromatography. In knee joint synovial biopsy samples from arthritic mice, 7alpha-OH-DHEA levels were 5-fold higher than in nonarthritic mice. Elevated levels of 7alpha-OH-DHEA were accompanied by an increase in CYP7B mRNA expression and were positively correlated with disease severity. In serum, no differences in 7alpha-OH-DHEA levels were observed between arthritic and nonarthritic mice. Incubation of FLS with IL-1beta resulted in a dose-dependent increase in 7alpha-OH-DHEA formation. In addition, IL-1beta enhanced CYP7B mRNA and CYP7B protein levels in FLS. Disease progression in CIA is correlated with enhanced CYP7B activity, which leads to locally enhanced 7alpha-OH-DHEA levels. Elevated IL-1beta levels within the arthritic joint may regulate this increase in CYP7B activity. Copyright 2004 American College of Rheumatology

  16. Mass spectrometry reveals thioredoxin-1 as a new partner of ADAM17 that can modulate its sheddase activity

    Energy Technology Data Exchange (ETDEWEB)

    Aragao, A.Z.B.; Simabuco, F.M.; Smetana, J.H.C. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Yokoo, S.; Paes Leme, A.F. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Rodrigues, E.; Mercadante, A.Z. [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

    2012-07-01

    Full text: ADAMs are a family of membrane-associated metalloproteinases with a complex multi-domain structure: a metalloproteinase domain, a disintegrin domain, a cysteine-rich region, an epidermal growth factor-like repeat, a transmembrane domain and a cytoplasmic tail. These proteases are responsible for shedding the ectodomains of cell surface proteins, modulating regulatory mechanisms. Many ADAMs are highly associated with tumorigenesis and tumor progression. The aim of this study is identify novel binding partners that can modulate ADAM17 activation via cytoplasmatic domain. We performed the cloning and overexpression of the ADAM17 cytoplasmic tail in HEK-293 cell line and the ligands were determined by LC-MS/MS after proteins immunoprecipitation (IP) with anti-FLAG M2 Affinity Gel (Sigma). Thioredoxin-1 (Trx-1) and others ligands were identified at least in two independent experiments, and this binding is independent of phosphorylation. The IP of Trx-1 was confirmed by Western blot, furthermore Trx-1 immunolocalized with full length ADAM17-HA and cytoplasmic tail-FLAG recombinant proteins in HEK293 and HeLa cells. Trx-1 is part of the system peroxiredoxin/thioredoxin/thioredoxin reductase, one of the mechanisms by which cells maintain the reduced cellular environment, inactivating the reactive oxygen species (ROS). We investigate whether ADAM17 activity is modulate by Trx-1 on AP reporter assay that was performed using HEK293 and SCC-9 cells transfected stably with HB-EGF-AP in co-transfection with transient recombinant Trx-1-HA. The results indicate that Trx-1 can modulate negatively the activity or maturation of ADAM17 in presence of PMA, which is known to increase ROS. In summary, this study identifies Trx-1 and suggest that this protein can modulate ADAM17 activity in normal and tumorigenic cells lines. (author)

  17. Mass spectrometry reveals thioredoxin-1 as a new partner of ADAM17 that can modulate its sheddase activity

    International Nuclear Information System (INIS)

    Aragao, A.Z.B.; Simabuco, F.M.; Smetana, J.H.C.; Yokoo, S.; Paes Leme, A.F.; Rodrigues, E.; Mercadante, A.Z.

    2012-01-01

    Full text: ADAMs are a family of membrane-associated metalloproteinases with a complex multi-domain structure: a metalloproteinase domain, a disintegrin domain, a cysteine-rich region, an epidermal growth factor-like repeat, a transmembrane domain and a cytoplasmic tail. These proteases are responsible for shedding the ectodomains of cell surface proteins, modulating regulatory mechanisms. Many ADAMs are highly associated with tumorigenesis and tumor progression. The aim of this study is identify novel binding partners that can modulate ADAM17 activation via cytoplasmatic domain. We performed the cloning and overexpression of the ADAM17 cytoplasmic tail in HEK-293 cell line and the ligands were determined by LC-MS/MS after proteins immunoprecipitation (IP) with anti-FLAG M2 Affinity Gel (Sigma). Thioredoxin-1 (Trx-1) and others ligands were identified at least in two independent experiments, and this binding is independent of phosphorylation. The IP of Trx-1 was confirmed by Western blot, furthermore Trx-1 immunolocalized with full length ADAM17-HA and cytoplasmic tail-FLAG recombinant proteins in HEK293 and HeLa cells. Trx-1 is part of the system peroxiredoxin/thioredoxin/thioredoxin reductase, one of the mechanisms by which cells maintain the reduced cellular environment, inactivating the reactive oxygen species (ROS). We investigate whether ADAM17 activity is modulate by Trx-1 on AP reporter assay that was performed using HEK293 and SCC-9 cells transfected stably with HB-EGF-AP in co-transfection with transient recombinant Trx-1-HA. The results indicate that Trx-1 can modulate negatively the activity or maturation of ADAM17 in presence of PMA, which is known to increase ROS. In summary, this study identifies Trx-1 and suggest that this protein can modulate ADAM17 activity in normal and tumorigenic cells lines. (author)

  18. The Enzymatic and Structural Basis for Inhibition of Echinococcus granulosus Thioredoxin Glutathione Reductase by Gold(I).

    Science.gov (United States)

    Salinas, Gustavo; Gao, Wei; Wang, Yang; Bonilla, Mariana; Yu, Long; Novikov, Andrey; Virginio, Veridiana G; Ferreira, Henrique B; Vieites, Marisol; Gladyshev, Vadim N; Gambino, Dinorah; Dai, Shaodong

    2017-12-20

    New drugs are needed to treat flatworm infections that cause severe human diseases such as schistosomiasis. The unique flatworm enzyme thioredoxin glutathione reductase (TGR), structurally different from the human enzyme, is a key drug target. Structural studies of the flatworm Echinococcus granulosus TGR, free and complexed with Au I -MPO, a novel gold inhibitor, together with inhibition assays were performed. Au I -MPO is a potent TGR inhibitor that achieves 75% inhibition at a 1:1 TGR:Au ratio and efficiently kills E. granulosus in vitro. The structures revealed salient insights: (i) unique monomer-monomer interactions, (ii) distinct binding sites for thioredoxin and the glutaredoxin (Grx) domain, (iii) a single glutathione disulfide reduction site in the Grx domain, (iv) rotation of the Grx domain toward the Sec-containing redox active site, and (v) a single gold atom bound to Cys 519 and Cys 573 in the Au I -TGR complex. Structural modeling suggests that these residues are involved in the stabilization of the Sec-containing C-terminus. Consistently, Cys→Ser mutations in these residues decreased TGR activities. Mass spectroscopy confirmed these cysteines are the primary binding site. The identification of a primary site for gold binding and the structural model provide a basis for gold compound optimization through scaffold adjustments. The structural study revealed that TGR functions are achieved not only through a mobile Sec-containing redox center but also by rotation of the Grx domain and distinct binding sites for Grx domain and thioredoxin. The conserved Cys 519 and Cys 573 residues targeted by gold assist catalysis through stabilization of the Sec-containing redox center. Antioxid. Redox Signal. 27, 1491-1504.

  19. NADPH-thioredoxin reductase C mediates the response to oxidative stress and thermotolerance in the cyanobacterium Anabaena sp. PCC7120.

    Directory of Open Access Journals (Sweden)

    ANA MARÍA SÁNCHEZ-RIEGO

    2016-08-01

    Full Text Available NTRC (NADPH-thioredoxin reductase C is a bimodular enzyme composed of an NADPH-thioredoxin reductase and a thioredoxin domain extension in the same protein. In plants, NTRC has been described to be involved in the protection of the chloroplast against oxidative stress damage through reduction of the 2-Cys peroxiredoxin (2-Cys Prx as well as through other functions related to redox enzyme regulation. In cyanobacteria, the Anabaena NTRC has been characterized in vitro, however nothing was known about its in vivo function. In order to study that, we have generated the first knockout mutant strain (∆ntrC, apart from the previously described in Arabidopsis. Detailed characterization of this strain reveals a differential sensitivity to oxidative stress treatments with respect to the wild-type Anabaena strain, including a higher level of ROS (reactive oxygen species in normal growth conditions. In the mutant strain, different oxidative stress treatments such as hydrogen peroxide, methyl-viologen or high light irradiance provoke an increase in the expression of genes related to ROS detoxification, including AnNTRC and peroxiredoxin genes, with a concomitant increase in the amount of AnNTRC and 2-Cys Prx. Moreover, the role of AnNTRC in the antioxidant response is confirmed by the observation of a pronounced overoxidation of the 2-Cys Prx and a time-delay recovery of the reduced form of this protein upon oxidative stress treatments. Our results suggest the participation of this enzyme in the peroxide detoxification in Anabaena. In addition, we describe the role of Anabaena NTRC in thermotolerance, by the appearance of high molecular mass AnNTRC complexes, showing that the mutant strain is more sensitive to high temperature treatments.

  20. The function and properties of the iron-sulfur center in spinach ferredoxin: Thioredoxin reductase: A new biological role for iron-sulfur clusters

    Energy Technology Data Exchange (ETDEWEB)

    Staples, C.R.; Ameyibor, E.; Fu, Weiguang; Johnson, M.K. [Univ. of Georgia, Athens, GA (United States)] [and others

    1996-09-03

    Thioredoxin reduction in chloroplasts in catalyzed by a unique class of disulfide reductases which use a [2Fe-2S]{sup 2+/+} ferredoxin as the electron donor and contain an Fe-S cluster as the sole prosthetic group in addition to the active-site disulfide. The nature, properties, and function of the Fe-S cluster in spinach ferredoxin: thioredoxin reductase (FTR) have been investigated by the combination of UV/visible absorption, variable-temperature magnetic circular dichroism (MCD), EPR, and resonance Raman (RR) spectroscopies. 66 refs., 5 figs., 1 tab.

  1. [Cloning, Expression and Immunodiagnostic Evaluation of the Fasciola gigantica Thioredoxin Peroxidase].

    Science.gov (United States)

    Wang, Yue-qi; Zhou, Yan; Cheng, Na; Chen, Mu-xin; Ai, Lin; Liu, Yu-hua; Zhang, Jian-guo; Luo, Jia-jun; Xu, Xue-nian

    2015-04-01

    To immunoscreen the gene encoding thioredoxin peroxidase (TPx) from a cDNA library made from adult Fasciola gigantica worms, clone and express the gene, and evaluate the immunodiagnostic value of TPx recombinant protein. The A ZAP cDNA library was immunoscreened with pooled serum of fascioliasis gigantica patients. The obtained positive clones were sequenced and analyzed by multiple sequence alignment. The full-length (rFgTPx) and N-termianal truncated (rFgTPx_nt) sequence of FgTPx was subcloned into prokaryotic plasmid pET28a(+) with a non-fusion expression technique, respectively. The recombinant proteins of rFgTPx and rFgTPx_nt were purified by His-bind affinity column (Ni-NTA). rFgTPx and rFgTPx_nt were used in indirect ELISA to test the antibody response of the serum samples. Sera of 27 fascioliasis gigantica patients, 15 patients with schistosomaisis japonica, 15 clonorchiasis sinensis patients, and 32 healthy donors were tested by using the recombinant protein based ELISA. The TPx recombinant proteins were obtained through expression, purification and renaturation, the relative molecular mass of rFgTPx and rFgTPx_nt were Mr 30,000 and Mr 26,000, respectively. The total diagnostic coincidence rate, sensitivity and specificity of rFgTPx_nt-based ELISA was 87.6% (78/89), 66.7% (18/27), and 96.8% (60/62), respectively. The cross reaction with Schistosoma japonicum and Clonorchis sinensis was 0 and 1/15 for rFgTPx_nt, respectively. Before and after treatment, A450 value of the serum samples from fascioliasis patients was 0.233 ± 0.088 and 0.129 ± 0.072, respectively (t = 4.27, P Fasciola gigantica infection.

