Science.gov

Sample records for 7-dehydrocholesterol reductase gene

  1. Rapid suppression of 7-dehydrocholesterol reductase activity in keratinocytes by vitamin D.

    PubMed

    Zou, Ling; Porter, Todd D

    2015-04-01

    7-Dehydrocholesterol (7DHC) serves as the sterol substrate for both cholesterol and vitamin D3 (cholecalciferol) synthesis. The pivotal enzyme in these two pathways is 7-dehydrocholesterol reductase (DHCR7), which converts 7DHC to cholesterol. Treatment of adult human epidermal keratinocytes (HEKa) with 10μM cholecalciferol resulted in a rapid decrease in DHCR7 activity (19% of control activity at 2h). This loss of activity was observed only in HEKa cells, a primary cell line cultured from normal human skin, and not in an immortalized skin cell line (HaCaT cells) nor in two hepatoma cell lines. The decrease in DHCR7 activity was not due to direct inhibition or to dephosphorylation of the enzyme, and enzyme protein levels were not decreased. 25-Hydroxyvitamin D3 had a lesser effect on DHCR7 activity, while 1α,25-dihydroxyvitamin D3 had no effect on DHCR7, indicating that the vitamin D receptor is not involved. Treatment with cholecalciferol did not lead to the accumulation of 7-dehydrocholesterol, and a 50% decrease in lanosterol synthesis in these cells suggests that cholecalciferol down-regulates the entire cholesterolgenic pathway. As vitamin D has been reported to be an inhibitor of hedgehog (Hh) signaling through Smo, we tested the effect of cyclopamine, an established inhibitor of the Hh pathway, on DHCR7 activity. Cyclopamine (10μM) also rapidly decreased DHCR7 activity (50% of control activity at 3h), suggesting that vitamin D3 may modulate DHCR7 activity and cholesterol/vitamin D3 synthesis by inhibiting hedgehog signaling. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

  2. Investigation of 7-dehydrocholesterol reductase pathway to elucidate off-target prenatal effects of pharmaceuticals: a systematic review

    PubMed Central

    Boland, M R; Tatonetti, N P

    2016-01-01

    Mendelian diseases contain important biological information regarding developmental effects of gene mutations that can guide drug discovery and toxicity efforts. In this review, we focus on Smith–Lemli–Opitz syndrome (SLOS), a rare Mendelian disease characterized by compound heterozygous mutations in 7-dehydrocholesterol reductase (DHCR7) resulting in severe fetal deformities. We present a compilation of SLOS-inducing DHCR7 mutations and the geographic distribution of those mutations in healthy and diseased populations. We observed that several mutations thought to be disease causing occur in healthy populations, indicating an incomplete understanding of the condition and highlighting new research opportunities. We describe the functional environment around DHCR7, including pharmacological DHCR7 inhibitors and cholesterol and vitamin D synthesis. Using PubMed, we investigated the fetal outcomes following prenatal exposure to DHCR7 modulators. First-trimester exposure to DHCR7 inhibitors resulted in outcomes similar to those of known teratogens (50 vs 48% born-healthy). DHCR7 activity should be considered during drug development and prenatal toxicity assessment. PMID:27401223

  3. UV-activated 7-dehydrocholesterol-coated titanium implants promote differentiation of human umbilical cord mesenchymal stem cells into osteoblasts.

    PubMed

    Satué, María; Ramis, Joana M; Monjo, Marta

    2016-01-01

    Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23℃. Further, we investigated in vitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants.

  4. Aripiprazole and trazodone cause elevations of 7-dehydrocholesterol in the absence of Smith-Lemli-Opitz Syndrome.

    PubMed

    Hall, Patricia; Michels, Virginia; Gavrilov, Dimitar; Matern, Dietrich; Oglesbee, Devin; Raymond, Kimiyo; Rinaldo, Piero; Tortorelli, Silvia

    2013-01-01

    Screening for Smith-Lemli-Opitz Syndrome (SLOS) using elevated 7-dehydrocholesterol (7DHC) as a marker is sensitive, but not always specific. Elevations of 7DHC can be seen in patients who do not have a defect in 7-dehydrocholesterol reductase. These results have often been attributed to medication artifacts, but specific causes have not been well reported. We examined the medical records of patients with elevated 7DHC to determine if they had been diagnosed with SLOS; and if they had not, to identify any common medications that may have caused the elevations. We found three individuals who were affected with SLOS, and 22 with elevated 7DHC in the absence of SLOS. Seven of these individuals underwent molecular testing which showed no mutations, while the other 15 were excluded based on clinical findings and other testing. The medication history of these individuals revealed aripiprazole and trazodone as common medications to all the false positive results.

  5. A novel pathway for sequential transformation of 7-dehydrocholesterol and expression of the P450scc system in mammalian skin.

    PubMed

    Slominski, Andrzej; Zjawiony, Jordan; Wortsman, Jacobo; Semak, Igor; Stewart, Jeremy; Pisarchik, Alexander; Sweatman, Trevor; Marcos, Josep; Dunbar, Chuck; C Tuckey, Robert

    2004-11-01

    Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.

  6. A novel pathway for sequential transformation of 7-dehydrocholesterol and expression of the P450scc system in mammalian skin

    PubMed Central

    Slominski, Andrzej; Zjawiony, Jordan; Wortsman, Jacobo; Semak, Igor; Stewart, Jeremy; Pisarchik, Alexander; Sweatman, Trevor; Marcos, Josep; Dunbar, Chuck; Tuckey, Robert C.

    2005-01-01

    Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB. PMID:15511223

  7. A highly sensitive method for analysis of 7-dehydrocholesterol for the study of Smith-Lemli-Opitz syndrome.

    PubMed

    Liu, Wei; Xu, Libin; Lamberson, Connor; Haas, Dorothea; Korade, Zeljka; Porter, Ned A

    2014-02-01

    We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD "ene" reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis.

  8. A highly sensitive method for analysis of 7-dehydrocholesterol for the study of Smith-Lemli-Opitz syndrome[S

    PubMed Central

    Liu, Wei; Xu, Libin; Lamberson, Connor; Haas, Dorothea; Korade, Zeljka; Porter, Ned A.

    2014-01-01

    We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD “ene” reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis. PMID:24259532

  9. A colorimetric assay for 7-dehydrocholesterol with potential application to screening for Smith-Lemli-Opitz syndrome.

    PubMed

    Xiong, Quanbo; Ruan, Benfang; Whitby, Frank G; Tuohy, Richard P; Belanger, Thomas L; Kelley, Richard I; Wilson, William K; Schroepfer, George J

    2002-05-01

    Smith-Lemli-Opitz syndrome (SLOS; MIM 270400) is a genetic disorder characterized by hypocholesterolemia and elevated 7-dehydrocholesterol (7DHC) levels resulting from mutations affecting 7-dehydrocholesterol reductase. We describe a colorimetric assay for 7DHC with potential application to large-scale screening for SLOS. Reaction of 7DHC and its esters with the Liebermann-Burchard reagent resulted in a brief initial absorbance at 510 nm (pink color) followed by an absorbance at 620 nm (blue color) after 2 min, while cholesterol samples were essentially colorless. The assay could identify typical SLOS blood samples by their pink color and increased absorbance at 620 nm after 2 min. Colorimetric identification of mild SLOS cases requires monitoring of the transient absorbance at 510 nm, which must be detected immediately after rapid, consistent mixing of the reagents. The need for special mixing devices and rigorous validation precludes sporadic use of the assay for diagnosing suspected SLOS cases. We also studied the stability of 7DHC in dried SLOS blood spots on Guthrie cards, which are widely used for archiving neonatal blood. Decomposition of 7DHC was effectively retarded by storage at low temperature and by precoating of the cards with antioxidants. The combined results provide a foundation for development of a simple, automated test for SLOS screening.

  10. Expression of human CYP27A1 in B. megaterium for the efficient hydroxylation of cholesterol, vitamin D3 and 7-dehydrocholesterol.

    PubMed

    Ehrhardt, Maximilian; Gerber, Adrian; Hannemann, Frank; Bernhardt, Rita

    2016-01-20

    In the current work the ability of Bacillus megaterium to take up hydrophobic substrates and efficiently express eukaryotic membrane proteins was utilized for establishing a CYP27A1-based biocatalyst. The human mitochondrial cytochrome P450CYP27A1 was co-expressed with its redox partners adrenodoxin reductase (Adr) and adrenodoxin (Adx). CYP27A1 could be localized at the cell's polyhydroxybutyrate (PHB) granules, carbon storage serving organelle-like vesicles that can take up cholesterol, resulting in bioreactor-like structures in B. megaterium . The resulting whole cell system allowed the efficient biotechnological conversion of the CYP27A1 substrates cholesterol, 7-dehydrocholesterol (7-DHC) and vitamin D3. After 48 h, nearly 100% of cholesterol was metabolized producing a final concentration of 113.14 mg/l 27-hydroxycholesterol (27-HC). Moreover, 70% of vitamin D3 was converted into 25-hydroxyvitamin D3 (25-OH-D3) with a final concentration of 80.81 mg/l. Also more than 97% of 7-DHC were found to be metabolized into two products, corresponding to 26/27-hydroxy-7-dehydrocholesterol (P1) and 25-hydroxy-7-dehydrocholesterol (P2). To our knowledge this is the first CYP27A1-based whole-cell system, allowing the efficient and low-cost production of pharmaceutically interesting metabolites of this enzyme from relatively cheap substrates.

  11. Increasing cholesterol synthesis in 7-dehydrosterol reductase (DHCR7) deficient mouse models through gene transfer

    PubMed Central

    Matabosch, Xavier; Ying, Lee; Serra, Montserrat; Wassif, Christopher A.; Porter, Forbes D.; Shackleton, Cedric; Watson, Gordon

    2010-01-01

    Smith-Lemli-Opitz syndrome (SLOS) is caused by deficiency in the terminal step of cholesterol biosynthesis: the conversion of 7-dehydrocholesterol (7DHC) to cholesterol (C), catalyzed by 7-dehydrocholesterol reductase (DHCR7). This disorder exhibits several phenotypic traits including dysmorphia and mental retardation with a broad range of severity. There are few proven treatment options. That most commonly used is a high cholesterol diet that seems to enhance the quality of life and improve behavioral characteristics of patients, although these positive effects are controversial. The goal of our study was to investigate the possibility of restoring DHCR7 activity by gene transfer. We constructed an adeno-associated virus (AAV) vector containing the DHCR7 gene. After we infused this vector into affected mice, the introduced DHCR7 gene could be identified in liver, mRNA was expressed and a functional enzyme was produced. Evidence of functionality came from the ability to partially normalize the serum ratio of 7DHC/C in treated animals, apparently by increasing cholesterol production with concomitant decrease in 7DHC precursor. By five weeks after treatment the mean ratio (for 7 animals) had fallen to 0.05 while the ratio for untreated littermate controls had risen to 0.14. This provides proof of principle that gene transfer can ameliorate the genetic defect causing SLOS and provides a new experimental tool for studying the pathogenesis of this disease. If effective in humans, it might also offer a possible alternative to exogenous cholesterol therapy. However, it would not offer a complete cure for the disorder as many of the negative implications of defective synthesis are already established during prenatal development. PMID:20800683

  12. Synthesis of [3 alpha-3H]7-dehydrocholesterol via stable tritiated 4-phenyl-1,2,4-triazoline-3,5-dione derivative.

    PubMed

    Batta, A K; Salen, G; Tint, G S; Honda, A; Shefer, S

    1997-11-01

    Synthesis of [3 alpha-3H]7-dehydrocholesterol is described via protection of the 5,7-diene system in 7-dehydrocholesterol as the Diels-Alder adduct with 4-phenyl-1,2,4-triazoline-3,5-dione followed by oxidation of the hydroxyl group to give the 3-oxo adduct. Reduction of the keto adduct with [3H]sodium borohydride produced the adduct of [3 alpha-3H]7-dehydrocholesterol from which the radiolabeled sterol was obtained via treatment with lithium aluminum hydride. The advantage of the method is that highly labeled [3 alpha-3H]7-dehydrocholesterol can be prepared. Further, unlike 7-dehydrocholesterol, its adduct with 4-phenyl-1,2,4-triazoline-3,5-dione is stable and can be stored. This allows the preparation of small batches of [3 alpha-3H]7-dehydrocholesterol for immediate use in biological experiments, and losses due to decomposition of excess radiolabeled 7-dehydrocholesterol are minimized.

  13. The effects of 7-dehydrocholesterol on the structural properties of membranes

    NASA Astrophysics Data System (ADS)

    Liu, Yingzhe; Chipot, Christophe; Shao, Xueguang; Cai, Wensheng

    2011-10-01

    Smith-Lemli-Opitz syndrome, a congenital and developmental malformation disease, is typified by abnormal accumulation of 7-dehydrocholesterol (7DHC), the immediate precursor of cholesterol (CHOL), and depletion thereof. Knowledge of the effect of 7DHC on the biological membrane is, however, still fragmentary. In this study, large-scale atomistic molecular dynamics simulations, employing two distinct force fields, have been conducted to elucidate differences in the structural properties of a hydrated dimyristoylphosphatidylcholine bilayer due to CHOL and 7DHC. The present series of results indicate that CHOL and 7DHC possess virtually the same ability to condense and order membranes. Furthermore, the condensing and ordering effects are shown to be strengthened at increasing sterol concentrations.

  14. 7-Dehydrocholesterol metabolites produced by sterol 27-hydroxylase (CYP27A1) modulate liver X receptor activity.

    PubMed

    Endo-Umeda, Kaori; Yasuda, Kaori; Sugita, Kazuyuki; Honda, Akira; Ohta, Miho; Ishikawa, Minoru; Hashimoto, Yuichi; Sakaki, Toshiyuki; Makishima, Makoto

    2014-03-01

    7-Dehydrocholesterol (7-DHC) is a common precursor of vitamin D3 and cholesterol. Although various oxysterols, oxygenated cholesterol derivatives, have been implicated in cellular signaling pathways, 7-DHC metabolism and potential functions of its metabolites remain poorly understood. We examined 7-DHC metabolism by various P450 enzymes and detected three metabolites produced by sterol 27-hydroxylase (CYP27A1) using high-performance liquid chromatography. Two were further identified as 25-hydroxy-7-DHC and 26/27-hydroxy-7-DHC. These 7-DHC metabolites were detected in serum of a patient with Smith-Lemli-Opitz syndrome. Luciferase reporter assays showed that 25-hydroxy-7-DHC activates liver X receptor (LXR) α, LXRβ and vitamin D receptor and that 26/27-hydroxy-7-DHC induces activation of LXRα and LXRβ, although the activities of both compounds on LXRs were weak. In a mammalian two-hybrid assay, 25-hydroxy-7-DHC and 26/27-hydroxy-7-DHC induced interaction between LXRα and a coactivator fragment less efficiently than a natural LXR agonist, 22(R)-hydroxycholesterol. These 7-DHC metabolites did not oppose agonist-induced LXR activation and interacted directly to LXRα in a manner distinct from a potent agonist. These findings indicate that the 7-DHC metabolites are partial LXR activators. Interestingly, 25-hydroxy-7-DHC and 26/27-hydroxy-7-DHC suppressed mRNA expression of sterol regulatory element-binding protein 1c, an LXR target gene, in HepG2 cells and HaCaT cells, while they weakly increased mRNA levels of ATP-binding cassette transporter A1, another LXR target, in HaCaT cells. Thus, 7-DHC is catabolized by CYP27A1 to metabolites that act as selective LXR modulators.

  15. Mechanism and stereoselectivity of biologically important oxygenation reactions of the 7-dehydrocholesterol radical.

    PubMed

    Rajeev, Ramanan; Sunoj, Raghavan B

    2013-07-19

    The mechanism of free radical oxygenation of 7-dehydrocholesterol (7-DHC), one of the biologically important sterols, is investigated by using density functional theory. The energetic origin of the product distribution and the stereoelectronic factors involved in various mechanistic pathways are delineated. The addition of triplet molecular oxygen to two types of conjugatively stabilized radicals, generated by the removal of the reactive allylic hydrogens from C9 or C14 positions, respectively denoted as H9 and H14 pathways, is studied. The distortion-interaction analysis of the C-O bond formation transition states suggests that the energetic preference toward the α prochiral face stems from reduced skeletal distortions of the cholesterol backbone as compared to that in the corresponding β prochiral face. This insight derived through a detailed quantitative analysis of the stereocontrolling transition states suggests that the commonly found interpretations solely based on steric interactions between the incoming oxygen and the protruding angular methyl groups (C10, C13 methyls) in the β face calls for adequate refinement. The relative energies of the transition states for molecular oxygen addition to C9, C5, and C14 (where spin densities are higher) and the ensuing products thereof are in agreement with the experimentally reported distribution of oxygenated 7-DHCs.

  16. Oxysterols from Free Radical Chain Oxidation of 7-Dehydrocholesterol: Product and Mechanistic Studies

    PubMed Central

    Xu, Libin; Korade, Zeljka; Porter, Ned A.

    2010-01-01

    Free radical chain oxidation of highly oxidizable 7-dehydrocholesterol (7-DHC) initiated by 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile) was carried out at 37°C in benzene for 24 hours. Fifteen oxysterols derived from 7-DHC were isolated and characterized with 1D- and 2D-NMR spectroscopy and mass spectrometry. A mechanism that involves abstraction of hydrogen atoms at C-9 and/or C-14 is proposed to account for the formation of all of the oxysterols and the reaction progress profile. In either the H-9 or H-14 mechanism, a pentadienyl radical intermediate is formed after abstraction of H-9 or H-14 by a peroxyl radical. This step is followed by the well-precedented transformations observed in peroxidation reactions of polyunsaturated fatty acids such as oxygen addition, peroxyl radical 5-exo cyclization, and SHi carbon radical attack on the peroxide bond. The mechanism for peroxidation of 7-DHC also accounts for the formation of numerous oxysterol natural products isolated from fungal species, marine sponges, and cactaceous species. In a cell viability test, the oxysterol mixture from 7-DHC peroxidation was found to be cytotoxic to Neuro2a neuroblastoma cells in the micromolar concentration range. We propose that the high reactivity of 7-DHC and the oxysterols generated from its peroxidation may play important roles in the pathogenesis of Smith-Lemli-Opitz syndrome (SLOS), X-linked dominant chondrodysplasia punctata (CDPX2), and cerebrotendinous xanthomatosis (CTX), all of these being metabolic disorders having an elevated level of 7-DHC. PMID:20121089

  17. 7-Dehydrocholesterol (7-DHC), But Not Cholesterol, Causes Suppression of Canonical TGF-β Signaling and Is Likely Involved in the Development of Atherosclerotic Cardiovascular Disease (ASCVD).

    PubMed

    Huang, Shuan Shian; Liu, I-Hua; Chen, Chun-Lin; Chang, Jia-Ming; Johnson, Frank E; Huang, Jung San

    2016-11-16

    For several decades, cholesterol has been thought to cause ASCVD. Limiting dietary cholesterol intake has been recommended to reduce the risk of the disease. However, several recent epidemiological studies do not support a relationship between dietary cholesterol and/or blood cholesterol and ASCVD. Consequently, the role of cholesterol in atherogenesis is now uncertain. Much evidence indicates that TGF-β, an anti-inflammatory cytokine, protects against ASCVD and that suppression of canonical TGF-β signaling (Smad2-dependent) is involved in atherogenesis. We had hypothesized that cholesterol causes ASCVD by suppressing canonical TGF-β signaling in vascular endothelium. To test this hypothesis, we determine the effects of cholesterol, 7-dehydrocholesterol (7-DHC; the biosynthetic precursor of cholesterol), and other sterols on canonical TGF-β signaling. We use Mv1Lu cells (a model cell system for studying TGF-β activity) stably expressing the Smad2-dependent luciferase reporter gene. We demonstrate that 7-DHC (but not cholesterol or other sterols) effectively suppresses the TGF-β-stimulated luciferase activity. We also demonstrate that 7-DHC suppresses TGF-β-stimulated luciferase activity by promoting lipid raft/caveolae formation and subsequently recruiting cell-surface TGF-β receptors from non-lipid raft microdomains to lipid rafts/caveolae where TGF-β receptors become inactive in transducing canonical signaling and undergo rapid degradation upon TGF-β binding. We determine this by cell-surface (125) I-TGF-β-cross-linking and sucrose density gradient ultracentrifugation. We further demonstrate that methyl-β-cyclodextrin (MβCD), a sterol-chelating agent, reverses 7-DHC-induced suppression of TGF-β-stimulated luciferase activity by extrusion of 7-DHC from resident lipid rafts/caveolae. These results suggest that 7-DHC, but not cholesterol, promotes lipid raft/caveolae formation, leading to suppression of canonical TGF-β signaling and atherogenesis. J

  18. Profiling and Imaging Ion Mobility-Mass Spectrometry Analysis of Cholesterol and 7-Dehydrocholesterol in Cells Via Sputtered Silver MALDI

    NASA Astrophysics Data System (ADS)

    Xu, Libin; Kliman, Michal; Forsythe, Jay G.; Korade, Zeljka; Hmelo, Anthony B.; Porter, Ned A.; McLean, John A.

    2015-06-01

    Profiling and imaging of cholesterol and its precursors by mass spectrometry (MS) are important in a number of cholesterol biosynthesis disorders, such as in Smith-Lemli-Opitz syndrome (SLOS), where 7-dehydrocholesterol (7-DHC) is accumulated in affected individuals. SLOS is caused by defects in the enzyme that reduces 7-DHC to cholesterol. However, analysis of sterols is challenging because these hydrophobic olefins are difficult to ionize for MS detection. We report here sputtered silver matrix-assisted laser desorption/ionization (MALDI)-ion mobility-MS (IM-MS) analysis of cholesterol and 7-DHC. In comparison with liquid-based AgNO3 and colloidal Ag nanoparticle (AgNP), sputtered silver NP (10-25 nm) provided the lowest limits-of-detection based on the silver coordinated [cholesterol + Ag]+ and [7-DHC + Ag]+ signals while minimizing dehydrogenation products ([M + Ag-2H]+). When analyzing human fibroblasts that were directly grown on poly-L-lysine-coated ITO glass plates with this technique, in situ, the 7-DHC/cholesterol ratios for both control and SLOS human fibroblasts are readily obtained. The m/z of 491 (specific for [7-DHC + 107Ag]+) and 495 (specific for [cholesterol + 109Ag]+) were subsequently imaged using MALDI-IM-MS. MS images were co-registered with optical images of the cells for metabolic ratio determination. From these comparisons, ratios of 7-DHC/cholesterol for SLOS human fibroblasts are distinctly higher than in control human fibroblasts. Thus, this strategy demonstrates the utility for diagnosing/assaying the severity of cholesterol biosynthesis disorders in vitro.

  19. Isolation, sequence identification and tissue expression profile of two novel soybean (glycine max) genes-vestitone reductase and chalcone reductase.

    PubMed

    Liu, G Y

    2009-09-01

    The complete mRNA sequences of two soybean (glycine max) genes-vestitone reductase and chalcone reductase, were amplified using the rapid amplification of cDNA ends methods. The sequence analysis of these two genes revealed that soybean vestitone reductase gene encodes a protein of 327 amino acids which has high homology with the vestitone reductase of Medicago sativa (77%). The soybean chalcone reductase gene encodes a protein of 314 amino acids that has high homology with the chalcone reductase of kudzu vine (88%) and medicago sativa (83%). The expression profiles of the soybean vestitone reductase and chalcone reductase genes were studied and the results indicated that these two soybean genes were differentially expressed in detected soybean tissues including leaves, stems, roots, inflorescences, embryos and endosperm. Our experiment established the foundation for further research on these two soybean genes.

  20. Dissimilatory Nitrite Reductase Genes from Autotrophic Ammonia-Oxidizing Bacteria

    PubMed Central

    Casciotti, Karen L.; Ward, Bess B.

    2001-01-01

    The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of β-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene from Nitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA and amoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria. PMID:11319103

  1. Differential Cytotoxic Effects of 7-Dehydrocholesterol-derived Oxysterols on Cultured Retina-derived Cells: Dependence on Sterol Structure, Cell Type, and Density

    PubMed Central

    Pfeffer, Bruce A.; Xu, Libin; Porter, Ned A.; Rao, Sriganesh Ramachandra; Fliesler, Steven J.

    2016-01-01

    Tissue accumulation of 7-dehydrocholesterol (7DHC) is a hallmark of Smith-Lemli-Opitz Syndrome (SLOS), a human inborn error of the cholesterol (CHOL) synthesis pathway. Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS, which exhibits a retinal degeneration characterized by selective loss of photoreceptors and associated functional deficits, Müller cell hypertrophy, and engorgement of the retinal pigment epithelium (RPE) with phagocytic inclusions. We evaluated the relative effects of four 7DHC-derived oxysterols on three retina-derived cell types in culture, with respect to changes in cellular morphology and viability. 661W (photoreceptor-derived) cells, rMC-1 (Müller glia-derived) cells, and normal diploid monkey RPE (mRPE) cells were incubated for 24 h with dose ranges of either 7-ketocholesterol (7kCHOL), 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD), 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), or 4β-hydroxy-7-dehydrocholesterol (4HDHC); CHOL served as a negative control (same dose range), along with appropriate vehicle controls, while staurosporine (Stsp) was used as a positive cytotoxic control. For 661W cells, the rank order of oxysterol potency was: EPCD > 7kCHOL >> DHCEO > 4HDHC ≈ CHOL. EC50 values were higher for confluent vs. subconfluent cultures. 661W cells exhibited much higher sensitivity to EPCD and 7kCHOL than either rMC-1 or mRPE cells, with the latter being the most robust when challenged, either at confluence or in sub-confluent cultures. When tested on rMC-1 and mRPE cells, EPCD was again an order of magnitude more potent than 7kCHOL in compromising cellular viability. Hence, 7DHC-derived oxysterols elicit differential cytotoxicity that is dose-, cell type-, and cell density-dependent. These results are consistent with the observed progressive, photoreceptor-specific retinal degeneration in the rat SLOS model, and support the hypothesis that 7DHC-derived oxysterols are causally linked to that

  2. Differential cytotoxic effects of 7-dehydrocholesterol-derived oxysterols on cultured retina-derived cells: Dependence on sterol structure, cell type, and density.

    PubMed

    Pfeffer, Bruce A; Xu, Libin; Porter, Ned A; Rao, Sriganesh Ramachandra; Fliesler, Steven J

    2016-04-01

    Tissue accumulation of 7-dehydrocholesterol (7DHC) is a hallmark of Smith-Lemli-Opitz Syndrome (SLOS), a human inborn error of the cholesterol (CHOL) synthesis pathway. Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS, which exhibits a retinal degeneration characterized by selective loss of photoreceptors and associated functional deficits, Müller cell hypertrophy, and engorgement of the retinal pigment epithelium (RPE) with phagocytic inclusions. We evaluated the relative effects of four 7DHC-derived oxysterols on three retina-derived cell types in culture, with respect to changes in cellular morphology and viability. 661W (photoreceptor-derived) cells, rMC-1 (Müller glia-derived) cells, and normal diploid monkey RPE (mRPE) cells were incubated for 24 h with dose ranges of either 7-ketocholesterol (7kCHOL), 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD), 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), or 4β-hydroxy-7-dehydrocholesterol (4HDHC); CHOL served as a negative control (same dose range), along with appropriate vehicle controls, while staurosporine (Stsp) was used as a positive cytotoxic control. For 661W cells, the rank order of oxysterol potency was: EPCD > 7kCHOL > DHCEO > 4HDHC ≈ CHOL. EC50 values were higher for confluent vs. subconfluent cultures. 661W cells exhibited much higher sensitivity to EPCD and 7kCHOL than either rMC-1 or mRPE cells, with the latter being the most robust when challenged, either at confluence or in sub-confluent cultures. When tested on rMC-1 and mRPE cells, EPCD was again an order of magnitude more potent than 7kCHOL in compromising cellular viability. Hence, 7DHC-derived oxysterols elicit differential cytotoxicity that is dose-, cell type-, and cell density-dependent. These results are consistent with the observed progressive, photoreceptor-specific retinal degeneration in the rat SLOS model, and support the hypothesis that 7DHC-derived oxysterols are causally linked to that

  3. A routine method for cholesterol and 7-dehydrocholesterol analysis in dried blood spot by GC-FID to diagnose the Smith-Lemli-Opitz syndrome.

    PubMed

    Gelzo, Monica; Clericuzio, Stefano; Barone, Rosalba; D'Apolito, Oceania; Dello Russo, Antonio; Corso, Gaetano

    2012-10-15

    This work was aimed to implement a fast and simple method to quantify cholesterol (CHOL) and 7-dehydrocholesterol (7-DHC) in dried blood spot (DBS) to diagnose the Smith-Lemli-Opitz syndrome (SLOS), an inborn error of CHOL biosynthesis. We developed and validated a GC-FID method for separation and quantification of underivatized CHOL and 7-DHC using a DBS disc of 6mm with a run time of 9 min. Correlation coefficients (r) of calibration curves ranged from 0.998 to 0.999 for CHOL and from 0.997 to 0.998 for 7-DHC. Within-day and between-day imprecision (CV%), accuracy (%), carry-over, and extraction efficacy (%) were also evaluated for validation. CHOL and 7-DHC were analyzed in DBS and plasma samples from 8 SLOS patients and 30 unaffected subjects. In SLOS patients, 7-DHC/CHOL ratios in DBS and plasma samples ranged from 0.035 to 1.448 and from 0.012 to 0.926, respectively. Results from calibration curves, quality controls and patient samples reveal that the method is suitable to analyze DBS to screen patients affected by SLOS.

  4. Induction of a massive endoplasmic reticulum and perinuclear space expansion by expression of lamin B receptor mutants and the related sterol reductases TM7SF2 and DHCR7.

    PubMed

    Zwerger, Monika; Kolb, Thorsten; Richter, Karsten; Karakesisoglou, Iakowos; Herrmann, Harald

    2010-01-15

    Lamin B receptor (LBR) is an inner nuclear membrane protein involved in tethering the nuclear lamina and the underlying chromatin to the nuclear envelope. In addition, LBR exhibits sterol reductase activity. Mutations in the LBR gene cause two different human diseases: Pelger-Huët anomaly and Greenberg skeletal dysplasia, a severe chrondrodystrophy causing embryonic death. Our study aimed at investigating the effect of five LBR disease mutants on human cultured cells. Three of the tested LBR mutants caused a massive compaction of chromatin coincidental with the formation of a large nucleus-associated vacuole (NAV) in several human cultured cell lines. Live cell imaging and electron microscopy revealed that this structure was generated by the separation of the inner and outer nuclear membrane. During NAV formation, nuclear pore complexes and components of the linker of nucleoskeleton and cytoskeleton complex were lost in areas of membrane separation. Concomitantly, a large number of smaller vacuoles formed throughout the cytoplasm. Notably, forced expression of the two structurally related sterol reductases transmembrane 7 superfamily member 2 and 7-dehydrocholesterol reductase caused, even in their wild-type form, a comparable phenotype in susceptible cell lines. Hence, LBR mutant variants and sterol reductases can severely interfere with the regular organization of the nuclear envelope and the endoplasmic reticulum.

  5. Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2.

    PubMed Central

    Wilkinson, J Q; Crawford, N M

    1991-01-01

    Chlorate, the chlorine analog of nitrate, is a herbicide that has been used to select mutants impaired in the process of nitrate assimilation. In Arabidopsis thaliana, mutations at any one of eight distinct loci confer resistance to chlorate. The molecular identities of the genes at these loci are not known; however, one of these loci--chl3--maps very near the nitrate reductase structural gene NIA2. Through the isolation, characterization, and genetic analysis of new chlorate-resistant mutants generated by gamma irradiation, we have been able to demonstrate that the CHL3 gene and the NIA2 gene are identical. Three new chlorate-resistant mutants were identified that had deletions of the entire NIA2 gene. These nia2 null mutants were viable and still retained 10% of wild-type nitrate reductase activity in the leaves of the plants. All three deletion mutations were found to be new alleles of chl3. Introduction of the NIA2 gene back into these chl3 mutants by Agrobacterium-mediated transformation partially complemented their mutant phenotype. From these data, we conclude that Arabidopsis has at least two functional nitrate reductase genes and that the NIA2 gene product accounts for the majority of the leaf nitrate reductase activity and chlorate sensitivity of Arabidopsis plants. PMID:1840922

  6. Sequential metabolism of 7-dehydrocholesterol to steroidal 5,7-dienes in adrenal glands and its biological implication in the skin.

    PubMed

    Slominski, Andrzej T; Zmijewski, Michal A; Semak, Igor; Sweatman, Trevor; Janjetovic, Zorica; Li, Wei; Zjawiony, Jordan K; Tuckey, Robert C

    2009-01-01

    Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3betaHSD for 7DHP (V(m)/K(m)) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC-->22(OH)7DHC-->20,22(OH)(2)7DHC-->7DHP, with potential further metabolism of 7DHP mediated by 3betaHSD or CYP17, depending on mammalian species. The 5-7 dienal intermediates of the pathway can be a source

  7. Sequential Metabolism of 7-Dehydrocholesterol to Steroidal 5,7-Dienes in Adrenal Glands and Its Biological Implication in the Skin

    PubMed Central

    Slominski, Andrzej T.; Zmijewski, Michal A.; Semak, Igor; Sweatman, Trevor; Janjetovic, Zorica; Li, Wei; Zjawiony, Jordan K.; Tuckey, Robert C.

    2009-01-01

    Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3βHSD for 7DHP (Vm/Km) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC→22(OH)7DHC→20,22(OH)27DHC→7DHP, with potential further metabolism of 7DHP mediated by 3βHSD or CYP17, depending on mammalian species. The 5–7 dienal intermediates of the pathway can be a source of

  8. Cloning and characterization of the methyl coenzyme M reductase genes from Methanobacterium thermoautotrophicum.

    PubMed Central

    Bokranz, M; Bäumner, G; Allmansberger, R; Ankel-Fuchs, D; Klein, A

    1988-01-01

    The genes coding for methyl coenzyme M reductase were cloned from a genomic library of Methanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, alpha, beta, and gamma. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes. Images PMID:2448287

  9. Effects of galactose feeding on aldose reductase gene expression.

    PubMed Central

    Wu, R R; Lyons, P A; Wang, A; Sainsbury, A J; Chung, S; Palmer, T N

    1993-01-01

    Aldose reductase (AR) is implicated in the pathogenesis of the diabetic complications and osmotic cataract. AR has been identified as an osmoregulatory protein, at least in the renal medulla. An outstanding question relates to the response of AR gene expression to diet-induced galactosemia in extrarenal tissues. This paper shows that AR gene expression in different tissues is regulated by a complex multifactorial mechanism. Galactose feeding in the rat is associated with a complex and, on occasions, multiphasic pattern of changes in AR mRNA levels in kidney, testis, skeletal muscle, and brain. These changes are not in synchrony with the temporal sequence of changes in tissue galactitol, galactose, and myoinositol concentrations. Moreover, galactose feeding results in changes in tissue AR activities that are not related, temporally or quantitatively, to the alterations in tissue AR mRNA or galactitol levels. It is concluded that AR gene expression and tissue AR activities are regulated by mechanisms that are not purely dependent on nonspecific alterations in intracellular metabolite concentrations. This conclusion is supported by the finding that chronic xylose feeding, despite being associated with intracellular xylitol accumulation, does not result in alterations in AR mRNA levels, at least in the kidney. PMID:8325980

  10. Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

    PubMed Central

    Cohen, G; Yanko, M; Mislovati, M; Argaman, A; Schreiber, R; Av-Gay, Y; Aharonowitz, Y

    1993-01-01

    The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated. Images PMID:8349555

  11. Amplification and loss of dihydrofolate reductase genes in a Chinese hamster ovary cell line

    SciTech Connect

    Kaufman, R.J.; Schimke, R.T.

    1981-12-01

    During stepwise increases in the methotrexate concentration in culture medium, the authors selected Chinese hamster ovary cells that contained elevated dihydrofolate reductase levels which were proportional to the number of dihydrofolate reductase gene copies (i.e., gene amplification). The authors studied the dihydrofolate reductase levels in individual cells that underwent the initial steps of methotrexate resistance by using the fluorescence-activated cell sorter technique. Such cells constituted a heterogeneous population with differing dihydrofolate reductase levels, and they characteristically lost the elevated enzyme levels when they were grown in the absence of methotrexate. The progeny of individual cells with high enzyme levels behaved differently and could lose all or variable numbers of the amplified genes.

  12. Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves.

    PubMed

    Bogs, Jochen; Downey, Mark O; Harvey, John S; Ashton, Anthony R; Tanner, Gregory J; Robinson, Simon P

    2005-10-01

    Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.

  13. Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae

    SciTech Connect

    Takuma, Shinya; Nakashima, Noriyuki; Tantirungkij, Manee

    1991-12-31

    A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast, Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced in Saccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation in P steps were inducible. S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol by S. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

  14. 5,10-Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) gene polymorphisms and adult meningioma risk.

    PubMed

    Zhang, Jun; Zhou, Yan-Wen; Shi, Hua-Ping; Wang, Yan-Zhong; Li, Gui-Ling; Yu, Hai-Tao; Xie, Xin-You

    2013-11-01

    The causes of meningiomas are not well understood. Folate metabolism gene polymorphisms have been shown to be associated with various human cancers. It is still controversial and ambiguous between the functional polymorphisms of folate metabolism genes 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTRR), and methionine synthase reductase (MTR) and risk of adult meningioma. A population-based case–control study involving 600 meningioma patients (World Health Organization [WHO] Grade I, 391 cases; WHO Grade II, 167 cases; WHO Grade III, 42 cases) and 600 controls was done for the MTHFR C677T and A1298C, MTRR A66G, and MTR A2756G variants in Chinese Han population. The folate metabolism gene polymorphisms were determined by using a polymerase chain reaction–restriction fragment length polymorphism assay. Meningioma cases had a significantly lower frequency of MTHFR 677 TT genotype [odds ratio (OR) = 0.49, 95 % confidence interval (CI) 0.33–0.74; P = 0.001] and T allele (OR = 0.80, 95 % CI 0.67–0.95; P = 0.01) than controls. A significant association between risk of meningioma and MTRR 66 GG (OR = 1.41, 95 % CI 1.02–1.96; P = 0.04) was also observed. When stratifying by the WHO grade of meningioma, no association was found. Our study suggested that MTHFR C677T and MTRR A66G variants may affect the risk of adult meningioma in Chinese Han population.

  15. Genetic Diversity of Benzoyl Coenzyme A Reductase Genes Detected in Denitrifying Isolates and Estuarine Sediment Communities

    PubMed Central

    Song, Bongkeun; Ward, Bess B.

    2005-01-01

    Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from Thauera aromatica, Rhodopseudomonas palustris, and Azoarcus evansii. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to α-, β-, or γ-Proteobacteria as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the bcr type (found in T. aromatica and R. palustris) or the bzd type (found in A. evansii). All the Thauera strains and the isolates from the genera Acidovorax, Bradyrhizobium, Paracoccus, Ensifer, and Pseudomonas had bcr-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from Azarocus strains were assigned to the bzd type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems. PMID:15812036

  16. Components of glycine reductase from Eubacterium acidaminophilum. Cloning, sequencing and identification of the genes for thioredoxin reductase, thioredoxin and selenoprotein PA.

    PubMed

    Lübbers, M; Andreesen, J R

    1993-10-15

    The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (grdA) and the first 23 amino acids of the large subunit of protein PC of glycine reductase (grdC) belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklandii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.

  17. Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines

    SciTech Connect

    Kaufman, R.J.; Brown, P.C.; Schimke, R.T.