  2. Characteristics of Three Thioredoxin Genes and Their Role in Chilling Tolerance of Harvested Banana Fruit.

    Science.gov (United States)

    Wu, Fuwang; Li, Qing; Yan, Huiling; Zhang, Dandan; Jiang, Guoxiang; Jiang, Yueming; Duan, Xuewu

    2016-09-09

    Thioredoxins (Trxs) are small proteins with a conserved redox active site WCGPC and are involved in a wide range of cellular redox processes. However, little information on the role of Trx in regulating low-temperature stress of harvested fruit is available. In this study, three full-length Trx cDNAs, designated MaTrx6, MaTrx9 and MaTrx12, were cloned from banana (Musa acuminata) fruit. Phylogenetic analysis and protein sequence alignments showed that MaTrx6 was grouped to h2 type with a typical active site of WCGPC, whereas MaTrx9 and MaTrx12 were assigned to atypical cys his-rich Trxs (ACHT) and h3 type with atypical active sites of GCAGC and WCSPC, respectively. Subcellular localization indicated that MaTrx6 and MaTrx12 were located in the plasma membrane and cytoplasm, respectively, whereas MaTrx9 showed a dual cytoplasmic and chloroplast localization. Application of ethylene induced chilling tolerance of harvested banana fruit, whereas 1-MCP, an inhibitor of ethylene perception, aggravated the development of chilling injury. RT-qPCR analysis showed that expression of MaTrx12 was up-regulated and down-regulated in ethylene- and 1-MCP-treated banana fruit at low temperature, respectively. Furthermore, heterologous expression of MaTrx12 in cytoplasmic Trx-deficient Saccharomyces cerevisiae strain increased the viability of the strain under H₂O₂. These results suggest that MaTrx12 plays an important role in the chilling tolerance of harvested banana fruit, possibly by regulating redox homeostasis.

  3. Roles of thioredoxin in nitric oxide-dependent preconditioning-induced tolerance against MPTP neurotoxin

    International Nuclear Information System (INIS)

    Chiueh, C.C.; Andoh, Tsugunobu; Chock, P. Boon

    2005-01-01

    Hormesis, a stress tolerance, can be induced by ischemic preconditioning stress. In addition to preconditioning, it may be induced by other means, such as gas anesthetics. Preconditioning mechanisms, which may be mediated by reprogramming survival genes and proteins, are obscure. A known neurotoxicant, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causes less neurotoxicity in the mice that are preconditioned. Pharmacological evidences suggest that the signaling pathway of ·NO-cGMP-PKG (protein kinase G) may mediate preconditioning phenomenon. We developed a human SH-SY5Y cell model for investigating · NO-mediated signaling pathway, gene regulation, and protein expression following a sublethal preconditioning stress caused by a brief 2-h serum deprivation. Preconditioned human SH-SY5Y cells are more resistant against severe oxidative stress and apoptosis caused by lethal serum deprivation and 1-mehtyl-4-phenylpyridinium (MPP + ). Both sublethal and lethal oxidative stress caused by serum withdrawal increased neuronal nitric oxide synthase (nNOS/NOS1) expression and · NO levels to a similar extent. In addition to free radical scavengers, inhibition of nNOS, guanylyl cyclase, and PKG blocks hormesis induced by preconditioning. S-nitrosothiols and 6-Br-cGMP produce a cytoprotection mimicking the action of preconditioning tolerance. There are two distinct cGMP-mediated survival pathways: (i) the up-regulation of a redox protein thioredoxin (Trx) for elevating mitochondrial levels of antioxidant protein Mn superoxide dismutase (MnSOD) and antiapoptotic protein Bcl-2, and (ii) the activation of mitochondrial ATP-sensitive potassium channels [K(ATP)]. Preconditioning induction of Trx increased tolerance against MPP + , which was blocked by Trx mRNA antisense oligonucleotide and Trx reductase inhibitor. It is concluded that Trx plays a pivotal role in · NO-dependent preconditioning hormesis against MPTP/MPP +

  4. Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts*

    Science.gov (United States)

    Yoshida, Keisuke; Hara, Satoshi; Hisabori, Toru

    2015-01-01

    Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. PMID:25878252

  5. Overexpression of peroxiredoxin I and thioredoxin1 in human breast carcinoma

    Directory of Open Access Journals (Sweden)

    Kim Il-Han

    2009-06-01

    Full Text Available Abstract Background Peroxiredoxins (Prxs are a novel group of peroxidases containing high antioxidant efficiency. The mammalian Prx family has six distinct members (Prx I-VI in various subcellular locations, including peroxisomes and mitochondria, places where oxidative stress is most evident. The function of Prx I in particular has been implicated in regulating cell proliferation, differentiation, and apoptosis. Since thioredoxin1 (Trx1 as an electron donor is functionally associated with Prx I, we investigated levels of expression of both Prx I and Trx1. Methods We investigated levels of expression of both Prx I and Trx1 in breast cancer by real-time polymerase chain reaction (RT-PCR and Western blot. Results Levels of messenger RNA (mRNA for both Prx I and Trx1 in normal human breast tissue were very low compared to other major human tissues, whereas their levels in breast cancer exceeded that in other solid cancers (colon, kidney, liver, lung, ovary, prostate, and thyroid. Among members of the Prx family (Prx I-VI and Trx family (Trx1, Trx2, Prx I and Trx1 were preferentially induced in breast cancer. Moreover, the expression of each was associated with progress of breast cancer and correlated with each other. Western blot analysis of different and paired breast tissues revealed consistent and preferential expression of Prx I and Trx1 protein in breast cancer tissue. Conclusion Prx I and Trx1 are overexpressed in human breast carcinoma and the expression levels are associated with tumor grade. The striking induction of Prx I and Trx1 in breast cancer may enable their use as breast cancer markers.

  6. Serum C-reactive protein and thioredoxin levels in subjects with mildly reduced glomerular filtration rate

    Directory of Open Access Journals (Sweden)

    Ishimura Eiji

    2010-04-01

    Full Text Available Abstract Background Chronic kidney disease (CKD is a newly recognized high-risk condition for cardiovascular disease (CVD, and previous studies reported the changes in inflammation and oxidative stress in advanced stages of CKD. We compared the levels of serum biomarkers for inflammation and oxidative stress between subjects with normal and mildly reduced glomerular filtration rate (GFR. Methods The subjects were 182 participants of a health check-up program including those with normal (≥ 90 mL/min/1.73 m2, N = 79 and mildly reduced eGFR (60-89 mL/min/1.73 m2, N = 103 which was calculated based on serum creatinine, age and sex. We excluded those with reduced eGFR 2. No one had proteinuria. We measured serum levels of C-reactive protein (CRP and thioredoxin (TRX as the markers of inflammation and oxidative stress, respectively. Results As compared with subjects with normal eGFR, those with mildly reduced eGFR had increased levels of both CRP and TRX. Also, eGFR was inversely correlated with these biomarkers. The associations of eGFR with these biomarkers remained significant after adjustment for age and sex. When adjustment was done for eight possible confounders, CRP showed significant association with systolic blood pressure, high density lipoprotein cholesterol (HDL-C and non-HDL-C, whereas TRX was associated with sex significantly, and with eGFR and systolic blood pressure at borderline significance. Conclusions We showed the increased levels of CRP and TRX in subjects with mildly reduced eGFR. The eGFR-CRP link and the eGFR-TRX link appeared to be mediated, at least partly, by the alterations in blood pressure and plasma lipids in these subjects.

  7. Jab1/Csn5-Thioredoxin Signaling in Relapsed Acute Monocytic Leukemia under Oxidative Stress.

    Science.gov (United States)

    Zhou, Fuling; Pan, Yunbao; Wei, Yongchang; Zhang, Ronghua; Bai, Gaigai; Shen, Qiuju; Meng, Shan; Le, Xiao-Feng; Andreeff, Michael; Claret, Francois X

    2017-08-01

    Purpose: High levels of ROS and ineffective antioxidant systems contribute to oxidative stress, which affects the function of hematopoietic cells in acute myeloid leukemia (AML); however, the mechanisms by which ROS lead to malignant transformation in relapsed AML-M5 are not completely understood. We hypothesized that alterations in intracellular ROS would trigger AML-M5 relapse by activating the intrinsic pathway. Experimental Design: We studied ROS levels and conducted c-Jun activation domain-binding protein-1 ( JAB1/COPS5 ) and thioredoxin ( TRX ) gene expression analyses with blood samples obtained from 60 matched AML-M5 patients at diagnosis and relapse and conducted mechanism studies of Jab1's regulation of Trx in leukemia cell lines. Results: Our data showed that increased production of ROS and a low capacity of antioxidant enzymes were characteristics of AML-M5, both at diagnosis and at relapse. Consistently, increased gene expression levels of TRX and JAB1/COPS5 were associated with low overall survival rates in patients with AML-M5. In addition, stimulating AML-M5 cells with low concentrations of hydrogen peroxide led to increased Jab1 and Trx expression. Consistently, transfection of ectopic Jab1 into leukemia cells increased Trx expression, whereas silencing of Jab1 in leukemia cells reduced Trx expression. Mechanistically, Jab1 interacted with Trx and stabilized Trx protein. Moreover, Jab1 transcriptionally regulated Trx. Furthermore, depletion of Jab1 inhibited leukemia cell growth both in vitro and in vivo Conclusions: We identified a novel Jab1-Trx axis that is a key cellular process in the pathobiologic characteristics of AML-M5. Targeting the ROS/Jab1/Trx pathway could be beneficial in the treatment of AML-M5. Clin Cancer Res; 23(15); 4450-61. ©2017 AACR . ©2017 American Association for Cancer Research.