    1981-12-01

    The authors studied the loss and stabilization of dihydrofolate reductase genes in clones of a methotrexate-resistant murine S-180 cell line. These cells contained multiple copies of the dihydrofolate reductase gene which were associated with double minute chromosomes. The growth rate of these cells in the absence of methotrexate was inversely related to the degree of gene amplification (number of double minute chromosomes). Cells could both gain and lose genes as a result of an unequal distribution of double minute chromosomes into daughter cells at mitosis. The loss of amplified dihydrofolate reductase genes during growth in the absence of methotrexate resulted from the continual generation of cells containing lower numbers of double minute chromosomes. Because of the growth advantage of these cells, they became dominant in the population. They also studied an unstably resistant S-180 cell line (clone) that, after 3 years of continuous growth in methotrexate, generated cells containing stably amplified dihydrofolate reductase genes. These genes were present on one or more chromosomes, and they were retained in a stable state.

  18. A new cotton SDR family gene encodes a polypeptide possessing aldehyde reductase and 3-ketoacyl-CoA reductase activities.

    PubMed

    Pang, Yu; Song, Wen-Qiang; Chen, Fang-Yuan; Qin, Yong-Mei

    2010-03-01

    To understand regulatory mechanisms of cotton fiber development, microarray analysis has been performed for upland cotton (Gossypium hirsutum). Based on this, a cDNA (GhKCR3) encoding a polypeptide belonging to short-chain alcohol dehydrogenase/reductase family was isolated and cloned. It contains an open reading frame of 987 bp encoding a polypeptide of 328 amino acid residues. Following its overexpression in bacterial cells, the purified recombinant protein specifically uses NADPH to reduce a variety of short-chain aldehydes. A fragment between Gly180 and Gly191 was found to be essential for its catalytic activity. Though the GhKCR3 gene shares low sequence similarities to the ortholog of Saccharomyces cerevisiae YBR159w that encodes 3-ketoacyl-CoA reductase (KCR) catalyzing the second step of fatty acid elongation, it was surprisingly able to complement the yeast ybr159wDelta mutant. Gas chromatography-mass spectrometry analysis showed that very long-chain fatty acids, especially C26:0, were produced in the ybr159wDelta mutant cells expressing GhKCR3. Applying palmitoyl-CoA and malonyl-CoA as substrates, GhKCR3 showed KCR activity in vitro. Quantitative real time-PCR analysis indicated GhKCR3 transcripts accumulated in rapidly elongating fibers, roots, and stems. Our results suggest that GhKCR3 is probably a novel KCR contributing to very long-chain fatty acid biosynthesis in plants.

  19. Cloning and sequencing of the genes from Salmonella typhimurium encoding a new bacterial ribonucleotide reductase.

    PubMed Central

    Jordan, A; Gibert, I; Barbé, J

    1994-01-01

    A plasmid library of Salmonella typhimurium was used to complement a temperature-sensitive nrdA mutant of Escherichia coli. Complementation was obtained with two different classes of plasmids, one carrying the E. coli nrdAB-like genes and the second containing an operon encoding a new bacterial ribonucleotide reductase. Plasmids harboring these new reductase genes also enable obligately anaerobic nrdB::Mud1 E. coli mutants to grow in the presence of oxygen. This operon consists of two open reading frames, which have been designated nrdE (2,145 bp) and nrdF (969 bp). The deduced amino acid sequences of the nrdE and nrdF products include the catalytically important residues conserved in ribonucleotide reductase enzymes of class I and show 25 and 28% overall identity with the R1 and R2 protein, respectively, of the aerobic ribonucleoside diphosphate reductase of E. coli. The 3' end of the sequenced 4.9-kb fragment corresponds to the upstream region of the previously published proU operon of both S. typhimurium and E. coli, indicating that the nrdEF genes are at 57 min on the chromosomal maps of these two bacterial species. Analysis of the nrdEF and proU sequences demonstrates that transcription of the nrdEF genes is in the clockwise direction on the S. typhimurium and E. coli maps. Images PMID:8195103

  20. The role of enoyl reductase genes in phloridzin biosynthesis in apple.

    PubMed

    Dare, Andrew P; Tomes, Sumathi; Cooney, Janine M; Greenwood, David R; Hellens, Roger P

    2013-11-01

    Phloridzin is the predominant polyphenol in apple (Malus × domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro-p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple.

  1. Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency

    SciTech Connect

    Goyette, P.; Frosst, P.; Rosenblatt, D.S.; Rozen. R.

    1995-05-01

    5-Methyltetrahydrofolate, the major form of folate in plasma, is a carbon donor for the remethylation of homocysteine to methionine. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. Patients with an autosomal recessive severe deficiency of MTHFR have homocystinuria and a wide range of neurological and vascular disturbances. We have recently described the isolation of a cDNA for MTHFR and the identification of two mutations in patients with severe MTHFR deficiency. We report here the characterization of seven novel mutations in this gene: six missense mutations and a 5{prime} splice-site defect that activates a cryptic splice in the coding sequence. We also present a preliminary analysis of the relationship between genotype and phenotype for all nine mutations identified thus far in this gene. A nonsense mutation and two missense mutations (proline to leucine and threonine to methionine) in the homozygous state are associated with extremely low activity (0%-3%) and onset of symptoms within the 1st year of age. Other missense mutations (arginine to cysteine and arginine to glutamine) are associated with higher enzyme activity and later onset of symptoms. 19 refs., 4 figs., 2 tabs.

  2. Molecular characterization of genes encoding leucoanthocyanidin reductase involved in proanthocyanidin biosynthesis in apple

    PubMed Central

    Liao, Liao; Vimolmangkang, Sornkanok; Wei, Guochao; Zhou, Hui; Korban, Schuyler S.; Han, Yuepeng

    2015-01-01

    Proanthocyanidins (PAs) are the major component of phenolics in apple, but mechanisms involved in PA biosynthesis remain unclear. Here, the relationship between the PA biosynthesis and the expression of genes encoding leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) was investigated in fruit skin of one apple cultivar and three crabapples. Transcript levels of LAR1 and ANR2 genes were significantly correlated with the contents of catechin and epicatechin, respectively, which suggests their active roles in PA synthesis. Surprisingly, transcript levels for both LAR1 and LAR2 genes were almost undetectable in two crabapples that accumulated both flavan-3-ols and PAs. This contradicts the previous finding that LAR1 gene is a strong candidate regulating the accumulation of metabolites such as epicatechin and PAs in apple. Ectopic expression of apple MdLAR1 gene in tobacco suppresses expression of the late genes in anthocyanin biosynthetic pathway, resulting in loss of anthocyanin in flowers. Interestingly, a decrease in PA biosynthesis was also observed in flowers of transgenic tobacco plants overexpressing the MdLAR1 gene, which could be attributed to decreased expression of both the NtANR1 and NtANR2 genes. Our study not only confirms the in vivo function of apple LAR1 gene, but it is also helpful for understanding the mechanism of PA biosynthesis. PMID:25914714

  3. Molecular characterization of nosRZDFYLX genes coding for denitrifying nitrous oxide reductase of Bradyrhizobium japonicum.

    PubMed

    Velasco, Leonardo; Mesa, Socorro; Xu, Chang-Ai; Delgado, María J; Bedmar, Eulogio J

    2004-04-01

    The nosRZDFYLX gene cluster for the respiratory nitrous oxide reductase from Bradyrhizobium japonicum strain USDA110 has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ, the structural gene, nosD, nosF, nosY, nosL, and nosX were detected. The deduced amino acid sequence exhibited a high degree of similarity to other nitrous oxide reductases from various sources. The NosZ protein included a signal peptide for protein export. Mutant strains carrying either a nosZ or a nosR mutation accumulated nitrous oxide when cultured microaerobically in the presence of nitrate. Maximal expression of a P nosZ-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Microaerobic activation of the fusion required FixLJ and FixK(2).

  4. Assignment of the human dihydrofolate reductase gene to the q11. -->. q22 region of chromosome 5

    SciTech Connect

    Funanage, V.L.; Myoda, T.T.; Moses, P.A.; Cowell, H.R.

    1984-10-01

    Cells from a dihydrofolate reductase-deficit Chinese hamster ovary cell line were hybridized to human fetal skin fibroblast cells. Nineteen dihydrofolate reductase-positive hybrid clones were isolated and characterized. Cytogenetic and biochemical analyses of these clones have shown that the human dihydrofolate reductase (DHFR) gene is located on chromosome 5. Three of these hybrid cell lines contained different terminal deletions of chromosome 5. An analysis of the breakpoints of these deletions has demonstrated that the DHFR gene resides in the q11..-->..q22 region.

  5. Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans.

    PubMed

    Sandhu, S S; Kinghorn, J R; Rajak, R C; Unkles, S E

    2001-07-01

    An heterologous transformation system for entomopathogenic fungi B. bassiana and M. anisopliae was developed based on the use of A. nidulans nitrate reductase gene (niaD). B. bassiana and M. anisopliae niaD stable mutants were selected by treatment of protoplast with ethane methane sulphonate (EMS) and regenerated on chlorate medium. The cloned gene was capable of transforming B. bassiana and M. anisopliae at a frequency of 5.8 to 20 transformants per microg of DNA. Most of them were mitotically stable.

  6. Characterization of the lys2 gene of Penicillium chrysogenum encoding alpha-aminoadipic acid reductase.

    PubMed

    Casqueiro, J; Gutiérrez, S; Bañuelos, O; Fierro, F; Velasco, J; Martín, J F

    1998-09-01

    A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

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

    PubMed

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

    2016-07-01

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

  8. A conservative region of the mercuric reductase gene (mera) as a molecular marker of bacterial mercury resistance

    PubMed Central

    Sotero-Martins, Adriana; de Jesus, Michele Silva; Lacerda, Michele; Moreira, Josino Costa; Filgueiras, Ana Luzia Lauria; Barrocas, Paulo Rubens Guimarães

    2008-01-01

    The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II) to Hg0, which is dependent of the mercuric reductase enzyme (MerA) activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA) gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains. PMID:24031221

  9. The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

    PubMed Central

    Sjöberg, B M; Hahne, S; Mathews, C Z; Mathews, C K; Rand, K N; Gait, M J

    1986-01-01

    The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located. PMID:3530746

  10. narI region of the Escherichia coli nitrate reductase (nar) operon contains two genes.

    PubMed Central

    Sodergren, E J; DeMoss, J A

    1988-01-01

    In previous studies it has been established that in Escherichia coli the three known subunits of anaerobic nitrate reductase are encoded by the narGHI operon. From the nucleotide sequence of the narI region of the operon we conclude that, in addition to the narG and narH genes, the nar operon contains two other open reading frames (ORFs), ORF1 and ORF2, that encode proteins of 26.5 and 25.5 kilodaltons, respectively. Protein fusions to each of the genes in the operon showed that expression of all four genes was similarly regulated. The reading frames of ORF1 and ORF2 were verified, and the N-terminal sequence for the ORF1 fusion protein was determined. The nar operon therefore contains four genes designated and ordered as narGHJI. Images PMID:2832376

  11. Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing, and expression in Escherichia coli.

    PubMed Central

    Garcia, G E; Stadtman, T C

    1992-01-01

    Gene grdA, which encodes selenoprotein A of the glycine reductase complex from Clostridium sticklandii, was identified and characterized. This gene encodes a protein of 158 amino acids with a calculated M(r) of 17,142. The known sequence of 15 amino acids around the selenocysteine residue and the known carboxy terminus of the protein are correctly predicted by the nucleotide sequence. An opal termination codon (TGA) corresponding to the location of the single selenocysteine residue in the polypeptide was found in frame at position 130. The C. sticklandii grdA gene was inserted behind the tac promotor of an Escherichia coli expression vector. An E. coli strain transformed with this vector produced an 18-kDa polypeptide that was not detected in extracts of nontransformed cells. Affinity-purified anti-C. sticklandii selenoprotein A immunoglobulin G reacted specifically with this polypeptide, which was indistinguishable from authentic C. sticklandii selenoprotein A by immunological analysis. Addition of the purified expressed protein to glycine reductase protein components B and C reconstituted the active glycine reductase complex. Although synthesis of enzymically active protein A depended on the presence of selenium in the growth medium, formation of immunologically reactive protein did not. Moreover, synthesis of enzymically active protein in a transformed E. coli selD mutant strain indicated that there is a nonspecific mechanism of selenocysteine incorporation. These findings imply that mRNA secondary structures of C. sticklandii grdA are not functional for UGA-directed selenocysteine insertion in the E. coli expression system. Images PMID:1429431

  12. Clostridium sticklandii glycine reductase selenoprotein A gene: cloning, sequencing, and expression in Escherichia coli.

    PubMed

    Garcia, G E; Stadtman, T C

    1992-11-01

    Gene grdA, which encodes selenoprotein A of the glycine reductase complex from Clostridium sticklandii, was identified and characterized. This gene encodes a protein of 158 amino acids with a calculated M(r) of 17,142. The known sequence of 15 amino acids around the selenocysteine residue and the known carboxy terminus of the protein are correctly predicted by the nucleotide sequence. An opal termination codon (TGA) corresponding to the location of the single selenocysteine residue in the polypeptide was found in frame at position 130. The C. sticklandii grdA gene was inserted behind the tac promotor of an Escherichia coli expression vector. An E. coli strain transformed with this vector produced an 18-kDa polypeptide that was not detected in extracts of nontransformed cells. Affinity-purified anti-C. sticklandii selenoprotein A immunoglobulin G reacted specifically with this polypeptide, which was indistinguishable from authentic C. sticklandii selenoprotein A by immunological analysis. Addition of the purified expressed protein to glycine reductase protein components B and C reconstituted the active glycine reductase complex. Although synthesis of enzymically active protein A depended on the presence of selenium in the growth medium, formation of immunologically reactive protein did not. Moreover, synthesis of enzymically active protein in a transformed E. coli selD mutant strain indicated that there is a nonspecific mechanism of selenocysteine incorporation. These findings imply that mRNA secondary structures of C. sticklandii grdA are not functional for UGA-directed selenocysteine insertion in the E. coli expression system.

  13. Nucleotide sequence of the luxC gene encoding fatty acid reductase of the lux operon from Photobacterium leiognathi.

    PubMed

    Lin, J W; Chao, Y F; Weng, S F

    1993-02-26

    The nucleotide sequence of the luxC gene (EMBL Accession No. 65156) encoding fatty acid reductase (FAR) of the lux operon from Photobacterium leiognathi PL741 was determined and the encoded amino acid sequence deduced. The fatty acid reductase is a component of the fatty acid reductase complex. The complex is responsible for converting fatty acid to aldehyde which serves as the substrate in the luciferase-catalyzed bioluminescent reaction. The protein comprises 478 amino acid residues and has a calculated M(r) of 53,858. Alignment and comparison of the fatty acid reductase of P. leiognathi with that of Vibrio harveyi B392 and Vibrio fischeri ATCC 7744 shows that there is 70% and 59% amino acid residues identity, respectively.

  14. Imbalanced positive selection maintains the functional divergence of duplicated DIHYDROKAEMPFEROL 4-REDUCTASE genes

    PubMed Central

    Huang, Bing-Hong; Chen, Yi-Wen; Huang, Chia-Lung; Gao, Jian; Liao, Pei-Chun

    2016-01-01

    Gene duplication could be beneficial by functional division but might increase the risk of genetic load. The dynamics of duplicated paralogs number could involve recombination, positive selection, and functional divergence. Duplication of DIHYDROFLAVONOL 4-REDUCTASE (DFR) has been reported in several organisms and may have been retained by escape from adaptive conflict (EAC). In this study, we screened the angiosperm DFR gene focusing on a diversified genus Scutellaria to investigate how these duplicated genes are retained. We deduced that gene duplication involved multiple independent events in angiosperms, but the duplication of DFR was before the divergence of Scutellaria. Asymmetric positive selective pressures resulted in different evolutionary rates between the duplicates. Different numbers of regulatory elements, differential codon usages, radical amino acid changes, and differential gene expressions provide evidences of functional divergence between the two DFR duplicates in Scutellaria, implying adaptive subfunctionalization between duplicates. The discovery of pseudogenes accompanying a reduced replacement rate in one DFR paralogous gene suggested possibly leading to “loss of function” due to dosage imbalance after the transient adaptive subfunctionalization in the early stage of duplication. Notwithstanding, episodic gene duplication and functional divergence may be relevant to the diversification of ecological function of DFR gene in Scutellaria. PMID:27966614

  15. Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines.

    PubMed Central

    Brown, P C; Beverley, S M; Schimke, R T

    1981-01-01

    Murine 3T6 selected in increasing concentrations of methotrexate were unstable with respect to dihydrofolate reductase overproduction and methotrexate resistance when they are cultured in the absence of methotrexate. An analysis of the karyotypes of these resistant cells revealed the presence of numerous double minute chromosomes. We observed essentially identical kinetics of loss of dihydrofolate reductase gene sequences in total deoxyribonucleic acid and in deoxyribonucleic acid from fractions enriched in double minute chromosomes and in the numbers of double minute chromosomes per cell during reversion to methotrexate sensitivity, and this suggested that unstably amplified gene sequences were localized on double minute chromosomes. This conclusion ws also supported by an analysis of cell populations sorted according to dihydrofolate reductase enzyme contents, in which relative gene amplification and double minute chromosome content were related proportionally. Images PMID:6287217

  16. Enhancement of methotrexate resistance and dihydrofolate reductase gene amplification by treatment of mouse 3T6 cells with hydroxyurea.

    PubMed Central

    Brown, P C; Tlsty, T D; Schimke, R T

    1983-01-01

    We investigated various parameters associated with the initial selection of mouse 3T6 cells for resistance to single concentrations of methotrexate and characterized resistant colonies for the presence of additional (amplified) copies of the dihydrofolate reductase gene. Our results indicate that the frequency of occurrence of dihydrofolate reductase gene amplification varies with the selecting concentration of methotrexate and is highly variable between clonally derived sublines of mouse 3T6 cells. Second, we increased the frequency of occurrence of cells with amplified dihydrofolate reductase genes by transiently inhibiting DNA synthesis with hydroxyurea before the selection of cells in single concentrations of methotrexate. This effect was dependent on the concentration of hydroxyurea, the time of exposure to the drug, and the time interval between the removal of hydroxyurea and the selection of cells in methotrexate. Images PMID:6877240

  17. Gene expression signature based screening identifies ribonucleotide reductase as a candidate therapeutic target in Ewing sarcoma

    PubMed Central

    Goss, Kelli L.; Gordon, David J.

    2016-01-01

    There is a critical need in cancer therapeutics to identify targeted therapies that will improve outcomes and decrease toxicities compared to conventional, cytotoxic chemotherapy. Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. Although EWS-FLI1 is specific for cancer cells, and required for tumorigenesis, directly targeting this transcription factor has proven challenging. Consequently, targeting unique dependencies or key downstream mediators of EWS-FLI1 represent important alternative strategies. We used gene expression data derived from a genetically defined model of Ewing sarcoma to interrogate the Connectivity Map and identify a class of drugs, iron chelators, that downregulate a significant number of EWS-FLI1 target genes. We then identified ribonucleotide reductase M2 (RRM2), the iron-dependent subunit of ribonucleotide reductase (RNR), as one mediator of iron chelator toxicity in Ewing sarcoma cells. Inhibition of RNR in Ewing sarcoma cells caused apoptosis in vitro and attenuated tumor growth in an in vivo, xenograft model. Additionally, we discovered that the sensitivity of Ewing sarcoma cells to inhibition or suppression of RNR is mediated, in part, by high levels of SLFN11, a protein that sensitizes cells to DNA damage. This work demonstrates a unique dependency of Ewing sarcoma cells on RNR and supports further investigation of RNR inhibitors, which are currently used in clinical practice, as a novel approach for treating Ewing sarcoma. PMID:27557498

  18. Ethylene could influence ferric reductase, iron transporter, and H+-ATPase gene expression by affecting FER (or FER-like) gene activity.

    PubMed

    Lucena, Carlos; Waters, Brian M; Romera, F Javier; García, María José; Morales, María; Alcántara, Esteban; Pérez-Vicente, Rafael

    2006-01-01

    In previous works, it has been shown, by using ethylene inhibitors and precursors, that ethylene could participate in the regulation of the enhanced ferric reductase activity of Fe-deficient Strategy I plants. However, it was not known whether ethylene regulates the ferric reductase gene expression or other aspects related to this activity. This paper is a study of the effects of ethylene inhibitors and precursors on the expression of the genes encoding the ferric reductases and iron transporters of Arabidopsis thaliana (FRO2 and IRT1) and Lycopersicon esculentum (=Solanum lycopersicum) (FRO1 and IRT1) plants. The effects of ethylene inhibitors and precursors on the activity of the iron reductase and the iron transporter have been examined in parallel. Also studied were the effects of ethylene inhibitors and precursors on the expression of the H(+)-ATPase genes of cucumber (CsHA1 and CsHA2) and the transcription factor genes of tomato (LeFER) and Arabidopsis (AtFRU or AtFIT1, an LeFER homologue) that regulate ferric reductase, iron transporter, and H(+)-ATPse activity. The results obtained suggest that ethylene participates in the regulation of ferric reductase, the iron transporter, and H(+)-ATPase gene expression by affecting the FER (or FER-like) levels.

  19. Diversity of nitrous oxide reductase (nosZ) genes in continental shelf sediments

    SciTech Connect

    Scala, D.J.; Kerkhof, L.J.

    1999-04-01

    Diversity of the nitrous oxide reductase (nosZ) gene was examined in sediments obtained from the Atlantic Ocean and Pacific Ocean continental shelves. Approximately 1,100 bp of the nosZ gene were amplified via PCR, using nosZ gene-specific primers. Thirty-seven unique copies of the nosZ gene from these marine environments were characterized, increasing the nosZ sequence database fourfold. The average DNA similarity for comparisons between all 49 variants of the nosZ gene was 64% {+-} 10%. Alignment of the derived amino acid sequences confirmed the conservation of important structural motifs. A highly conserved region is proposed as the copper binding, catalytic site (Cu{sub z}) of the mature protein. Phylogenetic analysis demonstrated three major clusters of nosZ genes, with little overlap between environmental and culture-based groups. Finally, the two non-culture-based gene clusters generally corresponded to sampling location, implying that denitrifier communities may be restricted geographically.

  20. Distribution of common methylenetetrahydrofolate reductase gene mutations in patients with obstructive sleep apnoea.

    PubMed

    Bekci, T T; Kocak, N; Kesli, R

    2009-01-01

    Homocysteine levels have been investigated in patients with obstructive sleep apnoea syndrome (OSAS), a syndrome associated with a high level of comorbid cardiovascular disease (CVD). While significant increases in homocysteine levels have been observed in OSAS patients with CVD, no increases have been noted in OSAS patients without CVD. This study was designed to investigate the methylenetetrahydrofolate reductase (MTHFR) gene, which is essential for homocysteine metabolism and has been shown to have a causal role in the development of CVD. Eighty subjects, 30 diagnosed with OSAS by polysomnography and 50 controls (healthy volunteers with no symptoms of OSAS) were enrolled. Two mutations in the MTHFR gene were investigated using polymerase chain reactions and restriction fragment length polymorphisms. No significant differences were found in mean age, body mass index, homocysteine levels, or MTHFR allele or genotype distributions between patient and control groups.

  1. Combined choroidal neovascularization and hypopituitarism in a patient with homozygous mutation in methylenetetrahydrofolate reductase gene

    PubMed Central

    Aydogdu, Aydogan; Haymana, Cem; Baskoy, Kamil; Durukan, Ali H.; Ozgur, Gokhan; Azal, Omer

    2014-01-01

    We report a case of choroidal neovascularization (CNV) secondary to methylenetetrahydrofolate reductase (MTHFR) gene mutation in a 20-year-old male patient with hypopituitarism. Treatment with three consecutive injections of intravitreal ranibizumab (anti-vascular endothelial growth factor) resulted in significant improvement of the patient's vision and the appearance of the macula. A search of the literature produced no previously reported case of MTHFR gene mutation associated both CNV and possibly hypopituitarism. With hormone replacement therapy of hypopituitarism, acetyl salicylic acid 100 mg/day also was started. The patient was clinically stable both for CNV and other thromboembolic disorders over a 6-month follow-up and also 1-year follow-up period. PMID:24672570

  2. Isolation and characterization of a nitrite reductase gene and its use as a probe for denitrifying bacteria.

    PubMed Central

    Smith, G B; Tiedje, J M

    1992-01-01

    The dissimilatory nitrite reductase gene (nir) from denitrifying bacterium Pseudomonas stutzeri JM300 was isolated and sequenced. In agreement with recent sequence information from another strain of P. stutzeri (strain ZoBell), strain JM300 nir is the first gene in an operon and is followed immediately by a gene which codes for a tetraheme protein; 2.5 kb downstream from the nitrite reductase carboxyl terminus is the cytochrome c551 gene. P. stutzeri JM300 nir is 67% homologous to P. aeruginosa nir and 88% homologous to P. stutzeri ZoBell nir. Within the nitrite reductase promoter region is an fnr-like operator very similar to an operator upstream of a separate anaerobic pathway, that for arginine catabolism in P. aeruginosa. The denitrification genes in P. stutzeri thus may be under the same regulatory control as that found for other anaerobic pathways of pseudomonads. We have generated gene probes from restriction fragments within the nitrite reductase operon to evaluate their usefulness in ecology studies of denitrification. Probes generated from the carboxyl terminus region hybridized to denitrifying bacteria from five separate genera and did not cross-hybridize to any nondenitrifying bacteria among six genera tested. The denitrifier probes were successful in detecting denitrifying bacteria from samples such as a bioreactor consortium, aquifer microcosms, and denitrifying toluene-degrading enrichments. The probes also were used to reveal restriction fragment length polymorphism patterns indicating the diversity of denitrifiers present in these mixed communities. Images PMID:1539983

  3. Gene expression of monodehydroascorbate reductase and dehydroascorbate reductase during fruit ripening and in response to environmental stresses in acerola (Malpighia glabra).

    PubMed

    Eltelib, Hani A; Badejo, Adebanjo A; Fujikawa, Yukichi; Esaka, Muneharu

    2011-04-15

    Acerola (Malpighia glabra) is an exotic fruit cultivated primarily for its abundant ascorbic acid (AsA) content. The molecular mechanisms that regulate the metabolism of AsA in acerola have yet to be defined. Monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) are key enzymes of the ascorbate-glutathione cycle that maintain reduced pools of ascorbic acid and serve as important antioxidants. cDNAs encoding MDHAR and DHAR were isolated from acerola using RT-PCR and RACE. Phylogenetic trees associated acerola MDHAR and DHAR with other plant cytosolic MDHARs and DHARs. Expressions of the two genes correlated with their enzymatic activities and were differentially regulated during fruit ripening. Interestingly, MDHAR expression was only detected in overripe fruits, whereas the transcript level of DHAR was highest at the intermediate stage of fruit ripening. Under dark conditions, there was a sharp and significant decline in the total and reduced ascorbate contents, accompanied by a decrease in the level of transcripts and enzyme activities of the two genes in acerola leaves. MDHAR and DHAR transcripts and enzyme activities were significantly up-regulated in the leaves of acerola under cold and salt stress conditions, indicating that expression of both genes are transcriptionally regulated under these stresses.

  4. Folic acid rivals methylenetetrahydrofolate reductase (MTHFR) gene-silencing effect on MEPM cell proliferation and apoptosis.

    PubMed

    Xiao, Wen-Lin; Wu, Min; Shi, Bing

    2006-11-01

    It's clear that environmental factors play a role in the aetiology of orofacial clefting (OFC) and an important area of future research will be to unravel interactions that occur between candidate genes and environmental factors during early development of the embryo. Periconceptional folic acid supplementation may reduce the risk of OFC. Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene reduce availability of 5-methylenetetrahydrofolate, the predominant circulating form of folic acid. To determine the effect of MTHFR gene mutation on murine embryonic palatal mesenchymal (MEPM) cells and the interaction with folic acid supplement, we used RNAi study in the primary cultures of MEPM cells. The cells of MTHFR gene silencing grew slower and the apoptosis cell number was more than the cells of control. Supplement with 20 microg/ml folic acid was the best to preventing teratogenic effect of MTHFR gene silencing. By flow cytometry analysis of cell cycle, results were shown that the MEPM cells were retarded in G(0)/G(1) after MTHFR gene silencing. While using 20 microg/ml folic acid supplements could make cell transit the G(1)/S restriction point and the cells growth was close to normal level.

  5. Identification of a functional antioxidant responsive element in the promoter of the Chinese hamster carbonyl reductase 3 (Chcr3) gene.

    PubMed

    Miura, Takeshi; Taketomi, Ayako; Nakabayashi, Toshikatsu; Nishinaka, Toru; Terada, Tomoyuki

    2015-07-01

    CHCR3, a member of the short-chain dehydrogenase/reductase superfamily, is a carbonyl reductase 3 enzyme in Chinese hamsters. Carbonyl reductase 3 in humans has been believed to involve the metabolism and/or pharmacokinetics of anthracycline drugs, and the mechanism underlying the gene regulation has been investigated. In this study, the nucleotide sequence of the Chcr3 promoter was originally determined, and its promoter activity was characterised. The proximal promoter region is TATA-less and GC-rich, similar to the promoter region of human carbonyl reductase 3. Cobalt stimulated the transcriptional activity of the Chcr3 gene. The results of a luciferase gene reporter assay demonstrated that cobalt-induced stimulation required an antioxidant responsive element. Forced expression of Nrf2, the transcription factor that binds to antioxidant responsive elements, enhanced the transcriptional activity of the Chcr3 gene. These results suggest that cobalt induces the expression of the Chcr3 gene via the Nrf2-antioxidant responsive element pathway.

  6. Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

    PubMed Central

    Wang, P; Denoya, C D; Morgenstern, M R; Skinner, D D; Wallace, K K; Digate, R; Patton, S; Banavali, N; Schuler, G; Speedie, M K; Reynolds, K A

    1996-01-01

    We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid. PMID:8955309

  7. Diversity of assimilatory nitrate reductase genes from plankton and epiphytes associated with a seagrass bed.

    PubMed

    Adhitya, Anita; Thomas, Florence I M; Ward, Bess B

    2007-11-01

    Assimilatory nitrate reductase gene fragments were isolated from epiphytes and plankton associated with seagrass blades collected from Tampa Bay, Florida, USA. Nitrate reductase genes from diatoms (NR) and heterotrophic bacteria (nasA) were amplified by polymerase chain reaction (PCR) using two sets of degenerate primers. A total of 129 NR and 75 nasA clones from four clone libraries, two from each of epiphytic and planktonic components, were sequenced and aligned. In addition, genomic DNA sequences for the NR fragment were obtained from Skeletonema costatum and Thalassiosira weissflogii diatom cultures. Rarefaction analysis with an operational taxonomic unit cut-off of 6% indicated that diversity of the NR and nasA clone libraries were similar, and that sequencing of the clone libraries was not yet saturated. Phylogenetic analysis indicated that 121 of the 129 NR clones sequenced were similar to diatom sequences. Of the eight non-diatom sequences, four were most closely related to the sequence of Chlorella vulgaris. Introns were found in 8% of the Tampa Bay NR sequences; introns were also observed in S. costatum, but not T. weissflogii. Introns from within the same clone library exhibited close similarity in nucleotide sequence, position and length; the corresponding exon sequences were unique. Introns from within the same component were similar in position and length, but not in nucleotide sequence. These findings raise questions about the function of introns, and mechanisms or time evolution of intron formation. A large cluster of 14 of the 75 nasA sequences was similar to sequences from Vibrio species; other sequences were closely related to sequences from Alteromonas, alpha-proteobacteria and Marinomonas-like species. Biogeographically consistent patterns were observed for the nasA Tampa Bay sequences compared with sequences from other locations: for example, Tampa Bay sequences were similar to those from the South Atlantic Bight, but not the Barents Sea. The

  8. Folate metabolism gene 5,10-methylenetetrahydrofolate reductase (MTHFR) is associated with ADHD in myelomeningocele patients.

    PubMed

    Spellicy, Catherine J; Northrup, Hope; Fletcher, Jack M; Cirino, Paul T; Dennis, Maureen; Morrison, Alanna C; Martinez, Carla A; Au, Kit Sing

    2012-01-01

    The objective of this study was to examine the relation between the 5, 10-methylenetetrahydrofolate reductase (MTHFR) gene and behaviors related to attention- deficit/hyperactivity disorder (ADHD) in individuals with myelomeningocele. The rationale for the study was twofold: folate metabolizing genes, (e.g. MTHFR), are important not only in the etiology of neural tube defects but are also critical to cognitive function; and individuals with myelomeningocele have an elevated incidence of ADHD. Here, we tested 478 individuals with myelomeningocele for attention-deficit hyperactivity disorder behavior using the Swanson Nolan Achenbach Pelham-IV ADHD rating scale. Myelomeningocele participants in this group for whom DNAs were available were genotyped for seven single nucleotide polymorphisms (SNPs) in the MTHFR gene. The SNPs were evaluated for an association with manifestation of the ADHD phenotype in children with myelomeningocele. The data show that 28.7% of myelomeningocele participants exhibit rating scale elevations consistent with ADHD; of these 70.1% had scores consistent with the predominantly inattentive subtype. In addition, we also show a positive association between the SNP rs4846049 in the 3'-untranslated region of the MTHFR gene and the attention-deficit hyperactivity disorder phenotype in myelomeningocele participants. These results lend further support to the finding that behavior related to ADHD is more prevalent in patients with myelomeningocele than in the general population. These data also indicate the potential importance of the MTHFR gene in the etiology of the ADHD phenotype.

  9. Pseudomonas stutzeri Nitrite Reductase Gene Abundance in Environmental Samples Measured by Real-Time PCR

    PubMed Central

    Grüntzig, Verónica; Nold, Stephen C.; Zhou, Jizhong; Tiedje, James M.

    2001-01-01

    We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 106 gene copies (r2 = 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. Although P. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier. PMID:11157241

  10. Conservation of the genes for dissimilatory sulfite reductase from Desulfovibrio vulgaris and Archaeoglobus fulgidus allows their detection by PCR.

    PubMed Central

    Karkhoff-Schweizer, R R; Huber, D P; Voordouw, G

    1995-01-01

    The structural genes for dissimilatory sulfite reductase (desulfoviridin) from Desulfovibrio vulgaris Hilden-borough were cloned as a 7.2-kbp SacII DNA fragment. Nucleotide sequencing indicated the presence of a third gene, encoding a protein of only 78 amino acids, immediately downstream from the genes for the alpha and beta subunits (dsvA and dsvB). We designated this protein DsvD and the gene encoding it the dsvD gene. The alpha- and beta-subunit sequences are highly homologous to those of the dissimilatory sulfite reductase from Archaeoglobus fulgidus, a thermophilic archaeal sulfate reducer, which grows optimally at 83 degrees C. A gene with significant homology to dsvD was also found immediately downstream from the dsrAB genes of A. fulgidus. The remarkable conservation of gene arrangement and sequence across domain (bacterial versus archaeal) and physical (mesophilic versus thermophilic) boundaries indicates an essential role for DsvD in dissimilatory sulfite reduction and allowed the construction of conserved deoxyoligonucleotide primers for detection of the dissimilatory sulfite reductase genes in the environment. PMID:7887608

  11. Effects of methionine synthase and methylenetetrahydrofolate reductase gene polymorphisms on markers of one-carbon metabolism.

    PubMed

    Ho, Vikki; Massey, Thomas E; King, Will D

    2013-11-01

    Genetic and nutritional factors play a role in determining the functionality of the one-carbon (1C) metabolism cycle, a network of biochemical reactions critical to intracellular processes. Genes encoding enzymes for methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) may determine biomarkers of the cycle including homocysteine (HCY), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). MTHFR C677T is an established genetic determinant of HCY but less is known of its effect on SAM and SAH. Conversely, the relationship between MTR A2756G and HCY remains inconclusive, and its effect on SAM and SAH has only been previously investigated in a female-specific population. Folate and vitamin B12 are essential substrate and cofactor of 1C metabolism; thus, consideration of gene-nutrient interactions may clarify the role of genetic determinants of HCY, SAM and SAH. This cross-sectional study included 570 healthy volunteers from Kingston, Ontario, Ottawa, Ontario and Halifax, Nova Scotia, Canada. Least squares regression was used to examine the effects of MTR and MTHFR polymorphisms on plasma HCY, SAM and SAH concentrations; gene-gene and gene-nutrient interactions were considered with the inclusion of cross-products in the model. Main effects of MTR and MTHFR polymorphisms on HCY concentrations were observed; however, no gene-gene or gene-nutrient interactions were found. No association was observed for SAM. For SAH, interactions between MTR and MTHFR polymorphisms, and MTHFR polymorphism and serum folate were found. The findings of this research provide evidence that HCY and SAH, biomarkers of 1C metabolism, are influenced by genetic and nutritional factors and their interactions.

  12. The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration.

    PubMed

    Delgado, María J; Bonnard, Nathalie; Tresierra-Ayala, Alvaro; Bedmar, Eulogio J; Müller, Peter

    2003-12-01

    The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66.5 bp downstream of the centre of a putative FNR-like binding site.

  13. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    EPA Science Inventory

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  14. A study of chromosomal changes associated with amplified dihydrofolate reductase genes in rat hepatoma cells and their dedifferentiated variants

    PubMed Central

    1984-01-01

    We have examined the karyological consequences of dihydrofolate reductase gene amplification in a series of six rat hepatoma cell lines, all derived from the same clone. Cells of three of these lines express a series of liver-specific functions whereas those of three others fail to express these functions. Cells of each line have been subjected to stepwise selection for methotrexate resistance and, in most cases, resistance is associated with a 40-50-fold amplification of sequences hybridizing to a dihydrofolate reductase cDNA probe. In one line no modified chromosome is observed, whereas in two others the amplified genes are associated with an expanded chromosomal region. R- banding analysis of these karyotypes showed that few changes have occurred. These observations apply to two of the well-differentiated lines, and to a variant able to revert to the differentiated state. In contrast, in the two stably dedifferentiated hepatoma cell lines, amplified dihydrofolate reductase genes are found on large chromosomes of variable size, on ring chromosomes, and on chromosomes containing terminal, median, or multiple centromeres. We conclude that the nature of the chromosomal changes associated with dihydrofolate reductase gene amplification are the result of differences in cell lines rather than in the protocols employed for selection. PMID:6746737

  15. Comprehensive detection of phototrophic sulfur bacteria using PCR primers that target reverse dissimilatory sulfite reductase gene.

    PubMed

    Mori, Yumi; Purdy, Kevin J; Oakley, Brian B; Kondo, Ryuji

    2010-01-01

    A new set of primers for the detection of phototrophic sulfur bacteria in natural environments is described. The primers target the α-subunit of the reverse dissimilatory sulfite reductase gene (dsrA). PCR-amplification resulted in products of the expected size from all the phototrophic strains tested, including purple sulfur and green sulfur bacteria. Seventy-nine clones obtained from environmental DNA using the primers were sequenced and all found to be closely related to the dsrA of purple sulfur bacteria and green sulfur bacteria. This newly developed PCR assay targeting dsrA is rapid and simple for the detection of phototrophic sulfur bacteria in situ and superior to the use of culture-dependent techniques.

  16. Light-regulated expression of the nitrate-reductase and nitrite-reductase genes in tomato and in the phytochrome-deficient aurea mutant of tomato.