  8. The thioredoxin TRX-1 regulates adult lifespan extension induced by dietary restriction in Caenorhabditis elegans.

    Science.gov (United States)

    Fierro-González, Juan Carlos; González-Barrios, María; Miranda-Vizuete, Antonio; Swoboda, Peter

    2011-03-18

    Dietary restriction (DR) is the only environmental intervention known to extend adult lifespan in a wide variety of animal models. However, the genetic and cellular events that mediate the anti-aging programs induced by DR remain elusive. Here, we used the nematode Caenorhabditis elegans to provide the first in vivo evidence that a thioredoxin (TRX-1) regulates adult lifespan extension induced by DR. We found that deletion of the gene trx-1 completely suppressed the lifespan extension caused by mutation of eat-2, a genetic surrogate of DR in the worm. However, trx-1 deletion only partially suppressed the long lifespan caused by mutation of the insulin-like receptor gene daf-2 or by mutation of the sensory cilia gene osm-5. A trx-1::GFP translational fusion expressed from its own promoter in ASJ neurons (Ptrx-1::trx-1::GFP) rescued the trx-1 deletion-mediated suppression of the lifespan extension caused by mutation of eat-2. This rescue was not observed when trx-1::GFP was expressed from the ges-1 promoter in the intestine. In addition, overexpression of Ptrx-1::trx-1::GFP extended lifespan in wild type, but not in eat-2 mutants. trx-1 deletion almost completely suppressed the lifespan extension induced by dietary deprivation (DD), a non-genetic, nutrient-based model of DR in the worm. Moreover, DD upregulated the expression of a trx-1 promoter-driven GFP reporter gene (Ptrx-1::GFP) in ASJ neurons of aging adults, but not that of control Pgpa-9::GFP (which is also expressed in ASJ neurons). We propose that DR activates TRX-1 in ASJ neurons during aging, which in turn triggers TRX-1-dependent mechanisms to extend adult lifespan in the worm. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. The induction of thioredoxin-1 by epinephrine withdraws stress via interaction with β-arrestin-1

    Science.gov (United States)

    Jia, Jin-Jing; Zeng, Xian-Si; Zhou, Xiao-Shuang; Li, Ye; Bai, Jie

    2014-01-01

    Stress regulates a panel of important physiological functions and disease states. Epinephrine is produced under stresses threaten to homeostasis. Thioredoxin-1(Trx-1) is a redox regulating protein which is induced to resist stresses and related with various diseases. Thus, it is important to examine whether Trx-1 is induced by epinephrine and to understand the underlying molecular mechanisms that Trx-1 modulates epinephrine stress. Here, we show that the expression of Trx-1 was induced by epinephrine via β-adrenergic receptor/Cyclic AMP/protein kinase A (PKA) signaling pathway in PC12 cells. The down-regulation of Trx-1 by siRNA aggravated accumulation of γ-H2AX and further decreased expression of p53 by epinephrine. Accordingly, Trx-1 overexpression alleviated accumulation of γ-H2AX and restored the expressions of p53 and C/EBP homologous protein (CHOP) in the cortex, hippocampus and thymus of mice. Moreover, Trx-1 overexpression reduced the malondialdehyde concentration by epinephrine. We further explored the mechanism on p53 and γ-H2AX regulated by Trx-1. We found that overexpression of Trx-1 suppressed β-arrestin-1 expression through interaction with β-arrestin-1. Consequently, the downregulation of β-arrestin-1 suppressed the cell viability and the expressions of γ-H2AX and cyclin D1, and increased p53 expression. Taken together, our data suggest that Trx-1/β-arrestin-1 interaction may represent a novel endogenous mechanism on protecting against stress. PMID:25486571

  10. Auranofin inactivates Trichomonas vaginalis thioredoxin reductase and is effective against trichomonads in vitro and in vivo.

    Science.gov (United States)

    Hopper, Melissa; Yun, Jeong-Fil; Zhou, Bianhua; Le, Christine; Kehoe, Katelin; Le, Ryan; Hill, Ryan; Jongeward, Gregg; Debnath, Anjan; Zhang, Liangfang; Miyamoto, Yukiko; Eckmann, Lars; Land, Kirkwood M; Wrischnik, Lisa A

    2016-12-01

    Trichomoniasis, caused by the protozoan parasite Trichomonas vaginalis, is the most common, non-viral, sexually transmitted infection in the world, but only two closely related nitro drugs are approved for its treatment. New antimicrobials against trichomoniasis remain an urgent need. Several organic gold compounds were tested for activity against T. vaginalis thioredoxin reductase (TrxR) in cell-free systems as well as for activity against different trichomonads in vitro and in a murine infection model. The organic gold(I) compounds auranofin and chloro(diethylphenylphosphine)gold(I) inhibited TrxR in a concentration-dependent manner in assays with recombinant purified reductase and in cytoplasmic extracts of T. vaginalis transfected with a haemagglutinin epitope-tagged form of the reductase. Auranofin potently suppressed the growth of three independent clinical T. vaginalis isolates as well as several strains of another trichomonad (Tritrichomonas foetus) in a 24 h-assay, with 50% inhibitory concentrations of 0.7-2.5 µM and minimum lethal concentrations of 2-6 µM. The drug also compromised the ability of the parasite to overcome oxidant stress, supporting the notion that auranofin acts, in part, by inactivating TrxR-dependent antioxidant defences. Chloro(diethylphenylphosphine)gold(I) was 10-fold less effective against T. vaginalis in vitro than auranofin. Oral administration of auranofin for 4 days cleared the parasites in a murine model of vaginal T. foetus infection without displaying any apparent adverse effects. The approved human drug auranofin may be a promising agent as an alternative treatment of trichomoniasis in cases when standard nitro drug therapies have failed. Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  11. Prokaryotic Expression and Serodiagnostic Potential of Glyceraldehyde-3-Phosphate Dehydrogenase and Thioredoxin Peroxidase from Baylisascaris schroederi

    Directory of Open Access Journals (Sweden)

    Yu Li

    2017-10-01

    Full Text Available Baylisascaris schroederi, a roundworm parasite of giant pandas, badly affects the health of its hosts. Diagnosis of this disease currently depends mainly on sedimentation floatation and Polymerase Chain Reaction (PCR methods to detect the eggs. However, neither of these methods is suitable for diagnosis of early-stage panda baylisascariasis and no information on early diagnosis of this disease is available so far. Therefore, to develop an effective serologic diagnostic method, this study produced recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH and thioredoxin peroxidase (Tpx proteins from B. schroederi using a prokaryotic expression system. We determined the immunological characteristics of these proteins and their location in the parasite. Indirect enzyme-linked immunosorbent assays (ELISAs were established to detect B. schroederi infection in giant pandas based on GAPDH and Tpx respectively. The open reading frame of the GAPDH gene (1083 bp encoded a 39 kDa protein, while the predicted molecular weight of Tpx (588 bp was 21.6 kDa. Western-blotting analysis revealed that both recombinant proteins could be recognized with positive serum of pandas infected with B. schroederi. Immunohistochemical staining showed that the endogenous GAPDH of B. schroederi was widely distributed in the worm while Tpx was mainly localized in the muscle, eggs, gut wall, uterus wall and hypodermis. Serological tests showed that the GAPDH-based indirect ELISA had a sensitivity of 95.83% and specificity of 100%, while the test using Tpx as the antigen had sensitivity of 75% and specificity of 91.7%. Thus, B. schroederi Tpx is unsuitable as a diagnostic antigen for baylisascariasis, but B. schroederi GAPDH is a good candidate diagnostic antigen for B. schroederi in pandas.

  12. Engineering of metabolic control

    Science.gov (United States)

    Liao, James C.

    2004-03-16

    The invention features a method of producing heterologous molecules in cells under the regulatory control of a metabolite and metabolic flux. The method can enhance the synthesis of heterologous polypeptides and metabolites.

  13. Trypanosoma cruzi infection is a potent risk factor for non-alcoholic steatohepatitis enhancing local and systemic inflammation associated with strong oxidative stress and metabolic disorders.

    Directory of Open Access Journals (Sweden)

    Luisina I Onofrio

    2015-02-01

    Full Text Available The immune mechanisms underlying experimental non-alcoholic steatohepatitis (NASH, and more interestingly, the effect of T. cruzi chronic infection on the pathogenesis of this metabolic disorder are not completely understood.We evaluated immunological parameters in male C57BL/6 wild type and TLR4 deficient mice fed with a standard, low fat diet, LFD (3% fat as control group, or a medium fat diet, MFD (14% fat in order to induce NASH, or mice infected intraperitoneally with 100 blood-derived trypomastigotes of Tulahuen strain and also fed with LFD (I+LFD or MFD (I+MFD for 24 weeks. We demonstrated that MFD by itself was able to induce NASH in WT mice and that parasitic infection induced marked metabolic changes with reduction of body weight and steatosis revealed by histological studies. The I+MFD group also improved insulin resistance, demonstrated by homeostasis model assessment of insulin resistance (HOMA-IR analysis; although parasitic infection increased the triglycerides and cholesterol plasma levels. In addition, hepatic M1 inflammatory macrophages and cytotoxic T cells showed intracellular inflammatory cytokines which were associated with high levels of IL6, IFNγ and IL17 plasmatic cytokines and CCL2 chemokine. These findings correlated with an increase in hepatic parasite load in I+MFD group demonstrated by qPCR assays. The recruitment of hepatic B lymphocytes, NK and dendritic cells was enhanced by MFD, and it was intensified by parasitic infection. These results were TLR4 signaling dependent. Flow cytometry and confocal microscopy analysis demonstrated that the reactive oxygen species and peroxinitrites produced by liver inflammatory leukocytes of MFD group were also exacerbated by parasitic infection in our NASH model.We highlight that a medium fat diet by itself is able to induce steatohepatitis. Our results also suggest a synergic effect between damage associated with molecular patterns generated during NASH and parasitic infection

  14. In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production.

    Science.gov (United States)

    Park, Jong Myoung; Song, Hyohak; Lee, Hee Jong; Seung, Doyoung

    2013-09-01

    Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising bulk chemical with wide industrial applications, along with various byproducts. In this study, the in silico gene knockout simulation of K. oxytoca was carried out for 2,3-BD overproduction by inhibiting the formation of byproducts. The knockouts of ldhA and pflB genes were targeted with the criteria of maximization of 2,3-BD production and minimization of byproducts formation. The constructed K. oxytoca ΔldhA ΔpflB strain showed higher 2,3-BD yields and higher final concentrations than those obtained from the wild-type and ΔldhA strains. However, the simultaneous deletion of both genes caused about a 50 % reduction in 2,3-BD productivity compared with K. oxytoca ΔldhA strain. Based on previous studies and in silico investigation that the agitation speed during 2,3-BD fermentation strongly affected cell growth and 2,3-BD synthesis, the effect of agitation speed on 2,3-BD production was investigated from 150 to 450 rpm in 5-L bioreactors containing 3-L culture media. The highest 2,3-BD productivity (2.7 g/L/h) was obtained at 450 rpm in batch fermentation. Considering the inhibition of acetoin for 2,3-BD production, fed-batch fermentations were performed using K. oxytoca ΔldhA ΔpflB strain to enhance 2,3-BD production. Altering the agitation speed from 450 to 350 rpm at nearly 10 g/L of acetoin during the fed-batch fermentation allowed for the production of 113 g/L 2,3-BD, with a yield of 0.45 g/g, and for the production of 2.1 g/L/h of 2,3-BD.

  15. 2-Deoxyglucose induces the expression of thioredoxin interacting protein (TXNIP) by increasing O-GlcNAcylation – Implications for targeting the Warburg effect in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Shin Yee; Hagen, Thilo, E-mail: bchth@nus.edu.sg

    2015-10-02

    The high proliferation rate of cancer cells and the microenvironment in the tumor tissue require the reprogramming of tumor cell metabolism. The major mechanism of metabolic reprogramming in cancer cells is the Warburg effect, defined as the preferential utilization of glucose via glycolysis even in the presence of oxygen. Targeting the Warburg effect is considered as a promising therapeutic strategy in cancer therapy. In this regard, the glycolytic inhibitor 2-deoxyglucose (2DG) has been evaluated clinically. 2DG exerts its effect by directly inhibiting glycolysis at the level of hexokinase and phosphoglucoisomerase. In addition, 2DG is also known to induce the expression of thioredoxin interacting protein (TXNIP), a tumor suppressor protein and an important negative regulator of cellular glucose uptake. Hence, characterization of the mechanism through which 2DG regulates TXNIP expression may reveal novel approaches to target the Warburg effect in cancer cells. Therefore, in this study we sought to test various hypotheses for the mechanistic basis of the 2DG dependent TXNIP regulation. We have shown that 2DG induced TXNIP expression is independent of carbohydrate response element mediated transcription. Furthermore, the induction of TXNIP is neither dependent on the ability of 2DG to deplete cellular ATP nor to cause endoplasmic reticulum stress. We found that the 2DG induced TXNIP expression is at least in part dependent on the inhibition of the O-GlcNAcase enzyme and the accumulation of O-GlcNAc modified proteins. These results have implications for the identification of therapeutic targets to increase TXNIP expression in cancer. - Highlights: • 2DG increases TXNIP expression at the mRNA and protein level. • The effect of 2DG on TXNIP is independent of ChoRE mediated transcription. • 2DG induces TXNIP independent of ER stress induction and ATP depletion. • 2DG inhibits OGA and leads to accumulation of O-GlcNAcylated proteins. • The upregulation of

  16. Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1.