    PubMed

    Becker, T W; Foyer, C; Caboche, M

    1992-08-01

    The phytochrome-deficient aurea mutant of tomato (Lycopersicon esculentum (L.) Mill) was used to investigate if phytochrome plays a role in the regulation of nitrate-reductase (NR, EC 1.6.6.1) and nitrite-reductase (NiR, EC 1.7.7.1) gene expression. We show that the expression of the tomato NR and NiR genes is stimulated by light and that this light response is mediated by the photoreceptor phytochrome. The red-light response of the NR and NiR genes was reduced in etiolated aurea seedlings when compared to isogenic wild-type cotyledons. The relative levels of NR mRNA and NiR transcripts and their diurnal fluctuations were identical in mature white-light-grown leaves of the wild-type and of the aurea mutant. The transcript levels for cab and RbcS (genes for the chlorophyll-a/b-binding protein of PSII and the small subunit of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively) in aurea leaves grown in white light were indistinguishable from the respective transcript levels in the leaves of the wildtype grown under the same conditions. Despite a severe reduction in the chlorophyll content, the rate of net CO2 uptake by leaves of the aurea mutant was only slightly reduced when compared to the rate of net photosynthesis of wild-type leaves. This difference in the photosynthetic performances of wild-type and aurea mutant plants disappeared during aging of the plants. The increase in zeaxanthin and the concomitant decrease in violaxanthin in leaves of the aurea mutant compared with the same pigment levels in leaves of the wild-type indicate that the activity of the xanthophyll cycle is increased in aurea leaves as a consequence of the reduced CO2-fixation capacity of the mutant leaves.

  17. Cloning and expression analysis of cinnamoyl-CoA reductase (CCR) genes in sorghum

    PubMed Central

    Fan, Feifei; Wang, Lihua; Zhan, Qiuwen; Wu, Peijin; Du, Junli; Yang, Xiaocui; Liu, Yanlong

    2016-01-01

    Cinnamoyl-CoA reductase (CCR) is the first enzyme in the monolignol-specific branch of the lignin biosynthetic pathway. In this research, three sorghum CCR genes including SbCCR1, SbCCR2-1 and SbCCR2-2 were cloned and characterized. Analyses of the structure and phylogeny of the three CCR genes showed evolutionary conservation of the functional domains and divergence of function. Transient expression assays in Nicotiana benthamiana leaves demonstrated that the three CCR proteins were localized in the cytoplasm. The expression analysis showed that the three CCR genes were induced by drought. But in 48 h, the expression levels of SbCCR1 and SbCCR2-2 did not differ between CK and the drought treatment; while the expression level of SbCCR2-1 in the drought treatment was higher than in CK. The expression of the SbCCR1 and SbCCR2-1 genes was not induced by sorghum aphid [Melanaphis sacchari (Zehntner)] attack, but SbCCR2-2 was significantly induced by sorghum aphid attack. It is suggested that SbCCR2-2 is involved in the process of pest defense. Absolute quantitative real-time PCR revealed that the three CCR genes were mainly expressed in lignin deposition organs. The gene copy number of SbCCR1 was significantly higher than those of SbCCR2-1 and SbCCR2-2 in the tested tissues, especially in stem. The results provide new insight into the functions of the three CCR genes in sorghum. PMID:27231650

  18. Isolation and characterization of genes encoding leucoanthocyanidin reductase (FeLAR) and anthocyanidin reductase (FeANR) in buckwheat (Fagopyrum esculentum).

    PubMed

    Matsui, Katsuhiro; Hisano, Tomomi; Yasui, Yasuo; Mori, Masashi; Walker, Amanda R; Morishita, Toshikazu; Katsu, Kenjiro

    2016-10-20

    Proanthocyanidins (PAs) are a major group of flavonoids synthesized via the phenylpropanoid biosynthesis pathway, however the pathway has not been fully characterized in buckwheat. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are involved in the last steps of PA biosynthesis. To isolate the genes for these enzymes from buckwheat we performed PCR using degenerate primers and obtained cDNAs of ANR and LAR, which we designated FeANR and FeLAR1. A search for homologs in a buckwheat genome database with both sequences returned two more LAR sequences, designated FeLAR2 and FeLAR3. Linkage analysis with an F2 segregating population indicated that the three LAR loci were not genetically linked. We detected high levels of PAs in roots and cotyledons of buckwheat seedlings and in buds and flowers of mature plants. FeANR and FeLAR1-3 were expressed in most organs but had different expression patterns. Our findings would be useful for breeding and further analysis of PA synthesis and its regulation in buckwheat.

  19. Is methylenetetrahydrofolate reductase (MTHFR) gene A1298C polymorphism related with varicocele risk?

    PubMed

    Ucar, V B; Nami, B; Acar, H; Kilinç, M

    2015-02-01

    Varicocele is one of the main reasons for male infertility the exact aetiology of which remains unclear. Methylenetetrahydrofolate reductase (MTHFR) is important for DNA synthesis and methylation, which has a key role during spermatogenesis. Numerous literature suggests that the MTHFR polymorphism may be genetic risk factors for male infertility. In this study, we evaluated C677T and A1298C MTHFR gene polymorphism frequency in patients with varicocele and normal men. A total of 107 varicocele patients and 109 fertile healthy individuals were included. Genotyping of the MTHFR gene in C677T and A1298C base pairs carried out by using real-time PCR technique and afterwards, the statistical analysis accomplished. There is a statistical difference for the frequency of 1298AA genotype in patients with varicocele compared with normal controls (P = 0.0051, OR = 2.2750). Instead, subsequently, 1298/A allel frequency in patient group was significantly higher in comparison with control group (P = 0.0174). According to our results, 1298AA genotype in MTHFR gene raises the risk of varicocele approximately 2.3 times more compared with men carrying other genotypes. The results show that genetic factors have an important role in the molecular basis of varicocele.

  20. Methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and susceptibility to ischemic stroke: a meta-analysis.

    PubMed

    Li, Pingping; Qin, Chao

    2014-02-10

    Associations between 5,10-methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism and ischemic stroke have been reported (Ariyaratnam et al., 2007; Banerjee et al., 2007; Casas et al., 2004), but the results of these studies are inconsistent. To investigate the possible associations between the MTHFR gene polymorphism and ischemic stroke, we performed a meta-analysis. Nineteen case-control studies associated with MTHFR gene C667T involving 2223 cases and 2936 controls were included. Heterogeneity among studies was evaluated with I(2) and Egger's test and an inverted funnel plot was used to assess publication bias. Odds ratio (OR) was observed to identify the associations. Statistically significant association with ischemic stroke was identified for allele T polymorphism of MTHFR [fixed-effects OR=1.28, 95% confidence interval (95% CI): 1.17-1.40, P<0.00001] and marginally significant association was detected with genotype CT of MTHFR (fixed-effects OR=1.13, 95% CI: 1.01-127, P=0.04) and genotype TT of MTHFR (fixed-effects OR=1.43, 95% CI: 1.20-1.70, P<0.001). The results suggested that the MTHFR C667T genetic polymorphism was significantly associated with increased risk of ischemic stroke.

  1. Expression and Enzyme Activity Detection of a Sepiapterin Reductase Gene from Musca domestica Larva.

    PubMed

    Tang, Yan; Pei, Zhihua; Liu, Lei; Wang, Dongfang; Kong, Lingcong; Liu, Shuming; Jiang, Xiuyun; Gao, Yunhang; Ma, Hongxia

    2017-02-01

    Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic acid hydroxylases and nitric oxide synthase. Sepiapterin reductase (SPR) catalyzes the final steps of BH4 biosynthesis. Studies on SPR from several insects and other organisms have been reported. However, thus far, enzyme activity of SPR in Musca domestica is kept unknown. In this study, 186 differentially expressed genes including SPR gene from Musca domestica (MDSPR) were screened in subtractive cDNA library. The MDSPR gene was cloned, and the recombinant MDSPI16 protein was expressed as a 51-kDa protein in soluble form. The MDSPR exhibited strong activity to the substrate sepiapterin (SP). The values of Vmax and Km of the MDSPR for SP were 6.83 μM/min and 23.48 μM, and the optimum temperature and pH of MDSPR were 50 °C and 4.0, respectively. This study provides new hypotheses and methods for the production of BH4 using insect-derived SPR.

  2. Molecular Properties and Functional Divergence of the Dehydroascorbate Reductase Gene Family in Lower and Higher Plants.

    PubMed

    Zhang, Yuan-Jie; Wang, Wei; Yang, Hai-Ling; Li, Yue; Kang, Xiang-Yang; Wang, Xiao-Ru; Yang, Zhi-Ling

    2015-01-01

    Dehydroascorbate reductase (DHAR), which reduces oxidized ascorbate, is important for maintaining an appropriate ascorbate redox state in plant cells. To date, genome-wide molecular characterization of DHARs has only been conducted in bryophytes (Physcomitrella patens) and eudicots (e.g. Arabidopsis thaliana). In this study, to gain a general understanding of the molecular properties and functional divergence of the DHARs in land plants, we further conducted a comprehensive analysis of DHARs from the lycophyte Selaginella moellendorffii, gymnosperm Picea abies and monocot Zea mays. DHARs were present as a small gene family in all of the land plants we examined, with gene numbers ranging from two to four. All the plants contained cytosolic and chloroplastic DHARs, indicating dehydroascorbate (DHA) can be directly reduced in the cytoplasm and chloroplast by DHARs in all the plants. A novel vacuolar DHAR was found in Z. mays, indicating DHA may also be reduced in the vacuole by DHARs in Z. mays. The DHARs within each species showed extensive functional divergence in their gene structures, subcellular localizations, and enzymatic characteristics. This study provides new insights into the molecular characteristics and functional divergence of DHARs in land plants.

  3. Genome-wide analysis of glutathione reductase (GR) genes from rice and Arabidopsis.

    PubMed

    Trivedi, Dipesh Kumar; Gill, Sarvajeet Singh; Yadav, Sandep; Tuteja, Narendra

    2013-02-01

    Plant cells and tissues remain always on risk under abiotic and biotic stresses due to increased production of reactive oxygen species (ROS). Plants protect themselves against ROS induced oxidative damage by the upregulation of antioxidant machinery. Out of many components of antioxidant machinery, glutathione reductase (GR, EC 1.6.4.2) and glutathione (GSH, γ-Glu-Cys-Gly) play important role in the protection of cell against oxidative damage. In stress condition, the GR helps in maintaining the reduced glutathione pool for strengthening the antioxidative processes in plants. Present study investigates genome wide analysis of GR from rice and Arabidopsis. We were able to identify 3 rice GR genes (LOC_Os02 g56850, LOC_Os03 g06740, LOC_Os10 g28000) and 2 Arabidopsis GR genes (AT3G54660, AT3G24170) from their respective genomes on the basis of their annotation as well as the presence of pyridine nucleotide-disulphide oxidoreductases class-I active site. The evolutionary relationship of the GR genes from rice and Arabidopsis genomes was analyzed using the multiple sequence alignment and phylogenetic tree. This revealed evolutionary conserved pyridine nucleotide-disulphide oxidoreductases class-I active site among the GR protein in rice and Arabidopsis. This study should make an important contribution to our better understanding of the GR under normal and stress condition in plants.

  4. Molecular Properties and Functional Divergence of the Dehydroascorbate Reductase Gene Family in Lower and Higher Plants

    PubMed Central

    Zhang, Yuan-Jie; Wang, Wei; Yang, Hai-Ling; Li, Yue; Kang, Xiang-Yang; Wang, Xiao-Ru; Yang, Zhi-Ling

    2015-01-01

    Dehydroascorbate reductase (DHAR), which reduces oxidized ascorbate, is important for maintaining an appropriate ascorbate redox state in plant cells. To date, genome-wide molecular characterization of DHARs has only been conducted in bryophytes (Physcomitrella patens) and eudicots (e.g. Arabidopsis thaliana). In this study, to gain a general understanding of the molecular properties and functional divergence of the DHARs in land plants, we further conducted a comprehensive analysis of DHARs from the lycophyte Selaginella moellendorffii, gymnosperm Picea abies and monocot Zea mays. DHARs were present as a small gene family in all of the land plants we examined, with gene numbers ranging from two to four. All the plants contained cytosolic and chloroplastic DHARs, indicating dehydroascorbate (DHA) can be directly reduced in the cytoplasm and chloroplast by DHARs in all the plants. A novel vacuolar DHAR was found in Z. mays, indicating DHA may also be reduced in the vacuole by DHARs in Z. mays. The DHARs within each species showed extensive functional divergence in their gene structures, subcellular localizations, and enzymatic characteristics. This study provides new insights into the molecular characteristics and functional divergence of DHARs in land plants. PMID:26684301

  5. The unique glutathione reductase from Xanthomonas campestris: Gene expression and enzyme characterization

    SciTech Connect

    Loprasert, Suvit . E-mail: suvit@cri.or.th; Whangsuk, Wirongrong; Sallabhan, Ratiboot; Mongkolsuk, Skorn

    2005-06-17

    The glutathione reductase gene, gor, was cloned from the plant pathogen Xanthomonas campestris pv. phaseoli. Its gene expression and enzyme characteristics were found to be different from those of previously studied homologues. Northern blot hybridization, promoter-lacZ fusion, and enzyme assay experiments revealed that its expression, unlike in Escherichia coli, is OxyR-independent and constitutive upon oxidative stress conditions. The deduced amino acid sequence shows a unique NADPH binding motif where the most highly conserved arginine residue, which is critical for NADPH binding, is replaced by glutamine. Interestingly, a search of the available Gor amino acid sequences from various sources, including other Xanthomonas species, revealed that this replacement is specific to the genus Xanthomonas. Recombinant Gor enzyme was purified and characterized, and was found to have a novel ability to use both, NADPH and NADH, as electron donor. A gor knockout mutant was constructed and shown to have increased expression of the organic peroxide-inducible regulator gene, ohrR.

  6. Sequence analysis of the oxidase/reductase genes upstream of the Rhodococcus erythropolis aldehyde dehydrogenase gene thcA reveals a gene organisation different from Mycobacterium tuberculosis.

    PubMed

    Nagy, I; De Mot, R

    1999-01-01

    The sequence of the DNA region upstream of the thiocarbamate-inducible aldehyde dehydrogenase gene thcA of Rhodococcus erythropolis NI86/21 was determined. Most of the predicted ORFs are related to various oxidases/reductases, including short-chain oxidases/reductases, GMC oxidoreductases, alpha-hydroxy acid oxidases (subfamily 1 flavin oxidases/dehydrogenases), and subfamily 2 flavin oxidases/dehydrogenases. One ORF is related to enzymes involved in biosynthesis of PQQ or molybdopterin cofactors. In addition, a putative member of the TetR family of regulatory proteins was identified. The substantial sequence divergence from functionally characterized enzymes precludes a reliable prediction about the probable function of these proteins at this stage. In Mycobacterium tuberculosis H37Rv, most of these ORFs have homologs that are also clustered in the genome, but some striking differences in gene organization were observed between Rhodococcus and Mycobacterium.

  7. Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils

    DOE PAGES

    Higgins, Steven A.; Welsh, Allana; Orellana, Luis H.; ...

    2016-03-11

    Members of the Fungi convert nitrate (NO3-) and nitrite (NO2-) to gaseous nitrous oxide (N2O) (denitrification), but the fungal contributions to N-loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N2O from added NO3- or NO2- in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, of which 151 produced N2O from NO2-. Novel PCR primersmore » targeting the p450nor gene that encodes the nitric oxide (NO) reductase responsible for N2O production in fungi yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54-98% amino acid identity to reference P450nor sequences within the phylum Ascomycota, and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from nitrite, whereas nirK (encoding the NO-forming nitrite reductase) was amplified in only 13-74% of the N2O-forming isolates using two separate nirK primer sets. Altogether, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation.« less

  8. Human carbonyl reductase (CBR) localized to band 21q22. 1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells

    SciTech Connect

    Lemieux, N. ); Malfoy, B. ); Forrest, G.L. )

    1993-01-01

    Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 2lq22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease. 28 refs., 1 fig., 1 tab.

  9. The Thiol Reductase Activity of YUCCA6 Mediates Delayed Leaf Senescence by Regulating Genes Involved in Auxin Redistribution

    PubMed Central

    Cha, Joon-Yung; Kim, Mi R.; Jung, In J.; Kang, Sun B.; Park, Hee J.; Kim, Min G.; Yun, Dae-Jin; Kim, Woe-Yeon

    2016-01-01

    Auxin, a phytohormone that affects almost every aspect of plant growth and development, is biosynthesized from tryptophan via the tryptamine, indole-3-acetamide, indole-3-pyruvic acid, and indole-3-acetaldoxime pathways. YUCCAs (YUCs), flavin monooxygenase enzymes, catalyze the conversion of indole-3-pyruvic acid (IPA) to the auxin (indole acetic acid). Arabidopsis thaliana YUC6 also exhibits thiol-reductase and chaperone activity in vitro; these activities require the highly conserved Cys-85 and are essential for scavenging of toxic reactive oxygen species (ROS) in the drought tolerance response. Here, we examined whether the YUC6 thiol reductase activity also participates in the delay in senescence observed in YUC6-overexpressing (YUC6-OX) plants. YUC6 overexpression delays leaf senescence in natural and dark-induced senescence conditions by reducing the expression of SENESCENCE-ASSOCIATED GENE 12 (SAG12). ROS accumulation normally occurs during senescence, but was not observed in the leaves of YUC6-OX plants; however, ROS accumulation was observed in YUC6-OXC85S plants, which overexpress a mutant YUC6 that lacks thiol reductase activity. We also found that YUC6-OX plants, but not YUC6-OXC85S plants, show upregulation of three genes encoding NADPH-dependent thioredoxin reductases (NTRA, NTRB, and NTRC), and GAMMA-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), encoding an enzyme involved in redox signaling. We further determined that excess ROS accumulation caused by methyl viologen treatment or decreased glutathione levels caused by buthionine sulfoximine treatment can decrease the levels of auxin efflux proteins such as PIN2-4. The expression of PINs is also reduced in YUC6-OX plants. These findings suggest that the thiol reductase activity of YUC6 may play an essential role in delaying senescence via the activation of genes involved in redox signaling and auxin availability. PMID:27242830

  10. The C677T mutation in the methylenetetrahydrofolate reductase gene among the Indonesian Javanese population.

    PubMed

    Sadewa, Ahmad Hamim; Sunarti; Sutomo, Retno; Hayashi, Chiyo; Lee, Myeong Jin; Ayaki, Hitoshi; Sofro, Abdul Salam M; Matsuo, Masafumi; Nishio, Hisahide

    2002-12-01

    The presence of the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene has been regarded as a genetic risk factor for coronary artery diseases and neural tube defects. Although the prevalence of this mutation has been reported from various ethnic populations, few data concerning Indonesian populations are available. We have investigated the frequency of the mutation in 68 Indonesian Javanese (residents of Java Island) and compared it with the data from 244 Japanese (residents of Honshu Island). The frequencies of the three genotypes in Javanese were C/C 0.84, C/T 0.16 and T/T 0.00, whereas those in Japanese were C/C 0.39, C/T 0.48 and T/T 0.13. The rarity of the T/T genotype in the Indonesian Javanese population may be due to malnutrition in pregnant women, because insufficient intake of folate is considered to be a survival disadvantage for fetuses with the T/T genotype. In conclusion, homozygosity for the C677T mutation in the MTHFR gene does not constitute a genetic risk factor for coronary artery diseases and neural tube defects in the Indonesian Javanese population.

  11. Vascular behcet and mutations in thrombogenic genes: methylene tetrahydrofolate reductase, factor V, and prothrombin.

    PubMed

    Dagan, Efrat; Baruch, Yoav; Fiorilli, Massimo; Rozenbaum, Michael; Rosner, Itzhak; Gershoni-Baruch, Ruth

    2012-01-01

    Vasculitis, thrombophlebitis, arterial aneurysms, and occlusions occur in about 25% of patients with Behçet's disease (BD). The common inherited gene defects, factor V (FV) 1691A (Leiden), methylene tetrahydrofolate reductase (MTHFR) 677T, and prothrombin 20210A, are known risk factors for thrombosis. The aim of the study was to evaluate the contribution of these mutations to thrombosis in Israeli patients with BD. Fifty-four patients with BD (n=54; 27 men and 27 women) underwent clinical and genetic evaluation. Most patients (n=43; 79.6%) were of Arab descent (31 sporadic and 12 familial cases from 4 families), and 11 patients (20.4%) were of Jewish descent (all sporadic cases). The FV Leiden mutation was identified in five patients (9.2%), and eight patients were MTHFR 677TT homozygotes (14.8%). None had the 20210A mutant prothrombin allele. No statistical differences between carriers and noncarriers with regards to demographic and disease manifestations were calculated. Arabs were diagnosed earlier than Jewish patients (25.8±11.6 compared with 37.2±10.7, p=0.01, respectively), but Jewish patients had, respectively, more events of deep vein thrombosis (DVT) compared with Arabs (3 of 11, 27.3% and 3 of 43, 7%, p=0.09). Thrombotic events in our patients with BD were not associated with variations in thrombophilic genes.

  12. The HIP1 binding site is required for growth regulation of the dihydrofolate reductase gene promoter.

    PubMed Central

    Means, A L; Slansky, J E; McMahon, S L; Knuth, M W; Farnham, P J

    1992-01-01

    The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to +11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the G1/S boundary. Images PMID:1545788

  13. Expression analysis of the fpr (ferredoxin-NADP{sup +} reductase) gene in Pseudomonas putida KT2440

    SciTech Connect

    Lee, Yunho; Pena-Llopis, Samuel; Kang, Yoon-Suk; Shin, Hyeon-Dong; Demple, Bruce; Madsen, Eugene L.; Jeon, Che Ok; Park, Woojun . E-mail: wpark@korea.ac.kr

    2006-01-27

    The ferredoxin-NADP{sup +} reductase (fpr) participates in cellular defense against oxidative damage. The fpr expression in Pseudomonas putida KT2440 is induced by oxidative and osmotic stresses. FinR, a LysR-type transcriptional factor near the fpr gene in the P. putida KT2440 genome, is required for induction of the fpr under both conditions. We have shown that the fpr and finR gene products can counteract the effects of oxidative and osmotic stresses. Interestingly, FinR-independent expression occurs either during a long period of incubation with paraquat or with high concentrations of oxidative stress agent. This result indicates that there may be additional regulators present in the P. putida KT2440 genome. In contrast to in vivo expression kinetics of fpr from the plant pathogen, Pseudomonas syringae, the fpr gene from P. putida KT2440 exhibited unusually prolonged expression after oxidative stress. Transcriptional fusion and Northern blot analysis studies indicated that the FinR is negatively autoregulated. Expression of the fpr promoter was higher in minimal media than in rich media during exponential phase growth. Consistent with this result, the fpr and finR mutants had a long lag phase in minimal media in contrast to wild-type growth characteristics. Antioxidants such as ascorbate could increase the growth rate of all tested strains in minimal media. This result confirmed that P. putida KT2440 experienced more oxidative stress during exponential growth in minimal media than in rich media. Endogenous promoter activity of the fpr gene is much higher during exponential growth than during stationary growth. These findings demonstrate new relationships between fpr, finR, and the physiology of oxidative stress in P. putida KT2440.

  14. Phylogenetic diversity of dissimilatory sulfite reductase genes from deep-sea cold seep sediment.

    PubMed

    Fukuba, Tatsuhiro; Ogawa, Mari; Fujii, Teruo; Naganuma, Takeshi

    2003-01-01

    The phylogenetic diversity of dissimilatory sulfite reductase (DSR, EC 1.8.99.3) alpha-subunit genes from a deep-sea cold seep was analyzed. Bulk genomic DNA was extracted from the cold seep sediment and used for amplification by polymerase chain reaction (PCR) of DSR alpha-subunit gene. Two sizes of PCR products, 1.4 kb (expected) and 1.3 kb (unexpected), were amplified. Sixteen clones of the 1.4-kb amplicons and 16 clones of 1.3-kb amplicons, a total of 32 clones, were obtained and grouped into operational DSR units (ODUs) based on restriction fragment length polymorphism (RFLP) by digestion with HaeIII and MboI. A total of 14 ODUs, i.e., 5 ODUs from 1.4-kb amplicon clones and 9 ODUs from 1.3-kb amplicon clones, were recovered. About 400 bp of the 5' ends of all the clones was sequenced and validated the RFLP-based ODU grouping. All the 5'-end 400-bp sequences of ODUs, even from the 1.3-kb amplicons, showed the characteristic DSR amino acid sequence motifs. The ODUs from 1.4-kb amplicons were closely related to the delta-Proteobacterial lineage with the DSR genes from epsilon-Proteobacterial epibionts of the hot vent worm Alvinella pompejana. The ODUs from 1.3-kb amplicons were mostly related to the unknown but possibly archaeal lineage. The diversity of the DSR genes may indicate the diversity of sulfate reducers in the seep sediment as well as the complexity of electron donors including methane.

  15. Molecular Characterization of Two Fatty Acyl-CoA Reductase Genes From Phenacoccus solenopsis (Hemiptera: Pseudococcidae).

    PubMed

    Li, Xiaolong; Zheng, Tianxiang; Zheng, Xiaowen; Han, Na; Chen, Xuexin; Zhang, Dayu

    2016-01-01

    Fatty acyl-CoA reductases (FARs) are key enzymes involved in fatty alcohol synthesis. Here, we cloned and characterized full-length cDNAs of two FAR genes from the cotton mealybug, Phenacoccus solenopsis. The results showed PsFAR I and PsFAR II cDNAs were 1,584 bp and 1,515 bp in length respectively. Both PsFAR I and PsFAR II were predicted to be located in the endoplasmic reticulum by Euk-mPLoc 2.0 approach. Both of them had a Rossmann folding region and a FAR_C region. Two conservative motifs were discovered in Rossmann folding region by sequence alignment including a NADPH combining motif, TGXXGG, and an active site motif, YXXXK. A phylogenetic tree made using MEGA 6.06 indicated that PsFAR I and PsFAR II were placed in two different branches. Gene expression analysis performed at different developmental stages showed that the expression of PsFar I is significantly higher than that of PsFar II in first and second instar nymphs and in male adults. Spirotetramat treatment at 125 mg/liter significantly increased the expression of PsFar I in third instar nymphs, but there was no effect in the expression of PsFar II Our results indicated these two FAR genes showed different expression patterns during insect development and after pesticide treatment, suggesting they play different roles in insect development and detoxification against pesticides.

  16. Structure and Promoter Characterization of Aldo-Keto Reductase Family 1 B10 Gene

    PubMed Central

    Liu, Ziwen; Zhong, Linlin; Krishack, Paulette A; Robbins, Sarah; Cao, Julia X; Zhao, Yupei; Chung, Stephen; Cao, Deliang

    2009-01-01

    Aldo-keto reductase family 1 member B10 (AKR1B10) is overexpressed in human hepatocellular carcinoma, lung squamous carcinoma, and lung adenocarcinoma in smokers. Our recent studies have showed that AKR1B10 plays a critical role in the growth and proliferation of cancer cells by detoxifying reactive carbonyls and regulating fatty acid biosynthesis. However, little is known about the regulatory mechanisms of AKR1B10 expression. In this study, we determined the structure of AKR1B10 gene and characterized its promoter. The results demonstrated that AKR1B10 consists of 10 exons and 9 introns, stretching approximately 13.8 kb. A 5′-RACE study determined the transcriptional start site of AKR1B10 at 320 bp upstream of the ATG translational start codon. A TATA-like (TAATAA) and a CAAT box are present from −145 to −140 bp and −193 to −190 bp upstream of the transcriptional start site, respectively. Motif analysis recognized multiple putative oncogenic and tumor suppressor protein binding sites in the AKR1B10 promoter, including c-Ets-1, C/EBP, AP-1, and p53, but osmolytic response elements were not found. A -4,091 bp of the 5′-flanking fragment of the AKR1B10 gene was capable of driving GFP and luciferase reporter gene expression in HepG2 cells derived from human hepatocellular carcinoma; progressive 5′-deletions revealed that a −255 bp fragment possesses full promoter activity. PMID:19236911

  17. Molecular Characterization of Two Fatty Acyl-CoA Reductase Genes From Phenacoccus solenopsis (Hemiptera: Pseudococcidae)

    PubMed Central

    Li, Xiaolong; Zheng, Tianxiang; Zheng, Xiaowen; Han, Na; Chen, Xuexin; Zhang, Dayu

    2016-01-01

    Fatty acyl-CoA reductases (FARs) are key enzymes involved in fatty alcohol synthesis. Here, we cloned and characterized full-length cDNAs of two FAR genes from the cotton mealybug, Phenacoccus solenopsis. The results showed PsFAR I and PsFAR II cDNAs were 1,584 bp and 1,515 bp in length respectively. Both PsFAR I and PsFAR II were predicted to be located in the endoplasmic reticulum by Euk-mPLoc 2.0 approach. Both of them had a Rossmann folding region and a FAR_C region. Two conservative motifs were discovered in Rossmann folding region by sequence alignment including a NADPH combining motif, TGXXGG, and an active site motif, YXXXK. A phylogenetic tree made using MEGA 6.06 indicated that PsFAR I and PsFAR II were placed in two different branches. Gene expression analysis performed at different developmental stages showed that the expression of PsFar I is significantly higher than that of PsFar II in first and second instar nymphs and in male adults. Spirotetramat treatment at 125 mg/liter significantly increased the expression of PsFar I in third instar nymphs, but there was no effect in the expression of PsFar II. Our results indicated these two FAR genes showed different expression patterns during insect development and after pesticide treatment, suggesting they play different roles in insect development and detoxification against pesticides. PMID:27271970

  18. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    PubMed

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  19. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    PubMed Central

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions. PMID:8597660

  20. Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum.

    PubMed

    Jargeat, Patricia; Rekangalt, David; Verner, Marie-Christine; Gay, Gilles; Debaud, Jean-Claude; Marmeisse, Roland; Fraissinet-Tachet, Laurence

    2003-06-01

    Symbiotic ectomycorrhizal fungi contribute to the nitrogen nutrition of their host-plants but little information is available on the molecular control of their nitrogen metabolism. We cloned and characterised genes encoding a nitrite reductase and a nitrate transporter in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. These two genes are divergently transcribed and linked to a previously cloned nitrate reductase gene, thus demonstrating that nitrate assimilation gene clusters occur in homobasidiomycetes. The nitrate transporter polypeptide (NRT2) is characterised by 12 transmembrane domains and presents both a long putative intracellular loop and a short C-terminal tail, two structural features which distinguish fungal high-affinity transporters from their plant homologues. In different wild-type genetic backgrounds, transcription of the two genes was repressed by ammonium and was strongly stimulated not only in the presence of nitrate but also in the presence of organic nitrogen sources or under nitrogen deficiency.

  1. Identification of an L-arabinose reductase gene in Aspergillus niger and its role in L-arabinose catabolism.

    PubMed

    Mojzita, Dominik; Penttilä, Merja; Richard, Peter

    2010-07-30

    The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K(m) for l-arabinose is 54 + or - 6 mm and for d-xylose 155 + or - 15 mm.

  2. Characteristic analysis of the luxG gene encoding the probable flavin reductase that resides in the lux operon of Photobacterium leiognathi.

    PubMed

    Lin, J W; Chao, Y F; Weng, S F

    1998-05-19

    Nucleotide sequence of the luxG gene (GenBank Accession No. AF053227) from Photobacterium leiognathi PL741 has been determined, and the encoded probable flavin reductase is deduced. The probable flavin reductase encoded by the luxG gene has a calculated M(r) 26,544 and comprises 235 amino acid residues. The probable flavin reductase like the NAD(P)H-flavin reductase might catalyze the reduction of flavins. Alignment and comparison of the probable flavin reductases from P. leiognathi PL741, ATCC 25521, P. phosphoreum, Vibrio fischeri, and V. harveyi show that they are homologous; there is 66% homologous (29.4% identity and 36.6% similarity). Also, the probable flavin reductase is homologous to the NAD(P)H-flavin reductase; it is perceived that the probable flavin reductase and the NAD(P)H-flavin reductase could be enzyme isoforms encoded by two genes of a multigene family for differential response functions. Functional analysis illustrates that the specific segment sequence lay inside and behind the luxG gene might form the potential hairpin loops omega gI, omega gII, omega o, and omega oT as mRNA stability loop or/and as the attenuator-like loop or the dynamic terminator-like block for sub-regulation in the lux operon. The gene order of the luxG gene in the lux operon and the lum operon is <--ter-lumQ-lumP-R&R-luxC-luxD-luxA-luxB-+ ++luxN-luxE-luxG--> (R&R: regulatory region; ter: transcriptional terminator), whereas the R&R is the regulatory region for the lum operon and the lux operon, and ter is the transcriptional terminator for the lum operon.

  3. Reduced impact of pyrimethamine drug pressure on Plasmodium malariae dihydrofolate reductase gene.

    PubMed

    Khim, Nimol; Kim, Saorin; Bouchier, Christiane; Tichit, Magali; Ariey, Frédéric; Fandeur, Thierry; Chim, Pheaktra; Ke, Sopheakvatey; Sum, Sarorn; Man, Somnang; Ratsimbasoa, Arsène; Durand, Rémy; Ménard, Didier

    2012-02-01

    Molecular investigations performed following the emergence of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum have allowed the identification of the dihydrofolate reductase (DHFR) enzyme as the target of pyrimethamine. Although clinical cases of Plasmodium malariae are not usually treated with antifolate therapy, incorrect diagnosis and the high frequency of undetected mixed infections has probably exposed non-P. falciparum parasites to antifolate therapy in many areas. In this context, we aimed to assess the worldwide genetic diversity of the P. malariae dhfr gene in 123 samples collected in Africa and Asia, areas with different histories of SP use. Among the 10 polymorphic sites found, we have observed 7 new mutations (K55E, S58R, S59A, F168S, N194S, D207G, and T221A), which led us to describe 6 new DHFR proteins. All isolates from African countries were classified as wild type, while new mutations and haplotypes were recognized as exclusive to Madagascar (except for the double mutations at nucleotides 341 and 342 [S114N] found in one Cambodian isolate). Among these nonsynonymous mutations, two were likely related to pyrimethamine resistance: S58R (corresponding to C59R in P. falciparum and S58R in Plasmodium vivax; observed in one Malagasy sample) and S114N (corresponding to S108N in P. falciparum and S117N in P. vivax; observed in three Cambodian samples).

  4. Reduced Impact of Pyrimethamine Drug Pressure on Plasmodium malariae Dihydrofolate Reductase Gene

    PubMed Central

    Khim, Nimol; Kim, Saorin; Bouchier, Christiane; Tichit, Magali; Ariey, Frédéric; Fandeur, Thierry; Chim, Pheaktra; Ke, Sopheakvatey; Sum, Sarorn; Man, Somnang; Ratsimbasoa, Arsène; Durand, Rémy

    2012-01-01

    Molecular investigations performed following the emergence of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum have allowed the identification of the dihydrofolate reductase (DHFR) enzyme as the target of pyrimethamine. Although clinical cases of Plasmodium malariae are not usually treated with antifolate therapy, incorrect diagnosis and the high frequency of undetected mixed infections has probably exposed non-P. falciparum parasites to antifolate therapy in many areas. In this context, we aimed to assess the worldwide genetic diversity of the P. malariae dhfr gene in 123 samples collected in Africa and Asia, areas with different histories of SP use. Among the 10 polymorphic sites found, we have observed 7 new mutations (K55E, S58R, S59A, F168S, N194S, D207G, and T221A), which led us to describe 6 new DHFR proteins. All isolates from African countries were classified as wild type, while new mutations and haplotypes were recognized as exclusive to Madagascar (except for the double mutations at nucleotides 341 and 342 [S114N] found in one Cambodian isolate). Among these nonsynonymous mutations, two were likely related to pyrimethamine resistance: S58R (corresponding to C59R in P. falciparum and S58R in Plasmodium vivax; observed in one Malagasy sample) and S114N (corresponding to S108N in P. falciparum and S117N in P. vivax; observed in three Cambodian samples). PMID:22123682

  5. Isolation and characterization of an enoyl-acyl carrier protein reductase gene from microalga Isochrysis galbana

    NASA Astrophysics Data System (ADS)

    Zheng, Minggang; Liang, Kepeng; Wang, Bo; Sun, Xiuqin; Yue, Yanyan; Wan, Wenwen; Zheng, Li

    2013-03-01

    In most bacteria, plants and algae, fatty acid biosynthesis is catalyzed by a group of freely dissociable proteins known as the type II fatty acid synthase (FAS II) system. In the FAS II system, enoylacyl carrier protein reductase (ENR) acts as a determinant for completing the cycles of fatty acid elongation. In this study, the cDNA sequence of ENR, designated as IgENR, was isolated from the microalga Isochrysis galbana CCMM5001. RACE (rapid amplification of cDNA ends) was used to isolate the full-length cDNA of IgENR (1 503 bp), which contains an open reading frame (ORF) of 1 044 bp and encodes a protein of 347 amino acids. The genomic DNA sequence of IgENR is interrupted by four introns. The putative amino acid sequence is homologous to the ENRs of seed plants and algae, and they contain common coenzymebinding sites and active site motifs. Under different stress conditions, real-time quantitative polymerase chain reaction (RT-qPCR) showed the expression of IgENR was upregulated by high temperature (35°C), and downregulated by depleted nitrogen (0 mol/L). To clarify the mechanism of lipids accumulating lipids, other genes involved in lipids accumulation should be studied.

  6. Ascorbic acid contents in transgenic potato plants overexpressing two dehydroascorbate reductase genes.

    PubMed

    Qin, Aiguo; Shi, Qinghua; Yu, Xianchang

    2011-03-01

    Ascorbic acid (AsA, vitamin C) is one of the most important nutritional quality factors in many horticultural crops and has many biological activities in the human body. Dehydroascorbate reductase (EC 1.8.5.1; DHAR) plays an important role in maintaining the normal level of ascorbic acid (AsA) by recycling oxidized ascorbic acid. To increase AsA content of potato, we isolated and characterized the cDNAs encoding two isoform DHARs localized in cytosol and chloroplast from potato, and developed two types of transgenic potato plants overexpressing cytosolic DHAR gene and chloroplastic DHAR, respectively. Incorporation of the transgene in the genome of potato was confirmed by PCR and real time RT-PCR. The overexpression of cytosolic DHAR significantly increased DHAR activities and AsA contents in potato leaves and tubers, whereas chloroplastic DHAR overexpression only increased DHAR activities and AsA contents in leaves, and did not change them in tubers. These results indicated that AsA content of potato can be elevated by enhancing recycling ascorbate via DHAR overexpression, moreover, cytosolic DHAR might play main important roles in improving the AsA contents of potato tubers.

  7. Molecular cloning and functional characterization of the anthocyanidin reductase gene from Vitis bellula.

    PubMed

    Zhu, Yue; Peng, Qing-Zhong; Li, Ke-Gang; Xie, De-Yu

    2014-08-01

    Anthocyanidin reductase (ANR) is an NADPH-/NADH-dependent enzyme that transfers two hydrides to anthocyanidins to produce three types of isomeric flavan-3-ols. This reductase forms the ANR pathway toward the biosynthesis of proanthocyanidins (PAs, which are also called condensed tannins). Here, we report cloning and functional characterization of an ANR (called VbANR) homolog from the leaves of Vitis bellula, a newly developed grape crop in southern China. The open reading frame (ORF) of VbANR is 1,017 bp in length and encodes 339 amino acids. A phylogenetic analysis and an alignment using 17 sequences revealed that VbANR is approximately 99.9 % identical to the ANR homolog from Vitis vinifera. The VbANR ORF is fused to the Trx gene containing a His-tag in the pET32a(+) vector to obtain a pET32a(+)-VbANR construct for expressing the recombinant VbANR. In vitro enzyme assays show that VbANR converts cyanidin, delphinidin, and pelargonidin to their corresponding flavan-3-ols. Enzymatic products include 2S,3R-trans- and 2R,3R-cis-flavan-3-ols isomers, such as (-)-catechin and (-)-epicatechin. In addition, the third compound that is observed from the enzymatic products is most likely a 2S,3S-cis-flavan-3-ol. To analyze the kinetics and optimize pH and temperature values, a UV spectrometry method was developed to quantify the concentrations of total enzymatic products. The optimum pH and temperature values are 4.0 and 40 °C, respectively. The K m , K cat, V max, and K cat/K m values for pelargonidin and delphinidin were similar. In comparison, VbANR exhibits a slightly lower affinity to cyanidin. VbANR uses both NADPH and NADH but prefers to employ NADPH. GFP fusion and confocal microscopy analyses revealed the cytosolic localization of VbANR. The overexpression of VbANR in ban mutants reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate that VbANR forms the ANR pathway, leading to the formation of three types of isomeric flavan-3-ols

  8. Detection and Diversity of Fungal Nitric Oxide Reductase Genes (p450nor) in Agricultural Soils

    PubMed Central

    Welsh, Allana; Orellana, Luis H.; Konstantinidis, Konstantinos T.; Chee-Sanford, Joanne C.; Sanford, Robert A.; Schadt, Christopher W.

    2016-01-01

    ABSTRACT Members of the Fungi convert nitrate (NO3−) and nitrite (NO2−) to gaseous nitrous oxide (N2O) (denitrification), but the fungal contributions to N loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations, and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N2O from added NO3− or NO2− in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, 151 of which produced N2O from NO2−. Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N2O production in fungi, yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54 to 98% amino acid identity with reference P450nor sequences within the phylum Ascomycota and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from NO2−, whereas nirK (encoding the NO-forming NO2− reductase) was amplified in only 13 to 74% of the N2O-forming isolates using two separate nirK primer sets. Collectively, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation. IMPORTANCE A comprehensive understanding of the microbiota controlling soil N loss and greenhouse gas (N2O) emissions is crucial for sustainable agricultural practices and addressing climate change concerns. We report the design and application of a novel PCR primer set targeting fungal p450nor, a biomarker for fungal N2O production, and demonstrate the utility of the new approach to assess fungal denitrification

  9. A bioinformatic analysis of ribonucleotide reductase genes in phage genomes and metagenomes

    PubMed Central

    2013-01-01

    Background Ribonucleotide reductase (RNR), the enzyme responsible for the formation of deoxyribonucleotides from ribonucleotides, is found in all domains of life and many viral genomes. RNRs are also amongst the most abundant genes identified in environmental metagenomes. This study focused on understanding the distribution, diversity, and evolution of RNRs in phages (viruses that infect bacteria). Hidden Markov Model profiles were used to analyze the proteins encoded by 685 completely sequenced double-stranded DNA phages and 22 environmental viral metagenomes to identify RNR homologs in cultured phages and uncultured viral communities, respectively. Results RNRs were identified in 128 phage genomes, nearly tripling the number of phages known to encode RNRs. Class I RNR was the most common RNR class observed in phages (70%), followed by class II (29%) and class III (28%). Twenty-eight percent of the phages contained genes belonging to multiple RNR classes. RNR class distribution varied according to phage type, isolation environment, and the host’s ability to utilize oxygen. The majority of the phages containing RNRs are Myoviridae (65%), followed by Siphoviridae (30%) and Podoviridae (3%). The phylogeny and genomic organization of phage and host RNRs reveal several distinct evolutionary scenarios involving horizontal gene transfer, co-evolution, and differential selection pressure. Several putative split RNR genes interrupted by self-splicing introns or inteins were identified, providing further evidence for the role of frequent genetic exchange. Finally, viral metagenomic data indicate that RNRs are prevalent and highly dynamic in uncultured viral communities, necessitating future research to determine the environmental conditions under which RNRs provide a selective advantage. Conclusions This comprehensive study describes the distribution, diversity, and evolution of RNRs in phage genomes and environmental viral metagenomes. The distinct distributions of

  10. The nitrate reductase-encoding gene of Volvox carteri: map location, sequence and induction kinetics.

    PubMed

    Gruber, H; Goetinck, S D; Kirk, D L; Schmitt, R

    1992-10-12

    The nitrate reductase (NR) structural gene (nitA) of Volvox carteri has been cloned and characterized. There is a single copy of this gene in the genome, and RFLP (restriction-fragment length polymorphism) analysis assigns it to the previously defined nitA/chlR locus on linkage group IX, 20-30 cM from the two beta-tubulin-encoding loci. Determination of the 5871-nt sequence of the coding region of genomic clones, and comparisons to a cDNA sequence, revealed ten introns and eleven exons that encode a 864-aa polypeptide. Detailed comparisons with higher-plant and fungal NRs indicate that, whereas the aa sequence is strongly conserved within functional domains for the flavin adenine dinucleotide-, heme- and molybdenum-pterin cofactor-binding sites, substantial differences in the aa sequence occur in the N-terminal end and the two inter-domain regions. Two potential transcription start points 439 and 452 nt upstream from the start codon and a polyadenylation signal 355 nt downstream from the stop codon have been identified by primer-extension analysis and cDNA sequencing, respectively. Accumulation of the nitA transcript is both induced by nitrate and repressed by ammonium and urea: after the organism is transferred from ammonium to nitrate as the nitrogen source, a 3.6-kb NR transcript is readily detectable on Northern blots by 10 min, reaches maximum abundance by 30 min, and then rapidly declines to an intermediate level that is subsequently maintained. Substantial induction by nitrate is observed at the end of the dark portion of the daily light/dark cycle, but the inductive response peaks in the first hour of the light period.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Ectopic Expression of a Basic Helix-Loop-Helix Gene Transactivates Parallel Pathways of Proanthocyanidin Biosynthesis. Structure, Expression Analysis, and Genetic Control of Leucoanthocyanidin 4-Reductase and Anthocyanidin Reductase Genes in Lotus corniculatus1[W

    PubMed Central

    Paolocci, Francesco; Robbins, Mark P.; Madeo, Laura; Arcioni, Sergio; Martens, Stefan; Damiani, Francesco

    2007-01-01

    Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis. PMID:17098849

  12. Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus.

    PubMed

    Paolocci, Francesco; Robbins, Mark P; Madeo, Laura; Arcioni, Sergio; Martens, Stefan; Damiani, Francesco

    2007-01-01

    Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

  13. Fine-tuned regulation of the dissimilatory nitrite reductase gene by oxygen and nitric oxide in Pseudomonas aeruginosa.

    PubMed

    Kuroki, Miho; Igarashi, Yasuo; Ishii, Masaharu; Arai, Hiroyuki

    2014-12-01

    Nitrite reductase (NIR) catalyses the reduction of nitrite to nitric oxide (NO) in the denitrification pathway. In Pseudomonas aeruginosa, expression of the gene encoding NIR (nirS) is induced by NO and is under control of the NO-sensing regulator DNR (dissimilatory nitrate respiration regulator). Because DNR is under control of the oxygen-sensing regulator ANR (anaerobic regulator of arginine deiminase and nitrate reductase), nirS is expressed only under low oxygen and anaerobic conditions. Both ANR and DNR are FNR (fumarate and nitrate reductase regulator)-type regulators and recognize the consensus FNR-binding motif. The motif of the nirS promoter is thought to be recognized only by DNR, and not by ANR. Here, mutant strains expressing either ANR or DNR were constructed and used to analyse the role of ANR and DNR in the activation of nirS expression. Analysis of transcriptional activity by microarray and quantitative reverse transcription polymerase chain reaction revealed that nirS is transcribed under low oxygen conditions in an ANR-dependent manner, although the expression level was 10-fold lower than that of the DNR-dependent expression. An artificial promoter containing the FNR-binding motif of the nirS promoter was also twofold upregulated by ANR. These results indicate that low-level expression of NIR in the presence of nitrite may provide NO as a trigger for the full expression of denitrification genes when oxygen is depleted.

  14. Diversity and abundance of nitrate reductase genes (narG and napA), nitrite reductase genes (nirS and nrfA), and their transcripts in estuarine sediments.

    PubMed

    Smith, Cindy J; Nedwell, David B; Dong, Liang F; Osborn, A Mark

    2007-06-01

    Estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrial-anthropogenic sources into marine ecosystems. Within estuarine sediments some microbially driven processes (denitrification and anammox) result in the net removal of nitrogen from the environment, while others (dissimilatory nitrate reduction to ammonium) do not. In this study, molecular approaches have been used to investigate the diversity, abundance, and activity of the nitrate-reducing communities in sediments from the hypernutrified Colne estuary, United Kingdom, via analysis of nitrate and nitrite reductase genes and transcripts. Sequence analysis of cloned PCR-amplified narG, napA, and nrfA gene sequences showed the indigenous nitrate-reducing communities to be both phylogenetically diverse and also divergent from previously characterized nitrate reduction sequences in soils and offshore marine sediments and from cultured nitrate reducers. In both the narG and nrfA libraries, the majority of clones (48% and 50%, respectively) were related to corresponding sequences from delta-proteobacteria. A suite of quantitative PCR primers and TaqMan probes was then developed to quantify phylotype-specific nitrate (narG and napA) and nitrite reductase (nirS and nrfA) gene and transcript numbers in sediments from three sites along the estuarine nitrate gradient. In general, both nitrate and nitrite reductase gene copy numbers were found to decline significantly (P < 0.05) from the estuary head towards the estuary mouth. The development and application, for the first time, of quantitative reverse transcription-PCR assays to quantify mRNA sequences in sediments revealed that transcript numbers for three of the five phylotypes quantified were greatest at the estuary head.

  15. Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum.

    PubMed

    Wankhede, Dhammaprakash Pandhari; Biswas, Dipul Kumar; Rajkumar, Subramani; Sinha, Alok Krishna

    2013-12-01

    Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

  16. Chlorate reductase is cotranscribed with cytochrome c and other downstream genes in the gene cluster for chlorate respiration of Ideonella dechloratans.

    PubMed

    Hellberg Lindqvist, Miriam; Nilsson, Thomas; Sundin, Pontus; Rova, Maria

    2015-03-01

    The chlorate-respiring bacterium Ideonella dechloratans is a facultative anaerobe that can use both oxygen and chlorate as terminal electron acceptors. The genes for the enzymes chlorate reductase (clrABDC) and chlorite dismutase, necessary for chlorate metabolism and probably acquired by lateral gene transfer, are located in a gene cluster that also includes other genes potentially important for chlorate metabolism. Among those are a gene for cytochrome c (cyc) whose gene product may serve as an electron carrier during chlorate reduction, a cofactor biosynthesis gene (mobB) and a predicted transcriptional regulator (arsR). Only chlorate reductase and chlorite dismutase have been shown to be expressed in vivo. Here, we report the in vivo production of a single polycistronic transcript covering eight open reading frames including clrABDC, cyc, mobB and arsR. Transcription levels of the cyc and clrA genes were compared to each other by the use of qRT-PCR in RNA preparations from cells grown under aerobic or chlorate reducing anaerobic conditions. The two genes showed the same mRNA levels under both growth regimes, indicating that no transcription termination occurs between them. Higher transcription levels were observed at growth without external oxygen supply. Implications for electron pathway integration following lateral gene transfer are discussed.

  17. Molecular study of the 5 {alpha}-reductase type 2 gene in three European families with 5 {alpha}-reductase deficiency

    SciTech Connect

    Boudon, C.; Lumbroso, S.; Lobaccaro, J.M. ||

    1995-07-01

    The molecular basis of 5{alpha}-reductase (5{alpha}R) deficiency was investigated in four patients from three European families. In the French family, the first patient was raised as a female, and gonadectomy was performed before puberty. The second sibling, also raised as female, differed in that gonadal removal was performed after the onset of pubertal masculinization. The other two patients, both from Polish families, developed masculinization of external genitalia during puberty. All patients developed a female sexual identity. In all cases, no known consanguinity or family history of 5{alpha}R deficiency was reported. The genomic DNAs of the patients were sequenced after polymerase chain reaction amplification of the five exons of the 5{alpha}R type 2 gene. We found two homozygous mutations responsible for gutamine to arginine and histidine to arginine substitution in families 1 and 3, respectively. In family 2, we found a heterozygous mutation responsible for an asparagine to serine substitution at position 193. The glutamine/arginine 126 mutation in the French family was previously reported in a Creole ethnic group, and the Polish histidine/arginine 231 mutation was previously reported in a patient from Chicago, Moreover, all of the mutations created new restriction sites, which were used to determine the kindred carrier status in the three families. Because 5{alpha}R deficiency is known to be heterogenous disease in terms of clinical and biochemical expression, our data suggest that molecular biology analysis of the type 2 gene could be an essential step in diagnosing 5{alpha}R deficiency. 22 refs., 3 figs., 1 tab.

  18. Impact of methionine synthase gene and methylenetetrahydrofolate reductase gene polymorphisms on the risk of sudden sensorineural hearing loss.

    PubMed

    Gross, Menachem; Friedman, Gideon; Eliashar, Ron; Koren-Morag, Nira; Goldschmidt, Neta; Atta, Iman Abou; Ben-Yehuda, Arie

    2006-01-01

    Idiopathic sudden sensorineural hearing loss (SSNHL) represents a frequently encountered otological disease of unknown etiology. In recent years, several inherited risk factors have been found in the pathogenesis of vascular diseases. In the present study, we determined whether specific polymorphism or the combination of polymorphisms in folate-dependent homocysteine metabolism genes can act as predisposing inherited vascular risk factors in the development of SSNHL. We conducted a prospective case-control study using DNA samples extracted from 81 patients diagnosed as suffering from SSNHL and 264 healthy control subjects. Three functional polymorphisms were analyzed by polymerase chain reaction amplification, restriction enzyme digestion, and DNA fragment separation by electrophoresis: methylenetetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C, and methionine synthase (MTR) A2756G polymorphisms. The prevalence of the homozygous genotype of MTR 2756GG in the SSNHL patients (9%) was significantly higher than in the control group (4%) (p = 0.011). The allelic frequency of the G allele of the MTR A2756G polymorphism among SSNHL patients (12.5%) was also significantly higher than in the control group (5%) (p = 0.033). The prevalence of patients possessing two polymorphisms (31%) and three polymorphisms (17%) in the SSNHL group was significantly higher than in the control group (23 and 9%, respectively; p = 0.019). The frequency of patients with a very high rank risk (double homozygous) was significantly higher in the SSNHL group, MTHFR 677TT/MTR 2675GG--7%, than the frequency of patients in the control group, MTHFR 677TT/MTR 2675GG--3% (p = 0.030). Certain polymorphisms in genes encoding enzymes in the folate-dependent homocysteine metabolism are associated with SSNHL. In our case-control study, a significant association between MTR 2756GG genotype and SSNHL was found which may represent an inherited vascular risk factor in the pathogenesis of SSNHL.

  19. C677T mutation in methylenetetrahydrofolate reductase gene and neural tube defects: should Japanese women undergo gene screening before pregnancy?

    PubMed

    Kondo, Atsuo; Fukuda, Hiromi; Matsuo, Takuya; Shinozaki, Keiko; Okai, Ikuyo

    2014-02-01

    We analyzed the role of maternal C677T mutation in methylenetetrahydrofolate reductase (MTHFR) gene on spina bifida development in newborns. A total of 115 mothers who had given birth to a spina bifida child (SB mothers) gave 10 mL of blood together with written informed consent. The genotype distribution of C677T mutation was assessed and compared with that of the 4517 control individuals. The prevalence of the homozygous genotype (TT) among SB mothers was not significantly different from that among the controls (odds ratio [OR] = 0.65; 95% confidence interval [CI] = 0.31-1.25; P = 0.182), suggesting that MTHFR 677TT genotype in Japan is not associated with spina bifida development in newborns. The T allele frequency was not increased in SB mothers (34.8%) as compared to that of the control individuals (38.2%). Further, the internationally reported association between the two groups was found to be similar in all 15 countries studied except the Netherlands, where the TT genotype was found to be a genetic risk factor for spina bifida. For the prevention of affected pregnancy every woman planning to conceive has to take folic acid supplements 400 μg a day and the government is asked to take action in implementing food fortification with folic acid in the near future. In conclusion, it is not necessary for Japanese women to undergo genetic screening C677T mutation of the MTHFR gene as a predictive marker for spina bifida prior to pregnancy, because the TT genotype is not a risk factor for having an affected infant.

  20. Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum.

    PubMed

    Velasco, L; Mesa, S; Delgado, M J; Bedmar, E J

    2001-10-31

    The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to other Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the sequence upstream from the structural nirK gene revealed the presence of an anaerobox located 83 base pairs from the putative translational start codon. Cells of strain GRK308, a nitrite reductase-deficient derivative of strain USDA110, were unable to grow when cultured under microaerobic conditions (1% O(2)) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Expression of beta-galactosidase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubation of the cells under oxygen-limiting conditions either with or without nitrate. Complementation of B. japonicum 9043 with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxygen-responsive two-component regulatory system FixLJ and on the Fnr/FixK-like DNA binding protein FixK(2).

  1. Positional cloning in Cryptococcus neoformans and its application for identification and cloning of the gene encoding methylenetetrahydrofolate reductase.

    PubMed

    Toh-E, Akio; Ohkusu, Misako; Shimizu, Kiminori; Kawamoto, Susumu

    2015-03-01

    Cryptococcus neoformans, a basidiomycetous human pathogenic yeast, has been widely used in research fields in medical mycology as well as basic biology. Gene cloning or identification of the gene responsible for a mutation of interest is a key step for functional analysis of a particular gene. The availability therefore, of the multiple methods for cloning is desirable. In this study, we proposed a method for a mapping-based gene identification/cloning (positional cloning) method in C. neoformans. To this end, we constructed a series of tester strains, one of whose chromosomes was labeled with the URA5 gene. A heterozygous diploid constructed by crossing one of the tester strains to a mutant strain of interest loses a chromosome(s) spontaneously, which is the basis for assigning a recessive mutant gene to a particular chromosome in the mitotic mapping method. Once the gene of interest is mapped to one of the 14 chromosomes, classical genetic crosses can then be performed to determine its more precise location. The positional information thus obtained can then be used to significantly narrow down candidate genes by referring to the Cryptococcus genome database. Each candidate gene is then examined whether it would complement the mutation. We successfully applied this method to identify CNA07390 encoding methylenetetrahydrofolate reductase as the gene responsible for a methionine-requiring mutant in our mutant collection.

  2. L-Methylfolate supplementation in a child with autism and methyltetrahydrofolate reductase, enzyme gene C677TT allele.

    PubMed

    Siscoe, Kim S; Lohr, W David

    2017-03-07

    Errors in folate metabolism may play a role in the pathology of autism spectrum disorders because of increased vulnerability to oxidative stress. We report a case where L-methylfolate supplementation improved symptoms of aggression and disruptive behavior in a child with autism who tested positive for the C677TT allele of the methyltetrahydrofolate reductase enzyme gene. To our knowledge, this is the first report of L-methylfolate administration in this situation. Further controlled studies of L-methylfolate in this population are warranted.

  3. Construction of a Specialized Cloning Strain of E. Coli for the Nitrate Reductase Genes of Haloferax Denitrificans

    NASA Technical Reports Server (NTRS)

    Johnson, Emmett

    1999-01-01

    This is the final report on Joint Research Interchange (NCC2-5011) "Construction of a Specialized Cloning Strain of E.. coli for the Nitrate Reductase Genes of Haloferax denitrificans." Originally the award was 11/l/93-10/31/95, but there were no-cost extensions made, because of a year Sabbatical at the Pasteur Institute in Paris and other leaves of 3 months each at the Pasteur Institute, during which work could not be done on this project, which extended the closing date to 10/30/98.

  4. Induction of methotrexate resistance by retroviral-mediated transfer of a mutant dihydrofolate reductase gene

    SciTech Connect

    Ricciardone, M.D.

    1986-01-01

    Methotrexate (MTX), a folate analog which inhibits the enzyme dihydrofolate reductase (DHFR), is an effective antineoplastic drug. However, MTX-induced myelosuppression limits the effectiveness of this agent. Selective induction of MTX resistance in bone marrow stem cells, prior to treatment with MTX, might prevent this toxicity and improve the therapeutic index of the drug. In these studies drug resistance was transferred to mouse and human bone marrow stem cells by retroviral expression vectors containing coding sequences of a mutant DHFR with a decreased affinity for MTX. Three retroviral expression vectors were analyzed. The CIS DR vector contained the mutant DHFR gene inserted into the replication-defective amphotropic 4070 virus, Cistor. The other vectors contained the mutant DHFR inserted into either the env region (SDHT1) or gag-pol region (SDHT2) of a replication-defective spleen focus-forming virus. All three constructs induced approximately a 200-fold resistance to MTX when transfected into NIH3T3 cells. Amphotropic infectious retroviruses were obtained by transfecting the mutant DHFR vectors into a packaging cell line, which supplied the gag, pol, and env proteins for virus production. Virus titers of 4.5 x 10/sup 3/ colony-forming units (CFU)/ml (CIS DR), 1.5 x 10/sup 4/ CFU/ml (SDHT2), and 5 x 10/sup 5/ CFU/ml (SDHT1) were measured by the transfer of MTX resistance to NIH3T3 cells. The amphotropic SDHT1 virus efficiently induced MTX resistance in cells of several species, including mouse NIH3T3 cells (5 x 10/sup 5/ CFU/ml), monkey CV1 cells (4 x 10/sup 3/ CFU/ml), and human MCF-7 cells (6 x 10/sup 4/ CFU/ml). When cocultured with SDHT1 virus-producing cells, both mouse and human bone marrow cells could be infected and rendered resistant to MTX. Mouse cytotoxic T lymphocytes and mouse helper T lymphocytes can also be made resistant to MTX.

  5. Methylenetetrahydrofolate reductase gene C677T polymorphism and breast cancer risk: Evidence for genetic susceptibility

    PubMed Central

    Kumar, Pradeep; Yadav, Upendra; Rai, Vandana

    2015-01-01

    There are several evidences supporting the role of 5–10 methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms in breast cancer (BC). Case control association studies on breast cancer have been repeatedly performed over the last two decades, but results are inconsistent. We performed a meta-analysis to confirm the association between MTHFR C677T polymorphism and BC risk. The articles were retrieved by searching the PubMed, Google Scholar, and Springer Link databases. Crude odds ratios (OR) with 95% confidence intervals (CIs) was used to assess the strength of association between C677T polymorphism and BC. Publication bias was assessed by Egger's and Begg-Mazumdar tests. Meta-analysis was performed with Open Meta Analyst. Total 75 studies with 31,315 cases and 35, 608 controls were found suitable for the inclusion in the present meta-analysis. The results of meta-analysis suggested that there were moderate significant association between C677T polymorphism and BC risk using overall comparisons in five genetic models (T vs. C: OR = 1.08, 95% CI = 1.03–1.13, p = < 0.001; TT + CT vs. CC: OR = 1.06, 95% CI = 1.02–1.09, p = < 0.001; TT vs. CC: OR = 1.17, 95% CI = 1.06–1.28, p = 0.001; CT vs. CC OR = 1.05, 95% CI = 1.01–1.08, p = 0.005; TT vs. CT + CC: OR = 1.12, 95% CI = 1.03–1.22, p = 0.005). In conclusion, results of present meta-analysis showed modest association between MTHFR C677T polymorphism with breast cancer in total studies. However, sub-group analysis results based on ethnicity showed strong significant association between TT genotype and breast cancer (TT vs. CC; OR°=°1.26; 95% CI: 1.06–1.51; p = 0.009) in Asian population but in Caucasian population such association was not observed (TT vs. CC; OR°=°1.08; 95% CI: 0.99–1.14; p = 0.05). PMID:26629412

  6. Methylenetetrahydrofolate reductase gene C677T polymorphism and breast cancer risk: Evidence for genetic susceptibility.

    PubMed

    Kumar, Pradeep; Yadav, Upendra; Rai, Vandana

    2015-12-01

    There are several evidences supporting the role of 5-10 methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms in breast cancer (BC). Case control association studies on breast cancer have been repeatedly performed over the last two decades, but results are inconsistent. We performed a meta-analysis to confirm the association between MTHFR C677T polymorphism and BC risk. The articles were retrieved by searching the PubMed, Google Scholar, and Springer Link databases. Crude odds ratios (OR) with 95% confidence intervals (CIs) was used to assess the strength of association between C677T polymorphism and BC. Publication bias was assessed by Egger's and Begg-Mazumdar tests. Meta-analysis was performed with Open Meta Analyst. Total 75 studies with 31,315 cases and 35, 608 controls were found suitable for the inclusion in the present meta-analysis. The results of meta-analysis suggested that there were moderate significant association between C677T polymorphism and BC risk using overall comparisons in five genetic models (T vs. C: OR = 1.08, 95% CI = 1.03-1.13, p = < 0.001; TT + CT vs. CC: OR = 1.06, 95% CI = 1.02-1.09, p = < 0.001; TT vs. CC: OR = 1.17, 95% CI = 1.06-1.28, p = 0.001; CT vs. CC OR = 1.05, 95% CI = 1.01-1.08, p = 0.005; TT vs. CT + CC: OR = 1.12, 95% CI = 1.03-1.22, p = 0.005). In conclusion, results of present meta-analysis showed modest association between MTHFR C677T polymorphism with breast cancer in total studies. However, sub-group analysis results based on ethnicity showed strong significant association between TT genotype and breast cancer (TT vs. CC; OR°=°1.26; 95% CI: 1.06-1.51; p = 0.009) in Asian population but in Caucasian population such association was not observed (TT vs. CC; OR°=°1.08; 95% CI: 0.99-1.14; p = 0.05).

  7. Characterization of the norCBQD genes, encoding nitric oxide reductase, in the nitrogen fixing bacterium Bradyrhizobium japonicum.

    PubMed

    Mesa, Socorro; Velasco, Leonardo; Manzanera, Maximino E; Delgado, María J; Bedmar, Eulogio J

    2002-11-01

    The genes norCBQD that encode the bc-type nitric oxide reductase from Bradyrhizobium japonicum USDA110 have been isolated and characterized. norC and norB encode the cytochrome c-containing subunit II and cytochrome b-containing subunit I of nitric oxide reductase, respectively. norQ encodes a protein with an ATP/GTP-binding motif, and the predicted norD gene product shows similarity with NorD from other denitrifiers. Mutational analysis indicates that the two structural norC and norB genes are required for microaerobic growth under nitrate-respiring conditions. A mutant strain lacking a functional norC gene also lacked the 16 kDa c-type cytochrome that is normally detectable by haem-staining of proteins from membranes of microaerobically grown wild-type cells. Expression of a transcriptional fusion of the nor promoter region to the reporter gene lacZ (P(norC)-lacZ) was not detected in aerobically grown cells of USDA110, but the fusion was induced threefold when the cells were cultured under microaerobic conditions (1% O(2)) with either nitrite or nitric oxide, and about 18-fold when nitrate was the N oxide present in the medium. The P(norC)-lacZ fusion was not expressed in the B. japonicum fixK(2) mutant strain 9043, but complementation of the mutant with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. The promoter region of the norCBQD genes has been characterized by primer extension. A major transcript initiates 45.5 bp downstream of the centre of a putative binding site for the transcription factor FixK(2).

  8. Expression of an isoflavone reductase-like gene enhanced by pollen tube growth in pistils of Solanum tuberosum.

    PubMed

    van Eldik, G J; Ruiter, R K; Colla, P H; van Herpen, M M; Schrauwen, J A; Wullems, G J

    1997-03-01

    Successful sexual reproduction relies on gene products delivered by the pistil to create an environment suitable for pollen tube growth. These compounds are either produced before pollination or formed during the interactions between pistil and pollen tubes. Here we describe the pollination-enhanced expression of the cp100 gene in pistils of Solanum tuberosum. Temporal analysis of gene expression revealed an enhanced expression already one hour after pollination and lasts more than 72 h. Increase in expression also occurred after touching the stigma and was not restricted to the site of touch but spread into the style. The predicted CP100 protein shows similarity to leguminous isoflavone reductases (IFRs), but belongs to a family of IFR-like NAD(P)H-dependent oxidoreductases present in various plant species.

  9. Gerbera hybrida (Asteraceae) imposes regulation at several anatomical levels during inflorescence development on the gene for dihydroflavonol-4-reductase.

    PubMed

    Helariutta, Y; Kotilainen, M; Elomaa, P; Teeri, T H

    1995-08-01

    In the ornamental cut flower plant Gerbera hybrida the spatial distribution of regulatory molecules characteristic of differentiation of the composite inflorescence is visualized as the various patterns of anthocyanin pigmentation of different varieties. In order to identify genes that the plant can regulate according to these anatomical patterns, we have analysed gene expression affecting two enzymatic steps, chalcone synthase (CHS) and dihydroflavonol-4-reductase (DFR), in five gerbera varieties with spatially restricted anthocyanin pigmentation patterns. The dfr expression profiles vary at the levels of floral organ, flower type and region within corolla during inflorescence development according to the anthocyanin pigmentation of the cultivars. In contrast, chs expression, although regulated in a tissue-specific manner during inflorescence development, varies only occasionally. The variation in the dfr expression profiles between the varieties reveals spatially specific gene regulation that senses the differentiation events characteristic of the composite inflorescence.

  10. Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice

    PubMed Central

    Wang, Xiu Jun; Chamberlain, Mark; Vassieva, Olga; Henderson, Colin J.; Wolf, C. Roland

    2005-01-01

    Cytochrome P450 reductase is the unique electron donor for microsomal cytochrome P450s; these enzymes play a major role in the metabolism of endogenous and xenobiotic compounds. In mice with a liver-specific deletion of cytochrome P450 reductase, hepatic cytochrome P450 activity is ablated, with consequent changes in bile acid and lipid homoeostasis. In order to gain insights into the metabolic changes resulting from this phenotype, we have analysed changes in hepatic mRNA expression using microarray analysis and real-time PCR. In parallel with the perturbations in bile acid levels, changes in the expression of key enzymes involved in cholesterol and lipid homoeostasis were observed in hepatic cytochrome P450 reductase null mice. This was characterized by a reduced expression of Cyp7b1, and elevation of Cyp7a1 and Cyp8b1 expression. The levels of mRNAs for other cytochrome P450 genes, including Cyp2b10, Cyp2c29, Cyp3a11 and Cyp3a16, were increased, demonstrating that endogenous factors play a role in regulating the expression of these proteins and that the increases are due, at least in part, to altered levels of transcripts. In addition, levels of mRNAs encoding genes involved in glycolysis and lipid transport were also increased; the latter may provide an explanation for the increased hepatic lipid content observed in the hepatic null mice. Serum testosterone and oestradiol levels were lowered, accompanied by significantly decreased expression of Hsd3b2 (3β-hydroxy-Δ5-steroid dehydrogenase-2), Hsd3b5 (3β-hydroxy-Δ5-steroid dehydrogenase-5) and Hsd11b1 (11β-hydroxysteroid dehydrogenase type 1), key enzymes in steroid hormone metabolism. These microarray data provide important insights into the control of metabolic pathways by the cytochrome system. PMID:15717863

  11. Large subunit of the ribonucleotide reductase gene is a virulent factor and plays a critical role in Marek's disease virus pathogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus (MDV) encodes a ribonucleotide reductase (RR) gene consisting of two subunits UL39 (RR1) and UL40 (RR2). Both RR1 and RR2 form an active holoenzyme and are necessary for enzyme activity. This gene was indentified by monoclonal antibody T81 in a gt11 MDV expression library and f...

  12. Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous

    PubMed Central

    Gutiérrez, María Soledad; Rojas, María Cecilia; Sepúlveda, Dionisia; Baeza, Marcelo; Cifuentes, Víctor; Alcaíno, Jennifer

    2015-01-01

    The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous. PMID:26466337

  13. RNAi silencing of the HaHMG-CoA reductase gene inhibits oviposition in the Helicoverpa armigera cotton bollworm.

    PubMed

    Wang, Zhijian; Dong, Yongcheng; Desneux, Nicolas; Niu, Changying

    2013-01-01

    RNA interference (RNAi) has considerable promise for developing novel pest control techniques, especially because of the threat of the development of resistance against current strategies. For this purpose, the key is to select pest control genes with the greatest potential for developing effective pest control treatments. The present study demonstrated that the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase; HMGR) gene is a potential target for insect control using RNAi. HMGR is a key enzyme in the mevalonate pathway in insects. A complete cDNA encoding full length HMGR (encoding an 837-aa protein) was cloned from Helicoverpa armigera (Lepidoptera: Noctuidae). The HaHMGR (H. armigera HMGR) knockdown using systemic RNAi in vivo inhibited the fecundity of the females, effectively inhibited ovipostion, and significantly reduced vitellogenin (Vg) mRNA levels. Moreover, the oviposition rate of the female moths was reduced by 98% by silencing HaHMGR compared to the control groups. One-pair experiments showed that both the proportions of valid mating and fecundity were zero. Furthermore, the HaHMGR-silenced females failed to lay eggs (approximate 99% decrease in oviposition) in the semi-field cage performance. The present study demonstrated the potential implications for developing novel pest management strategies using HaHMGR RNAi in the control of H. armigera and other insect pests.

  14. The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation.

    PubMed

    Tang, Ho Man; Liu, Sanzhen; Hill-Skinner, Sarah; Wu, Wei; Reed, Danielle; Yeh, Cheng-Ting; Nettleton, Dan; Schnable, Patrick S

    2014-02-01

    The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-L-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., ), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis.

  15. Gene Cloning and Characterization of Two NADH-Dependent 3-Quinuclidinone Reductases from Microbacterium luteolum JCM 9174

    PubMed Central

    Isotani, Kentaro; Kurokawa, Junji; Suzuki, Fumiko; Nomoto, Syunsuke; Negishi, Takashi; Matsuda, Michiko

    2013-01-01

    We used the resting-cell reaction to screen approximately 200 microorganisms for biocatalysts which reduce 3-quinuclidinone to optically pure (R)-(−)-3-quinuclidinol. Microbacterium luteolum JCM 9174 was selected as the most suitable organism. The genes encoding the protein products that reduced 3-quinuclidinone were isolated from M. luteolum JCM 9174. The bacC gene, which consists of 768 nucleotides corresponding to 255 amino acid residues and is a constituent of the bacilysin synthetic gene cluster, was amplified by PCR based on homology to known genes. The qnr gene consisted of 759 nucleotides corresponding to 252 amino acid residues. Both enzymes belong to the short-chain alcohol dehydrogenase/reductase (SDR) family. The genes were expressed in Escherichia coli as proteins which were His tagged at the N terminus, and the recombinant enzymes were purified and characterized. Both enzymes showed narrow substrate specificity and high stereoselectivity for the reduction of 3-quinuclidinone to (R)-(−)-3-quinuclidinol. PMID:23263947

  16. Identification of Aeromonas hydrophila Genes Preferentially Expressed after Phagocytosis by Tetrahymena and Involvement of Methionine Sulfoxide Reductases.

    PubMed

    Pang, Maoda; Lin, Xiaoqin; Liu, Jin; Guo, Changming; Gao, Shanshan; Du, Hechao; Lu, Chengping; Liu, Yongjie

    2016-01-01

    Free-living protozoa affect the survival and virulence evolution of pathogens in the environment. In this study, we explored the fate of Aeromonas hydrophila when co-cultured with the bacteriovorous ciliate Tetrahymena thermophila and investigated bacterial gene expression associated with the co-culture. Virulent A. hydrophila strains were found to have ability to evade digestion in the vacuoles of this protozoan. In A. hydrophila, a total of 116 genes were identified as up-regulated following co-culture with T. thermophila by selective capture of transcribed sequences (SCOTS) and comparative dot-blot analysis. A large proportion of these genes (42/116) play a role in metabolism, and some of the genes have previously been characterized as required for bacterial survival and replication within macrophages. Then, we inactivated the genes encoding methionine sulfoxide reductases, msrA, and msrB, in A. hydrophila. Compared to the wild-type, the mutants ΔmsrA and ΔmsrAB displayed significantly reduced resistance to predation by T. thermophila, and 50% lethal dose (LD50) determinations in zebrafish demonstrated that both mutants were highly attenuated. This study forms a solid foundation for the study of mechanisms and implications of bacterial defenses.

  17. Characterization of dihydroflavonol 4-reductase (DFR) genes and their association with cold and freezing stress in Brassica rapa.

    PubMed

    Ahmed, Nasar Uddin; Park, Jong-In; Jung, Hee-Jeong; Yang, Tae-Jin; Hur, Yoonkang; Nou, Ill-Sup

    2014-10-15

    Flavonoids including anthocyanins provide flower and leaf colors, as well as other derivatives that play diverse roles in plant development and interactions with the environment. Dihydroflavonol 4-reductase (DFR) is part of an important step in the flavonoid biosynthetic pathway of anthocyanins. This study characterized 12 DFR genes of Brassica rapa and investigated their association with anthocyanin coloration, as well as cold and freezing stress in several genotypes of B. rapa. Comparison of sequences of these genes with DFR gene sequences from other species revealed a high degree of homology. Constitutive expression of the genes in several pigmented and non-pigmented lines of B. rapa demonstrated correlation with anthocyanin accumulation for BrDFR8 and 9. Conversely, BrDFR2, 4, 8 and 9 only showed very high responses to cold stress in pigmented B. rapa samples. BrDFR1, 3, 5, 6 and 10 responded to cold and freezing stress treatments, regardless of pigmentation. BrDFRs were also shown to be regulated by two transcription factors, BrMYB2-2 and BrTT8, contrasting with anthocyanin accumulation and cold and freezing stress. Thus, the above results suggest that these genes are associated with anthocyanin biosynthesis and cold and freezing stress tolerance and might be useful resources for development of cold and/or freezing stress resistant Brassica crops with desirable colors as well. These findings may also facilitate exploration of the molecular mechanism that regulates anthocyanin biosynthesis and its response to abiotic stresses.

  18. [Clinical course of acute coronary syndrome in dependence on containing of homozystein and С677Т methylenetetrahydrofolate reductase gene polymorphism].

    PubMed

    Pristupa, L N; Grek, A V; Ataman, Iu A; Orlovskiy, A V; Opolonska, N A

    2015-01-01

    Nowadays to a numerous factors of IHD development risks hyperhomocysteinemia (HHc), C-reactive protein, fibrogen, as well as genetic disorders are relating. With development of IHD and its complications associated methylentetrahudrofolate reductase gene mutation of С677Т polymorphism. The purpose of the investigation was studying the connection between acute coronary syndrome severity (ACS) in dependence on plasma homocysteine containing and genotype by С677Т polymorphism MTHFR gene. Examined: 161 patients with ACS and 87 almost healthy people. Identification of 4th exon allelic polymorphism MTHFR С677Т gene (rs1801133) was conducted with method of polymerase chain reaction, the investigation of homocysteine containing with immunoenzymated method. The statistic analyze was performed with using of SPSS - 17 programme. According to results of study patients with ACS of homozygote by minor allele T С677Т MTHFR gene polymorphism by main allele C and heterozygote were associated with high homocysteine containing in plasma. While frequencies of T/T genotype was reliably higher in patients with ACS with segment ST elevation and complicated course compare with patients with ACS with segment ST elevation and non-complicated course and ACS without climbs of segment ST. Also, statistically reliable difference in genotypes distribution by C677T MTHFR gene polymorphism in dependence on homocysteine plasma level and clinical course of ACS severity were established.

  19. Identification of Aeromonas hydrophila Genes Preferentially Expressed after Phagocytosis by Tetrahymena and Involvement of Methionine Sulfoxide Reductases

    PubMed Central

    Pang, Maoda; Lin, Xiaoqin; Liu, Jin; Guo, Changming; Gao, Shanshan; Du, Hechao; Lu, Chengping; Liu, Yongjie

    2016-01-01

    Free-living protozoa affect the survival and virulence evolution of pathogens in the environment. In this study, we explored the fate of Aeromonas hydrophila when co-cultured with the bacteriovorous ciliate Tetrahymena thermophila and investigated bacterial gene expression associated with the co-culture. Virulent A. hydrophila strains were found to have ability to evade digestion in the vacuoles of this protozoan. In A. hydrophila, a total of 116 genes were identified as up-regulated following co-culture with T. thermophila by selective capture of transcribed sequences (SCOTS) and comparative dot-blot analysis. A large proportion of these genes (42/116) play a role in metabolism, and some of the genes have previously been characterized as required for bacterial survival and replication within macrophages. Then, we inactivated the genes encoding methionine sulfoxide reductases, msrA, and msrB, in A. hydrophila. Compared to the wild-type, the mutants ΔmsrA and ΔmsrAB displayed significantly reduced resistance to predation by T. thermophila, and 50% lethal dose (LD50) determinations in zebrafish demonstrated that both mutants were highly attenuated. This study forms a solid foundation for the study of mechanisms and implications of bacterial defenses. PMID:28083518

  20. Molecular characterization of the sor gene, which encodes the sulfur oxygenase/reductase of the thermoacidophilic Archaeum Desulfurolobus ambivalens.