    Directory of Open Access Journals (Sweden)

    Masahito Asada

    Full Text Available Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd or human dihydrofolate reductase (hdhfr. In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP and human dihydrofolate reductase (hDHFR, was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP and blasticidin-S deaminase (BSD. Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1 gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.

  17. Overexpression of Arabidopsis NADPH-dependent thioredoxin reductase C (AtNTRC) confers freezing and cold shock tolerance to plants

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Jeong Chan [National Institute of Ecology, 1210 Geumgang-ro, Maseo-myeon, Seocheon-gun 325-813 (Korea, Republic of); Lee, Sangmin [Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon (Korea, Republic of); Shin, Su Young [National Institute of Ecology, 1210 Geumgang-ro, Maseo-myeon, Seocheon-gun 325-813 (Korea, Republic of); Chae, Ho Byoung; Jung, Young Jun [Division of Applied Life Science - BK21+ program, PMBBRC, Gyeongsang National University, Jinju (Korea, Republic of); Jung, Hyun Suk [Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon (Korea, Republic of); Lee, Kyun Oh [Division of Applied Life Science - BK21+ program, PMBBRC, Gyeongsang National University, Jinju (Korea, Republic of); Lee, Jung Ro, E-mail: leejr73@nie.re.kr [National Institute of Ecology, 1210 Geumgang-ro, Maseo-myeon, Seocheon-gun 325-813 (Korea, Republic of); Department of Biochemistry and Biophysics, Texas A& M University, College Station, TX (United States); Lee, Sang Yeol, E-mail: sylee@gnu.ac.kr [Division of Applied Life Science - BK21+ program, PMBBRC, Gyeongsang National University, Jinju (Korea, Republic of)

    2015-08-07

    Overexpression of AtNTRC (AtNTRC{sup OE}) in Arabidopsis thaliana led to a freezing and cold stress tolerance, whereas a knockout mutant (atntrc) showed a stress-sensitive phenotype. Biochemical analyses showed that the recombinant AtNTRC proteins exhibited a cryoprotective activity for malate dehydrogenase and lactic dehydrogenase. Furthermore, conclusive evidence of its interaction with nucleic acids in vitro is provided here on the basis of gel shift and electron microscopy analysis. Recombinant AtNTRC efficiently protected RNA and DNA from RNase A and metal catalyzed oxidation damage, respectively. The C-terminal thioredoxin domain is required for the nucleic acid–protein complex formation. From these results, it can be hypothesized that AtNTRC, which is known to be an electron donor of peroxiredoxin, contributes the stability of macromolecules under cold stress. - Highlights: • AtNTRC has a cryoprotective activity in vitro. • Overexpression of AtNTRC increases tolerance to freezing and cold shock stresses. • Thioredoxin domain of AtNTRC protects nucleic acids in vitro. • AtNTRC inhibits protein aggregation under freezing stress in vitro.

  18. Overexpression of Arabidopsis NADPH-dependent thioredoxin reductase C (AtNTRC) confers freezing and cold shock tolerance to plants

    International Nuclear Information System (INIS)

    Moon, Jeong Chan; Lee, Sangmin; Shin, Su Young; Chae, Ho Byoung; Jung, Young Jun; Jung, Hyun Suk; Lee, Kyun Oh; Lee, Jung Ro; Lee, Sang Yeol

    2015-01-01

    Overexpression of AtNTRC (AtNTRC OE ) in Arabidopsis thaliana led to a freezing and cold stress tolerance, whereas a knockout mutant (atntrc) showed a stress-sensitive phenotype. Biochemical analyses showed that the recombinant AtNTRC proteins exhibited a cryoprotective activity for malate dehydrogenase and lactic dehydrogenase. Furthermore, conclusive evidence of its interaction with nucleic acids in vitro is provided here on the basis of gel shift and electron microscopy analysis. Recombinant AtNTRC efficiently protected RNA and DNA from RNase A and metal catalyzed oxidation damage, respectively. The C-terminal thioredoxin domain is required for the nucleic acid–protein complex formation. From these results, it can be hypothesized that AtNTRC, which is known to be an electron donor of peroxiredoxin, contributes the stability of macromolecules under cold stress. - Highlights: • AtNTRC has a cryoprotective activity in vitro. • Overexpression of AtNTRC increases tolerance to freezing and cold shock stresses. • Thioredoxin domain of AtNTRC protects nucleic acids in vitro. • AtNTRC inhibits protein aggregation under freezing stress in vitro

  19. Thioredoxin Reductase Activity may be More Important than GSH Level in Protecting Human Lens Epithelial Cells Against UVA Light

    Science.gov (United States)

    Padgaonkar, Vanita A.; Leverenz, Victor R.; Bhat, Aparna V.; Pelliccia, Sara E.; Giblin, Frank J.

    2014-01-01

    This study compares the abilities of the glutathione (GSH) and thioredoxin (Trx) antioxidant systems in defending cultured human lens epithelial cells (LECs) against UVA light. Levels of GSH were depleted with either L-buthionine-(S,R)-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB). CDNB treatment also inhibited the activity of thioredoxin reductase (TrxR). Two levels of O2, 3% and 20%, were employed during a 1 hr exposure of the cells to 25 J/cm2 of UVA radiation (338-400nm wavelength, peak at 365nm). Inhibition of TrxR activity by CDNB, combined with exposure to UVA light, produced a substantial loss of LECs and cell damage, with the effects being considerably more severe at 20% O2 compared to 3%. In contrast, depletion of GSH by BSO, combined with exposure to UVA light, produced only a slight cell loss, with no apparent morphological effects. Catalase was highly sensitive to UVA-induced inactivation, but was not essential for protection. Although UVA light presented a challenge for the lens epithelium, it was well-tolerated under normal conditions. The results demonstrate an important role for TrxR activity in defending the lens epithelium against UVA light, possibly related to the ability of the Trx system to assist DNA synthesis following UVA-induced cell damage. PMID:25495870

  20. Geranylgeranylacetone ameliorates lung ischemia/reperfusion injury by HSP70 and thioredoxin redox system: NF-kB pathway involved.

    Science.gov (United States)

    Cao, Weijun; Li, Manhui; Li, Jianxiong; Li, Chengwei; Xu, Xin; Gu, Weiqing

    2015-06-01

    Geranylgeranylacetone (GGA) has been clinically used as an anti-ulcer drug. In the present study, we explored the protective effects of GGA on lung ischemia/reperfusion injury (IRI) and the underlying mechanism. The results demonstrated that GGA ameliorated the lung biochemical and histological alterations induced by IRI, which was reversed by HSP70 inhibition. To further explore the mechanism of GGA action, we focused on NF-kB and thioredoxin (Trx) redox system. It was shown that GGA induced the HSP70 and Trx-1 expression, NF-kB nuclear translocation and activated thioredoxin reductase (TrxR). The Trx-1 expression and TrxR activity was suppressed by HSP70 and NF-kB inhibition, while the nuclear NF-kB p65 expression was suppressed by HSP70 inhibitor. These results indicated that GGA may protect rat lung against IRI by HSP70 and Trx redox system, in which NF-kB pathway may be involved. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Nurse-assessed metabolic monitoring: a file audit of risk factor prevalence and impact of an intervention to enhance measurement of waist circumference.

    Science.gov (United States)

    Rosenbaum, Simon; Nijjar, Sukh; Watkins, Andrew; Garwood, Natasha; Sherrington, Catherine; Tiedemann, Anne

    2014-06-01

    The aim of the present study was to: (i) document the prevalence of risk factors for non-communicable diseases among mental health consumers (inpatients) with various diagnoses; and (ii) audit the frequency of waist circumference (WC) documentation before and after an intervention that involved a single nurse-education session, and change in assessment-form design. The study was undertaken in a private psychiatric hospital in Sydney, Australia. Twenty-five nurses participated in the educational intervention. File audits were performed prior to intervention delivery (n = 60), and 3 months' (n = 60), and 9 months' (n = 60) post-intervention. Files were randomly selected, and demographic (age, diagnosis) and risk factor (WC, body mass index (BMI), smoking status, blood pressure) data were extracted. WC was higher in this cohort compared to published general population means, and only 19% of patients had a BMI within the healthy range. In total, 37% of patients smoked, while 31% were hypertensive. At baseline, none of the audited files reported WC, which increased to 35 of the 60 (58%) files audited at the 3-month follow up. At the 9-month follow up, 25 of the 60 (42%) files audited reported a WC. In the 120 post-intervention files audited, only two patients refused measurement. These results illustrate the poor physical health of inpatients, and suggest that nurse-assessed metabolic monitoring can be enhanced with minimal training. © 2014 Australian College of Mental Health Nurses Inc.

  2. Cannabidiol attenuates OGD/R-induced damage by enhancing mitochondrial bioenergetics and modulating glucose metabolism via pentose-phosphate pathway in hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Shanshan Sun

    2017-04-01

    Full Text Available Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD, a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen–glucose-deprivation/reperfusion (OGD/R model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers. Using the Seahorse XFe24 Extracellular Flux Analyzer, we found that CBD significantly improved basal respiration, ATP-linked oxygen consumption rate, and the spare respiratory capacity, and augmented glucose consumption in OGD/R-injured neurons. The activation of glucose 6-phosphate dehydrogenase and the preservation of the NADPH/NADP+ ratio implies that the pentose-phosphate pathway is stimulated by CBD, thus protecting hippocampal neurons from OGD/R injury. This study is the first to document the neuroprotective effects of CBD against OGD/R insult, which depend in part on attenuating oxidative stress, enhancing mitochondrial bioenergetics, and modulating glucose metabolism via the pentose-phosphate pathway, thus preserving both energy and the redox balance.

  3. Cannabidiol attenuates OGD/R-induced damage by enhancing mitochondrial bioenergetics and modulating glucose metabolism via pentose-phosphate pathway in hippocampal neurons.

    Science.gov (United States)

    Sun, Shanshan; Hu, Fangyuan; Wu, Jihong; Zhang, Shenghai

    2017-04-01

    Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD), a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen-glucose-deprivation/reperfusion (OGD/R) model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers. Using the Seahorse XF e 24 Extracellular Flux Analyzer, we found that CBD significantly improved basal respiration, ATP-linked oxygen consumption rate, and the spare respiratory capacity, and augmented glucose consumption in OGD/R-injured neurons. The activation of glucose 6-phosphate dehydrogenase and the preservation of the NADPH/NADP + ratio implies that the pentose-phosphate pathway is stimulated by CBD, thus protecting hippocampal neurons from OGD/R injury. This study is the first to document the neuroprotective effects of CBD against OGD/R insult, which depend in part on attenuating oxidative stress, enhancing mitochondrial bioenergetics, and modulating glucose metabolism via the pentose-phosphate pathway, thus preserving both energy and the redox balance. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Mechanism of thioredoxin-catalyzed disulfide reduction. Activation of the buried thiol and role of the variable active-site residues

    NARCIS (Netherlands)

    Carvalho, A.P.; Swart, M.; van Stralen, J.N.P.; Fernandes, P.A.; Ramos, M.E.; Bickelhaupt, F.M.

    2008-01-01

    Thioredoxins (Trx) are enzymes with a characteristic CXYC active-site motif that catalyze the reduction of disulfide bonds in other proteins. We have theoretically explored this reaction mechanism, both in the gas phase and in water, using density functional theory. The mechanism of disulfide

  5. Role of thioredoxin-1 in apoptosis induction by alpha-tocopheryl succinate and TNF-related apoptosis-inducing ligand in mesothelioma cells

    Czech Academy of Sciences Publication Activity Database

    Freeman, R.E.; Neužil, Jiří

    2006-01-01

    Roč. 580, č. 11 (2006), s. 2671-2676 ISSN 0014-5793 Institutional research plan: CEZ:AV0Z50520514 Keywords : apoptosis * malignant mesothelioma * thioredoxin-1 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.372, year: 2006

  6. In vitro activation of sigma-aminolevulinate dehydratase from far-red irradiated radish (Raphanus sativus L. ) seedlings by thioredoxin f

    Energy Technology Data Exchange (ETDEWEB)

    Balange, A.P. (Centre National de la Recherche Scientifique, Mont Saint Aignan (France). Laboratoire de Photobiologie); Lambert, C. (UER Scientifique de Luminy, Department de Biologie Moleculaire et Cellulaire, Marseille, France)

    1983-10-01

    sigma-Aminolevulinate dehydratase has been found to be activated in vitro by dithiotreitol and factors isolated from radish cotyledons grown under continuous far-red light. Cross experiments, between fructose 1-6 bisphosphatase system, and sigma-aminolevulinate dehydratase, show that these factors are functionally identical to thioredoxin f.

  7. Changes in serum thioredoxin among individuals chronically exposed to arsenic in drinking water

    International Nuclear Information System (INIS)

    Li, Yuanyuan; Gao, Yanhui; Zhao, Lijun; Wei, Yudan; Feng, Hongqi; Wang, Cheng; Wei, Wei; Ding, Yunpeng; Sun, Dianjun

    2012-01-01

    It is well known that oxidative damage plays a key role in the development of chronic arsenicosis. There is a complex set of mechanisms of redox cycling in vivo to protect cells from the damage. In this study, we examined the differences in the levels of serum thioredoxin1 (TRX1) among individuals exposed to different levels of arsenic in drinking water and detected early biomarkers of arsenic poisoning before the appearance of skin lesions. A total of 157 subjects from endemic regions of China were selected and divided into arsenicosis group with skin lesions (total intake of arsenic: 8.68–45.71 mg-year) and non-arsenicosis group without skin lesions, which further divided into low (0.00–1.06 mg-year), medium (1.37–3.55 mg-year), and high (4.26–48.13 mg-year) arsenic exposure groups. Concentrations of serum TRX1 were analyzed by an ELISA method. Levels of water arsenic and urinary speciated arsenics, including inorganic arsenic (iAs), monomethylated arsenic (MMA), and dimethylated arsenic (DMA), were determined by hydride generation atomic absorption spectrometry. Our results showed that the levels of serum TRX1 in arsenicosis patients were significantly higher than that of the subjects who were chronically exposed to arsenic, but without skin lesions. A positive correlation was seen between the levels of serum TRX1 and the total water arsenic intake or the levels of urinary arsenic species. The results of this study indicate that arsenic exposure could significantly change the levels of human serum TRX1, which can be detected before arsenic-specific dermatological symptoms occur. This study provides further evidence on revealing the mechanism of arsenic toxicity. -- Highlights: ► Three regions are selected as the areas affected by endemic arsenicosis of China. ► We first examine changes in serum TRX1 among individuals exposed to arsenic. ► A positive correlation was seen between serum TRX1 and total water arsenic intake. ► The same relationship

  8. Thioredoxin and impaired spatial learning and memory in the rats exposed to intermittent hypoxia

    Institute of Scientific and Technical Information of China (English)

    YANG Xiu-hong; LIU Hui-guo; LIU Xue; CHEN Jun-nan

    2012-01-01

    Background Obstructive sleep apnea (OSA) can cause cognitive dysfunction and may be a reversible cause of cognitive loss in patients with Alzheimer's disease (AD).Chronic exposure to intermittent hypoxia (IH),such as encountered in OSA,is marked by neurodegenerative changes in rat brain.We investigated the change of thioredoxin (Trx),spatial learning and memory in rats exposed to chronic intermittent hypoxia (CIH).Methods Forty healthy male Sprague-Dawley (SD) rats were randomly divided into four groups of ten each:a CIH+normal saline (CIH+NS group),a N-acetylcystein-treated CIH (CIH+NAC) group,a sham CIH group (sham CIH+NS),and a sham NAC-treated sham CIH (CIH+NAC) group.Spatial learning and memory in each group was assessed with the Morris water maze.Real-time PCR and Western blotting were used to examine mRNA and protein expression of Trx in the hippocampus tissue.The terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL) method was used to detect the apoptotic cells of the hippocampus CA1 region.Results ClH-rats showed impaired spatial learning and memory in the Morris water maze,including longer mean latencies for the target platform,reduced numbers of passes over the previous target platform and a smaller percentage of time spent in the target quadrant.Trx mRNA and protein levels were significantly decreased in the CIH-hippocampus,meanwhile,an elevated apoptotic index revealed apoptosis of hippocampal neurons of rats exposed to CIH.The rats,which acted better in the Morris water maze,showed higher levels of the Trx mRNA and protein in the hippocampus;apoptotic index of the neurons in the hippocampus of each group was negatively correlated with the Trx mRNA and protein levels.Conclusion The Trx deficit likely plays an important role in the impaired spatial learning and memory in the rats exposed to CIH and may work through the apoptosis of neurons in the hippocampus.

  9. Changes in serum thioredoxin among individuals chronically exposed to arsenic in drinking water

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuanyuan; Gao, Yanhui; Zhao, Lijun [Center for Endemic Disease Control, Chinese Center for Disease Control and Prevention, Harbin Medical University, Key Lab of Etiology and Epidemiology, Education Bureau of Hei Long Jiang Province and Ministry of Health (23618104), Harbin 150081 (China); Wei, Yudan [Department of Community Medicine, Mercer University School of Medicine, Macon 31207, GA (United States); Feng, Hongqi; Wang, Cheng; Wei, Wei; Ding, Yunpeng [Center for Endemic Disease Control, Chinese Center for Disease Control and Prevention, Harbin Medical University, Key Lab of Etiology and Epidemiology, Education Bureau of Hei Long Jiang Province and Ministry of Health (23618104), Harbin 150081 (China); Sun, Dianjun, E-mail: hrbmusdj@163.com [Center for Endemic Disease Control, Chinese Center for Disease Control and Prevention, Harbin Medical University, Key Lab of Etiology and Epidemiology, Education Bureau of Hei Long Jiang Province and Ministry of Health (23618104), Harbin 150081 (China)

    2012-02-15

    It is well known that oxidative damage plays a key role in the development of chronic arsenicosis. There is a complex set of mechanisms of redox cycling in vivo to protect cells from the damage. In this study, we examined the differences in the levels of serum thioredoxin1 (TRX1) among individuals exposed to different levels of arsenic in drinking water and detected early biomarkers of arsenic poisoning before the appearance of skin lesions. A total of 157 subjects from endemic regions of China were selected and divided into arsenicosis group with skin lesions (total intake of arsenic: 8.68–45.71 mg-year) and non-arsenicosis group without skin lesions, which further divided into low (0.00–1.06 mg-year), medium (1.37–3.55 mg-year), and high (4.26–48.13 mg-year) arsenic exposure groups. Concentrations of serum TRX1 were analyzed by an ELISA method. Levels of water arsenic and urinary speciated arsenics, including inorganic arsenic (iAs), monomethylated arsenic (MMA), and dimethylated arsenic (DMA), were determined by hydride generation atomic absorption spectrometry. Our results showed that the levels of serum TRX1 in arsenicosis patients were significantly higher than that of the subjects who were chronically exposed to arsenic, but without skin lesions. A positive correlation was seen between the levels of serum TRX1 and the total water arsenic intake or the levels of urinary arsenic species. The results of this study indicate that arsenic exposure could significantly change the levels of human serum TRX1, which can be detected before arsenic-specific dermatological symptoms occur. This study provides further evidence on revealing the mechanism of arsenic toxicity. -- Highlights: ► Three regions are selected as the areas affected by endemic arsenicosis of China. ► We first examine changes in serum TRX1 among individuals exposed to arsenic. ► A positive correlation was seen between serum TRX1 and total water arsenic intake. ► The same relationship

  10. Thioredoxin-1 Negatively Modulates ADAM17 Activity Through Direct Binding and Indirect Reductive Activity.

    Science.gov (United States)

    Granato, Daniela C; E Costa, Rute A P; Kawahara, Rebeca; Yokoo, Sami; Aragão, Annelize Z; Domingues, Romênia R; Pauletti, Bianca A; Honorato, Rodrigo V; Fattori, Juliana; Figueira, Ana Carolina M; Oliveira, Paulo S L; Consonni, Silvio R; Fernandes, Denise; Laurindo, Francisco; Hansen, Hinrich P; Paes Leme, Adriana F

    2018-02-27

    A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1 K72A and catalytic site mutant Trx-1 C32/35S rescued ADAM17 activity, although the interaction with Trx-1 C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1 C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1 K72A mutant showed similar oxidant levels to Trx-1 C32/35S , even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. This unexpected Trx-1 K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass

  11. Comparative analysis of two thioredoxin-like genes in black rockfish Sebastes schlegelii and their possible involvement in redox homeostasis and innate immune responses.

    Science.gov (United States)

    Kugapreethan, Roopasingam; Umasuthan, Navaneethaiyer; Wan, Qiang; Thulasitha, William Shanthakumar; Kim, Chul; Lee, Jehee

    2017-02-01

    Elevated levels of ROS can cause serious intracellular damages by reacting readily with nucleic acids, proteins and lipids, thus triggering tissue damage and cell death. Thioredoxin system is one of the principal factors that maintain the intracellular redox balance via its antioxidant property. In this study, we characterized two new thioredoxin isoforms (SsTXN-like 1 and SsMtTXN-like) from black rockfish, Sebastes schlegelii. The molecular and structural characteristics, as well as the evolutionary relationships of SsTXN-like 1 and SsMtTXN-like confirmed that they belong to the thioredoxin superfamily. A classical thioredoxin domain was found in both proteins with a conserved redox-active site CXYC, however, only the precursor of SsMtTXN-like protein possessed a mitochondrial targeting signal. The results from insulin disulfide reduction activity assay demonstrated that their recombinant proteins are capable of reducing the disulfide bonds of oxidatively damaged proteins via their oxidoreductase activities. The free radical scavenging activity assay revealed the prominent hydroxyl and DPPH scavenging activities of rSsTXN-like 1 and rSsMtTXN-like in a dose-dependent manner. Transcriptional studies showed a broad distribution of SsTXN-like 1 and SsMtTXN-like transcripts in all the examined tissues. Significant (p immune-related tissues after LPS, poly I:C and Streptococcus iniae challenges reflect their critical role in redox homeostasis in black rockfish. Taken together, SsTXN-like 1 and SsMtTXN-like, as two active members of thioredoxin superfamily, have significant antioxidant properties to housekeep the redox potential during various stress conditions and innate immune response of Sebastes schlegelii. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Thioredoxin A Is Essential for Motility and Contributes to Host Infection of Listeria monocytogenes via Redox Interactions

    Directory of Open Access Journals (Sweden)

    Changyong Cheng

    2017-06-01

    Full Text Available Microbes employ the thioredoxin system to defend against oxidative stress and ensure correct disulfide bonding to maintain protein function. Listeria monocytogenes has been shown to encode a putative thioredoxin, TrxA, but its biological roles and underlying mechanisms remain unknown. Here, we showed that expression of L. monocytogenes TrxA is significantly induced in bacteria treated with the thiol-specific oxidizing agent, diamide. Deletion of trxA markedly compromised tolerance of the pathogen to diamide, and mainly impaired early stages of infection in human intestinal epithelial Caco-2 cells. In addition, most trxA mutant bacteria were not associated with polymerized actin, and the rare bacteria that were associated with polymerized actin displayed very short tails or clouds during infection. Deletion or constitutive overexpression of TrxA, which was regulated by SigH, severely attenuated the virulence of the pathogen. Transcriptome analysis of L. monocytogenes revealed over 270 genes that were differentially transcribed in the ΔtrxA mutant compared to the wild-type, especially for the virulence-associated genes plcA, mpl, hly, actA, and plcB. Particularly, deletion of TrxA completely reduced LLO expression, and thereby led to a thoroughly impaired hemolytic activity. Expression of these virulence factors are positively regulated by the master regulator PrfA that was found here to use TrxA to maintain its reduced forms for activation. Interestingly, the trxA deletion mutant completely lacked flagella and was non-motile. We further confirmed that this deficiency is attributable to TrxA in maintaining the reduced intracellular monomer status of MogR, the key regulator for flagellar formation, to ensure correct dimerization. In summary, we demonstrated for the first time that L. monocytogenes thioredoxin A as a vital cellular reductase is essential for maintaining a highly reducing environment in the bacterial cytosol, which provides a

  13. Thioredoxin A Is Essential for Motility and Contributes to Host Infection of Listeria monocytogenes via Redox Interactions.