    PubMed Central

    Kletzin, A

    1992-01-01

    A 5.8-kbp HindIII fragment containing the sor gene which encodes the aerobically induced sulfur oxygenase/reductase of the thermoacidophilic, chemolithoautotrophic, and facultatively anaerobic archaeum Desulfurolobus ambivalens, was cloned in pUC18 by using an oligonucleotide derived from the N-terminal amino acid sequence for identification (pSOR-1/17). The native enzyme is a 550,000-molecular-weight oligomer composed of single 40,000-molecular-weight subunits; this oligomer is capable of the simultaneous oxidation and reduction of sulfur (A. Kletzin, J. Bacteriol. 171:1638-1643, 1989). From the fragment, 3,025 bp that contained the entire sor gene were sequenced. The sor gene encoded a protein with 309 amino acid residues (molecular weight, 35,317). The transcript length was determined by Northern RNA hybridization to be 960 to 1,020 nucleotides, and the transcriptional start site was mapped by primer extension analysis. The transcript of the sor gene in aerobically grown cells was amplified 38- to 42-fold relative to that in anaerobically grown cells. An initial transcriptional characterization of three neighboring genes of unknown function is also reported. Images PMID:1522063

  1. Molecular characterization and expression analysis of dihydroflavonol 4-reductase (DFR) gene in Saussurea medusa.

    PubMed

    Li, Houhua; Qiu, Jian; Chen, Fudong; Lv, Xiaofen; Fu, Chunxiang; Zhao, Dexiu; Hua, Xuejun; Zhao, Qiao

    2012-03-01

    Dihydroflavonol 4-reductase (DFR), which catalyzes the reduction of dihydroflavonols to leucoanthocyanins, is a key enzyme in the biosynthesis of anthocyanidins, proanthocyanidins, and other flavonoids of importance in plant development and human nutrition. This study isolated a full length cDNA encoding DFR, designated as SmDFR (GenBank Accession No. EF600682), by screening a cDNA library from a red callus line of Saussurea medusa, which is an endangered, traditional Chinese medicinal plant with high pharmacological value. SmDFR was functionally expressed in yeast (Saccharomyces cerevisiae) to confirm that SmDFR can readily reduce dihydroquercetin (DHQ) and dihydrokampferol (DHK), but it could not reduce dihydromyricetin (DHM). The deduced SmDFR structure shared extensive sequence similarity with previously characterized plant DFRs and phylogenetic analysis showed that it belonged to the plant DFR super-family. SmDFR also possessed flavanone 4-reductase (FNR) activity and can catalyze the conversion of eridictyol to luteoforol. Real-time PCR analysis showed that the expression level of SmDFR was higher in flowers compared with both leaves and roots. This work greatly enhances our knowledge of flavonoid biosynthesis in S. medusa and marks a major advance that could facilitate future genetic modification of S. medusa.

  2. The regulatory region controlling the nitrate-responsive expression of a nitrate reductase gene, NIA1, in Arabidopsis.

    PubMed

    Konishi, Mineko; Yanagisawa, Shuichi

    2011-05-01

    Nitrate reductase (NR) is the enzyme that catalyzes the first step of nitrate assimilation. It is well known that the expression of NR genes is rapidly induced in various plants by nitrate. Previously, the activity of a tobacco NR gene promoter was reported to be high in tobacco plants grown on medium containing ammonium as the sole nitrogen source, but low in tobacco plants grown on nitrate-containing medium. This cast some doubt on the role of the NR gene promoter in the nitrate-inducible expression of this gene. Furthermore, in previous studies, transformation with genomic fragments containing NR loci restored the reduced NR activity in NR mutants to a limited extent, suggesting a complex regulation of NR gene expression. Here, we show that although the 1.9 kb promoter of an NR gene in Arabidopsis, NIA1, is not activated by nitrate, the expression of a GUS (β-glucuronidase) reporter gene inserted between the 5'- and 3'-flanking sequences of the NIA1 coding region is strongly induced by nitrate. When the 3'-flanking sequence was fused downstream of the GUS gene under the control of the 35S minimal promoter, its expression was also strongly induced by nitrate. Furthermore, dissection analysis of the 3'-flanking region revealed that the sequence downstream of the transcriptional terminator rather than the 3'-untranslated region plays a role in nitrate-inducible expression, indicating a requirement for the 3'-flanking sequence for the nitrate-inducible transcription of NIA1. We also show that the 2.7 kb promoter sequence of NIA2, another NR gene of Arabidopsis, cannot direct nitrate-inducible expression.

  3. Dihydroflavonol 4-Reductase Genes from Freesia hybrida Play Important and Partially Overlapping Roles in the Biosynthesis of Flavonoids

    PubMed Central

    Li, Yueqing; Liu, Xingxue; Cai, Xinquan; Shan, Xiaotong; Gao, Ruifang; Yang, Song; Han, Taotao; Wang, Shucai; Wang, Li; Gao, Xiang

    2017-01-01

    Dihydroflavonol-4-reductase (DFR) is a key enzyme in the reduction of dihydroflavonols to leucoanthocyanidins in both anthocyanin biosynthesis and proanthocyanidin accumulation. In many plant species, it is encoded by a gene family, however, how the different copies evolve either to function in different tissues or at different times or to specialize in the use of different but related substrates needs to be further investigated, especially in monocot plants. In this study, a total of eight putative DFR-like genes were firstly cloned from Freesia hybrida. Phylogenetic analysis showed that they were classified into different branches, and FhDFR1, FhDFR2, and FhDFR3 were clustered into DFR subgroup, whereas others fell into the group with cinnamoyl-CoA reductase (CCR) proteins. Then, the functions of the three FhDFR genes were further characterized. Different spatio-temporal transcription patterns and levels were observed, indicating that the duplicated FhDFR genes might function divergently. After introducing them into Arabidopsis dfr (tt3-1) mutant plants, partial complementation of the loss of cyanidin derivative synthesis was observed, implying that FhDFRs could convert dihydroquercetin to leucocyanidin in planta. Biochemical assays also showed that FhDFR1, FhDFR2, and FhDFR3 could utilize dihydromyricetin to generate leucodelphinidin, while FhDFR2 could also catalyze the formation of leucocyanidin from dihydrocyanidin. On the contrary, neither transgenic nor biochemical analysis demonstrated that FhDFR proteins could reduce dihydrokaempferol to leucopelargonidin. These results were consistent with the freesia flower anthocyanin profiles, among which delphinidin derivatives were predominant, with minor quantities of cyanidin derivatives and undetectable pelargonidin derivatives. Thus, it can be deduced that substrate specificities of DFRs were the determinant for the categories of anthocyanins aglycons accumulated in F. hybrida. Furthermore, we also found that

  4. Quantification of Desulfovibrio vulgaris dissimilatory sulfite reductase gene expression during electron donor- and electron acceptor-limited growth.

    PubMed

    Villanueva, Laura; Haveman, Shelley A; Summers, Zara M; Lovley, Derek R

    2008-09-01

    Previous studies have suggested that levels of transcripts for dsrA, a gene encoding a subunit of the dissimilatory sulfite reductase, are not directly related to the rates of sulfate reduction in sediments under all conditions. This phenomenon was further investigated with chemostat-grown Desulfovibrio vulgaris. Under sulfate-limiting conditions, dsrA transcript levels increased as the bulk rates of sulfate reduction in the chemostat increased, but transcript levels were similar at all sulfate reduction rates under electron donor-limiting conditions. When both electron donor- and electron acceptor-limiting conditions were considered, there was a direct correspondence between dsrA transcript levels and the rates of sulfate reduction per cell. These results suggest that dsrA transcript levels may provide important information on the metabolic state of sulfate reducers.

  5. Mutations in cystathionine beta-synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans.

    PubMed

    Jakubowski, Hieronim; Boers, Godfried H J; Strauss, Kevin A

    2008-12-01

    Severely elevated plasma homocysteine (Hcy) levels observed in genetic disorders of Hcy metabolism are associated with pathologies in multiple organs and lead to premature death due to vascular complications. In addition to elevating plasma Hcy, mutations in cystathionine beta-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) gene lead to markedly elevated levels of circulating Hcy-thiolactone. The thiooester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein lysine residues (N-Hcy-protein), which may impair or alter the protein's function. However, it was not known whether genetic deficiencies in Hcy metabolism affect N-Hcy-protein levels in humans. Here we show that plasma N-Hcy-protein levels are significantly elevated in CBS- and MTHFR-deficient patients. We also show that CBS-deficient patients have significantly elevated plasma levels of prothrombotic N-Hcy-fibrinogen. These results provide a possible explanation for increased atherothrombosis observed in CBS-deficient patients.

  6. Expressions of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase genes are stimulated by recombinant platelet-derived growth factor isomers

    SciTech Connect

    Roth, M.; Emmons, L.R.; Perruchoud, A. ); Block, L.H. )

    1991-03-01

    The plausible role that platelet-derived growth factor (PDGF) has in the localized pathophysiological changes that occur in the arterial wall during development of atherosclerotic lesions led the authors to investigate the influence of recombinant (r)PDGF isomers -AA, -AB, and -BB on the expression of low density lipoprotein receptor (LDL-R) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG0CoA) reductase ((S)-mevalonate:NAD{sup +} oxidoreductase (CoA-acylating), EC 1.1.1.88) genes. In addition, they clarified the role of protein kinase C (PKC) in expression of the two genes in human skin fibroblasts and vascular smooth muscle cells. The various rPDGF isoforms are distinct in their ability to activate transcription of both genes: (i) both rPDGF-AA and -BB stimulate transcription of the LDL-R gene; in contrast, rPDGF-BB but not -AA, activates transcription of the HMG-CoA reductase gene; (ii) all recombinant isoforms of PDGF activate transcription of the c-fos gene; (iii) while rPDGF-dependent transcription of the lDL-R gene occurs independently of PKC, transcription of the HMG-CoA reductase gene appears to involve the action of that enzyme.

  7. The MET13 methylenetetrahydrofolate reductase gene is essential for infection-related morphogenesis in the rice blast fungus Magnaporthe oryzae.

    PubMed

    Yan, Xia; Que, Yawei; Wang, Hong; Wang, Congcong; Li, Ya; Yue, Xiaofeng; Ma, Zhonghua; Talbot, Nicholas J; Wang, Zhengyi

    2013-01-01

    Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae.

  8. The Bacillus subtilis ydjL (bdhA) Gene Encodes Acetoin Reductase/2,3-Butanediol Dehydrogenase ▿

    PubMed Central

    Nicholson, Wayne L.

    2008-01-01

    Bacillus subtilis is capable of producing 2,3-butanediol from acetoin by fermentation, but to date, the gene encoding the enzyme responsible, acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), has remained unknown. A search of the B. subtilis genome database with the amino acid sequences of functional AR/BDHs from Saccharomyces cerevisiae and Bacillus cereus resulted in the identification of a highly similar protein encoded by the B. subtilis ydjL gene. A knockout strain carrying a ydjL::cat insertion mutation was constructed, which (i) abolished 2,3-butanediol production in early stationary phase, (ii) produced no detectable AR or BDH activity in vitro, and (iii) accumulated the precursor acetoin in early stationary phase. The ydjL::cat mutation also affected the kinetics of lactate but not acetate production during stationary-phase cultivation with glucose under oxygen limitation. A very small amount of 2,3-butanediol was detected in very-late-stationary-phase (96-hour) cultures of the ydjL::cat mutant, suggesting the existence of a second gene encoding a minor AR activity. From the data, it is proposed that the major AR/BDH-encoding gene ydjL be renamed bdhA. PMID:18820069

  9. The MET13 Methylenetetrahydrofolate Reductase Gene Is Essential for Infection-Related Morphogenesis in the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Wang, Hong; Wang, Congcong; Li, Ya; Yue, Xiaofeng; Ma, Zhonghua; Talbot, Nicholas J.; Wang, Zhengyi

    2013-01-01

    Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae. PMID:24116181

  10. Role of methylenetetrahydrofolate reductase gene (MTHFR) 677C>T polymorphism in pediatric cerebrovascular disorders.

    PubMed

    Alsayouf, Hamza; Zamel, Khaled M; Heyer, Geoffrey L; Khuhro, A Latif; Kahwash, Samir B; de los Reyes, Emily C

    2011-03-01

    Homozygosity for the methylenetetrahydrofolate reductase (MTHFR) 677C>T mutation (MTHFR TT) has been linked to an increased risk for stroke, coronary artery disease, and migraine headaches. The authors analyzed the potential link between MTHFR 677C>T homozygosity and childhood stroke. A true association might facilitate screening, recurrence risk stratification, and treatment in patients with cerebrovascular disease. They performed a retrospective chart review of children tested for the MTHFR 677C>/T mutation; 533 patients underwent MTHFR testing, and 8% were homozygous for the MTHFR 677C>T mutation. There was no difference in the cohort compared with the prevalence in the general population. This suggests that the MTHFR 677 C>T polymorphism played a minimal role or no role in stroke risk. However, the data suggest that the MTHFR TT genotype may influence migraine susceptibility in children because there was a higher proportion of migraine patients (28.6%) with the MTHFR TT homozygous genotype.

  11. The Importance of Homozygous Polymorphisms of Methylenetetrahydrofolate Reductase Gene in Romanian Patients with Idiopathic Venous Thromboembolism

    PubMed Central

    Hotoleanu, Cristina; Trifa, Adrian; Popp, Radu; Fodor, Daniela

    2013-01-01

    Background: Methylenetetrahydrofolate reductase (MTHFR) polymorphisms have recently raised the interest as a possible thrombophilic factors. Aims: We aimed to assess the frequency of the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms in idiopathic venous thromboembolism (VTE) in a Romanian population and the associated risk of VTE. Study Design: We performed a case-control transversal study including 90 patients diagnosed with VTE and 75 sex- and age-matched controls. Methods: MTHFR C677T and A1298C polymorphisms were detected using PCR-RFLP method. Results: The homozygous MTHFR 677TT genotype, present in 18.8% of patients with VTE versus 6.6% of controls, was significantly associated with VTE (p= 0.021, OR= 3.26, 95%CI (1.141–9.313)). The heterozygous MTHFR A1298C genotype, presenting the highest prevalence in the VTE group (34.4%) as well as in controls (37.3%), was not associated with VTE (p=0.7). No associations were found for heterozygous MTHFR C677T (with a frequency of 32.2% in VTE and 37.3% in controls, p=0.492), respective homozygous MTHFR A1298C genotype (with a frequency of 1.1% in VTE and 2.6% in controls, p=0.456). Conclusion: Among MTHFR polymorphisms, only homozygosity for MTHFR 677TT may be considered a risk factor for VTE; the MTHFR A1298C polymorphism is not significantly associated with an increased risk of VTE. PMID:25207100

  12. Molecular analysis of the distribution and phylogeny of dissimilatory adenosine-5'-phosphosulfate reductase-encoding genes (aprBA) among sulfur-oxidizing prokaryotes.

    PubMed

    Meyer, Birte; Kuever, Jan

    2007-10-01

    Dissimilatory adenosine-5'-phosphosulfate (APS) reductase (AprBA) is a key enzyme of the dissimilatory sulfate-reduction pathway. Homologues have been found in photo- and chemotrophic sulfur-oxidizing prokaryotes (SOP), in which they are postulated to operate in the reverse direction, oxidizing sulfite to APS. Newly developed PCR assays allowed the amplification of 92-93 % (2.1-2.3 kb) of the APS reductase locus aprBA. PCR-based screening of 116 taxonomically divergent SOP reference strains revealed a distribution of aprBA restricted to photo- and chemotrophs with strict anaerobic or at least facultative anaerobic lifestyles, including Chlorobiaceae, Chromatiaceae, Thiobacillus, Thiothrix and invertebrate symbionts. In the AprBA-based tree, the SOP diverge into two distantly related phylogenetic lineages, Apr lineages I and II, with the proteins of lineage II (Chlorobiaceae and others) in closer affiliation to the enzymes of the sulfate-reducing prokaryotes (SRP). This clustering is discordant with the dissimilatory sulfite reductase (DsrAB) phylogeny and indicates putative lateral aprBA gene transfer from SRP to the respective SOB lineages. In support of lateral gene transfer (LGT), several beta- and gammaproteobacterial species harbour both aprBA homologues, the DsrAB-congruent 'authentic' and the SRP-related, LGT-derived gene loci, while some relatives possess exclusively the SRP-related apr genes as a possible result of resident gene displacement by the xenologue. The two-gene state might be an intermediate in the replacement of the resident essential gene. Collected genome data demonstrate the correlation between the AprBA tree topology and the composition/arrangement of the apr gene loci (occurrence of qmoABC or aprM genes) from SRP and SOP of lineages I and II. The putative functional role of the SRP-related APS reductases in photo- and chemotrophic SOP is discussed.

  13. Over-Expression of a Tobacco Nitrate Reductase Gene in Wheat (Triticum aestivum L.) Increases Seed Protein Content and Weight without Augmenting Nitrogen Supplying

    PubMed Central

    Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

    2013-01-01

    Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, “Nongda146” and “Jimai6358”, by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying. PMID:24040315

  14. Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA.

    PubMed Central

    Garcia, G E; Stadtman, T C

    1991-01-01

    The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon. PMID:1825826

  15. Selenoprotein A component of the glycine reductase complex from Clostridium purinolyticum: nucleotide sequence of the gene shows that selenocysteine is encoded by UGA.

    PubMed

    Garcia, G E; Stadtman, T C

    1991-03-01

    The gene encoding the selenoprotein A component of glycine reductase was isolated from Clostridium purinolyticum. The nucleotide sequence of this gene (grdA) was determined. The opal termination codon (TGA) was found in-frame at the position corresponding to the location of the selenocysteine residue in the gene product. A comparison of the nucleotide sequences and secondary mRNA structures corresponding to the selenoprotein A gene and the fdhF gene of Escherichia coli formate dehydrogenase shows that there is a similar potential for regulation of the specific insertion of selenocysteine at the UGA codon.

  16. Relationship between methylenetetrahydrofolate reductase (MTHFR) A1298C gene polymorphism and type 2 diabetic nephropathy risk: a meta-analysis.

    PubMed

    Zhang, Jie; Xiao, Yan; Zhang, Xian-Wen; Gao, Zhi-Qing; Han, Jing-Hui

    2014-07-01

    Relationship between methylenetetrahydrofolate reductase (MTHFR) A1298C gene polymorphism and type 2 diabetic nephropathy (T2DN) risk is still unclear. This study was performed to evaluate if there is an association between the MTHFR A1298C gene polymorphism and T2DN risk using meta-analysis. The relevant reports were searched and identified from PubMed, Cochrane Library on 1 October 2013, and eligible studies were included and synthesized. Eight reports were recruited into this meta-analysis for the association of the MTHFR A1298C gene polymorphism with T2DN risk. The MTHFR A1298C C allele or CC genotype was shown to be not associated with T2DN risk (C allele: OR = 0.76, 95% CI: 0.43-1.34, p = 0.34; CC genotype: OR = 1.18, 95% CI: 0.63-2.22, p = 0.60). Interestingly, AA genotype was associated with the T2DN risk (OR = 0.68, 95% CI: 0.49-0.96, p = 0.03). In the sensitivity analysis according to the Hardy-Weinberg equilibrium (HWE), the results were consistent with those in non-sensitivity analysis. However, in the sensitivity analysis according to the control source from hospital, sample size of case (≥ 100), sample size of case (<100), the MTHFR A1298C gene polymorphism was not associated with T2DN risk. In conclusion, the MTHFR A1298C gene polymorphism was not associated with T2DN risk. However, additional studies are required to firmly establish a correlation between the MTHFR A1298C gene polymorphism and T2DN risk.

  17. Identification and characterization of a gene that is coamplified with dihydrofolate reductase in a methotrexate-resistant CHO cell line

    SciTech Connect

    Foreman, P.K.; Hamlin, J.L. . School of Medicine)

    1989-03-01

    As part of an effort to characterize the spatial and functional relationships among genetic elements within the amplified dihydrofolate reductase (DHFR) domain in Chinese hamster cells, the authors have used a variation of the differential hybridization approach to identify cDNA clones whose genes are coamplified with DHFR in the methotrexate-resistant cell line, CHOC 400. Their initial screen was successful in isolating both DHFR and non-DHFR cDNAs. One of the non-DHFR cDNA clones, 2BE2121, hybridizes on Northern (RNA) blots to abundant 1,200- and 1,500-nucleotide (nt) transcripts which differ in the lengths of their 3' untranslated regions. The clone 2BE2121 contains a 789-nt open reading frame but does not appear to be related to any members of the protein or nucleic acid sequence databases. A second larger non-DHFR cDNA, II-19-211, was isolated that is transcribed from the same gene as 2BE2121 but contains only a small carboxyl-terminal portion of the open reading frame. II-19-211 may, therefore, represent either a splicing intermediate or an mRNA transcribed from a cryptic intragenic promoter. Hybridization to cosmids from DHFr domain shows that 2BE2121 is encoded by a gene --34 kilobases (kb) long. The 5'-most genomic fragment is less than 4 kb from an interamplicon injection. The 3' end of the 2BE2121 gene lies --75 kb downstream from the DHFR gene and --25 kb downstream from the proximal replication initiation site, and the transcriptional polarity is opposite to that of the leading strand of replication. Thus, both the DHFR and 2BE2121 genes are exceptions to the theory that transcription proceeds in the same direction as the leading strand of the replication fork.

  18. Identification and characterization of a gene that is coamplified with dihydrofolate reductase in a methotrexate-resistant CHO cell line.

    PubMed Central

    Foreman, P K; Hamlin, J L

    1989-01-01

    As part of an effort to characterize the spatial and functional relationships among genetic elements within the amplified dihydrofolate reductase (DHFR) domain in Chinese hamster cells, we have used a variation of the differential hybridization approach to identify cDNA clones whose genes are coamplified with DHFR in the methotrexate-resistant cell line, CHOC 400. Our initial screen was successful in isolating both DHFR and non-DHFR cDNAs. One of the non-DHFR cDNA clones, 2BE2121, hybridizes on Northern (RNA) blots to abundant 1,200- and 1,500-nucleotide (nt) transcripts which differ in the lengths of their 3' untranslated regions. The clone 2BE2121 contains a 789-nt open reading frame but does not appear to be related to any members of the protein or nucleic acid sequence databases. A second larger non-DHFR cDNA, II-19-211, was isolated that is transcribed from the same gene as 2BE2121 but contains only a small carboxyl-terminal portion of the open reading frame. II-19-211 may, therefore, represent either a splicing intermediate or an mRNA transcribed from a cryptic intragenic promoter. Hybridization to cosmids from the DHFR domain shows that 2BE2121 is encoded by a gene approximately 34 kilobases (kb) long. The 5'-most genomic fragment is less than 4 kb from an interamplicon junction. The 3' end of the 2BE2121 gene lies approximately 75 kb downstream from the DHFR gene and approximately 25 kb downstream from the proximal replication initiation site, and the transcriptional polarity is opposite to that of the leading strand of replication. Thus, both the DHFR and 2BE2121 genes are exceptions to the theory that transcription proceeds in the same direction as the leading strand of the replication fork. Images PMID:2725490

  19. Ribonucleotide Reduction in Mycobacterium tuberculosis: Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

    PubMed Central

    Dawes, Stephanie S.; Warner, Digby F.; Tsenova, Liana; Timm, Juliano; McKinney, John D.; Kaplan, Gilla; Rubin, Harvey; Mizrahi, Valerie

    2003-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a ΔnrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O2-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O2 restriction. However, survival of the ΔnrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F1 mice infected with the ΔnrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model. PMID:14573627

  20. Design and evaluation of primers targeting genes encoding NO-forming nitrite reductases: implications for ecological inference of denitrifying communities

    PubMed Central

    Bonilla-Rosso, Germán; Wittorf, Lea; Jones, Christopher M.; Hallin, Sara

    2016-01-01

    The detection of NO-forming nitrite reductase genes (nir) has become the standard when studying denitrifying communities in the environment, despite well-known amplification biases in available primers. We review the performance of 35 published and 121 newly designed primers targeting the nirS and nirK genes, against sequences from complete genomes and 47 metagenomes from three major habitats where denitrification is important. There were no optimal universal primer pairs for either gene, although published primers targeting nirS displayed up to 75% coverage. The alternative is clade-specific primers, which show a trade-off between coverage and specificity. The test against metagenomic datasets showed a distinct performance of primers across habitats. The implications of clade-specific nir primers choice and their performance for ecological inference when used for quantitative estimates and in sequenced-based community ecology studies are discussed and our phylogenomic primer evaluation can be used as a reference along with their environmental specificity as a guide for primer selection. Based on our results, we also propose a general framework for primer evaluation that emphasizes the testing of coverage and phylogenetic range using full-length sequences from complete genomes, as well as accounting for environmental range using metagenomes. This framework serves as a guideline to simplify primer performance comparisons while explicitly addressing the limitations and biases of the primers evaluated. PMID:27966627

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

    PubMed Central

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

    2010-01-01

    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 immunocytochemistry were used to demonstrate that Trx and TrxR are expressed in the cytoplasm and in the nuclei of MCF-7 human breast cancer cells. More importantly, endogenously expressed ERα, Trx, and TrxR interact and ERα and TrxR associate with the native, estrogen-responsive pS2 and progesterone receptor genes in MCF-7 cells. RNA interference assays demonstrated that Trx and TrxR differentially influence estrogen-responsive gene expression and that together, 17β-estradiol, Trx, and TrxR alter hydrogen peroxide (H2O2) levels in MCF-7 cells. Our findings suggest that Trx and TrxR are multifunctional proteins that, in addition to modulating H2O2 levels and transcription factor activity, aid ERα in regulating the expression of estrogen-responsive genes in target cells. PMID:19620238

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

    PubMed

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

    2009-12-01

    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 alpha (ERalpha). Western analysis and immunocytochemistry were used to demonstrate that Trx and TrxR are expressed in the cytoplasm and in the nuclei of MCF-7 human breast cancer cells. More importantly, endogenously expressed ERalpha, Trx, and TrxR interact and ERalpha and TrxR associate with the native, estrogen-responsive pS2 and progesterone receptor genes in MCF-7 cells. RNA interference assays demonstrated that Trx and TrxR differentially influence estrogen-responsive gene expression and that together, 17beta-estradiol, Trx, and TrxR alter hydrogen peroxide (H(2)O(2)) levels in MCF-7 cells. Our findings suggest that Trx and TrxR are multifunctional proteins that, in addition to modulating H(2)O(2) levels and transcription factor activity, aid ERalpha in regulating the expression of estrogen-responsive genes in target cells.

  3. Molecular characterization and expression profile of methionine sulfoxide reductase gene family in maize (Zea mays) under abiotic stresses.

    PubMed

    Zhu, Jiantang; Ding, Pengcheng; Li, Qingqing; Gao, YanKun; Chen, Fanguo; Xia, Guangmin

    2015-05-15

    Methionine (Met) oxidation to methionine sulfoxide (MetSO) is a common form of damage caused by reactive oxygen species (ROS) accumulation via various environmental stresses. Methionine sulfoxide reductase (MSR) repairs oxidized Met and protects organisms from oxidative damage. Two types of MSR, A and B, have been identified based on substrate stereo specificity; they share no sequence similarity. In the present study, we characterized six genes encoding the putative MSR from two public databases. We compared them with MSRs from 6 species, and evaluated molecular characterization, phylogenetic analysis, tertiary structure and conserved motifs. On the basis of in silico and the qRT-PCR experimental data, we analyzed cDNA sequences and expression patterns of ZmMSR genes in different organs in maize. We found that ZmMSR genes were induced by polyethylene glycol (PEG) and NaCl, both known to generate oxidative stress. The results show that MSRs are conserved in different species, suggesting that MSRs across different species share common mechanisms related to diverse defense responses.

  4. Staying green postharvest: how three mutations in the Arabidopsis chlorophyll b reductase gene NYC1 delay degreening by distinct mechanisms.

    PubMed

    Jibran, Rubina; Sullivan, Kerry L; Crowhurst, Ross; Erridge, Zoe A; Chagné, David; McLachlan, Andrew R G; Brummell, David A; Dijkwel, Paul P; Hunter, Donald A

    2015-11-01

    Stresses such as energy deprivation, wounding and water-supply disruption often contribute to rapid deterioration of harvested tissues. To uncover the genetic regulation behind such stresses, a simple assessment system was used to detect senescence mutants in conjunction with two rapid mapping techniques to identify the causal mutations. To demonstrate the power of this approach, immature inflorescences of Arabidopsis plants that contained ethyl methanesulfonate-induced lesions were detached and screened for altered timing of dark-induced senescence. Numerous mutant lines displaying accelerated or delayed timing of senescence relative to wild type were discovered. The underlying mutations in three of these were identified using High Resolution Melting analysis to map to a chromosomal arm followed by a whole-genome sequencing-based mapping method, termed 'Needle in the K-Stack', to identify the causal lesions. All three mutations were single base pair changes and occurred in the same gene, NON-YELLOW COLORING1 (NYC1), a chlorophyll b reductase of the short-chain dehydrogenase/reductase (SDR) superfamily. This was consistent with the mutants preferentially retaining chlorophyll b, although substantial amounts of chlorophyll b were still lost. The single base pair mutations disrupted NYC1 function by three distinct mechanisms, one by producing a termination codon, the second by interfering with correct intron splicing and the third by replacing a highly conserved proline with a non-equivalent serine residue. This non-synonymous amino acid change, which occurred in the NADPH binding domain of NYC1, is the first example of such a mutation in an SDR protein inhibiting a physiological response in plants.

  5. Folate Pathway Gene Methylenetetrahydrofolate Reductase C677T Polymorphism and Alzheimer Disease Risk in Asian Population.

    PubMed

    Rai, Vandana

    2016-07-01

    The association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and susceptibility to Alzheimers disease (AD) was controversial in previous studies. The present meta-analysis was designed to investigate the association of MTHFR C677T polymorphism with AD. Nine studies were identified by search of PubMed, Google Scholar, Elsevier, Springer Link databases, up to January 2013. Odds ratios (ORs) with corresponding 95 % confidence interval (CI) were calculated using fixed effects model or random effects model. All statistical analysis was done by Mix version 1.7. MTHFR C677T polymorphism had a significant association with susceptibility to AD in all genetic models (for T vs C: OR 1.29, 95 % CI 1.15-1.44, p < 0.0001; for TT + CT vs CC: OR 1.38, 95 % CI 1.16-1.364, p = 0.0002; for TT vs CC: OR 1.60, 95 % CI 1.25-2.04, p = 0.0001; for CT vs CC: OR 1.28, 95 % CI 1.1-1.53, p < 0.008; for TT vs CT + CC: OR 1.37, 95 % CI 1.12-1.67, p = 0.002). Results from present meta-analysis supported that the MTHFR C677T polymorphism was associated with an increased risk of AD in Asian population.

  6. High diversity of bacterial mercuric reductase genes from surface and sub-surface floodplain soil (Oak Ridge, USA).

    PubMed

    Oregaard, Gunnar; Sørensen, Søren J

    2007-09-01

    DNA was extracted from different depth soils (0-5, 45-55 and 90-100 cm below surface) sampled at Lower East Fork Poplar Creek floodplain (LEFPCF), Oak Ridge (TN, USA). The presence of merA genes, encoding the mercuric reductase, the key enzyme in detoxification of mercury in bacteria, was examined by PCR targeting Actinobacteria, Firmicutes or beta/gamma-Proteobacteria. beta/gamma-Proteobacteria merA genes were successfully amplified from all soils, whereas Actinobacteria were amplified only from surface soil. merA clone libraries were constructed and sequenced. beta/gamma-Proteobacteria sequences revealed high diversity in all soils, but limited vertical similarity. Less than 20% of the operational taxonomic units (OTU) (DNA sequences > or = 95% identical) were shared between the different soils. Only one of the 62 OTU was > or = 95% identical to a GenBank sequence, highlighting that cultivated bacteria are not representative of what is found in nature. Fewer merA sequences were obtained from the Actinobacteria, but these were also diverse, and all were different from GenBank sequences. A single clone was most closely related to merA of alpha-Proteobacteria. An alignment of putative merA genes of genome sequenced mainly marine alpha-Proteobacteria was used for design of merA primers. PCR amplification of soil alpha-Proteobacteria isolates and sequencing revealed that they were very different from the genome-sequenced bacteria (only 62%-66% identical at the amino-acid level), although internally similar. In light of the high functional diversity of mercury resistance genes and the limited vertical distribution of shared OTU, we discuss the role of horizontal gene transfer as a mechanism of bacterial adaptation to mercury.

  7. Construction of a modular dihydrofolate reductase cDNA gene: Analysis of signals utilized for efficient expression

    SciTech Connect

    Kaufman, J.; Sharp, P.A.

    1982-11-01

    Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome, DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR/sup -/ cells to the DHFR/sup +/ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFr mRNA produced in amplified lines indicated the following: (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA generated a recombinant capable of transforming cells to the DHFR/sup +/ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer 72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.

  8. Overexpression of D-xylose reductase (xyl1) gene and antisense inhibition of D-xylulokinase (xyiH) gene increase xylitol production in Trichoderma reesei.

    PubMed

    Hong, Yuanyuan; Dashtban, Mehdi; Kepka, Greg; Chen, Sanfeng; Qin, Wensheng

    2014-01-01

    T. reesei is an efficient cellulase producer and biomass degrader. To improve xylitol production in Trichoderma reesei strains by genetic engineering, two approaches were used in this study. First, the presumptive D-xylulokinase gene in T. reesei (xyiH), which has high homology to known fungi D-xylulokinase genes, was silenced by transformation of T. reesei QM9414 strain with an antisense construct to create strain S6-2-2. The expression of the xyiH gene in the transformed strain S6-2-2 decreased at the mRNA level, and D-xylulokinase activity decreased after 48 h of incubation. This led to an increase in xylitol production from undetectable levels in wild-type T. reesei QM9414 to 8.6 mM in S6-2-2. The T. reesei Δxdh is a xylose dehydrogenase knockout strain with increased xylitol production compared to the wild-type T. reesei QM9414 (22.8 mM versus undetectable). The copy number of the xylose reductase gene (xyl1) in T. reesei Δxdh strain was increased by genetic engineering to create a new strain Δ9-5-1. The Δ9-5-1 strain showed a higher xyl1 expression and a higher yield of xylose reductase, and xylitol production was increased from 22.8 mM to 24.8 mM. Two novel strains S6-2-2 and Δ9-5-1 are capable of producing higher yields of xylitol. T. reesei has great potential in the industrial production of xylitol.

  9. Diversity, abundance, and distribution of NO-forming nitrite reductase-encoding genes in deep-sea subsurface sediments of the South China Sea.

    PubMed

    Li, M; Hong, Y; Cao, H; Klotz, M G; Gu, J-D

    2013-03-01

    In marine ecosystems, both nitrite-reducing bacteria and anaerobic ammonium-oxidizing (anammox) bacteria, containing different types of NO-forming nitrite reductase-encoding genes, contribute to the nitrogen cycle. The objectives of study were to reveal the diversity, abundance, and distribution of NO-forming nitrite reductase-encoding genes in deep-sea subsurface environments. Results showed that higher diversity and abundance of nirS gene than nirK and Scalindua-nirS genes were evident in the sediments of the South China Sea (SCS), indicating bacteria containing nirS gene dominated the NO-forming nitrite-reducing microbial community in this ecosystem. Similar diversity and abundance distribution patterns of both nirS and Scalindua-nirS genes were detected in this study sites, but different from nirK gene. Further statistical analyses also showed both nirS and Scalindua-nirS genes respond similarly to environmental factors, but differed from nirK gene. These results suggest that bacteria containing nirS and Scalindua-nirS genes share similar niche in deep-sea subsurface sediments of the SCS, but differed from those containing nirK gene, indicating that community structures of nitrite-reducing bacteria are segregated by the functional modules (NirS vs. NirK) rather than the competing processes (anammox vs. classical denitrification).

  10. Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling.

    PubMed

    Treusch, Alexander H; Leininger, Sven; Kletzin, Arnulf; Schuster, Stephan C; Klenk, Hans-Peter; Schleper, Christa

    2005-12-01

    Mesophilic crenarchaeota are frequently found in terrestrial and marine habitats worldwide, but despite their considerable abundance the physiology of these as yet uncultivated archaea has remained unknown. From a 1.2 Gb large-insert environmental fosmid library of a calcareous grassland soil, a 43 kb genomic fragment was isolated with a ribosomal RNA that shows its affiliation to group 1.1b of crenarchaeota repeatedly found in soils. The insert encoded a homologue of a copper-containing nitrite reductase with an unusual C-terminus that encoded a potential amicyanin-like electron transfer domain as well as two proteins related to subunits of ammonia monooxygenases or particulate methane monooxygenases (AmoAB/PmoAB) respectively. Expression of nirK and the amoA-like gene was shown by reverse transcription polymerase chain reaction (PCR) analyses in soil samples, the latter being found at higher levels when the soil was incubated with ammonia (measured by quantitative PCR). Further variants of both genes were amplified from soil samples and were found in the environmental database from the Sargasso Sea plankton. Taken together, our findings suggest that mesophilic terrestrial and marine crenarchaeota might be capable of ammonia oxidation under aerobic and potentially also under anaerobic conditions.

  11. Survival and risk of relapse of acute lymphoblastic leukemia in a Mexican population is affected by dihydrofolate reductase gene polymorphisms

    PubMed Central

    GÓMEZ-GÓMEZ, YAZMÍN; ORGANISTA-NAVA, JORGE; SAAVEDRA-HERRERA, MÓNICA VIRGINIA; RIVERA-RAMÍREZ, ANA BERTHA; TERÁN-PORCAYO, MARCO ANTONIO; DEL CARMEN ALARCÓN-ROMERO, LUZ; ILLADES-AGUIAR, BERENICE; LEYVA-VÁZQUEZ, MARCO ANTONIO

    2012-01-01

    Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. Polymorphisms in the gene coding for DHFR have been associated with adverse event treatment. This study evaluated the effect of the -A317G and C829T polymorphisms in the DHFR gene on survival and risk of relapse of ALL. Seventy patients with ALL and 100 healthy individuals were genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. An association between the polymorphisms and the risk of relapse was found (p<0.05); patients with the -317G/G genotype were found to have an 8.55 (95% CI 1.84–39.70) higher chance of relapse and carriers of the 829T/T genotype had a 14.0 (95% CI 1.13–172.63) higher chance of relapse. Other variables, such as age and leukocyte count, were associated (p<0.05) with the risk of relapse of the disease. Individuals with the G/G and T/T genotype of the -A317G and C829T polymorphisms had poorer survival compared to other genotype groups (log-rank test; p<0.05). Although preliminary, these data seem to suggest a role for the DHFR polymorphisms in the risk of relapse of ALL and the mortality risk in these patients. PMID:22969948

  12. Molecular cloning, heterologous expression, and primary structure of the structural gene for the copper enzyme nitrous oxide reductase from denitrifying Pseudomonas stutzeri.