    Science.gov (United States)

    Cheng, Changyong; Dong, Zhimei; Han, Xiao; Wang, Hang; Jiang, Li; Sun, Jing; Yang, Yongchun; Ma, Tiantian; Shao, Chunyan; Wang, Xiaodu; Chen, Zhongwei; Fang, Weihuan; Freitag, Nancy E; Huang, Huarong; Song, Houhui

    2017-01-01

    Microbes employ the thioredoxin system to defend against oxidative stress and ensure correct disulfide bonding to maintain protein function. Listeria monocytogenes has been shown to encode a putative thioredoxin, TrxA, but its biological roles and underlying mechanisms remain unknown. Here, we showed that expression of L. monocytogenes TrxA is significantly induced in bacteria treated with the thiol-specific oxidizing agent, diamide. Deletion of trxA markedly compromised tolerance of the pathogen to diamide, and mainly impaired early stages of infection in human intestinal epithelial Caco-2 cells. In addition, most trxA mutant bacteria were not associated with polymerized actin, and the rare bacteria that were associated with polymerized actin displayed very short tails or clouds during infection. Deletion or constitutive overexpression of TrxA, which was regulated by SigH, severely attenuated the virulence of the pathogen. Transcriptome analysis of L. monocytogenes revealed over 270 genes that were differentially transcribed in the Δ trxA mutant compared to the wild-type, especially for the virulence-associated genes plcA, mpl, hly, actA , and plcB . Particularly, deletion of TrxA completely reduced LLO expression, and thereby led to a thoroughly impaired hemolytic activity. Expression of these virulence factors are positively regulated by the master regulator PrfA that was found here to use TrxA to maintain its reduced forms for activation. Interestingly, the trxA deletion mutant completely lacked flagella and was non-motile. We further confirmed that this deficiency is attributable to TrxA in maintaining the reduced intracellular monomer status of MogR, the key regulator for flagellar formation, to ensure correct dimerization. In summary, we demonstrated for the first time that L. monocytogenes thioredoxin A as a vital cellular reductase is essential for maintaining a highly reducing environment in the bacterial cytosol, which provides a favorable condition for

  14. Ginger consumption enhances the thermic effect of food and promotes feelings of satiety without affecting metabolic and hormonal parameters in overweight men: A pilot study

    Science.gov (United States)

    Mansour, Muhammad S.; Ni, Yu-Ming; Roberts, Amy L.; Kelleman, Michael; RoyChoudhury, Arindam; St-Onge, Marie-Pierre

    2012-01-01

    Objective Evidence suggests that ginger consumption has anti-inflammatory, anti-hypertensive, glucose-sensitizing, and stimulatory effects on the gastrointestinal tract. This study assessed the effects of a hot ginger beverage on energy expenditure, feelings of appetite and satiety and metabolic risk factors in overweight men. Methods Ten men, age 39.1 ± 3.3 y and body mass index (BMI) 27.2 ± 0.3 kg/m2, participated in this randomized crossover study. Resting state energy expenditure was measured using indirect calorimetry and for 6 h after consumption of a breakfast meal with or without 2 g ginger powder dissolved in a hot water beverage. Subjective feelings of satiety were assessed hourly using visual analog scales (VAS) and blood samples were taken fasted and for 3 h after breakfast consumption. Results There was no significant effect of ginger on total resting energy expenditure (P = 0.43) or respiratory quotient (P = 0.41). There was a significant effect of ginger on thermic effect of food (ginger vs control = 42.7 ± 21.4 kcal/d, P = 0.049) but the area under the curve was not different (P = 0.43). VAS ratings showed lower hunger (P = 0.002), lower prospective food intake (P = 0.004) and greater fullness (P = 0.064) with ginger consumption versus control. There were no effects of ginger on glucose, insulin, lipids, or inflammatory markers. Conclusions The results, showing enhanced thermogenesis and reduced feelings of hunger with ginger consumption, suggest a potential role of ginger in weight management. Additional studies are necessary to confirm these findings. PMID:22538118

  15. The thermodynamics of protein aggregation reactions may underpin the enhanced metabolic efficiency associated with heterosis, some balancing selection, and the evolution of ploidy levels.

    Science.gov (United States)

    Ginn, B R

    2017-07-01

    Identifying the physical basis of heterosis (or "hybrid vigor") has remained elusive despite over a hundred years of research on the subject. The three main theories of heterosis are dominance theory, overdominance theory, and epistasis theory. Kacser and Burns (1981) identified the molecular basis of dominance, which has greatly enhanced our understanding of its importance to heterosis. This paper aims to explain how overdominance, and some features of epistasis, can similarly emerge from the molecular dynamics of proteins. Possessing multiple alleles at a gene locus results in the synthesis of different allozymes at reduced concentrations. This in turn reduces the rate at which each allozyme forms soluble oligomers, which are toxic and must be degraded, because allozymes co-aggregate at low efficiencies. The model developed in this paper can explain how heterozygosity impacts the metabolic efficiency of an organism. It can also explain why the viabilities of some inbred lines seem to decline rapidly at high inbreeding coefficients (F > 0.5), which may provide a physical basis for truncation selection for heterozygosity. Finally, the model has implications for the ploidy level of organisms. It can explain why polyploids are frequently found in environments where severe physical stresses promote the formation of soluble oligomers. The model can also explain why complex organisms, which need to synthesize aggregation-prone proteins that contain intrinsically unstructured regions (IURs) and multiple domains because they facilitate complex protein interaction networks (PINs), tend to be diploid while haploidy tends to be restricted to relatively simple organisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Thioredoxin plays a key role in retinal neuropathy prior to endothelial damage in diabetic mice

    OpenAIRE

    Ren, Xiang; Li, Chen; Liu, Junli; Zhang, Chenghong; Fu, Yuzhen; Wang, Nina; Ma, Haiying; Lu, Heyuan; Kong, Hui; Kong, Li

    2017-01-01

    Diabetes is a chronic metabolic syndrome that results in changes in carbohydrate, lipid and protein metabolism. With diabetes for a long time, it increases the risk of diabetic retinopathy (DR) and long-term morbidity and mortality. Moreover, emerging evidence suggests that neuron damage occurs earlier than microvascular complications in DR patients, but the underlying mechanism is unclear. We investigated diabetes-induced retinal neuropathy and elucidated key molecular events to identify new...

  17. Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

    DEFF Research Database (Denmark)

    Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

    2008-01-01

    Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity...... of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly......, protein extract of embryos from germinated barley seeds was treated +/- Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (-Trx) samples were then chemically reduced and labeled...

  18. An improved synthesis of α-13C glycine and heteronuclear NMR studies of its incorporation into thioredoxin

    International Nuclear Information System (INIS)

    Wishart, D.S.; Sykes, B.D.; Richards, F.M.

    1992-01-01

    We present an improved method to easily prepare gram quantities of α- 13 C glycine beginning from K 13 CN. The four step synthesis involves the production of an N, N-diphenyl-cyanoformamidine intermediate through the coupling of cyanide to N, N-diphenylcarbodiimide. Subsequent reduction by LiAlH 4 and hydrolysis of the resulting amidine produces fully enriched α- 13 C labelled glycine with a 45-50% yield. This relatively fast and simple synthesis uses only commonly available compounds and requires no special equipment, making the process easy to perform in any well equipped biochemistry laboratory. We further demonstrate that the product may be used, without extensive purification, to specifically label bacterially expressed proteins (E. coli thioredoxin) through standard biosynthetic procedures. We also show that the 13 C glycine-labelled protein may be readily analyzed using commonly available heteronuclear NMR techniques. Complete assignments for all 9 glycines of native E. coli thoredoxin are presented. (Author)

  19. Rare sugar D-allose strongly induces thioredoxin-interacting protein and inhibits osteoclast differentiation in Raw264 cells.

    Science.gov (United States)

    Yamada, Kana; Noguchi, Chisato; Kamitori, Kazuyo; Dong, Youyi; Hirata, Yuko; Hossain, Mohammad A; Tsukamoto, Ikuko; Tokuda, Masaaki; Yamaguchi, Fuminori

    2012-02-01

    Oxidative stress modulates the osteoclast differentiation via redox systems, and thioredoxin 1 (Trx) promotes the osteoclast formation by regulating the activity of transcription factors. The function of Trx is known to be regulated by its binding partner, thioredoxin-interacting protein (TXNIP). We previously reported that the expression of TXNIP gene is strongly induced by a rare sugar D-allose. In this study, we tested the hypothesis that D-allose could inhibit the osteoclast differentiation by regulating the Trx function. We used a murine Raw264 cell line that differentiates to the osteoclast by the receptor activator of nuclear factor-κB ligand (RANKL) treatment. The effect of sugars was evaluated by tartrate-resistant acid phosphatase staining. The expression and localization of TXNIP and Trx protein were examined by Western blotting and immunohistochemisty. The activity of the nuclear factor-κB, nuclear factor of activated T cells, and activator protein 1 transcription factors was measured by the luciferase reporter assay. The addition of D-allose (25 mmol/L) inhibited the osteoclast differentiation down to 9.53% ± 1.27% of a receptor activator of nuclear factor-κB ligand-only treatment. During the osteoclast differentiation, a significant increase of TNXIP was observed by D-allose treatment. The immunohistochemical analysis showed that both Trx and TXNIP existed in the nucleus in preosteoclasts and osteoclasts. Overexpression of TXNIP by plasmid transfection also inhibited the osteoclast formation, indicating the functional importance of TXNIP for the osteoclast differentiation. Transcriptional activity of the activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells, known to be modulated by Trx, were inhibited by D-allose. In conclusion, our data indicate that D-allose is a strong inhibitor of the osteoclast differentiation, and this effect could be caused by TXNIP induction and a resulting inhibition of the Trx function

  20. The conformational stability and biophysical properties of the eukaryotic thioredoxins of Pisum sativum are not family-conserved.

    Directory of Open Access Journals (Sweden)

    David Aguado-Llera

    2011-02-01

    Full Text Available Thioredoxins (TRXs are ubiquitous proteins involved in redox processes. About forty genes encode TRX or TRX-related proteins in plants, grouped in different families according to their subcellular localization. For instance, the h-type TRXs are located in cytoplasm or mitochondria, whereas f-type TRXs have a plastidial origin, although both types of proteins have an eukaryotic origin as opposed to other TRXs. Herein, we study the conformational and the biophysical features of TRXh1, TRXh2 and TRXf from Pisum sativum. The modelled structures of the three proteins show the well-known TRX fold. While sharing similar pH-denaturations features, the chemical and thermal stabilities are different, being PsTRXh1 (Pisum sativum thioredoxin h1 the most stable isoform; moreover, the three proteins follow a three-state denaturation model, during the chemical-denaturations. These differences in the thermal- and chemical-denaturations result from changes, in a broad sense, of the several ASAs (accessible surface areas of the proteins. Thus, although a strong relationship can be found between the primary amino acid sequence and the structure among TRXs, that between the residue sequence and the conformational stability and biophysical properties is not. We discuss how these differences in the biophysical properties of TRXs determine their unique functions in pea, and we show how residues involved in the biophysical features described (pH-titrations, dimerizations and chemical-denaturations belong to regions involved in interaction with other proteins. Our results suggest that the sequence demands of protein-protein function are relatively rigid, with different protein-binding pockets (some in common for each of the three proteins, but the demands of structure and conformational stability per se (as long as there is a maintained core, are less so.

  1. Growth hormone alters the glutathione S-transferase and mitochondrial thioredoxin systems in long-living Ames dwarf mice.

    Science.gov (United States)

    Rojanathammanee, Lalida; Rakoczy, Sharlene; Brown-Borg, Holly M

    2014-10-01

    Ames dwarf mice are deficient in growth hormone (GH), prolactin, and thyroid-stimulating hormone and live significantly longer than their wild-type (WT) siblings. The lack of GH is associated with stress resistance and increased longevity. However, the mechanism underlying GH's actions on cellular stress defense have yet to be elucidated. In this study, WT or Ames dwarf mice were treated with saline or GH (WT saline, Dwarf saline, and Dwarf GH) two times daily for 7 days. The body and liver weights of Ames dwarf mice were significantly increased after 7 days of GH administration. Mitochondrial protein levels of the glutathione S-transferase (GST) isozymes, K1 and M4 (GSTK1 and GSTM4), were significantly higher in dwarf mice (Dwarf saline) when compared with WT mice (WT saline). GH administration downregulated the expression of GSTK1 proteins in dwarf mice. We further investigated GST activity from liver lysates using different substrates. Substrate-specific GST activity (bromosulfophthalein, dichloronitrobenzene, and 4-hydrox-ynonenal) was significantly reduced in GH-treated dwarf mice. In addition, GH treatment attenuated the activity of thioredoxin and glutaredoxin in liver mitochondria of Ames mice. Importantly, GH treatment suppressed Trx2 and TrxR2 mRNA expression. These data indicate that GH has a role in stress resistance by altering the functional capacity of the GST system through the regulation of specific GST family members in long-living Ames dwarf mice. It also affects the regulation of thioredoxin and glutaredoxin, factors that regulate posttranslational modification of proteins and redox balance, thereby further influencing stress resistance. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Electrical stimulation of the ventromedial hypothalamus enhances both fat utilization and metabolic rate that precede and parallel the inhibition of feeding behavior

    NARCIS (Netherlands)

    Ruffin, MP; Nicolaidis, S

    1999-01-01

    The effects of ventromedial hypothalamic (VMH) stimulation on various metabolic parameters in freely moving animals were measured using a specific indirect calorimetric chamber associated with a quantitative measurement of locomotor activity, which allows the separate measurement of locomotor energy

  3. Moderate-to-high-intensity training and a hypocaloric Mediterranean diet enhance endothelial progenitor cells and fitness in subjects with the metabolic syndrome.

    Science.gov (United States)

    Fernández, Juan Marcelo; Rosado-Álvarez, Daniel; Da Silva Grigoletto, Marzo Edir; Rangel-Zúñiga, Oriol Alberto; Landaeta-Díaz, Leslie Lorena; Caballero-Villarraso, Javier; López-Miranda, José; Pérez-Jiménez, Francisco; Fuentes-Jiménez, Francisco

    2012-09-01

    A reduction in EPC (endothelial progenitor cell) number could explain the development and progression of atherosclerosis in the MetS (metabolic syndrome). Although much research in recent years has focused on the Mediterranean dietary pattern and the MetS, the effect of this diet with/without moderate-to-high-intensity endurance training on EPCs levels and CrF (cardiorespiratory fitness) remains unclear. In the present study, the objective was to assess the effect of a Mediterranean diet hypocaloric model with and without moderate-to-high-intensity endurance training on EPC number and CrF of MetS patients. Thus 45 MetS patients (50-66 years) were randomized to a 12-week intervention with the hypocaloric MeD (Mediterranean diet) or the MeDE (MeD plus moderate-to-high-intensity endurance training). Training included two weekly supervised sessions [80% MaxHR (maximum heart rate); leg and arm pedalling] and one at-home session (65-75% MaxHR; walking controlled by heart rate monitors). Changes in: (i) EPC number [CD34(+)KDR(+) (kinase insert domain-containing receptor)], (ii) CrF variables and (iii) MetS components and IRH (ischaemic reactive hyperaemia) were determined at the end of the study. A total of 40 subjects completed all 12 weeks of the study, with 20 in each group. The MeDE led to a greater increase in EPC numbers and CrF than did the MeD intervention (P ≤ 0.001). In addition, a positive correlation was observed between the increase in EPCs and fitness in the MeDE group (r=0.72; r(2)=0.52; P ≤ 0.001). Body weight loss, insulin sensitivity, TAGs (triacylglycerols) and blood pressure showed a greater decrease in the MeDE than MeD groups. Furthermore, IRH was only improved after the MeDE intervention. In conclusion, compliance with moderate-to-high-intensity endurance training enhances the positive effects of a model of MeD on the regenerative capacity of endothelium and on the fitness of MetS patients.

  4. Nitrogen Metabolism and Growth Enhancement in Tomato Plants Challenged with Trichoderma harzianum Expressing the Aspergillus nidulans Acetamidase amdS Gene

    Science.gov (United States)

    Domínguez, Sara; Rubio, M. Belén; Cardoza, Rosa E.; Gutiérrez, Santiago; Nicolás, Carlos; Bettiol, Wagner; Hermosa, Rosa; Monte, Enrique

    2016-01-01

    Trichoderma is a fungal genus that includes species that are currently being used as biological control agents and/or as biofertilizers. In addition to the direct application of Trichoderma spp. as biocontrol agents in plant protection, recent studies have focused on the beneficial responses exerted on plants, stimulating the growth, activating the defenses, and/or improving nutrient uptake. The amdS gene, encoding an acetamidase of Aspergillus, has been used as a selectable marker for the transformation of filamentous fungi, including Trichoderma spp., but the physiological effects of the introduction of this gene into the genome of these microorganisms still remains unexplored. No evidence of amdS orthologous genes has been detected within the Trichoderma spp. genomes and the amdS heterologous expression in Trichoderma harzianum T34 did not affect the growth of this fungus in media lacking acetamide. However, it did confer the ability for the fungus to use this amide as a nitrogen source. Although a similar antagonistic behavior was observed for T34 and amdS transformants in dual cultures against Rhizoctonia solani, Botrytis cinerea, and Fusarium oxysporum, a significantly higher antifungal activity was detected in amdS transformants against F. oxysporum, compared to that of T34, in membrane assays on media lacking acetamide. In Trichoderma-tomato interaction assays, amdS transformants were able to promote plant growth to a greater extent than the wild-type T34, although compared with this strain the transformants showed similar capability to colonize tomato roots. Gene expression patterns from aerial parts of 3-week-old tomato plants treated with T34 and the amdS transformants have also been investigated using GeneChip Tomato Genome Arrays. The downregulation of defense genes and the upregulation of carbon and nitrogen metabolism genes observed in the microarrays were accompanied by (i) enhanced growth, (ii) increased carbon and nitrogen levels, and (iii) a

  5. Nitrogen metabolism and growth enhancement in tomato plants challenged with Trichoderma harzianum expressing the Aspergillus nidulans acetamidase amdS gene

    Directory of Open Access Journals (Sweden)

    Sara Domínguez

    2016-08-01

    Full Text Available Trichoderma is a fungal genus that includes species that are currently being used as biological control agents and/or as biofertilizers. In addition to the direct application of Trichoderma spp. as biocontrol agents in plant protection, recent studies have focused on the beneficial responses exerted on plants, stimulating the growth, activating the defenses, and/or improving nutrient uptake. The amdS gene, encoding an acetamidase of Aspergillus, has been used as a selectable marker for the transformation of filamentous fungi, including Trichoderma spp., but the physiological effects of the introduction of this gene into the genome of these microorganisms still remains unexplored. No evidence of amdS orthologous genes has been detected within the Trichoderma spp. genomes and the amdS heterologous expression in T. harzianum T34 did not affect the growth of this fungus in media lacking acetamide. However, it did confer the ability for the fungus to use this amide as a nitrogen source. Although a similar antagonistic behavior was observed for T34 and amdS transformants in dual cultures against Rhizoctonia solani, Botrytis cinerea and Fusarium oxysporum, a significantly higher antifungal activity was detected in amdS transformants against F. oxysporum, compared to that of T34, in membrane assays on media lacking acetamide. In Trichoderma-tomato interaction assays, amdS transformants were able to promote plant growth to a greater extent than the wild-type T34, although compared with this strain the transformants showed similar capability to colonize tomato roots. Gene expression patterns from aerial parts of 3-week-old tomato plants treated with T34 and the amdS transformants have also been investigated using GeneChip Tomato Genome Arrays. The downregulation of defense genes and the upregulation of carbon and nitrogen metabolism genes observed in the microarrays were accompanied by i enhanced growth, ii increased carbon and nitrogen levels and iii a

  6. Nitrogen Metabolism and Growth Enhancement in Tomato Plants Challenged with Trichoderma harzianum Expressing the Aspergillus nidulans Acetamidase amdS Gene.

    Science.gov (United States)

    Domínguez, Sara; Rubio, M Belén; Cardoza, Rosa E; Gutiérrez, Santiago; Nicolás, Carlos; Bettiol, Wagner; Hermosa, Rosa; Monte, Enrique

    2016-01-01

    Trichoderma is a fungal genus that includes species that are currently being used as biological control agents and/or as biofertilizers. In addition to the direct application of Trichoderma spp. as biocontrol agents in plant protection, recent studies have focused on the beneficial responses exerted on plants, stimulating the growth, activating the defenses, and/or improving nutrient uptake. The amdS gene, encoding an acetamidase of Aspergillus, has been used as a selectable marker for the transformation of filamentous fungi, including Trichoderma spp., but the physiological effects of the introduction of this gene into the genome of these microorganisms still remains unexplored. No evidence of amdS orthologous genes has been detected within the Trichoderma spp. genomes and the amdS heterologous expression in Trichoderma harzianum T34 did not affect the growth of this fungus in media lacking acetamide. However, it did confer the ability for the fungus to use this amide as a nitrogen source. Although a similar antagonistic behavior was observed for T34 and amdS transformants in dual cultures against Rhizoctonia solani, Botrytis cinerea, and Fusarium oxysporum, a significantly higher antifungal activity was detected in amdS transformants against F. oxysporum, compared to that of T34, in membrane assays on media lacking acetamide. In Trichoderma-tomato interaction assays, amdS transformants were able to promote plant growth to a greater extent than the wild-type T34, although compared with this strain the transformants showed similar capability to colonize tomato roots. Gene expression patterns from aerial parts of 3-week-old tomato plants treated with T34 and the amdS transformants have also been investigated using GeneChip Tomato Genome Arrays. The downregulation of defense genes and the upregulation of carbon and nitrogen metabolism genes observed in the microarrays were accompanied by (i) enhanced growth, (ii) increased carbon and nitrogen levels, and (iii) a

  7. Spirulina platensis Improves Mitochondrial Function Impaired by Elevated Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and Enhances Insulin Sensitivity in Equine Metabolic Syndrome (EMS) Horses.