    PubMed Central

    Viebrock, A; Zumft, W G

    1988-01-01

    The nos genes of Pseudomonas stutzeri are required for the anaerobic respiration of nitrous oxide, which is part of the overall denitrification process. A nos-coding region of ca. 8 kilobases was cloned by plasmid integration and excision. It comprised nosZ, the structural gene for the copper-containing enzyme nitrous oxide reductase, genes for copper chromophore biosynthesis, and a supposed regulatory region. The location of the nosZ gene and its transcriptional direction were identified by using a series of constructs to transform Escherichia coli and express nitrous oxide reductase in the heterologous background. Plasmid pAV5021 led to a nearly 12-fold overexpression of the NosZ protein compared with that in the P. stutzeri wild type. The complete sequence of the nosZ gene, comprising 1,914 nucleotides, together with 282 nucleotides of 5'-flanking sequences and 238 nucleotides of 3'-flanking sequences was determined. An open reading frame coded for a protein of 638 residues (Mr, 70,822) including a presumed signal sequence of 35 residues for protein export. The presequence is in conformity with the periplasmic location of the enzyme. Another open reading frame of 2,097 nucleotides, in the opposite transcriptional direction to that of nosZ, was excluded by several criteria from representing the coding region for nitrous oxide reductase. Codon usage for nosZ of P. stutzeri showed a high G + C content in the degenerate codon position (83.9% versus an average of 60.2%) and relaxed codon usage for the Glu codon, characteristic features of Pseudomonas genes from other species. E. coli nitrous oxide reductase was purified to homogeneity. It had the Mr of the P. stutzeri enzyme but lacked the copper chromophore. Images PMID:3049543

  13. [Association between methylene-tetrahydrofolate reductase gene polymorphisms and chronic myeloid leukemia].

    PubMed

    Dorgham, Samia; Aberkane, Meriem; Boughrara, Wefa; Antar Soltan, Badra; Mehalhal, Nemra; Touhami, Hadj; Sidimansour, Noureddine; Merad Boudia, Nadia; Louhibi, Lotfi; Boudjema, Abdallah

    2014-09-01

    Methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme of folate metabolism. Few studies were reported about its relationship with chronic myeloid leukemia (CML). We conducted a case-control study analyzing the prevalence of the polymorphisms MTHFR C677T and MTHFR A1298C in Algerians CML patients. Using TaqMan(®) allelic discrimination assay, we investigate MTHFR C677T and A1298C polymorphism distribution in 90 cases of CML and 100 healthy subjects. The frequencies of 677T alleles and genotypes 677TT and 677CT were significantly higher in cases than in control (P = 1E-6; OR = 6.77 [4.22-10.86]) and (P = 1E-6; OR = 10.38 [4.56-23.6]) respectively. Also, the frequencies of 1298C alleles and genotypes 1298CC and 1298AC were higher in cases (P = 9 E-6; OR = 2.65 [1.71-4.10]) and (P = 0.008; OR = 2.22 [1.21-4.06]) respectively. We report also the higher significance of the haplotype 677T/1298A and 677T/1298C in cases (P = 0.007; OR = 2.57 [1.26-5.24]) and (P = 5 E-6, OR = 6.91 [2.7646-17.2899]) respectively. Our results demonstrate that 677T and 1298C alleles are both associated with an increased risk of CML in Algeria.

  14. [Molecular characterization of a HMG-CoA reductase gene from a rare and endangered medicinal plant, Dendrobium officinale].

    PubMed

    Zhang, Lin; Wang, Ji-Tao; Zhang, Da-Wei; Zhang, Gang; Guo, Shun-Xing

    2014-03-01

    The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.

  15. Induction of aldose reductase gene expression in LEC rats during the development of the hereditary hepatitis and hepatoma.

    PubMed

    Takahashi, M; Hoshi, A; Fujii, J; Miyoshi, E; Kasahara, T; Suzuki, K; Aozasa, K; Taniguchi, N

    1996-04-01

    We examined age-related changes in the protein and the mRNA expression of aldose reductase in livers of Long-Evans with a cinnamon-like color (LEC) rats, which develop hereditary hepatitis and hepatoma with aging, using Long-Evans with an agouti color rats as controls. The levels of the protein and mRNA of aldose reductase increased after 20 weeks, at the stage of acute hepatitis, and were maintained at 60 weeks of age, while those of aldehyde reductase seemed to be constant at all ages. The expression of aldose reductase was marked in cancerous lesions in hepatoma-bearing LEC rat liver compared to uninvolved surrounding tissues. These results indicated that elevation of aldose reductase accompanied hepatocarcinogenesis and may be related to the acquisition of immortality of the cancer cells through detoxifying cytotoxic aldehyde compounds.

  16. Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells.

    PubMed

    Carothers, A M; Urlaub, G; Ellis, N; Chasin, L A

    1983-04-11

    Overlapping recombinant lambda 1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been isolated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly repeated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolutionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.

  17. Methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and diabetic nephropathy susceptibility in patients with type 2 diabetes mellitus.

    PubMed

    Zhou, Tian-Biao; Drummen, Gregor P C; Jiang, Zong-Pei; Li, Hong-Yan

    2015-01-01

    Methylenetetrahydrofolate reductase (MTHFR) is a crucial enzyme that regulates nucleotide synthesis and DNA methylation. The MTHFR C677T gene polymorphism (rs1801133), a C → T transition at nucleotide 677 in exon 4, is a common gene variant of MTHFR and has been implicated in diabetic nephropathy, albeit with inconsistent results. Here, we performed a meta-analysis to assess the common effect size of this polymorphism on DN susceptibility. Case-control studies on the association of the MTHFR C677T gene polymorphism with DN risk were retrieved from databases up to August 1, 2013, and eligible studies were recruited into the meta-analysis and further analyzed. Of 132 studies, 33 were identified as suitable for this analysis. The results showed that T allele and TT genotype were distinctly associated with DN susceptibility in the overall population and Asians, and might be a risk factor in Caucasians and Africans (T allele: Overall population: p < 0.00001, Asians: p = 0.0002, Caucasians: p = 0.02, Africans: p < 0.00001; TT genotype: Overall population: p < 0.00001, Asians: p = 0.0003, Caucasians: p = 0.008, Africans: p = 0.0003). Furthermore, the analysis suggested that the CC genotype might play a protective role against DN onset in patients with type 2 diabetes for the overall population, Asians, Caucasian and Africans. However, due to the limited sample size in the African population, these results should be interpreted with care. In conclusion, the MTHFR C677T T allele or TT genotype might be a significant genetic molecular marker to determine the risk of DN in patients with type 2 diabetes and help to develop suitable disease prevention and management strategies.

  18. Role of 677C→T polymorphism a single substitution in methylenetetrahydrofolate reductase (MTHFR) gene in North Indian infertile men.

    PubMed

    Naqvi, Hena; Hussain, Syed Rizwan; Ahmad, Mohammad Kaleem; Mahdi, Farzana; Jaiswar, Shyam Pyari; Shankhwar, Satya Narayain; Mahdi, Abbas Ali

    2014-02-01

    Failure or severe difficulty in conceiving a child is surprisingly common, worldwide problem. Half of these cases are due to male factors with defects in sperm (1 in 15 men) being the single most common cause. Also about 60-75 % of male infertility cases are idiopathic, since the molecular mechanisms underlying the defects remain unknown. DNA methylation is crucial for spermatogenesis and high methylenetetrahydrofolate reductase (MTHFR) activity in adult testis than other organs in mouse, signifies its critical role in spermatogenesis. According to recent findings there is a correlation of epigenetic regulation of several imprinted genes with disturbed spermatogenesis and fertility. Consequently any change in the MTHFR gene sequence can modify the spermatogenesis including transmission of infertility to the carriers. The aim of the study is to analyze the distribution of the single nucleotide polymorphism C677T in the MTHFR gene in 637 North Indian infertile patients and 364 fertile North Indian men as controls by using PCR-RFLP technique and Chi Square test for statistical analysis. The average MTHFR 677CC, 677CT, 677TT genotype frequencies of total infertile men were 70.17, 24.17, 5.65 % in infertile men and 75.27, 21.7, 2.74 % in controls, respectively. The average frequency of the MTHFR 677T allele was 17.73 % in infertile men as compared to 13.59 % in controls. The statistical difference was significant. Disease risk was found 2.27-folds increased in patients who were carrying T allele. We found an association of C677T polymorphism with male infertility and that it may be a genetic risk factor for male infertility in North Indian population.

  19. Association between the methylenetetrahydrofolate reductase gene C677T polymorphism and sudden sensorineural hearing loss: a meta-analysis.

    PubMed

    Shu, Jingcheng; Yin, Shihua; Tan, An-Zhou; He, Meirong

    2015-09-01

    A variety of epidemiological studies have evaluated the association between methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism and sudden sensorineural hearing loss (SSNHL), but the results were inconsistent. The aim of this meta-analysis was to clarify more accurately the association of this polymorphism with SSNHL. A systematic literature search of the associated studies up to May 1, 2014, was conducted using the following electronic databases: PubMed, Embase, Medline, and the China National Knowledge Infrastructure. Statistical analyses were performed by STATA12.0 software, with odds ratios (ORs) and their 95 % confidence intervals (CIs). Six eligible studies including covering 1,271 objects were identified. A pooled analysis of these studies showed no significant association between C677T polymorphism and risk of SSNHL: T vs. C (OR = 1.334, POR = 0.105); TT vs. CC (OR = 1.580, POR = 0.231); CT vs. CC (OR = 1.500, POR = 0.123); TT vs. CC + CT (OR = 1.326, POR = 0.293); and TT + CT vs. CC (OR = 1.540, POR = 0.102). But in subgroup analysis, a significant association was found in European populations (T vs. C, OR = 1.542, 95 % CI 1.008-2.359, P = 0.046; TT vs. CT + CC, OR = 1.856, 95 % CI 1.245-2.767, P = 0.002). There was no significant association in any model in the Asian populations. The present meta-analysis suggests that MTHFR gene C677T polymorphism is significantly associated with increased risk of SSNHL disease in European populations, but no statistically significant association was found between the MTHFR C677T gene mutation and SSNHL in Asian. Further large and well-designed studies are needed to confirm this association.

  20. Serum homocysteine, vitamin B12, folic acid levels and methylenetetrahydrofolate reductase (MTHFR) gene polymorphism in vitiligo.

    PubMed

    Yasar, Ali; Gunduz, Kamer; Onur, Ece; Calkan, Mehmet

    2012-01-01

    The aim of this study was to determine serum vitamin B12, folic acid and homocysteine (Hcy) levels as well as MTHFR (C677, A1298C) gene polymorphisms in patients with vitiligo, and to compare the results with healthy controls. Forty patients with vitiligo and 40 age and sex matched healthy subjects were studied. Serum vitamin B12 and folate levels were determined by enzyme-linked immunosorbent assay. Plasma Hcy levels and MTHFR polymorphisms were determined by chemiluminescence and real time PCR methods, respectively. Mean serum vitamin B12 and Hcy levels were not significantly different while folic acid levels were significantly lower in the control group. There was no significant relationship between disease activity and vitamin B12, folic acid and homocystein levels. No significant difference in C677T gene polymorphism was detected. Heterozygote A1298C gene polymorphism in the patient group was statistically higher than the control group. There was no significant relationship between MTHFR gene polymorphisms and vitamin B12, folic acid and homocysteine levels. In conclusion, vitamin B12, folate and Hcy levels are not altered in vitiligo and MTHFR gene mutations (C677T and A1298C) do not seem to create susceptibility for vitiligo.

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

    PubMed Central

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

    2015-01-01

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

  2. Triclosan Resistome from Metagenome Reveals Diverse Enoyl Acyl Carrier Protein Reductases and Selective Enrichment of Triclosan Resistance Genes

    PubMed Central

    Khan, Raees; Kong, Hyun Gi; Jung, Yong-Hoon; Choi, Jinhee; Baek, Kwang-Yeol; Hwang, Eul Chul; Lee, Seon-Woo

    2016-01-01

    Triclosan (TCS) is a widely used antimicrobial agent and TCS resistance is considered to have evolved in diverse organisms with extensive use of TCS, but distribution of TCS resistance has not been well characterized. Functional screening of the soil metagenome in this study has revealed that a variety of target enoyl acyl carrier protein reductases (ENR) homologues are responsible for the majority of TCS resistance. Diverse ENRs similar to 7-α-hydroxysteroid dehydrogenase (7-α-HSDH), FabG, or the unusual YX7K-type ENR conferred extreme tolerance to TCS. The TCS-refractory 7-α HSDH-like ENR and the TCS-resistant YX7K-type ENR seem to be prevalent in human pathogenic bacteria, suggesting that a selective enrichment occurred in pathogenic bacteria in soil. Additionally, resistance to multiple antibiotics was found to be mediated by antibiotic resistance genes that co-localize with TCS resistance determinants. Further comparative analysis of ENRs from 13 different environments has revealed a huge diversity of both prototypic and metagenomic TCS-resistant ENRs, in addition to a selective enrichment of TCS-resistant specific ENRs in presumably TCS-contaminated environments with reduced ENR diversity. Our results suggest that long-term extensive use of TCS can lead to the selective emergence of TCS-resistant bacterial pathogens, possibly with additional resistance to multiple antibiotics, in natural environments. PMID:27577999

  3. Mercuric reductase genes (merA) and mercury resistance plasmids in High Arctic snow, freshwater and sea-ice brine.

    PubMed

    Møller, Annette K; Barkay, Tamar; Hansen, Martin A; Norman, Anders; Hansen, Lars H; Sørensen, Søren J; Boyd, Eric S; Kroer, Niels

    2014-01-01

    Bacterial reduction in Hg(2+) to Hg(0) , mediated by the mercuric reductase (MerA), is important in the biogeochemical cycling of Hg in temperate environments. Little is known about the occurrence and diversity of merA in the Arctic. Seven merA determinants were identified among bacterial isolates from High Arctic snow, freshwater and sea-ice brine. Three determinants in Bacteriodetes, Firmicutes and Actinobacteria showed < 92% (amino acid) sequence similarity to known merA, while one merA homologue in Alphaproteobacteria and 3 homologues from Betaproteobacteria and Gammaproteobacteria were > 99% similar to known merA's. Phylogenetic analysis showed the Bacteroidetes merA to be part of an early lineage in the mer phylogeny, whereas the Betaproteobacteria and Gammaproteobacteria merA appeared to have evolved recently. Several isolates, in which merA was not detected, were able to reduce Hg(2+) , suggesting presence of unidentified merA genes. About 25% of the isolates contained plasmids, two of which encoded mer operons. One plasmid was a broad host-range IncP-α plasmid. No known incompatibility group could be assigned to the others. The presence of conjugative plasmids, and an incongruent distribution of merA within the taxonomic groups, suggests horizontal transfer of merA as a likely mechanism for High Arctic microbial communities to adapt to changing mercury concentration.

  4. The relationship between methylenetetrahydrofolate reductase C677T gene polymorphism and diabetic nephropathy in Croatian type 2 diabetic patients.

    PubMed

    Tomić, Nives Gojo; Marusić, Srećko; Bozikov, Velimir; Kusec, Rajko; Bacić-Vrca, Vesna; Tadić, Mario

    2013-09-01

    Methylenetetrahydrofolate reductase (MTHFR) polymorphism has been shown to be associated with the development of diabetic nephropathy in many ethnic groups. In this study, we examined the correlation between MTHFR C677T polymorphism and microalbuminuria in patients with diabetes mellitus type 2 in Croatian patients. 85 patients with diabetes mellitus type 2 were recruited. Patients were classified into two groups--with and without diabetic nephropathy according to urinary albumin excretion rate in urine collected during 24 hours. The C677T genotype was determined by real-time PCR analysis. The genotype frequencies were CC 36.5%, CT 42.3% and TT 21.2% in diabetic patients without nephropathy versus CC 39.4%, CT 45.4% and TT 15.2% in those with nephropathy. There was no statistically significant difference in allele distribution between patients with nephropathy and those without (p = 0.788). Our study did not show a correlation between mutations in the MTHFR gene and diabetic nephropathy in Croatian patients. Diabetic nephropathy is influenced by multiple risk factors which can modify the importance of MTHFR polymorphism in its development.

  5. Detection and diversity of fungal nitric oxide reductase genes (p450nor) in agricultural soils

    SciTech Connect

    Higgins, Steven A.; Welsh, Allana; Orellana, Luis H.; Konstantinidis, Konstantinos T.; Chee-Sanford, Joanne C.; Sanford, Robert A.; Schadt, Christopher W.; Löffler, Frank E.

    2016-03-11

    Members of the Fungi convert nitrate (NO3-) and nitrite (NO2-) to gaseous nitrous oxide (N2O) (denitrification), but the fungal contributions to N-loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N2O from added NO3- or NO2- in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, of which 151 produced N2O from NO2-. Novel PCR primers targeting the p450nor gene that encodes the nitric oxide (NO) reductase responsible for N2O production in fungi yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54-98% amino acid identity to reference P450nor sequences within the phylum Ascomycota, and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from nitrite, whereas nirK (encoding the NO-forming nitrite reductase) was amplified in only 13-74% of the N2O-forming isolates using two separate nirK primer sets. Altogether, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation.

  6. [C677T polymorphism of the methylentetrahydrofolate reductase gene in mothers of children affected with neural tube defects].

    PubMed

    Morales de Machín, Alisandra; Méndez, Karile; Solís, Ernesto; Borjas de Borjas, Lisbeth; Bracho, Ana; Hernández, María Luisa; Negrón, Aimara; Delgado, Wilmer; Sánchez, Yanira

    2015-09-01

    Neural tube defects (NTD) are the most common congenital anomalies of the central nervous system, with a multifactorial pattern of inheritance, presumably involving the interaction of several genetic and environmental factors. The methylenetetrahydrofolate reductase (MTHFR) gene 677C>T polymorphism has been implicated as a risk factor for NTD. The main objective of this research was to investigate the association of the 677C>T polymorphism of the MTHFR gene as a genetic risk factor for NTD. Molecular analysis was performed in DNA samples from 52 mothers with antecedent of NTD offspring and from 119 healthy control mothers. Using the Polymerase Chain Reaction, a 198 bases pairs fragment was digested with the restriction enzyme Hinfi. 677T MTHFR allele frequencies for the problem and the control groups were 51.92% and 34.45%, respectively, and 677C MTHFR allele frequencies were 48.08% and 65.55%, respectively. There were significant differences in allele (p: 0.002) and genotype (p: 0.007) frequencies between these two groups. The odds ratio (OR) to the TT genotype vs. the CC genotype was estimated as OR: 4.9 [95% CI: 1,347-6.416] p: 0.002; CT+TT vs. CC: OR: 2.9 [95% CI: 1.347-6.416] p: 0.005; TT vs. CT+CC: OR: 2.675 [95% CI: 1,111-6.441] p: 0.024. The data presented in this study support the relationship between MTHFR 677C>T polymorphism and risk in mothers with antecedent of NTD offspring.

  7. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects?

    PubMed Central

    van der Put, N M; Gabreëls, F; Stevens, E M; Smeitink, J A; Trijbels, F J; Eskes, T K; van den Heuvel, L P; Blom, H J

    1998-01-01

    Recently, we showed that homozygosity for the common 677(C-->T) mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, causing thermolability of the enzyme, is a risk factor for neural-tube defects (NTDs). We now report on another mutation in the same gene, the 1298(A-->C) mutation, which changes a glutamate into an alanine residue. This mutation destroys an MboII recognition site and has an allele frequency of .33. This 1298(A-->C) mutation results in decreased MTHFR activity (one-way analysis of variance [ANOVA] P < .0001), which is more pronounced in the homozygous than heterozygous state. Neither the homozygous nor the heterozygous state is associated with higher plasma homocysteine (Hcy) or a lower plasma folate concentration-phenomena that are evident with homozygosity for the 677(C-->T) mutation. However, there appears to be an interaction between these two common mutations. When compared with heterozygosity for either the 677(C-->T) or 1298(A-->C) mutations, the combined heterozygosity for the 1298(A-->C) and 677(C-->T) mutations was associated with reduced MTHFR specific activity (ANOVA P < .0001), higher Hcy, and decreased plasma folate levels (ANOVA P <.03). Thus, combined heterozygosity for both MTHFR mutations results in similar features as observed in homozygotes for the 677(C-->T) mutation. This combined heterozygosity was observed in 28% (n =86) of the NTD patients compared with 20% (n =403) among controls, resulting in an odds ratio of 2.04 (95% confidence interval: .9-4.7). These data suggest that the combined heterozygosity for the two MTHFR common mutations accounts for a proportion of folate-related NTDs, which is not explained by homozygosity for the 677(C-->T) mutation, and can be an additional genetic risk factor for NTDs. PMID:9545395

  8. A Meta-Analysis of Association between Methylenetetrahydrofolate Reductase Gene (MTHFR) 677C/T Polymorphism and Diabetic Retinopathy

    PubMed Central

    Luo, Shasha; Wang, Furu; Shi, Chao; Wu, Zhifeng

    2016-01-01

    Aims: To shed light on the conflicting findings of the association between the methylenetetrahydrofolate reductase gene (MTHFR) 677C/T polymorphism and the risk of diabetic retinopathy (DR), a meta-analysis was conducted. Methods: A predefined search was performed on 1747 DR cases and 3146 controls from 18 published studies by searching electronic databases and reference lists of relevant articles. A random-effects or fixed-effects model was used to estimate the sizes of overall and stratification effects of the MTHFR 677C/T polymorphism on the risk of DR, as appropriate. Results: Risks were evaluated by odds ratios (OR) with 95% confidence intervals (95% CI). We found a significant association between the MTHFR 677C/T polymorphism and the risk of DR for each genetic model (recessive model: OR = 1.67; 95% CI: 1.19–2.40 and dominant model: OR = 1.71; 95% CI: 1.28–2.28; respectively). In stratified analysis; we further found that the Asian group with both types of diabetes mellitus (DM) showed a significant association with genetic models (recessive model: OR = 2.16; 95% CI: 1.75–2.60 and dominant model: OR = 1.98; 95% CI: 1.42–2.76; respectively). Conclusions: Our study suggested that the MTHFR 677C/T polymorphism may contribute to DR development, especially in Asian populations. Prospective and additional genome-wide association studies (GWAS) are needed to clarify the real role of the MTHFR gene in determining susceptibility to DR. PMID:27517946

  9. Real-Time PCR for Dihydrofolate Reductase Gene Single-Nucleotide Polymorphisms in Plasmodium vivax Isolates

    PubMed Central

    Brega, Sara; de Monbrison, Frédérique; Severini, Carlo; Udomsangpetch, Rachanee; Sutanto, Inge; Ruckert, Paul; Peyron, François; Picot, Stéphane

    2004-01-01

    Mutations in the dhfr gene of Plasmodium vivax (pvdhfr) are associated with resistance to the antifolate antimalarial drugs. Polymorphisms in the pvdhfr gene were assessed by hybridization probe technology on the LightCycler instrument with 134 P. vivax-infected blood samples from Turkey (n = 24), Azerbaijan (n = 39), Thailand (n = 16), Indonesia (n = 53), and travelers (n = 19). Double mutations (S58R and S117N) or quadruple mutations (F57L/I, S58R, T61M, and S117N) in the pvdhfr genes were found in all Thai samples (100%). pvdhfr mutant-type alleles were significantly more common in samples from travelers (42%) than in those from patients from Indonesia (5%). Surprisingly, the pvdhfr single-mutation allele (S117N) was identified at a high frequency in parasites from Turkey and Azerbaijan (71 and 36%, respectively), where sulfadoxine-pyrimethamine is not recommended for the treatment of P. vivax malaria by the World Health Organization and the Malaria National Programs. PMID:15215112

  10. Gene-wide association study between the methylenetetrahydrofolate reductase gene (MTHFR) and schizophrenia in the Japanese population, with an updated meta-analysis on currently available data.

    PubMed

    Yoshimi, Akira; Aleksic, Branko; Kawamura, Yukiko; Takahashi, Nagahide; Yamada, Shinnosuke; Usui, Hinako; Saito, Shinichi; Ito, Yoshihito; Iwata, Nakao; Inada, Toshiya; Noda, Yukihiro; Yamada, Kiyofumi; Ozaki, Norio

    2010-12-01

    Methylenetetrahydrofolate reductase (MTHFR) is a critical molecule for single-carbon transfer reactions. Recent evidence suggests that polymorphisms of MTHFR are related to neural tube deficits and the pathogenesis of schizophrenia. While several studies have demonstrated associations between the gene encoding the MTHFR (MTHFR) polymorphisms and schizophrenia, these studies lack consistency. Therefore, we conducted a gene-wide association study (patients with schizophrenia = 696, control subjects = 747) and performed imputation analysis. Additionally, we performed meta-analysis on currently available data from 18 studies for two common functional polymorphisms (rs1801131 and rs1801133). There were no significant associations with schizophrenia in the single marker analysis for the seven tagging SNPs of MTHFR. In the haplotypic analysis, a nominally significant association was observed between the haplotypes, which included four SNPs (rs1801133, rs17421511, rs17037396, and rs9651118) and the schizophrenic patients. Additionally, the imputation analysis demonstrated there were several associated markers on the MTHFR chromosomal region. However, confirmatory analyses of three tagging SNPs (rs1801133, rs17037396, and rs9651118) and the top SNP (rs17421511) for the imputation results (patients with schizophrenia = 797, control subjects = 1025) failed to replicate the haplotypic analysis and the imputation results. These findings suggest that MTHFR polymorphisms are unlikely to be related to the development of schizophrenia in the Japanese population. However, since our meta-analysis results demonstrated strong support for association of rs1801133 with schizophrenia, further replication studies based on a gene-wide approach need to be considered.

  11. Fatty acyl-CoA reductase

    SciTech Connect

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

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

  12. Cloning and expression analysis of interferon-gamma-inducible-lysosomal thiol reductase gene in large yellow croaker (Pseudosciaena crocea).

    PubMed

    Zheng, Wenbiao; Chen, Xinhua

    2006-05-01

    In mammals, interferon-gamma-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. Here, we reported the cloning of a GILT gene homologue from the spleen of large yellow croaker, a marine fish (LycGILT). The full-length cDNA of LycGILT gene is 1033 nucleotides (nt) encoding a protein of 256 amino acids (aa), with a putative molecular weight of 28.9 kDa. The deduced protein is highly homologous to that of mammalian and zebrafish GILTs and shares 54.1% sequence identity to that of zebrafish and 43.2-39.2% sequence identity to that of various mammals. The deduced LycGILT possesses the typical structural feature of mammalian GILT, including an active-site CXXC motif, a GILT signature sequence CQHGX2ECX2NX4C, and other six cysteines responsible for the formation of disulfide bonds in the C-terminus. Genomic analysis revealed that LycGILT gene, spanning a 3159nt fragment, contained seven exons interrupted by six introns and exhibited a similar exon-intron organization to human and mouse GILT genes except for a slightly more compact intron arrangement. The LycGILT expression is obviously up-regulated in spleen and kidney after immunization with inactivated trivalent bacterial vaccine consisting of Vibrio alginolyticus, V. paraphaemolyticus, and Aeromonas hydrophila although it also is constitutively expressed in liver, gills, brain, and heart, suggesting that LycGILT may be involved in the immune response to bacterial challenge in large yellow croaker. A search of NCBI sequence data with LycGILT cDNA identified a pufferfish (fugu rubrides) GILT homologue cDNA and its genomic DNA sequence, where two putative interferon-gamma activation sites (GAS) were found within the promoter region. This provided evidence that a fish GILT homologue like

  13. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    PubMed

    Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

  14. Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in Senegal.

    PubMed

    Ndiaye, D; Daily, J P; Sarr, O; Ndir, O; Gaye, O; Mboup, S; Wirth, D F

    2005-11-01

    Senegal recently (2004) switched to sulfadoxine-pyrimethamine (SP) with amodiaquine as first line therapy for malaria in response to increasing chloroquine resistance. In anticipation of emerging resistance to SP as a result of this change in drug pressure, we set out to define the baseline prevalence of SP-associated mutations in the dhfr and dhps genes in Plasmodium falciparum using geographically diverse and longitudinally collected samples. A total of 153 blood samples were analysed from patients (5 years or older) with mild malaria after informed consent was obtained. Longitudinal samples were collected between 2000 and 2003 in Pikine, a suburb of Dakar. Geographically diverse site sampling was carried out in 2003. The mutation prevalence in DHFR codons 51, 59 and 108 is 65%, 61% and 78% in Pikine, 2003. The overall prevalence of the triple mutation that is associated with high-level pyrimethamine resistance is 61%. The mutation prevalence rate in DHPS codons 436 and 437 is 21% and 40%, respectively. There is significant geographic variation in genotypic resistance, as samples from Pikine in 2003 had higher mutation prevalence in the pfdhfr and pfdhps genes compared to samples from Tambacounda (P < 0.015). In summary, this study demonstrates a high background prevalence of SP resistance mutations already present in P. falciparum in Senegal.

  15. The Sorghum Gene for Leaf Color Changes upon Wounding (P) Encodes a Flavanone 4-Reductase in the 3-Deoxyanthocyanidin Biosynthesis Pathway

    PubMed Central

    Kawahigashi, Hiroyuki; Kasuga, Shigemitsu; Sawada, Yuji; Yonemaru, Jun-ichi; Ando, Tsuyu; Kanamori, Hiroyuki; Wu, Jianzhong; Mizuno, Hiroshi; Momma, Mitsuru; Fujimoto, Zui; Hirai, Masami Yokota; Matsumoto, Takashi

    2016-01-01

    Upon wounding or pathogen invasion, leaves of sorghum [Sorghum bicolor (L.) Moench] plants with the P gene turn purple, whereas leaves with the recessive allele turn brown or tan. This purple phenotype is determined by the production of two 3-deoxyanthocyanidins, apigeninidin and luteolinidin, which are not produced by the tan-phenotype plants. Using map-based cloning in progeny from a cross between purple Nakei-MS3B (PP) and tan Greenleaf (pp) cultivars, we isolated this gene, which was located in a 27-kb genomic region around the 58.1 Mb position on chromosome 6. Four candidate genes identified in this region were similar to the maize leucoanthocyanidin reductase gene. None of them was expressed before wounding, and only the Sb06g029550 gene was induced in both cultivars after wounding. The Sb06g029550 protein was detected in Nakei-MS3B, but only slightly in Greenleaf, in which it may be unstable because of a Cys252Tyr substitution. A recombinant Sb06g029550 protein had a specific flavanone 4-reductase activity, and converted flavanones (naringenin or eriodictyol) to flavan-4-ols (apiforol or luteoforol) in vitro. Our data indicate that the Sb06g029550 gene is involved in the 3-deoxyanthocyanidin synthesis pathway. PMID:26994288

  16. The Sorghum Gene for Leaf Color Changes upon Wounding (P) Encodes a Flavanone 4-Reductase in the 3-Deoxyanthocyanidin Biosynthesis Pathway.

    PubMed

    Kawahigashi, Hiroyuki; Kasuga, Shigemitsu; Sawada, Yuji; Yonemaru, Jun-Ichi; Ando, Tsuyu; Kanamori, Hiroyuki; Wu, Jianzhong; Mizuno, Hiroshi; Momma, Mitsuru; Fujimoto, Zui; Hirai, Masami Yokota; Matsumoto, Takashi

    2016-05-03

    Upon wounding or pathogen invasion, leaves of sorghum [Sorghum bicolor (L.) Moench] plants with the P gene turn purple, whereas leaves with the recessive allele turn brown or tan. This purple phenotype is determined by the production of two 3-deoxyanthocyanidins, apigeninidin and luteolinidin, which are not produced by the tan-phenotype plants. Using map-based cloning in progeny from a cross between purple Nakei-MS3B (PP) and tan Greenleaf (pp) cultivars, we isolated this gene, which was located in a 27-kb genomic region around the 58.1 Mb position on chromosome 6. Four candidate genes identified in this region were similar to the maize leucoanthocyanidin reductase gene. None of them was expressed before wounding, and only the Sb06g029550 gene was induced in both cultivars after wounding. The Sb06g029550 protein was detected in Nakei-MS3B, but only slightly in Greenleaf, in which it may be unstable because of a Cys252Tyr substitution. A recombinant Sb06g029550 protein had a specific flavanone 4-reductase activity, and converted flavanones (naringenin or eriodictyol) to flavan-4-ols (apiforol or luteoforol) in vitro Our data indicate that the Sb06g029550 gene is involved in the 3-deoxyanthocyanidin synthesis pathway.

  17. Functional nucleotide excision repair is required for the preferential removal of N-ethylpurines from the transcribed strand of the dihydrofolate reductase gene of Chinese hamster ovary cells.

    PubMed Central

    Sitaram, A; Plitas, G; Wang, W; Scicchitano, D A

    1997-01-01

    Transcription-coupled repair of DNA adducts is an essential factor that must be considered when one is elucidating biological endpoints resulting from exposure to genotoxic agents. Alkylating agents comprise one group of chemical compounds which modify DNA by reacting with oxygen and nitrogen atoms in the bases of the double helix. To discern the role of transcription-coupled DNA repair of N-ethylpurines present in discrete genetic domains, Chinese hamster ovary cells were exposed to N-ethyl-N-nitrosourea, and the clearance of the damage from the dihydrofolate reductase gene was investigated. The results indicate that N-ethylpurines were removed from the dihydrofolate reductase gene of nucleotide excision repair-proficient Chinese hamster ovary cells; furthermore, when repair rates in the individual strands were determined, a statistically significant bias in the removal of ethyl-induced, alkali-labile sites was observed, with clearance occurring 30% faster from the transcribed strand than from its nontranscribed counterpart at early times after exposure. In contrast, removal of N-ethylpurines was observed in the dihydrofolate reductase locus in cells that lacked nucleotide excision repair, but both strands were repaired at the same rate, indicating that transcription-coupled clearance of these lesions requires the presence of active nucleotide excision repair. PMID:9001209

  18. Excision of transposable elements from the chalcone isomerase and dihydroflavonol 4-reductase genes may contribute to the variegation of the yellow-flowered carnation (Dianthus caryophyllus).

    PubMed

    Itoh, Yoshio; Higeta, Daisuke; Suzuki, Akane; Yoshida, Hiroyuki; Ozeki, Yoshihiro

    2002-05-01

    In the "Rhapsody" cultivar of the carnation, which bears white flowers variegated with red flecks and sectors, a transposable element, dTdic1, belonging to the Ac/Ds superfamily, was found within the dihydroflavonol 4-reductase (DFR) gene. The red flecks and sectors of "Rhapsody" may be attributable to a reversion to DFR activity after the excision of dTdic1. The yellow color of the carnation petals is attributed to the synthesis and accumulation of chalcone 2'-glucoside. In several of the carnation cultivars that bear yellow flowers variegated with white flecks and sectors, both the chalcone isomerase (CHI) and DFR genes are disrupted by dTdic1.

  19. Gene Expression and Silencing Studies in Phytophthora infestans Reveal Infection-Specific Nutrient Transporters and a Role for the Nitrate Reductase Pathway in Plant Pathogenesis

    PubMed Central

    Ah-Fong, Audrey M. V.; Davis, Carol; Andreeva, Kalina; Judelson, Howard S.

    2016-01-01

    To help learn how phytopathogens feed from their hosts, genes for nutrient transporters from the hemibiotrophic potato and tomato pest Phytophthora infestans were annotated. This identified 453 genes from 19 families. Comparisons with a necrotrophic oomycete, Pythium ultimum var. ultimum, and a hemibiotrophic fungus, Magnaporthe oryzae, revealed diversity in the size of some families although a similar fraction of genes encoded transporters. RNA-seq of infected potato tubers, tomato leaves, and several artificial media revealed that 56 and 207 transporters from P. infestans were significantly up- or down-regulated, respectively, during early infection timepoints of leaves or tubers versus media. About 17 were up-regulated >4-fold in both leaves and tubers compared to media and expressed primarily in the biotrophic stage. The transcription pattern of many genes was host-organ specific. For example, the mRNA level of a nitrate transporter (NRT) was about 100-fold higher during mid-infection in leaves, which are nitrate-rich, than in tubers and three types of artificial media, which are nitrate-poor. The NRT gene is physically linked with genes encoding nitrate reductase (NR) and nitrite reductase (NiR), which mobilize nitrate into ammonium and amino acids. All three genes were coregulated. For example, the three genes were expressed primarily at mid-stage infection timepoints in both potato and tomato leaves, but showed little expression in potato tubers. Transformants down-regulated for all three genes were generated by DNA-directed RNAi, with silencing spreading from the NR target to the flanking NRT and NiR genes. The silenced strains were nonpathogenic on leaves but colonized tubers. We propose that the nitrate assimilation genes play roles both in obtaining nitrogen for amino acid biosynthesis and protecting P. infestans from natural or fertilization-induced nitrate and nitrite toxicity. PMID:27936244

  20. Polymorphisms in methylenetetrahydrofolate reductase gene and risk of non-Hodgkin lymphoma in a multi-ethnic population.

    PubMed

    Suthandiram, Sujatha; Gan, Gin Gin; Zain, Shamsul Mohd; Haerian, Batoul Sadat; Bee, Ping Chong; Lian, Lay Hoong; Chang, Kian Meng; Ong, Tee Chuan; Mohamed, Zahurin

    2014-05-01

    An imbalance in folate metabolism can adversely affect DNA synthesis and methylation systems which can lead to susceptibility to non-Hodgkin lymphoma (NHL). Whether single nucleotide polymorphisms (SNPs) and their haplotypes in the methylenetetrahydrofolate reductase (MTHFR) are associated with NHL, remain inconclusive. We investigated the association between MTHFR C677T and A1298C SNPs and NHL risk in a population which is made up of Malay, Chinese and Indian ethnic subgroups. A total of 372 NHL patients and 722 controls were genotyped using the Sequenom MassARRAY platform. Our results of the pooled subjects failed to demonstrate significant association between the MTHFR C677T and A1298C SNPs with NHL and its subtypes. The results were in agreement with the previous meta-analyses. In the Indian ethnic subgroup however, single locus analysis of MTHFR A1298C appears to confer risk to NHL (Odds ratio (OR) 1.91, 95% confidence interval (95% CI) 1.22-3.00, P=0.006). The risk is almost doubled in homozygous carrier of MTHFR 1298CC (OR 4.03, 95% CI 1.56-10.43, P=0.004). Haplotype analysis revealed higher frequency of CC in the Indian NHL patients compared with controls (OR 1.86, 95% CI 1.18-2.93, P=0.007). There is lack of evidence to suggest an association between MTHFR C677T and A1298C with the risk of NHL in the Malays and Chinese. In the Indians however, the MTHFR A1298C confers risk to NHL. This study suggests ethnicity modifies the relationship between polymorphisms in the folate-metabolizing gene and NHL.

  1. Molecular Cloning and Characterization of Three Genes Encoding Dihydroflavonol-4-Reductase from Ginkgo biloba in Anthocyanin Biosynthetic Pathway

    PubMed Central

    Hua, Cheng; Linling, Li; Shuiyuan, Cheng; Fuliang, Cao; Feng, Xu; Honghui, Yuan; Conghua, Wu

    2013-01-01

    Dihydroflavonol-4-reductase (DFR, EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs) were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species. PMID:23991027

  2. Vertical distribution of nitrite reductase genes (nir S) in continental margin sediments of the Gulf of Mexico.

    PubMed

    Tiquia, Sonia M; Masson, Steven A; Devol, Allan

    2006-12-01

    Marine sediments account for up to 66% of the loss of nitrogen load to coastal areas. Sedimentary denitrification is the main sink for fixed nitrogen in the global nitrogen budget, and thus it is important to understand the structure and composition of denitrifying communities. To understand the structure and composition of denitrifying communities, the diversity of nitrite reductase (nirS) genes from sediments along the Gulf of Mexico was examined using a PCR-based cloning approach. Sediments were collected at three different depths (0-0.5, 4-5 and 19-21 cm). Geochemical analysis revealed decreasing nitrate and oxygen concentrations with increasing sediment depth. This trend coincided with the decrease in diversity of denitrifying bacteria. LIBSHUFF analysis indicated that the clone library in the shallowest sediment (depth, 0-0.5 cm) was significantly different from that in the deepest sediment (depth, 19-21 cm), and that the deeper sediments (depths of 4-5 and 19-21 cm) were significantly similar. Community structural shifts were evident between the shallowest (oxic zone) and deepest (anoxic zone) sediments. Community changes within the deepest sediments were more subtle, with the presence of different nirS clone sequences gradually becoming dominant or, alternatively, decreasing with depth. The changes in community structure at this depth are possibly driven by nutrient availability, with lower quality sources of carbon and energy leading to the disappearance of nirS sequences common in the top layer. The majority of recovered nirS sequences were phylogenetically divergent relative to known denitrifying bacteria in the database.