    Science.gov (United States)

    Nawrocka, Daria; Kornicka, Katarzyna; Śmieszek, Agnieszka; Marycz, Krzysztof

    2017-08-03

    Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)-the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs' and IECs' morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome.

  8. Lactococcus lactis TrxD represents a subgroup of thioredoxins prevalent in Gram-positive bacteria containing WCXDC active site motifs

    DEFF Research Database (Denmark)

    Björnberg, Olof; Efler, Petr; Epie, Denis Ebong

    2014-01-01

    Three protein disulfide reductases of the thioredoxin superfamily from the industrially important Gram-positive Lactococcus lactis (LlTrxA, LlTrxD and LlNrdH) are compared to the "classical" thioredoxin from Escherichia coil (EcTrx1). LlTrxA resembles EcTrx1 with a WCGPC active site motif and other...... capacity to reduce insulin disulfides and their exposed active site thiol is alkylated at a similar rate at pH 7.0. LlTrxD on the other hand, is alkylated by iodoacetamide at almost 100 fold higher rate and shows no activity towards insulin disulfides. LlTrxA, LlTrxD and L1NrdH are all efficiently reduced...

  9. GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea: Possible role of the thioredoxin system as a functional backup for GSR.

    Directory of Open Access Journals (Sweden)

    Chul Han

    Full Text Available Glutathione reductase (GSR, a key member of the glutathione antioxidant defense system, converts oxidized glutathione (GSSG to reduced glutathione (GSH and maintains the intracellular glutathione redox state to protect the cells from oxidative damage. Previous reports have shown that Gsr deficiency results in defects in host defense against bacterial infection, while diquat induces renal injury in Gsr hypomorphic mice. In flies, overexpression of GSR extended lifespan under hyperoxia. In the current study, we investigated the roles of GSR in cochlear antioxidant defense using Gsr homozygous knockout mice that were backcrossed onto the CBA/CaJ mouse strain, a normal-hearing strain that does not carry a specific Cdh23 mutation that causes progressive hair cell degeneration and early onset of hearing loss. Gsr-/- mice displayed a significant decrease in GSR activity and GSH/GSSG ratios in the cytosol of the inner ears. However, Gsr deficiency did not affect ABR (auditory brainstem response hearing thresholds, wave I amplitudes or wave I latencies in young mice. No histological abnormalities were observed in the cochlea of Gsr-/- mice. Furthermore, there were no differences in the activities of cytosolic glutathione-related enzymes, including glutathione peroxidase and glutamate-cysteine ligase, or the levels of oxidative damage markers in the inner ears between WT and Gsr-/- mice. In contrast, Gsr deficiency resulted in increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears. Therefore, under normal physiological conditions, GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea. Given that the thioredoxin system is known to reduce GSSG to GSH in multiple species, our findings suggest that the thioredoxin system can support GSSG reduction in the mouse peripheral auditory system.

  10. The Anti-Apoptotic Properties of APEX1 in the Endothelium Require the First 20 Amino Acids and Converge on Thioredoxin-1.

    Science.gov (United States)

    Dyballa-Rukes, Nadine; Jakobs, Philipp; Eckers, Anna; Ale-Agha, Niloofar; Serbulea, Vlad; Aufenvenne, Karin; Zschauer, Tim-Christian; Rabanter, Lothar L; Jakob, Sascha; von Ameln, Florian; Eckermann, Olaf; Leitinger, Norbert; Goy, Christine; Altschmied, Joachim; Haendeler, Judith

    2017-04-20

    The APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has a disordered N-terminus, a redox, and a DNA repair domain. APEX1 has anti-apoptotic properties, which have been linked to both domains depending on cell type and experimental conditions. As protection against apoptosis is a hallmark of vessel integrity, we wanted to elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells and, if so, what the underlying mechanisms are. APEX1 inhibits apoptosis in endothelial cells by reducing Cathepsin D (CatD) cleavage, potentially by binding to the unprocessed form. Diminished CatD activation results in increased Thioredoxin-1 protein levels leading to reduced Caspase 3 activation. Consequently, apoptosis rates are decreased. This depends on the first twenty amino acids in APEX1, because APEX1 (21-318) induces CatD activity, decreases Thioredoxin-1 protein levels, and, thus, increases Caspase 3 activity and apoptosis. Along the same lines, APEX1 (1-20) inhibits Caspase 3 cleavage and apoptosis. Furthermore, re-expression of Thioredoxin-1 via lentiviral transduction rescues endothelial cells from APEX1 (21-318)-induced apoptosis. In an in vivo model of restenosis, which is characterized by oxidative stress, endothelial activation, and smooth muscle cell proliferation, Thioredoxin-1 protein levels are reduced in the endothelium of the carotids. APEX1 acts anti-apoptotic in endothelial cells. This anti-apoptotic effect depends on the first 20 amino acids of APEX1. As proper function of the endothelium during life span is a hallmark for individual health span, a detailed characterization of the functions of the APEX1N-terminus is required to understand all its cellular properties. Antioxid. Redox Signal. 26, 616-629.

  11. Intake of Hydrolyzed Casein is Associated with Reduced Body Fat Accretion and Enhanced Phase II Metabolism in Obesity Prone C57BL/6J Mice

    Science.gov (United States)

    Clausen, Morten Rahr; Zhang, Xumin; Yde, Christian C.; Ditlev, Ditte B.; Lillefosse, Haldis H.; Madsen, Lise; Kristiansen, Karsten; Liaset, Bjørn; Bertram, Hanne C.

    2015-01-01

    The amount and form of dietary casein have been shown to affect energy metabolism and lipid accumulation in mice, but the underlying mechanisms are not fully understood. We investigated 48 hrs urinary metabolome, hepatic lipid composition and gene expression in male C57BL/6J mice fed Western diets with 16 or 32 energy% protein in the form of extensively hydrolyzed or intact casein. LC-MS based metabolomics revealed a very strong impact of casein form on the urinary metabolome. Evaluation of the discriminatory metabolites using tandem mass spectrometry indicated that intake of extensively hydrolyzed casein modulated Phase II metabolism associated with an elevated urinary excretion of glucuronic acid- and sulphate conjugated molecules, whereas glycine conjugated molecules were more abundant in urine from mice fed the intact casein diets. Despite the differences in the urinary metabolome, we observed no differences in hepatic expression of genes involved in Phase II metabolism, but it was observed that expression of Abcc3 encoding ATP binding cassette c3 (transporter of glucuronic acid conjugates) was increased in livers of mice fed hydrolyzed casein. As glucuronic acid is derived from glucose and sulphate is derived from cysteine, our metabolomic data provided evidence for changes in carbohydrate and amino acid metabolism and we propose that this modulation of metabolism was associated with the reduced glucose and lipid levels observed in mice fed the extensively hydrolyzed casein diets. PMID:25738501

  12. Characterization of hemin-binding protein 35 (HBP35 in Porphyromonas gingivalis: its cellular distribution, thioredoxin activity and role in heme utilization

    Directory of Open Access Journals (Sweden)

    Abiko Yoshimitsu

    2010-05-01

    Full Text Available Abstract Background The periodontal pathogen Porphyromonas gingivalis is an obligate anaerobe that requires heme for growth. To understand its heme acquisition mechanism, we focused on a hemin-binding protein (HBP35 protein, possessing one thioredoxin-like motif and a conserved C-terminal domain, which are proposed to be involved in redox regulation and cell surface attachment, respectively. Results We observed that the hbp35 gene was transcribed as a 1.1-kb mRNA with subsequent translation resulting in three proteins with molecular masses of 40, 29 and 27 kDa in the cytoplasm, and one modified form of the 40-kDa protein on the cell surface. A recombinant 40-kDa HBP35 exhibited thioredoxin activity in vitro and mutation of the two putative active site cysteine residues abolished this activity. Both recombinant 40- and 27-kDa proteins had the ability to bind hemin, and growth of an hbp35 deletion mutant was substantially retarded under hemin-depleted conditions compared with growth of the wild type under the same conditions. Conclusion P. gingivalis HBP35 exhibits thioredoxin and hemin-binding activities and is essential for growth in hemin-depleted conditions suggesting that the protein plays a significant role in hemin acquisition.

  13. Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein.

    Science.gov (United States)

    Shaikhali, Jehad; Davoine, Céline; Brännström, Kristoffer; Rouhier, Nicolas; Bygdell, Joakim; Björklund, Stefan; Wingsle, Gunnar

    2015-06-15

    The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combination with specific TFs as GeBPL. © The Authors Journal compilation © 2015 Biochemical Society.

  14. Metabolic Syndrome

    Science.gov (United States)

    Metabolic syndrome is a group of conditions that put you at risk for heart disease and diabetes. These conditions ... agree on the definition or cause of metabolic syndrome. The cause might be insulin resistance. Insulin is ...

  15. Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP+-dependent dehydrogenases of the pentose phosphate pathway

    International Nuclear Information System (INIS)

    Rodrigues, Juan; Branco, Vasco; Lu, Jun; Holmgren, Arne; Carvalho, Cristina

    2015-01-01

    Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP + -dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI 50 : 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg 2+ > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite alleviates the

  16. Toxicological effects of thiomersal and ethylmercury: Inhibition of the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway

    Energy Technology Data Exchange (ETDEWEB)

    Rodrigues, Juan, E-mail: juanricardorodrigues@gmail.com [Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (Portugal); Laboratory of Biochemistry, Faculty of Pharmacy, Central University of Venezuela (Venezuela, Bolivarian Republic of); Branco, Vasco [Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (Portugal); Lu, Jun; Holmgren, Arne [Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet (Sweden); Carvalho, Cristina, E-mail: cristina.carvalho@ff.ulisboa.pt [Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa (Portugal)

    2015-08-01

    Mercury (Hg) is a strong toxicant affecting mainly the central nervous, renal, cardiovascular and immune systems. Thiomersal (TM) is still in use in medical practice as a topical antiseptic and as a preservative in multiple dose vaccines, routinely given to young children in some developing countries, while other forms of mercury such as methylmercury represent an environmental and food hazard. The aim of the present study was to determine the effects of thiomersal (TM) and its breakdown product ethylmercury (EtHg) on the thioredoxin system and NADP{sup +}-dependent dehydrogenases of the pentose phosphate pathway. Results show that TM and EtHg inhibited the thioredoxin system enzymes in purified suspensions, being EtHg comparable to methylmercury (MeHg). Also, treatment of neuroblastoma and liver cells with TM or EtHg decreased cell viability (GI{sub 50}: 1.5 to 20 μM) and caused a significant (p < 0.05) decrease in the overall activities of thioredoxin (Trx) and thioredoxin reductase (TrxR) in a concentration- and time-dependent manner in cell lysates. Compared to control, the activities of Trx and TrxR in neuroblastoma cells after EtHg incubation were reduced up to 60% and 80% respectively, whereas in hepatoma cells the reduction was almost 100%. In addition, the activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also significantly inhibited by all mercurials, with inhibition intensity of Hg{sup 2+} > MeHg ≈ EtHg > TM (p < 0.05). Cell incubation with sodium selenite alleviated the inhibitory effects on TrxR and glucose-6-phosphate dehydrogenase. Thus, the molecular mechanism of toxicity of TM and especially of its metabolite EtHg encompasses the blockage of the electrons from NADPH via the thioredoxin system. - Highlights: • TM and EtHg inhibit Trx and TrxR both in purified suspensions and cell lysates. • TM and EtHg also inhibit the activities of G6PDH and 6PGDH in cell lysates, • Co-exposure to selenite alleviates

  17. Tumor Metabolism of Malignant Gliomas

    Energy Technology Data Exchange