  3. Artemisinic acid inhibits melanogenesis through downregulation of C/EBP α-dependent expression of HMG-CoA reductase gene.

    PubMed

    Lee, Jongsung; Lee, Jienny; Jung, Eunsun; Cho, Jae Youl; Park, Deokhoon

    2013-01-01

    Cholesterol is associated with the regulation of melanogenesis which is the major physiological defense against solar irradiation. The present study was designed to determine the effects of artemisinic acid on melanogenesis and its mechanisms of action in human epidermal melanocytes. In this study, we found that artemisinic acid inhibited melanin content. The mRNA levels of microphthalmia-associated transcription factor (MITF) and its downstream genes tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 were reduced by artemisinic acid treatment. Additionally, the mRNA levels of melanogenesis-related genes (c-KIT, stem cell factor (SCF), and macrophage migration inhibitory factor (MIF)) were down-regulated by artemisinic acid. Furthermore, cAMP production and protein kinase A (PKA) activity were suppressed by artemisinic acid. Moreover, attempts to elucidate a possible mechanism underlying the artemisinic acid-mediated effects revealed that artemisinic acid regulated melanogenesis by inhibiting cholesterol synthesis through downregulation of the hydroxymethylglutaryl CoA (HMG CoA) reductase gene, which was mediated through reduced expression of the CCAAT/enhancer-binding protein (C/EBP) α gene. Taken together, these findings indicate that the inhibition of melanogenesis by artemisinic acid occurs through reduced expression of the HMG CoA reductase gene, which is mediated by C/EBP α inhibition and suggest that artemisinic acid may be useful as a hyperpigmentation inhibitor.

  4. Methylenetetrahydrofolate reductase gene polymorphisms and the risk of anencephaly in Mexico.

    PubMed

    Muñoz, Julia Blanco; Lacasaña, Marina; Cavazos, Ricardo García; Borja-Aburto, Victor Hugo; Galavíz-Hernández, Carlos; Garduño, Clemente Aguilar

    2007-06-01

    The precise etiology of neural tube defects (NTDs) is not known. There is some evidence that mutations in MTHFR gene provide susceptibility to NTDs in some populations; however, other studies have not found this association. One of the problems with previous studies is that they treat NTDs as a homogeneous group, when specific defects could have different etiologies. We conducted a case-control study specifically for anencephaly, based on the Mexican Epidemiological Surveillance System of Neural Tube Defects to evaluate its association with maternal MTHFR 677C > T and 1298A > C polymorphisms, in three states with high frequencies of NTDs: Puebla, Estado de México and Guerrero. We interviewed and collected blood samples from 118 case mothers and 112 control mothers. The questionnaire included information on their reproductive history, socioeconomic characteristics, prenatal care, tobacco and alcohol use, presence of chronic diseases, acute illnesses and fever, consumption of multivitamins and drugs during the periconceptional period. After adjusting for potential confounders, the risk from the mutated homozygous mothers (677TT genotype) was significantly higher than that from mothers with 677CC genotype (OR 3.16, 95% CI 1.29-7.73); in the case of the heterozygous mothers, an increased risk of anencephaly was observed, even though this was not statistically significant (OR 1.81 95% CI 0.78-4.25). The association found between maternal 677TT genotype and anencephaly and the elevated presence of the 677T allele among Mexican women of fertile age urges intensifying folic acid supplementation which has proved to modify this genetic risk in other populations.

  5. Association of methylenetetrahydrofolate reductase gene 677C > T polymorphism and Down syndrome.

    PubMed

    Costa-Lima, Marcelo Aguiar; Amorim, Márcia Rodrigues; Orioli, Iêda Maria

    2013-03-01

    The association between Down syndrome (DS) and maternal polymorphisms in genes encoding folic acid metabolizing enzymes remains a controversial issue. A meta-analysis was performed to evaluate the association of maternal MTHFR 677C > T polymorphism and the risk of having a child with DS. Case-control studies were screened from major literature databases. Twenty articles from 13 countries worldwide, with a total of 2,101 DS and 2,702 control mothers, attended the inclusion criteria. We found a 50 % increase for the association of maternal homozygous TT genotype and DS in both fixed (OR = 1.51; 95 % CI 1.22-1.87) and random effects models (OR 1.54; 95 % 1.15-2.05). Similarly, a significant pooled OR was found for the heterozygote CT, with an OR 1.26; 95 % CI 1.10-1.43 (fixed effects model) and OR 1.28; 95 % 1.08-1.51 (random effects model). As ultra-violet B solar radiation highly depends on latitude, and can promote, in less pigmented skin, intravascular folate photolysis, we stratified the analysis by latitude region, defining as Tropical (between 23.5(°) S and 23.5(°) N), Sub-Tropical (between 23.5(°) and 40(°) N and S), and Northern (≥ 40(o) N). Significant association was only found for Sub-Tropical area, both using fixed and random effect models. In conclusion, MTHFR 677C > T polymorphism is a moderate risk factor for DS for some populations, and populations located in Sub-Tropical region seem to be at greater risk. Latitude, ethnicity, skin pigmentation, and red blood cell folate are important variables to be considered in future studies.

  6. Identification of three IFN-γ inducible lysosomal thiol reductase (GILT)-like genes in mud crab Scylla paramamosain with distinct gene organizations and patterns of expression.

    PubMed

    Huang, Wen-Shu; Duan, Li-Peng; Huang, Bei; Zhou, Li-Hong; Liang, Ying; Tu, Chen-Ling; Zhang, Fang-Fang; Nie, Pin; Wang, Tiehui

    2015-10-01

    Vertebrate gamma-interferon inducible lysosomal thiol reductase (GILT) is an IFN-γ-inducible protein and is involved in MHCII-restricted antigen processing and cross-presentation of MHCI-restricted antigens in adaptive immunity. Outside of the endocytic MHC pathway, GILT regulates the cellular redox state, inhibits T cell activation, neutralizes extracellular pathogens and is also a host factor of some bacterial pathogens. In this report, we isolated and characterized three divergent GILT-like genes, GILT1, GILT2 and GILT3, which share only 30.9-40.4% identities in a crustacean mud crab Scylla paramamosain. Whilst the crab GILT1 and GILT3 possess four and five exons, respectively, the GILT2 is intronless, suggesting that GILT2 may arise from a recent retroposition event. The invertebrate GILT-like genes have diverse gene organizations and may be evolved in a species/lineage-specific manner as suggested by phylogenetic tree analysis. The amino acid sequences equivalent to human mature GILT are well conserved, including the GILT signature and nine of the ten cysteine residues that potentially form 5 disulfide bonds in human GILT, across the animal kingdom. However, most invertebrate GILT-like molecules lack the human-type N-terminal propeptide, as well as the human-type C-terminal with a conserved cysteine residue, suggesting differences in post translational processing and mode of action. All the three GILT-like genes are highly expressed in the hepatopancreas and up-regulated by pathogenic bacterial infection suggesting a role in immune defense against bacterial diseases. This study may provide the basis for further investigation of the expanding functions of GILT-like molecules in immunity and other physiological processes in mud crabs and other animals.

  7. Identification of the yqhE and yafB Genes Encoding Two 2,5-Diketo-d-Gluconate Reductases in Escherichia coli

    PubMed Central

    Yum, Do-Young; Lee, Bong-Yong; Pan, Jae-Gu

    1999-01-01

    The identification of a gene (yiaE) encoding 2-ketoaldonate reductase (2KR) in our previous work led to the hypothesis that Escherichia coli has other ketogluconate reductases including 2,5-diketo-d-gluconate reductase (25DKGR) and to study of the related ketogluconate metabolism. By using the deduced amino acid sequences of 5-diketo-d-gluconate reductase (5KDGR) of Gluconobacter oxydans and 25DKGR of Corynebacterium sp., protein databases were screened to detect homologous proteins. Among the proteins of E. coli, an oxidoreductase encoded by yjgU and having 56% similarity to 5KDGR of G. oxydans and two hypothetical oxidoreductases encoded by yqhE and yafB and having 49.8 and 42% similarity, respectively, to 25DKGR of Corynebacterium sp. were detected. Recently, the yjgU gene was identified as encoding 5KDGR and renamed idnO (C. Bausch, N. Peekhaus, C. Utz, T. Blais, E. Murray, T. Lowary, and T. Conway, J. Bacteriol. 180:3704–3710, 1998). The pathways involved in the metabolism of ketogluconate by E. coli have been predicted by biochemical analysis of purified enzymes and chemical analysis of the pathway intermediates. The gene products of yqhE and yafB were identified as 25DKGR-A, and 25DKGR-B, respectively, catalyzing the reduction of 25KDG to 2-keto-l-gulonate (2KLG). The native 25DKGR-A, 25DKGR-B, and 5KDGR had apparent molecular weights of about 30,000, 30,000, and 54,000, respectively. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, all three enzymes showed protein bands with a molecular weight of about 29,000, which indicated that 25DKGR-A, 25DKGR-B, and 5KDGR may exist as monomeric, monomeric, and dimeric proteins, respectively. The optimum pHs for reduction were 7.5, 7.0, and 8.0, respectively. The 5KDGR was active with NADH, whereas 25DKGR-A and 25DKGR-B were active with NADPH as a preferred electron donor. 25DKG can be converted to 5KDG by 2KR, which is then reduced to d-gluconate by 5KDGR. The pathways were compared with those of

  8. Quantification of nitrogen reductase and nitrite reductase genes in soil of thinned and clear-cut Douglas-fir stands by using real-time PCR.

    PubMed

    Levy-Booth, David J; Winder, Richard S

    2010-11-01

    The abundance of nifH, nirS, and nirK gene fragments involved in nitrogen (N) fixation and denitrification in thinned second-growth Douglas-fir (Pseudotsuga menziesii subsp. menziesii [Mirb.] Franco) forest soil was investigated by using quantitative real-time PCR. Prokaryotic N cycling is an important aspect of N availability in forest soil. The abundance of universal nifH, Azotobacter sp.-specific nifH (nifH-g1), nirS, and nirK gene fragments in unthinned control and 30, 90, and 100% thinning treatments were compared at two long-term research sites on Vancouver Island, Canada. The soil was analyzed for organic matter (OM), total carbon (C), total N, NH₄-N, NO₃-N, and phosphorus (P). The soil horizon accounted for the greatest variation in nutrient status, followed by the site location. The 30% thinning treatment was associated with significantly greater nifH-g1 abundance than the control treatment in one site; at the same site, nirS in the mineral soil horizon was significantly reduced by thinning. The abundance of nirS genes significantly correlated with the abundance of nirK genes. In addition, significant correlations were observed between nifH-g1 abundance and C and N in the organic horizon and between nirS and nirK and N in the mineral horizon. Overall, no clear influence of tree thinning on nifH, nirS, and nirK was observed. However, soil OM, C, and N were found to significantly influence N-cycling gene abundance.

  9. bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants.

    PubMed

    Burke, D H; Alberti, M; Hearst, J E

    1993-04-01

    We present the nucleotide and deduced amino acid sequences of four contiguous bacteriochlorophyll synthesis genes from Rhodobacter capsulatus. Three of these genes code for enzymes which catalyze reactions common to the chlorophyll synthesis pathway and therefore are likely to be found in plants and cyanobacteria as well. The pigments accumulated in strains with physically mapped transposon insertion mutations are analyzed by absorbance and fluorescence spectroscopy, allowing us to assign the genes as bchF, bchN, bchB, and bchH, in that order. bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. The other three genes are required for portions of the pathway that are shared with chlorophyll synthesis, and they were expected to be common to both pathways. bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL), a reaction that has been observed in all major groups of photosynthetic organisms except angiosperms, where only the light-dependent reaction has been clearly established. The purple bacterial and plant enzymes show 35% identity between the amino acids coded by bchN and chlN (gidA) and 49% identity between the amino acids coded by bchL and chlL (frxC). Furthermore, bchB is 33% identical to ORF513 from the Marchantia polymorpha chloroplast. We present arguments in favor of the probable role of ORF513 (chlB) in protochlorophyllide reduction in the dark. The further similarities of all three subunits of protochlorophyllide reductase and the three subunits of chlorin reductase in bacteriochlorophyll synthesis suggest that the two reductase systems are derived from a common ancestor.

  10. Is the C677T polymorphism in methylenetetrahydrofolate reductase gene or plasma homocysteine a risk factor for diabetic peripheral neuropathy in Chinese individuals?☆

    PubMed Central

    Wang, Hongli; Fan, Dongsheng; Hong, Tianpei

    2012-01-01

    The present study enrolled 251 diabetic patients, including 101 with neuropathy and 150 without neuropathy. Of the 150 patients, 100 had no complications, such as retinopathy, nephropathy, or neuropathy. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to identify methylenetetrahydrofolate reductase gene variants. Plasma homocysteine levels were also measured. Homocysteine levels and the frequency of hyperhomocysteinemia were significantly higher in patients with diabetic peripheral neuropathy compared with diabetic patients without neuropathy (P < 0.05). In logistic regression analysis with neuropathy as the dependent variable, the frequency of C677T in methylenetetrahydrofolate reductase was significantly higher in patients with diabetic peripheral neuropathy compared with patients without diabetic complications. Homocysteine levels were significantly higher in patients with diabetic peripheral neuropathy carrying the 677T allele and low folic acid levels. In conclusion, hyperhomocysteinemia is an independent risk factor for diabetic neuropathy in Chinese patients with diabetes. The C677T polymorphism in methylenetetrahydrofolate reductase and low folic acid levels may be risk factors for diabetic peripheral neuropathy in Chinese patients with diabetes. PMID:25538764

  11. Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102.

    PubMed Central

    Kikuchi, Y; Nagata, Y; Hinata, M; Kimbara, K; Fukuda, M; Yano, K; Takagi, M

    1994-01-01

    The nucleotide sequence of the downstream region of the bph operon from Pseudomonas sp. strain KKS102 was determined. Two open reading frames (ORF1 and ORF2) were found in this region, and the deduced amino acid sequence of ORF2 showed homology with the sequences of four ferredoxin reductases of dioxygenase systems. When this region was inserted just upstream of the bph operon, which does not contain a gene encoding ferredoxin reductase, biphenyl dioxygenase activity was detected. The 24- and 44-kDa polypeptides predicted from the two open reading frames were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Crude extract which contained the products of ORF2 and bphA1A2A3 showed cytochrome c reduction activity. These data clearly suggest that ORF2 encodes ferredoxin reductase. The deduced amino acid sequence of ORF1 does not show significant homology with the sequences of any other proteins in the SWISS-PROT data bank, and the function of ORF1 is unknown. Images PMID:8132464

  12. Is the C677T polymorphism in methylenetetrahydrofolate reductase gene or plasma homocysteine a risk factor for diabetic peripheral neuropathy in Chinese individuals?

    PubMed

    Wang, Hongli; Fan, Dongsheng; Hong, Tianpei

    2012-10-25

    The present study enrolled 251 diabetic patients, including 101 with neuropathy and 150 without neuropathy. Of the 150 patients, 100 had no complications, such as retinopathy, nephropathy, or neuropathy. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to identify methylenetetrahydrofolate reductase gene variants. Plasma homocysteine levels were also measured. Homocysteine levels and the frequency of hyperhomocysteinemia were significantly higher in patients with diabetic peripheral neuropathy compared with diabetic patients without neuropathy (P < 0.05). In logistic regression analysis with neuropathy as the dependent variable, the frequency of C677T in methylenetetrahydrofolate reductase was significantly higher in patients with diabetic peripheral neuropathy compared with patients without diabetic complications. Homocysteine levels were significantly higher in patients with diabetic peripheral neuropathy carrying the 677T allele and low folic acid levels. In conclusion, hyperhomocysteinemia is an independent risk factor for diabetic neuropathy in Chinese patients with diabetes. The C677T polymorphism in methylenetetrahydrofolate reductase and low folic acid levels may be risk factors for diabetic peripheral neuropathy in Chinese patients with diabetes.

  13. Arsenic and phosphate rock impacted the abundance and diversity of bacterial arsenic oxidase and reductase genes in rhizosphere of As-hyperaccumulator Pteris vittata.

    PubMed

    Han, Yong-He; Fu, Jing-Wei; Xiang, Ping; Cao, Yue; Rathinasabapathi, Bala; Chen, Yanshan; Ma, Lena Q

    2017-01-05

    Microbially-mediated arsenic (As) transformation in soils affects As speciation and plant uptake. However, little is known about the impacts of As on bacterial communities and their functional genes in the rhizosphere of As-hyperaccumulator Pteris vittata. In this study, arsenite (AsIII) oxidase genes (aroA-like) and arsenate (AsV) reductase genes (arsC) were amplified from three soils, which were amended with 50mgkg(-1) As and/or 1.5% phosphate rock (PR) and grew P. vittata for 90 d. The aroA-like genes in the rhizosphere were 50 times more abundant than arsC genes, consistent with the dominance of AsV in soils. According to functional gene alignment, most bacteria belonged to α-, β- and γ-Proteobacteria. Moreover, aroA-like genes showed a higher biodiversity than arsC genes based on clone library analysis and could be grouped into nine clusters based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Besides, AsV amendment elevated aroA-like gene diversity, but decreased arsC gene diversity. Redundancy analysis indicated that soil pH, available Ca and P, and AsV concentration were key factors driving diverse compositions in aroA-like gene community. This work identified new opportunities to screen for As-oxidizing and/or -reducing bacteria to aid phytoremediation of As-contaminated soils.

  14. Thioredoxin reductase.

    PubMed Central

    Mustacich, D; Powis, G

    2000-01-01

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

  15. Sex Differences in Ethanol's Anxiolytic Effect and Chronic Ethanol Withdrawal Severity in Mice with a Null Mutation of the 5α-Reductase Type 1 Gene.

    PubMed

    Tanchuck-Nipper, Michelle A; Ford, Matthew M; Hertzberg, Anna; Beadles-Bohling, Amy; Cozzoli, Debra K; Finn, Deborah A

    2015-05-01

    Manipulation of endogenous levels of the GABAergic neurosteroid allopregnanolone alters sensitivity to some effects of ethanol. Chronic ethanol withdrawal decreases activity and expression of 5α-reductase-1, an important enzyme in allopregnanolone biosynthesis encoded by the 5α-reductase-1 gene (Srd5a1). The present studies examined the impact of Srd5a1 deletion in male and female mice on several acute effects of ethanol and on chronic ethanol withdrawal severity. Genotype and sex did not differentially alter ethanol-induced hypothermia, ataxia, hypnosis, or metabolism, but ethanol withdrawal was significantly lower in female versus male mice. On the elevated plus maze, deletion of the Srd5a1 gene significantly decreased ethanol's effect on total entries versus wildtype (WT) mice and significantly decreased ethanol's anxiolytic effect in female knockout (KO) versus WT mice. The limited sex differences in the ability of Srd5a1 genotype to modulate select ethanol effects may reflect an interaction between developmental compensations to deletion of the Srd5a1 gene with sex hormones and levels of endogenous neurosteroids.

  16. Thyroid vein thrombosis in a young woman with methylenetetrahydrofolate reductase gene mutation receiving cyproterone acetate/ethinylestradiol treatment for polycystic ovary syndrome: a case report.

    PubMed

    Nazli, Yunus; Colak, Necmettin; Aksoy, Omer N; Cakir, Omer

    2011-06-01

    Thyroid vein thrombosis is a rare variety of venous thrombosis. The use of oral contraceptives and heterozygous methylenetetrahydrofolate reductase (MTHFR) gene mutation could have synergistic effects on the onset of venous thromboembolism. We report a 28-year-old woman with thyroid vein thrombosis. The thrombosis was possibly provoked by a combination of heterozygous MTHFR gene mutation and the use of a combined oral contraceptive (COC) containing cyproterone acetate/ethinylestradiol (CPA/EE) for polycystic ovary syndrome (PCOS). The patient was treated successfully with low molecular weight heparin and oral anticoagulation. The thyroid vein thrombosis resolved and the patient was advised to maintain lifelong use of anticoagulation therapy. Our report emphasizes that the combination of heterozygous MTHFR gene mutation, PCOS, and the use of a COC-containing CPA/EE could increase risk of thyroid vein thrombosis in young adults.

  17. Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli.

    PubMed

    Blasco, F; Pommier, J; Augier, V; Chippaux, M; Giordano, G

    1992-01-01

    Two membrane-bound nitrate reductases, NRA and NRZ, exist in Escherichia coli. Both isoenzymes are composed of three structural subunits, alpha, beta, and gamma encoded by narG/narZ, narH/narY and narI/narV, respectively. The genes are in transcription units which also contain a fourth gene encoding a polypeptide, delta, which is not part of the final enzyme. A strain which is devoid of, or does not express, the nar genes, was used to investigate the role of the delta and gamma polypeptides in the formation and/or processing of the nitrate reductase. When only the alpha and beta polypeptides are produced, an (alpha beta) complex exists which is inactive and soluble. When the alpha, beta and delta polypeptides are produced, the (alpha beta) complex is active with artificial donors such as benzyl viologen but is soluble. When the alpha, beta and gamma polypeptides are produced, the (alpha beta) complex is inactive but partially binds the membrane. It was concluded that the gamma polypeptide is involved in the binding of the (alpha beta) complex to the membrane while the delta polypeptide is indispensable for the (alpha beta) nitrate reductase activity. The activation by the delta polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (alpha beta) complex was shown to contain the four iron-sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme. The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the delta polypeptide promotes the correct assembly of the alpha and beta subunits. Although this corresponds to the definition of a chaperone protein this possibility has been rejected. In this study we have also demonstrated that the delta or gamma polypeptide encoded by one nar operon can be substituted successfully for by its respective counterpart from the other nar operon to give an active membrane bound

  18. Combined effect of polymorphisms in type III 5-α reductase and androgen receptor gene with the risk of benign prostatic hyperplasia in Korea

    PubMed Central

    Lee, Chung Lyul; Lee, Jaegeun; Na, Yong Gil; Song, Ki Hak

    2016-01-01

    We evaluated whether type III 5-alpha reductase (SRD5A3; steroid reductase 5-alpha 3) polymorphism was associated with susceptibility of benign prostate hyperplasia (BPH) and the combined effects in BPH risk between the type of short tandem repeat (STR) in SRD5A3 and the length of trinucleotide (CAG) repeats in androgen receptor (AR) gene. We compared the length of AC repeats in STR region of SRD5A3 gene and a CAG repeat in AR in 188 BPH patients who underwent transurethral resection of prostate (TURP) and 98 controls by polymerase chain reaction-based methods. We defined short type was less than 21 copies of AC repeats. The odds ratio for BPH between the men with at least one of short type and with both large types of STR in SRD5A3 gene was 3.10 (95% confidence interval [CI], 1.87–5.16; P=0.000). And BPH was 2.35 times more likely to occur in with less than 23 copies of CAG repeats than men equal or greater than 23 copies in AR gene (95% CI, 1.18–2.36; P=0.016). The men with the large type of STR and ≥23 copies of CAG repeats have 5.3 times BPH risk compared to the reference group with the at least one of the short type of STR and <23 copies (P<0.000). In conclusion, these results suggest that shorter AC repeats of SRD5A3 gene and shorter CAG repeats of AR gene were associated with an increased risk for BPH. However, the interaction between above two factors was not affected in risk of BPH. PMID:27807533

  19. Methylenetetrahydrofolate reductase gene polymorphisms are associated with ischemic and hemorrhagic stroke: Dual effect of MTHFR polymorphisms C677T and A1298C.

    PubMed

    Sazci, Ali; Ergul, Emel; Tuncer, Nese; Akpinar, Gurler; Kara, Ihsan

    2006-12-11

    Hyperhomocysteinemia is an independent risk factor for ischemic stroke. The enzyme methylenetetrahydrofolate reductase (MTHFR) plays a critical role in modulating the levels of plasma homocysteine. Two polymorphisms in the MTHFR gene, C677T, A1298C result in reduced enzyme activity. The mechanisms of ischemic and hemorrhagic stroke are not well understood. Although controversial, previous studies have shown evidence of causality of both stroke subtypes in patients with methylenetetrahydrofolate reductase gene polymorphisms. Therefore, we examined whether the C677T and A1298C polymorphisms of MTHFR gene are genetic risk factors for both ischemic and hemorrhagic stroke in a Turkish Caucasian population. In a case-control study, 120 total unrelated stroke patients (92 ischemic stroke, 28 hemorrhagic stroke), and 259 healthy controls were genotyped for C677T and A1298C polymorphisms of the MTHFR gene using a PCR-RFLP based-method. The MTHFR 1298C allele (chi(2)=8.589; P=0.014), C1298C genotype (OR=2.544; P=0.004), and C677C/C1298C compound genotype (OR=3.020; P=0.001) were associated with overall stroke. The MTHFR 1298C allele (chi(2)=11.166; P=0.004), C1298C genotype (OR=2.950; P=0.001), and C677C/C1298C compound genotype (OR=3.463, P=0.0001) were strongly associated with ischemic stroke. Interestingly however, the MTHFR 677T allele (chi(2)=6.033; P=0.049), T677T genotype (OR=3.120; P=0.014), and T677T/A1298A compound genotype (OR=4.211; P=0.002) were associated with hemorrhagic stroke. In conclusion, the C677T and A1298C polymorphisms of the MTHFR gene are genetic risk factors for hamorrhagic and ischemic stroke respectively, independent of other atherothrombotic risk factors.

  20. Clustered Genes Encoding 2-Keto-l-Gulonate Reductase and l-Idonate 5-Dehydrogenase in the Novel Fungal d-Glucuronic Acid Pathway.

    PubMed

    Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

    2017-01-01

    D-Glucuronic acid is a biomass component that occurs in plant cell wall polysaccharides and is catabolized by saprotrophic microorganisms including fungi. A pathway for D-glucuronic acid catabolism in fungal microorganisms is only partly known. In the filamentous fungus Aspergillus niger, the enzymes that are known to be part of the pathway are the NADPH requiring D-glucuronic acid reductase forming L-gulonate and the NADH requiring 2-keto-L-gulonate reductase that forms L-idonate. With the aid of RNA sequencing we identified two more enzymes of the pathway. The first is a NADPH requiring 2-keto-L-gulonate reductase that forms L-idonate, GluD. The second is a NAD(+) requiring L-idonate 5-dehydrogenase forming 5-keto-gluconate, GluE. The genes coding for these two enzymes are clustered and share the same bidirectional promoter. The GluD is an enzyme with a strict requirement for NADP(+)/NADPH as cofactors. The kcat for 2-keto-L-gulonate and L-idonate is 21.4 and 1.1 s(-1), and the Km 25.3 and 12.6 mM, respectively, when using the purified protein. In contrast, the GluE has a strict requirement for NAD(+)/NADH. The kcat for L-idonate and 5-keto-D-gluconate is 5.5 and 7.2 s(-1), and the Km 30.9 and 8.4 mM, respectively. These values also refer to the purified protein. The gluD deletion resulted in accumulation of 2-keto-L-gulonate in the liquid cultivation while the gluE deletion resulted in reduced growth and cessation of the D-glucuronic acid catabolism.

  1. Clustered Genes Encoding 2-Keto-l-Gulonate Reductase and l-Idonate 5-Dehydrogenase in the Novel Fungal d-Glucuronic Acid Pathway

    PubMed Central

    Kuivanen, Joosu; Arvas, Mikko; Richard, Peter

    2017-01-01

    D-Glucuronic acid is a biomass component that occurs in plant cell wall polysaccharides and is catabolized by saprotrophic microorganisms including fungi. A pathway for D-glucuronic acid catabolism in fungal microorganisms is only partly known. In the filamentous fungus Aspergillus niger, the enzymes that are known to be part of the pathway are the NADPH requiring D-glucuronic acid reductase forming L-gulonate and the NADH requiring 2-keto-L-gulonate reductase that forms L-idonate. With the aid of RNA sequencing we identified two more enzymes of the pathway. The first is a NADPH requiring 2-keto-L-gulonate reductase that forms L-idonate, GluD. The second is a NAD+ requiring L-idonate 5-dehydrogenase forming 5-keto-gluconate, GluE. The genes coding for these two enzymes are clustered and share the same bidirectional promoter. The GluD is an enzyme with a strict requirement for NADP+/NADPH as cofactors. The kcat for 2-keto-L-gulonate and L-idonate is 21.4 and 1.1 s-1, and the Km 25.3 and 12.6 mM, respectively, when using the purified protein. In contrast, the GluE has a strict requirement for NAD+/NADH. The kcat for L-idonate and 5-keto-D-gluconate is 5.5 and 7.2 s-1, and the Km 30.9 and 8.4 mM, respectively. These values also refer to the purified protein. The gluD deletion resulted in accumulation of 2-keto-L-gulonate in the liquid cultivation while the gluE deletion resulted in reduced growth and cessation of the D-glucuronic acid catabolism. PMID:28261181

  2. Resolution of two native monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes

    SciTech Connect

    Simpson, Philippa J.L.; McKinzie, Audra A.; Codd, Rachel

    2010-07-16

    Research highlights: {yields} Two monomeric 90 kDa nitrate reductase active proteins from Shewanella gelidimarina. {yields} Sequence of napA from napEDABC-type operon and napA from NapDAGHB-type operon. {yields} Isolation of NAP as NapA or NapAB correlated with NapA P47E amino acid substitution. -- Abstract: The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90 kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90 kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90 kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90 kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

  3. Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii 1

    PubMed Central

    Galván, Aurora; Cárdenas, Jacobo; Fernández, Emilio

    1992-01-01

    In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities. PMID:16668656

  4. Short hairpin RNA targeted to dihydrofolate reductase enhances the immunoglobulin G expression in gene-amplified stable Chinese hamster ovary cells.

    PubMed

    Wu, Suh-Chin; Hong, Willy W L; Liu, Jin-Hwang

    2008-09-08

    The dihydrofolate reductase (dhfr)/methotrexate (MTX) selection is a common method to conduct gene amplification in stable clones of Chinese hamster ovary (CHO) cells. We previously reported the use of a short hairpin RNA (shRNA) vector targeted to the dhfr gene resulted in improving the intracellular antigen expression in gene-amplified stable CHO cells [Hong, W.W., Wu, S.C., 2007. A novel RNA silencing vector to improve antigen expression and stability in Chinese hamster ovary cells. Vaccine 25 (20), 4103-4111]. Here we investigated the use of the dhfr-targeted shRNA vector for immunoglobulin G (IgG) expression in gene-amplified stable CHO cells. With the use of the dhfr-targeted shRNA vector, the gene-amplified CHO/dhFr(-) cells were found to increase IgG expression at 1.0 microM MTX by more than 100% and to improve the genomic stability of IgG expression in MTX-free cultures by approximately 30%. The use of the dhfr-targeted shRNA vector can enhance the IgG expression in the gene-amplified stable CHO cells and uphold the IgG expression in MTX-free cultures. Utilizing the dhfr-targeted shRNA vector may provide an alternative way to maneuver CHO cell factories for IgG production in cultures.

  5. An event of alternative splicing affects the expression of the NTRC gene, encoding NADPH-thioredoxin reductase C, in seed plants.

    PubMed

    Nájera, Victoria A; González, María Cruz; Pérez-Ruiz, Juan Manuel; Cejudo, Francisco Javier

    2017-05-01

    The NTRC gene encodes a NADPH-dependent thioredoxin reductase with a joint thioredoxin domain, exclusive of photosynthetic organisms. An updated search shows that although most species harbor a single copy of the NTRC gene, two copies were identified in different species of the genus Solanum, Glycine max and the moss Physcomitrella patens. The phylogenetic analysis of NTRCs from different sources produced a tree with the major groups of photosynthetic organisms: cyanobacteria, algae and land plants, indicating the evolutionary success of the NTRC gene among photosynthetic eukaryotes. An event of alternative splicing affecting the expression of the NTRC gene was identified, which is conserved in seed plants but not in algae, bryophytes and lycophytes. The alternative splicing event results in a transcript with premature stop codon, which would produce a truncated form of the enzyme. The standard splicing/alternative splicing (SS/AS) transcripts ratio was higher in photosynthetic tissues from Arabidopsis, Brachypodium and tomato, in line with the higher content of the NTRC polypeptide in these tissues. Moreover, environmental stresses such as cold or high salt affected the SS/AS ratio of the NTRC gene transcripts in Brachypodium seedlings. These results suggest that the alternative splicing of the NTRC gene might be an additional mechanism for modulating the content of NTRC in photosynthetic and non-photosynthetic tissues of seed plants.

  6. C677T polymorphism of the methylenetetrahydrofolate reductase gene does not affect folic acid, vitamin B12, and homocysteine serum levels in Turkish children with neural tube defects.

    PubMed

    Erdogan, M O; Yildiz, S H; Solak, M; Eser, O; Cosar, E; Eser, B; Koken, R; Buyukbas, S

    2010-06-22

    Association between neural tube defects (NTDs) and C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene was suspected, because the MTHFR gene codes for a key enzyme in folate metabolism. Its deficiency usually leads to significant reductions in plasma concentrations of folate, vitamin B(12) and methionine, whereas homocysteine levels are increased. We examined folate, vitamin B(12) and homocysteine serum concentrations and polymorphism of the C677T MTHFR gene in Turkish children with neural tube defects. Thirty-three children with NTDs, 26 mothers and 48 healthy individuals were studied. C677T MTHFR polymorphism was determined by melting curve analyses (LightCycler). The levels of folate, vitamin B(12) and homocysteine serum concentrations in NTDs were evaluated and compared, along with information concerning alleles of the MTHFR gene. C677T allele frequencies in NTD children and their mothers were similar to those found in controls. Serum folate and vitamin B(12) concentrations were significantly higher in NTD children than that of controls. Serum homocysteine concentrations were not significantly higher in NTD children and mothers. We concluded that C677T MTHFR gene polymorphism does not affect folic acid, vitamin B(12) and homocysteine metabolism in Turkish children with NTDs. C677T polymorphism of the MTHFR gene cannot be regarded as a major risk factor for NTDs in Turkish children.

  7. Evaluation of the roles that alkyl hydroperoxide reductase and Ohr play in organic peroxide-induced gene expression and protection against organic peroxides in Xanthomonas campestris.

    PubMed

    Vattanaviboon, Paiboon; Whangsuk, Wirongrong; Panmanee, Warunya; Klomsiri, Chananat; Dharmsthiti, Saovanee; Mongkolsuk, Skorn

    2002-11-29

    Alkyl hydroperoxide reductase (ahpC) and organic hydroperoxide resistance (ohr) are distinct genes, structurally and regulatory, but have similar physiological functions. In Xanthomonas campestris pv. phaseoli inactivation of either gene results in increased sensitivity to killing with organic peroxides. An ahpC1-ohr double mutant was highly sensitive to both growth inhibition and killing treatment with organic peroxides. High level expression of ahpC or ohr only partially complemented the phenotype of the double mutant, suggesting that these genes function synergistically, but through different pathways, to protect Xanthomonas from organic peroxide toxicity. Functional analyses of Ohr and AhpC abilities to degrade organic hydroperoxides revealed that both Ohr and AhpC could degrade tert-butyl hydroperoxide (tBOOH) while the former was more efficient at degrading cumene hydroperoxide (CuOOH). Expression analysis of these genes in the mutants showed no compensatory alterations in the levels of AhpC or Ohr. However, CuOOH induced expression of these genes in the mutants was affected. CuOOH induced ahpC expression was higher in the ohr mutant than in the parental strain; in contrast, the ahpC mutation has no effect on the level of induced ohr expression. These analyses reveal complex physiological roles and expression patterns of seemingly functionally similar genes.

  8. Localization of TDPX1, a human homologue of the yeast thioredoxin-dependent peroxide reductase gene (TPX), to chromosome 13q12

    SciTech Connect

    Pahl, P.; Berger, R.; Hart, I. |

    1995-04-10

    Reactive oxygen species and free radicals that are produced during normal metabolism can potentially damage cellular macromolecules. Defenses against such damage include a number of antioxidant enzymes that specifically target the removal or dismutation of the reactive agent. We report here the isolation and regional mapping of a human gene, TDPX1, that encodes an enzyme homologous to a yeast thioredoxin-dependent peroxide reductase (thioredoxin peroxidase, TPX). The human TDPX1 coding sequence was determined from the product of a polymerase chain reaction (PCR) amplification of human cDNA. Based on PCR analysis of DNA from a human/rodent somatic cell hybrid panel, the TDPX1 locus was assigned to chromosome 13. Further localization of the locus to 13q12 was accomplished by fluorescence in situ hybridization analysis, using as a probe DNA from a yeast artificial chromosome (YAC) that contains the TDPX1 gene. It was also determined by PCR analysis of various YACs that the TDPX1 locus is in the region of the dinucleotide repeat markers D13S289 and D13S290. This regional mapping localizes the TDPX1 gene to a genomic region recently shown to contain the breast cancer susceptibility gene BRCA2 and a gene associated with a form of muscular dystrophy. Oxygen radical metabolism has been hypothesized to be important for cancer, muscular dystrophy, and other disorders, so TDPX1 should be considered a candidate gene for these diseases. 33 refs., 2 figs., 1 tab.

  9. Cloning of the cytochrome p450 reductase (crtR) gene and its involvement in the astaxanthin biosynthesis of Xanthophyllomyces dendrorhous

    PubMed Central

    Alcaíno, Jennifer; Barahona, Salvador; Carmona, Marisela; Lozano, Carla; Marcoleta, Andrés; Niklitschek, Mauricio; Sepúlveda, Dionisia; Baeza, Marcelo; Cifuentes, Víctor

    2008-01-01

    Background The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid with high commercial interest. The proposed biosynthetic route in this organism is isopentenyl-pyrophosphate (IPP) → geranyleranyl pyrophosphate (GGPP) → phytoene → lycopene → β-carotene → astaxanthin. Recently, it has been published that the conversion of β-carotene into astaxanthin requires only one enzyme, astaxanthin synthase or CrtS, encoded by crtS gene. This enzyme belongs to the cytochrome P450 protein family. Results In this work, a crtR gene was isolated from X. dendrorhous yeast, which encodes a cytochrome P450 reductase (CPR) that provides CrtS with the necessary electrons for substrate oxygenation. We determined the structural organization of the crtR gene and its location in the yeast electrophoretic karyotype. Two transformants, CBSTr and T13, were obtained by deleting the crtR gene and inserting a hygromycin B resistance cassette. The carotenoid composition of the transformants was altered in relation to the wild type strain. CBSTr forms yellow colonies because it is unable to produce astaxanthin, hence accumulating β-carotene. T13 forms pale colonies because its astaxanthin content is reduced and its β-carotene content is increased. Conclusion In addition to the crtS gene, X. dendrorhous requires a novel gene, crtR, for the conversion of β-carotene to astaxanthin. PMID:18837978

  10. A Root-Preferential DFR-Like Gene Encoding Dihydrokaempferol Reductase Involved in Anthocyanin Biosynthesis of Purple-Fleshed Sweet Potato

    PubMed Central

    Liu, Xiaoqiang; Xiang, Min; Fan, Yufang; Yang, Chunxian; Zeng, Lingjiang; Zhang, Qitang; Chen, Min; Liao, Zhihua

    2017-01-01

    Purple-fleshed sweet potato is good for health due to rich anthocyanins in tubers. Although the anthocyanin biosynthetic pathway is well understood in up-ground organs of plants, the knowledge on anthocyanin biosynthesis in underground tubers is limited. In the present study, we isolated and functionally characterized a root-preferential gene encoding dihydrokaempferol reductase (IbDHKR) from purple-fleshed sweet potato. IbDHKR showed highly similarity with the reported dihydroflavonol reductases in other plant species at the sequence levels and the NADPH-binding motif and the substrate-binding domain were also found in IbDHKR. The tissue profile showed that IbDHKR was expressed in all the tested organs, but with much higher level in tuber roots. The expression level of IbDHKR was consistent with the anthocyanin content in sweet potato organs, suggesting that tuber roots were the main organs to synthesize anthocyanins. The recombinant 44 kD IbDHKR was purified and fed by three different dihydroflavonol substrates including dihydrokaempferol (DHK), dihydroquerctin, and dihydromyrecetin. The substrate feeding assay indicated that only DHK could be accepted as substrate by IbDHKR, which was reduced to leucopelargonidin confirmed by LC-MS. Finally, IbDHKR was overexpressed in transgenic tobacco. The IbDHKR-overexpression tobacco corolla was more highly pigmented and contained higher level of anthocyanins than the wild-type tobacco corolla. In summary, IbDHKR was a root-preferential gene involved in anthocyanin biosynthesis and its encoding protein, specifically catalyzing DHK reduction to yield leucopelargonidin, was a candidate gene for engineering anthocyanin biosynthetic pathway. PMID:28293252

  11. Susceptibility to diabetic neuropathy in patients with insulin dependent diabetes mellitus is associated with a polymorphism at the 5' end of the aldose reductase gene

    PubMed Central

    Heesom, A.; Millward, A.; Demaine, A.

    1998-01-01

    OBJECTIVES—There is evidence that the polyol pathway is involved in the pathogenesis of diabetic neuropathy. Aldose reductase (ALR2) is the first and rate limiting enzyme of this pathway and recent studies have suggested that polymorphisms in and around the gene are associated with the development of diabetic microvascular disease. The aim was to examine the role of ALR2 in the susceptibility to diabetic neuropathy in patients with insulin dependent diabetes mellitus (IDDM).
METHODS—One hundred and fifty nine British white patients with IDDM and 102 normal healthy controls were studied using the polymerase chain reaction to test for a highly polymorphic microsatellite marker 2.1 kilobase (kb) upstream of the initiation site of the ALR2 gene.
RESULTS—Seven alleles were detected (Z-6, Z-4, Z-2, Z, Z+2, Z+4, and Z+6). There was a highly significant decrease in the frequency of the Z+2 allele in those patients with overt neuropathy compared with those with no neuropathy after 20 years duration of diabetes (14.1% v 38.2%, χ2 =17.3, p<0.00001). A similar difference was also found between the neuropathy group and those patients who have had diabetes for< five years with no overt neuropathy (14.1% v 30.2%, χ2=9.0, p<0.0025). The neuropathy group also had a significant decrease in the frequency of the Z/Z+2 genotype compared with those patients who have no neuropathy after 20 years duration of diabetes (14.0% v 44.7%, χ2=13.0, p<0.0005).
CONCLUSION—These results suggest that the aldose reductase gene is intimately involved in the pathogenesis of diabetic neuropathy.

 PMID:9489533

  12. Transgenic Cotton Plants Expressing Double-stranded RNAs Target HMG-CoA Reductase (HMGR) Gene Inhibits the Growth, Development and Survival of Cotton Bollworms.

    PubMed

    Tian, Geng; Cheng, Linlin; Qi, Xuewei; Ge, Zonghe; Niu, Changying; Zhang, Xianlong; Jin, Shuangxia

    2015-01-01

    RNA interference (RNAi) has been developed as a powerful technique in the research of functional genomics as well as plant pest control. In this report, double-stranded RNAs (dsRNA) targeting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, which catalyze a rate-limiting enzymatic reaction in the mevalonate pathway of juvenile hormone (JH) synthesis in cotton bollworm, was expressed in cotton plants via Agrobacterium tumefaciens-mediated transformation. PCR and Sothern analysis revealed the integration of HMGR gene into cotton genome. RT-PCR and qRT-PCR confirmed the high transcription level of dsHMGR in transgenic cotton lines. The HMGR expression both in transcription and translation level was significantly downregulated in cotton bollworms (helicoverpa armigera) larvae after feeding on the leaves of HMGR transgenic plants. The transcription level of HMGR gene in larvae reared on transgenic cotton leaves was as much as 80.68% lower than that of wild type. In addition, the relative expression level of vitellogenin (Vg, crucial source of nourishment for offspring embryo development) gene was also reduced by 76.86% when the insect larvae were fed with transgenic leaves. The result of insect bioassays showed that the transgenic plant harboring dsHMGR not only inhibited net weight gain but also delayed the growth of cotton bollworm larvae. Taken together, transgenic cotton plant expressing dsRNAs successfully downregulated HMGR gene and impaired the development and survival of target insect, which provided more option for plant pest control.

  13. Laser Microdissection and Spatiotemporal Pinoresinol-Lariciresinol Reductase Gene Expression Assign the Cell Layer-Specific Accumulation of Secoisolariciresinol Diglucoside in Flaxseed Coats

    PubMed Central

    Fang, Jingjing; Ramsay, Aïna; Renouard, Sullivan; Hano, Christophe; Lamblin, Frédéric; Chabbert, Brigitte; Mesnard, François; Schneider, Bernd

    2016-01-01

    The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed (Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum (LuPLR1), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase (GUS) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS. The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats. PMID:27917190

  14. The Gene Encoding Dihydroflavonol 4-Reductase Is a Candidate for the anthocyaninless Locus of Rapid Cycling Brassica rapa (Fast Plants Type)

    PubMed Central

    Wendell, Douglas L.; Vaziri, Anoumid; Shergill, Gurbaksh

    2016-01-01

    Rapid cycling Brassica rapa, also known as Wisconsin Fast Plants, are a widely used organism in both K-12 and college science education. They are an excellent system for genetics laboratory instruction because it is very easy to conduct genetic crosses with this organism, there are numerous seed stocks with variation in both Mendelian and quantitative traits, they have a short generation time, and there is a wealth of educational materials for instructors using them. Their main deficiency for genetics education is that none of the genetic variation in RCBr has yet been characterized at the molecular level. Here we present the first molecular characterization of a gene responsible for a trait in Fast Plants. The trait under study is purple/nonpurple variation due to the anthocyaninless locus, which is one of the Mendelian traits most frequently used for genetics education with this organism. We present evidence that the DFR gene, which encodes dihyroflavonol 4-reductase, is the candidate gene for the anthocyaninless (ANL) locus in RCBr. DFR shows complete linkage with ANL in genetic crosses with a total of 948 informative chromosomes, and strains with the recessive nonpurple phenotype have a transposon-related insertion in the DFR which is predicted to disrupt gene function. PMID:27548675

  15. The Gene Encoding Dihydroflavonol 4-Reductase Is a Candidate for the anthocyaninless Locus of Rapid Cycling Brassica rapa (Fast Plants Type).

    PubMed

    Wendell, Douglas L; Vaziri, Anoumid; Shergill, Gurbaksh

    2016-01-01

    Rapid cycling Brassica rapa, also known as Wisconsin Fast Plants, are a widely used organism in both K-12 and college science education. They are an excellent system for genetics laboratory instruction because it is very easy to conduct genetic crosses with this organism, there are numerous seed stocks with variation in both Mendelian and quantitative traits, they have a short generation time, and there is a wealth of educational materials for instructors using them. Their main deficiency for genetics education is that none of the genetic variation in RCBr has yet been characterized at the molecular level. Here we present the first molecular characterization of a gene responsible for a trait in Fast Plants. The trait under study is purple/nonpurple variation due to the anthocyaninless locus, which is one of the Mendelian traits most frequently used for genetics education with this organism. We present evidence that the DFR gene, which encodes dihyroflavonol 4-reductase, is the candidate gene for the anthocyaninless (ANL) locus in RCBr. DFR shows complete linkage with ANL in genetic crosses with a total of 948 informative chromosomes, and strains with the recessive nonpurple phenotype have a transposon-related insertion in the DFR which is predicted to disrupt gene function.

  16. Free-floating thrombus of the carotid artery with a homozygous methylenetetrahydrofolate reductase gene mutation: a case report.

    PubMed

    Colak, Necmettin; Nazli, Yunus; Kosehan, Dilek; Alpay, Mehmet Fatih; Cakir, Omer

    2013-02-01

    Free-floating thrombus (FFT) of the carotid artery is a rare condition of currently unknown etiology. We describe a symptomatic patient with an FFT in the left common carotid artery. A duplex ultrasonography scan showed the presence of a mobile floating thrombus moving in cyclical motion with the cardiac cycles in the left common carotid artery. During emergency surgery, an FFT was seen at this location and removed. No underlying wall defect was seen at the time of surgery. In a genetic screening test, TT homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T genetic polymorphisms was detected. The patient recovered uneventfully, with no neurogical events. Lifelong anticoagulant therapy was recommended. An aggressive surgical approach is recommended in the patient to prevent embolic episodes.

  17. Expression of genes encoding chalcone synthase, flavanone 3-hydroxylase and dihydroflavonol 4-reductase correlates with flavanol accumulation during heartwood formation in Juglans nigra.

    PubMed

    Beritognolo, Isacco; Magel, Elisabeth; Abdel-Latif, Amani; Charpentier, Jean-Paul; Jay-Allemand, Christian; Breton, Christian

    2002-04-01

    Heartwood formation is generally characterized by the accumulation of phenolic substances that increase the natural color and durability of wood. Although there is evidence that these substances are synthesized in aging sapwood cells, little is known about heartwood formation at the molecular level. We monitored seasonal changes in flavanol concentration across the stems of 23-year-old Juglans nigra L. trees by sampling growth rings extending from the differentiating xylem to the heartwood. We also analyzed expression of phenylpropanoid and flavonoid structural genes in these samples. In the sapwood-heartwood transition zone, flavanol accumulation was correlated with the transcription levels of the chalcone synthase (CHS) and flavanone 3-hydroxylase (F3H) genes. We also observed correlations between flavanol accumulation and the amount of dihydroflavonol 4-reductase (DFR) gene transcript in October, January and May. Although transcription of phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL) genes did not correlate with flavanol accumulation, PAL genes were strongly expressed in the transition zone of samples collected in autumn, suggesting that their transcription in these tissues contributes to phenolic biosynthesis. Western immunoblotting showed that accumulation of CHS protein correlated with the amount of CHS gene transcript, whereas accumulation of PAL protein did not correlate with the the transcription levels PAL genes. Preliminary analyses revealed that PAL and CHS activities were higher in the transition zone than in the inner sapwood in autumn, winter, and spring. Thus, CHS activity could be regulated mainly at the transcriptional level, whereas post-translational modifications could modulate PAL activity. We conclude that flavanols are synthesized de novo in J. nigra sapwood cells that are undergoing transformation to heartwood.

  18. Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus.

    PubMed

    Thierry, D; Vaucheret, H

    1996-12-01

    The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing.

  19. Diversity and Abundance of Ammonia-Oxidizing Archaeal Nitrite Reductase (nirK) Genes in Estuarine Sediments of San Francisco Bay

    NASA Astrophysics Data System (ADS)

    Reji, L.; Lee, J. A.; Damashek, J.; Francis, C. A.

    2013-12-01

    Nitrification, the microbially-mediated aerobic oxidation of ammonia to nitrate via nitrite, is an integral component of the global biogeochemical nitrogen cycle. The first and rate-limiting step of nitrification, ammonia oxidation, is carried out by two distinct microbial groups: ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Molecular ecological studies targeting the amoA gene have revealed the abundance and ubiquity of AOA in terrestrial as well as aquatic environments. In addition to the ammonia oxidation machinery that includes the amoA gene, AOA also encode a gene for copper-containing nitrite reductase (nirK). The distribution patterns and functional role of nirK in AOA remain mostly unknown; proposed functions include the indirect involvement in ammonia oxidation through the production of nitric oxide during nitrite reduction, and (2) nitrite detoxification. In the present study, the diversity and abundance of archaeal nirK genes in estuarine sediments were investigated using quantitative polymerase chain reaction, cloning and sequencing approaches. In sediment samples collected from the San Francisco Bay estuary, two archaeal nirK variants (AnirKa and AnirKb) were amplified using specific primer sets. Overall, AnirKa was observed to be significantly more abundant than AnirKb in the sediment samples, with variation in relative abundance spanning two to three orders of magnitude between sampling sites. Phylogenetic analysis revealed a number of unique archaeal nirK sequence types, as well as many that clustered with sequences from previous estuarine studies and cultured AOA isolates, such as Nitrosopumilus maritimus. This study yielded new insights into the diversity and abundance of archaeal nirK genes in estuarine sediments, and highlights the importance of further investigating the physiological role of this gene in AOA, as well as its suitability as a marker gene for studying AOA in the environment.

  20. The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina.

    PubMed

    Sen, Gulseren; Eryilmaz, Isil Ezgi; Ozakca, Dilek

    2014-02-01

    In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes.

  1. Control of dihydrofolate reductase messenger ribonucleic acid production

    SciTech Connect

    Leys, E.J.; Kellems, R.E.

    1981-11-01

    The authors used methotrexate-resistant mouse cells in which dihydrofolate reductase levels are approximately 500 times normal to study the effect of growth stimulation on dihydrofolate reductase gene expression. As a result of growth stimulation, the relative rate of dihydrofolate reductase protein synthesis increased threefold, reaching a maximum between 25 and 30 h after stimulation. The relative rate of dihydrofolate reductase messenger ribonucleic acid production (i.e., the appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm) increased threefold after growth stimulation and was accompanied by a corresponding increase in the relative steady-state level of dihydrofolate reductase ribonucleic acid in the nucleus. However, the increase in the nuclear level of dihydrofolate reductase ribonucleic acid was not accompanied by a significant increase in the relative rate of transcription of the dihydrofolate reductase genes. These data indicated that the relative rate of appearance of dihydrofolate reductase messenger ribonucleic acid in the cytoplasm depends on the relative stability of the dihydrofolate reductase ribonucleic acid sequences in the nucleus and is not dependent on the relative rate of transcription of the dihydrofolate reductase genes.

  2. Pseudomonas aeruginosa IscR-Regulated Ferredoxin NADP(+) Reductase Gene (fprB) Functions in Iron-Sulfur Cluster Biogenesis and Multiple Stress Response

    PubMed Central

    Romsang, Adisak; Duang-nkern, Jintana; Wirathorn, Wilaiwan; Vattanaviboon, Paiboon; Mongkolsuk, Skorn

    2015-01-01

    P. aeruginosa (PAO1) has two putative genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. Here, the regulation of fprB expression and the protein’s physiological roles in [4Fe-4S] cluster biogenesis and stress protection are characterized. The fprB mutant has defects in [4Fe-4S] cluster biogenesis, as shown by reduced activities of [4Fe-4S] cluster-containing enzymes. Inactivation of the gene resulted in increased sensitivity to oxidative, thiol, osmotic and metal stresses compared with the PAO1 wild type. The increased sensitivity could be partially or completely suppressed by high expression of genes from the isc operon, which are involved in [Fe-S] cluster biogenesis, indicating that stress sensitivity in the fprB mutant is partially caused by a reduction in levels of [4Fe-4S] clusters. The pattern and regulation of fprB expression are in agreement with the gene physiological roles; fprB expression was highly induced by redox cycling drugs and diamide and was moderately induced by peroxides, an iron chelator and salt stress. The stress-induced expression of fprB was abolished by a deletion of the iscR gene. An IscR DNA-binding site close to fprB promoter elements was identified and confirmed by specific binding of purified IscR. Analysis of the regulation of fprB expression supports the role of IscR in directly regulating fprB transcription as a transcription activator. The combination of IscR-regulated expression of fprB and the fprB roles in response to multiple stressors emphasizes the importance of [Fe-S] cluster homeostasis in both gene regulation and stress protection. PMID:26230408

  3. Water-Soluble Compounds from Lentinula edodes Influencing the HMG-CoA Reductase Activity and the Expression of Genes Involved in the Cholesterol Metabolism.

    PubMed

    Gil-Ramírez, Alicia; Caz, Víctor; Smiderle, Fhernanda R; Martin-Hernandez, Roberto; Largo, Carlota; Tabernero, María; Marín, Francisco R; Iacomini, Marcello; Reglero, Guillermo; Soler-Rivas, Cristina

    2016-03-09

    A water extract from Lentinula edodes (LWE) showed HMG-CoA reductase inhibitory activity but contained no statins. NMR indicated the presence of water-soluble α- and β-glucans and fucomannogalactans. Fractions containing derivatives of these polysaccharides with molecular weight down to approximately 1 kDa still retained their inhibitory activity. Once digested LWE was applied to Caco2 in transport experiments, no significant effect was noticed on the modulation of cholesterol-related gene expression. But, when the lower compartment of the Caco2 monolayer was applied to HepG2, some genes were modulated (after 24 h). LWE was also administrated to normo- and hypercholesterolemic mice, and no significant lowering of serum cholesterol levels was observed; but reduction of triglycerides in liver was observed. However, LWE supplementation modulated the transcriptional profile of some genes involved in the cholesterol metabolism similarly to simvastatin, suggesting that it could hold potential as a hypolipidemic/hypocholesterolemic extract, although further dose-dependent studies should be carried out.

  4. Digitalis purpurea P5 beta R2, encoding steroid 5 beta-reductase, is a novel defense-related gene involved in cardenolide biosynthesis.

    PubMed

    Pérez-Bermúdez, Pedro; García, Aurelio A Moya; Tuñón, Iñaki; Gavidia, Isabel

    2010-02-01

    The stereospecific 5 beta-reduction of progesterone is a required step for cardiac glycoside biosynthesis in foxglove plants. Recently, we have isolated the gene P5 beta R, and here we investigate the function and regulation of P5 beta R2, a new progesterone 5 beta-reductase gene from Digitalis purpurea. P5 beta R2 cDNA was isolated from a D. purpurea cDNA library and further characterized at the biochemical, structural and physiological levels. Like P5 beta R, P5 beta R2 catalyzes the 5 beta-reduction of the Delta(4) double bond of several steroids and is present in all plant organs. Under stress conditions or on treatment with chemical elicitors, P5 beta R expression does not vary, whereas P5 beta R2 is highly responsive. P5 beta R2 expression is regulated by ethylene and hydrogen peroxide. The correlation between P5 beta R2 expression and cardenolide formation demonstrates the key role of this gene in cardenolide biosynthesis, and therefore in the chemical defense of foxglove plants.

  5. Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

    PubMed Central

    Xu, Jun-Wei; Xu, Yi-Ning

    2012-01-01

    Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway. PMID:22941092

  6. Expression of an Antirrhinum dihydroflavonol reductase gene results in changes in condensed tannin structure and accumulation in root cultures of Lotus corniculatus (bird's foot trefoil).

    PubMed

    Bavage, A D; Davies, I G; Robbins, M P; Morris, P

    1997-11-01

    Condensed tannins (proanthocyanidins) are an important factor in the nutritive and dietary quality of many forage crops. We report here experiments aimed at altering the levels and monomer composition of condensed tannins (CTs) in 'hairy root' cultures of Lotus corniculatus (bird's foot trefoil) using genetic manipulation. An Antirrhinum majus dihydroflavonol reductase (DFR) cDNA was expressed in sense in L. corniculatus and CT levels in transgenic root cultures were analysed. Two co-transformed lines were noted with decreased CT content relative to controls and these levels were comparable with antisense-DFR phenotypes. In ADFR10, a co-transformed line with the highest CT levels, CT structure was altered in a manner consistent with the substrate specificity of the introduced gene; that is an increase in pro-pelargonidin monomers noted after hydrolysis of CTs. RT-PCR confirmed the expression of endogenous DFR gene(s) in both putatively co-suppressed lines and also in ADFR10. Analysis of selected root culture lines indicated that the monomer composition of CTs did not change during growth and development but that levels of CTs varied in a regulated manner.

  7. Silencing of SlFTR-c, the catalytic subunit of ferredoxin:thioredoxin reductase, induces pathogenesis-related genes and pathogen resistance in tomato plants.

    PubMed

    Lim, Chan Ju; Kim, Woong Bom; Lee, Bok-Sim; Lee, Ha Youn; Kwon, Tae-Ho; Park, Jeong Mee; Kwon, Suk-Yoon

    2010-09-03

    As a heterodimeric protein, ferredoxin:thioredoxin reductase (FTR) catalyses the light-dependant activation of several photosynthetic enzymes. The active site of the catalytic subunit of FTR contains a redox-active disulfide and a [4Fe-4S] center. We isolated the catalytic subunit gene of FTR, designated SlFTR-c, from tomato (Solanum lycopersicum L.). SlFTR-c transcripts were detected in all tissues examined, including roots, leaves, flowers, fruits, and seeds. Interestingly, virus-induced gene silencing (VIGS) of SlFTR-c resulted in necrotic lesions with typical cell death symptoms and reactive oxygen species (ROS) production in tomato leaves. Moreover, these SlFTR-c-silenced plants displayed enhanced disease resistance against bacterial pathogens, specifically Pseudomonas syringae pv. tomato DC3000, by the induction of defense-related genes (SlPR-1, SlPR-2, SlPR-5, SlGlucA, SlChi3, and SlChi9). Taken together, it seems that SlFTR-c works as a regulator of programmed cell death (PCD) and pathogen resistance in tomato plants.

  8. Priming with NO controls redox state and prevents cadmium-induced general up-regulation of methionine sulfoxide reductase gene family in Arabidopsis.

    PubMed

    Méndez, Andrea A E; Pena, Liliana B; Benavides, María P; Gallego, Susana M

    2016-12-01

    In the present study we evaluated the pre-treatment (priming) of Arabidopsis thaliana plants with sodium nitroprusside (SNP), a NO-donor, as an interesting approach for improving plant tolerance to cadmium stress. We focused on the cell redox balance and on the methionine sulfoxide reductases (MSR) family as a key component of such response. MSR catalyse the reversible oxidation of MetSO residues back to Met. Five MSRA genes and nine MSRB genes have been identified in A. thaliana, coding for proteins with different subcellular locations. After treating 20 days-old A. thaliana (Col 0) plants with 100 μM CdCl2, increased protein carbonylation in leaf tissue, lower chlorophyll content and higher levels of reactive oxygen species (ROS) in chloroplasts were detected, together with increased accumulation of all MSR transcripts evaluated. Further analysis showed reduction in guaiacol peroxidase activity (GPX) and increased catalase (CAT) activity, with no effect on ascorbate peroxidase (APX) activity. Pre-exposition of plants to 100 μM SNP before cadmium treatment restored redox balance; this seems to be linked to a better performance of antioxidant defenses. Our results indicate that NO priming may be acting as a modulator of plant antioxidant system by interfering in oxidative responses and by preventing up-regulation of MSR genes caused by metal exposure.

  9. Characterization of a cinnamoyl-CoA reductase 1 (CCR1) mutant in maize: effects on lignification, fibre development, and global gene expression

    PubMed Central

    Tamasloukht, Barek; Wong Quai Lam, Mary Sarah-Jane; Martinez, Yves; Tozo, Koffi; Barbier, Odile; Jourda, Cyril; Jauneau, Alain; Borderies, Gisèle; Balzergue, Sandrine; Renou, Jean-Pierre; Huguet, Stéphanie; Martinant, Jean Pierre; Tatout, Christophe; Lapierre, Catherine; Barrière, Yves; Goffner, Deborah; Pichon, Magalie

    2011-01-01

    Cinnamoyl-CoA reductase (CCR), which catalyses the first committed step of the lignin-specific branch of monolignol biosynthesis, has been extensively characterized in dicot species, but few data are available in monocots. By screening a Mu insertional mutant collection in maize, a mutant in the CCR1 gene was isolated named Zmccr1–. In this mutant, CCR1 gene expression is reduced to 31% of the residual wild-type level. Zmccr1– exhibited enhanced digestibility without compromising plant growth and development. Lignin analysis revealed a slight decrease in lignin content and significant changes in lignin structure. p-Hydroxyphenyl units were strongly decreased and the syringyl/guaiacyl ratio was slightly increased. At the cellular level, alterations in lignin deposition were mainly observed in the walls of the sclerenchymatic fibre cells surrounding the vascular bundles. These cell walls showed little to no staining with phloroglucinol. These histochemical changes were accompanied by an increase in sclerenchyma surface area and an alteration in cell shape. In keeping with this cell type-specific phenotype, transcriptomics performed at an early stage of plant development revealed the down-regulation of genes specifically associated with fibre wall formation. To the present authors’ knowledge, this is the first functional characterization of CCR1 in a grass species. PMID:21493812

  10. Functional Analyses of Caffeic Acid O-Methyltransferase and Cinnamoyl-CoA-Reductase Genes from Perennial Ryegrass (Lolium perenne)[W

    PubMed Central

    Tu, Yi; Rochfort, Simone; Liu, Zhiqian; Ran, Yidong; Griffith, Megan; Badenhorst, Pieter; Louie, Gordon V.; Bowman, Marianne E.; Smith, Kevin F.; Noel, Joseph P.; Mouradov, Aidyn; Spangenberg, German

    2010-01-01

    Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference–mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production. PMID:20952635

  11. Resolution of two native monomeric 90kDa nitrate reductase active proteins from Shewanella gelidimarina and the sequence of two napA genes.

    PubMed

    Simpson, Philippa J L; McKinzie, Audra A; Codd, Rachel

    2010-07-16

    The reduction of nitrate to nitrite in the bacterial periplasm occurs in the 90kDa NapA subunit of the periplasmic nitrate reductase (NAP) system. Most Shewanella genomes contain two nap operons: napEDABC and napDAGHB, which is an unusual feature of this genus. Two native, monomeric, 90kDa nitrate reductase active proteins were resolved by hydrophobic interaction chromatography from aerobic cultures of Shewanella gelidimarina replete with reduced nitrogen compounds. The 90kDa protein obtained in higher yield was characterized as NapA by electronic absorption and electron paramagnetic resonance spectroscopies and was identified by LC/MS/MS and MALDI-TOF/TOF MS as NapA from the napEDABC-type operon. The other 90kDa protein, which was unstable and produced in low yields, was posited as NapA from the napDAGHB-type operon. Two napA genes have been sequenced from the napEDABC-type and napDAGHB-type operons of S. gelidimarina. Native NAP from S. putrefaciens was resolved as one NapA monomer and one NapAB heterodimer. Two amino acid substitutions in NapA correlated with the isolation of NAP as a NapA monomer or a NapAB heterodimer. The resolution of native, redox-active NapA isoforms in Shewanella provides new insight into the respiratory versatility of this genus, which has implications in bioremediation and the assembly of microbial fuel cells.

  12. Cloning and characterization of subunit genes of ribonucleotide reductase, a cell-cycle-regulated enzyme, from Plasmodium falciparum.

    PubMed Central

    Chakrabarti, D; Schuster, S M; Chakrabarti, R

    1993-01-01

    Ribonucleotide reductase (EC 1.17.4.1; RNR), a cell-cycle-regulated enzyme, catalyzes the rate-limiting step in the de novo synthesis of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. The important role of the RNR in DNA synthesis and cell division makes this enzyme an excellent target for chemotherapy. However, nothing is known about this enzyme from the malaria parasite Plasmodium falciparum. We have isolated cDNA clones encoding both the large and small RNR subunits. The sequences of full-length clones of the large and small RNR subunits revealed an open reading frame encoding 806 and 349 amino acids, respectively, and showed significant identity with other RNR sequences in the data base. RNA blot analysis showed that the size of the large and small RNR subunit transcripts are 5.4 kb and 2.2 kb, respectively. Both the RNR subunit transcripts fluctuate in level during the cell cycle, reaching a peak preceding maximal DNA synthesis activity. An oligodeoxynucleotide phosphorothioate that is complementary to sequences around the translational initiation codon of the small RNR subunit showed significant inhibition of growth, as measured by the inhibition in DNA synthesis. Images Fig. 2 PMID:8265664

  13. Expression of a deregulated tobacco nitrate reductase gene in potato increases biomass production and decreases nitrate concentration in all organs.

    PubMed

    Djennane, Samia; Quilleré, Isabelle; Leydecker, Marie-Thérèse; Meyer, Christian; Chauvin, Jean-Eric

    2004-09-01

    We investigated the physiological consequences for nitrogen metabolism and growth of the deregulated expression of an N-terminal-deleted tobacco nitrate reductase in two lines of potato (Solanum tuberosum L. cv Safrane). The transgenic plants showed a higher biomass accumulation, especially in tubers, but a constant nitrogen content per plant. This implies that the transformed lines had a reduced nitrogen concentration per unit of dry weight. A severe reduction in nitrate concentrations was also observed in all organs, but was more apparent in tubers where nitrate was almost undetectable in the transgenic lines. In leaves and roots, but not tubers, this nitrate decrease was accompanied by a statistically significant increase in the level of malate, which acts as a counter-anion for nitrate reduction. Apart from glutamine in tubers, no major changes in amino acid concentration were seen in leaves, roots or tubers. We conclude that enhancement of nitrate reduction rate leads to higher biomass production, probably by allowing a better allocation of N-resources to photosynthesis and C-metabolism.

  14. Multilocus Phylogenetic Study of the Scheffersomyces Yeast Clade and Characterization of the N-Terminal Region of Xylose Reductase Gene

    PubMed Central

    Urbina, Hector; Blackwell, Meredith

    2012-01-01

    Many of the known xylose-fermenting (X-F) yeasts are placed in the Scheffersomyces clade, a group of ascomycete yeasts that have been isolated from plant tissues and in association with lignicolous insects. We formally recognize fourteen species in this clade based on a maximum likelihood (ML) phylogenetic analysis using a multilocus dataset. This clade is divided into three subclades, each of which exhibits the biochemical ability to ferment cellobiose or xylose. New combinations are made for seven species of Candida in the clade, and three X-F taxa associated with rotted hardwood are described: Scheffersomyces illinoinensis (type strain NRRL Y-48827T  =  CBS 12624), Scheffersomyces quercinus (type strain NRRL Y-48825T  =  CBS 12625), and Scheffersomyces virginianus (type strain NRRL Y-48822T  =  CBS 12626). The new X-F species are distinctive based on their position in the multilocus phylogenetic analysis and biochemical and morphological characters. The molecular characterization of xylose reductase (XR) indicates that the regions surrounding the conserved domain contain mutations that may enhance the performance of the enzyme in X-F yeasts. The phylogenetic reconstruction using XYL1 or RPB1 was identical to the multilocus analysis, and these loci have potential for rapid identification of cryptic species in this clade. PMID:22720049

  15. Molecular genetic analysis in mild hyperhomocysteinemia: A common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease

    SciTech Connect

    Kluijtmans, L.A.J.; Heuvel, L.P.W.J. van den; Stevens, E.M.B.

    1996-01-01

    Mild hyperhomocysteinemia is an established risk factor for cardiovascular disease. Genetic aberrations in the cystathionine P-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR) genes may account for reduced enzyme activities and elevated plasma homocysteine levels. In 15 unrelated Dutch patients with homozygous CBS deficiency, we observed the 833T{yields}C (1278T) mutation in 50% of the alleles. Very recently, we identified a common mutation (677C{yields}T; A{yields}V) in the MTHFR gene, which, in homozygous state, is responsible for the thermolabile phenotype and which is associated with decreased specific MTHFR activity and elevated homocysteine levels. We screened 60 cardiovascular patients and 111 controls for these two mutations, to determine whether these mutations are risk factors for premature cardiovascular disease. Heterozygosity for the 833T{yields}C mutation in the CBS gene was observed in one individual of the control group but was absent in patients with premature cardiovascular disease. Homozygosity for the 677C-{yields}T mutation in the MTHFR gene was found in 9 (15%) of 60 cardiovascular patients and in only 6 ({approximately}5%) of 111 control individuals (odds ratio 3.1 [95% confidence interval 1.0-9.21]). Because of both the high prevalence of the 833T-{yields}C mutation among homozygotes for CBS deficiency and its absence in 60 cardiovascular patients, we may conclude that heterozygosity for CBS deficiency does not appear to be involved in premature cardiovascular disease. However, a frequent homozygous mutation in the MTHFR gene is associated with a threefold increase in risk for premature cardiovascular disease. 35 refs., 3 figs., 1 tab.

  16. Molecular genetic analysis in mild hyperhomocysteinemia: a common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease.

    PubMed Central

    Kluijtmans, L. A.; van den Heuvel, L. P.; Boers, G. H.; Frosst, P.; Stevens, E. M.; van Oost, B. A.; den Heijer, M.; Trijbels, F. J.; Rozen, R.; Blom, H. J.

    1996-01-01

    Mild hyperhomocysteinemia is an established risk factor for cardiovascular disease. Genetic aberrations in the cystathionine beta-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR) genes may account for reduced enzyme activities and elevated plasma homocysteine levels. In 15 unrelated Dutch patients with homozygous CBS deficiency, we observed the 833T-->C (I278T) mutation in 50% of the alleles. Very recently, we identified a common mutation (677C-->T; A-->V) in the MTHFR gene, which, in homozygous state, is responsible for the thermolabile phenotype and which is associated with decreased specific MTHRF activity and elevated homocysteine levels. We screened 60 cardiovascular patients and 111 controls for these two mutations, to determine whether these mutations are risk factors for premature cardiovascular disease. Heterozygosity for the 833T-->C mutation in the CBS gene was observed in one individual of the control group but was absent in patients with premature cardiovascular disease. Homozygosity for the 677C-->T mutation in the MTHFR gene was found in (15%) of 60 cardiovascular patients and in only 6 (approximately 5%) of 111 control individuals (odds ratio 3.1 [95% confidence interval 1.0-9.2]). Because of both the high prevalence of the 833T-->C mutation among homozygotes for CBS deficiency and its absence in 60 cardiovascular patients, we may conclude that heterozygosity for CBS deficiency does not appear to be involved in premature cardiovascular disease. However, a frequent homozygous mutation in the MTHFR gene is associated with a threefold increase in risk for premature cardiovascular disease. PMID:8554066

  17. Xylose reductase from the thermophilic fungus Talaromyces emersonii: cloning and heterologous expression of the native gene (Texr) and a double mutant (TexrK271R + N273D) with altered coenzyme specificity.

    PubMed

    Fernandes, Sara; Tuohy, Maria G; Murray, Patrick G

    2009-12-01

    Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene encoding xylose reductase (Texr) was isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli and purified to homogeneity. Texr encodes a 320 amino acid protein with a molecular weight of 36 kDa, which exhibited high sequence identity with other xylose reductase sequences and was shown to be a member of the aldoketoreductase (AKR) superfamily with a preference for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as coenzyme. Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed mutagenesis. The TeXR K271R+N273D double mutant displayed an altered coenzyme preference with a 16-fold improvement in NADH utilization relative to the wild type and therefore has the potential to reduce redox imbalance of xylose fermentation in recombinant S. cerevisiae strains. Expression of Texr was shown to be inducible by the same carbon sources responsible for the induction of genes encoding enzymes relevant to lignocellulose hydrolysis, suggesting a coordinated expression of intracellular and extracellular enzymes relevant to hydrolysis and metabolism of pentose sugars in T. emersonii in adaptation to its natural habitat. This indicates a potential advantage in survival and response to a nutrient-poor environment.

  18. Dicarbonyl/L-xylulose reductase: a potential biomarker identified by laser-capture microdissection-micro serial analysis of gene expression of human prostate adenocarcinoma.

    PubMed

    Cho-Vega, Jeong Hee; Tsavachidis, Spiridon; Do, Kim-Anh; Nakagawa, Junichi; Medeiros, L Jeffrey; McDonnell, Timothy J

    2007-12-01

    To identify genes involved in prostate carcinogenesis, we used laser-capture microdissection-micro serial analysis of gene expression to construct libraries of paired cancer and normal cells from human tissue samples. After computational comparison of the two libraries, we identified dicarbonyl/l-xylulose reductase (DCXR), an enzyme that catalyzes alpha-dicarbonyl and l-xylulose, as being significantly up-regulated in prostate cancer cells. The specificity of DCXR up-regulation for prostate cancer tissues was confirmed by quantitative real-time reverse transcriptase-PCR, virtual Northern blot, and Western blot analyses. Furthermore, DCXR expression at the protein level was assessed using fresh-frozen tissues and a tissue microarray consisting of 46 cases of organ-confined early-stage prostate cancer and 29 cases of chemohormonally treated prostate cancer. In most normal prostate epithelial cells, DCXR was expressed at low levels and was localized predominantly in the cytoplasmic membrane. In contrast, in virtually all grades of early-stage prostate cancer and in all chemohormonally treated cases, DCXR was strikingly overexpressed and was localized predominantly in the cytoplasm and nucleus. In all samples, the stromal cells were completely devoid of DCXR expression. Based on these findings, we suggest that DCXR overexpression has the potential to be an additional useful biomarker for prostate cancer.

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

    PubMed Central

    Cao, Yan; Zhao, Bin; Han, Yanhui; Zhang, Juan; Li, Xuezhen; Qiu, Chunhui; Wu, Xiujuan; Hong, Yang; Ai, Dezhou; Lin, Jiaojiao; Fu, Zhiqiang

    2013-01-01

    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% (P < 0.01) of worm reduction and 40.38–44.51% (P < 0.01) 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. PMID:23509820

  20. Massive pulmonary embolism associated with Factor V Leiden, prothrombin, and methylenetetrahydrofolate reductase gene mutations in a young patient on oral contraceptive pills: a case report.

    PubMed

    Charafeddine, Khalil M; Mahfouz, Rami A; Ibrahim, Georges Y; Taher, Ali T; Hoballah, Jamal J; Taha, Assad M

    2010-10-01

    Factor V Leiden (Factor V G1691A), prothrombin gene mutation G20210A, and homozygous C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene are known to predispose venous thromboembolism (VTE). We present herein a rare case of a young woman heterozygous for these mutations and taking oral contraceptive pills for less than 2 months, diagnosed to have massive deep venous thrombosis and bilateral pulmonary embolism. The patient was managed for 10 days in the hospital and discharged home on oral anticoagulants. This case suggests that screening for these factors in people with family history of thrombosis and in relatives of patients with these mutations is highly recommended to prevent fatal consequences. In addition, a new guideline for treatment and prophylaxis with anticoagulant for these patients and others who are at risk of developing VTE (American College of Chest Physicians [ACCP] guidelines-Chest 2008) has been published recently. Our recommendation is to promote for the internationally published algorithms through their application, where necessary, to prevent any future thrombotic morbidity or mortality incidents.

  1. Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from hazel (Corylus avellana L. Gasaway).

    PubMed

    Wang, Yechun; Guo, Binhui; Zhang, Fei; Yao, Hongyan; Miao, Zhiqi; Tang, Kexuan

    2007-11-30

    The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR (designated as CgHMGR, GenBank accession number EF206343) from hazel (Corylus avellana L. Gasaway), a taxol-producing plant species. The full-length cDNA of CgHMGR was 2064 bp containing a 1704-bp ORF encoding 567 amino acids. Bioinformatic analyses revealed that the deduced CgHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of CgHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis of beta-carotene, indicating that CgHMGR encoded a functional protein. The cloning, characterization and functional analysis of CgHMGR gene will enable us to further understand the role of CgHMGR involved in taxol biosynthetic pathway in C. avellana at molecular level.

  2. Triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P: purification, characterization, gene cloning, and overexpression of a functional protein in Escherichia coli.

    PubMed

    Jang, Moon-Sun; Lee, Young-Mi; Kim, Cheorl-Ho; Lee, Jai-Heon; Kang, Dong-Woo; Kim, Seok-Jo; Lee, Young-Choon

    2005-12-01

    We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60 degrees C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.

  3. Cloning and expression analysis of interferon-γ-inducible-lysosomal thiol reductase gene in South African clawed frog (Xenopus laevis).

    PubMed

    Cui, Xian-wei; Xiao, Wen; Ke, Zhen; Liu, Xia; Xu, Xing-zhou; Zhang, Shuang-quan

    2011-12-01

    In this study, an interferon-γ-inducible-lysosomal thiol reductase (GILT) homologue has been cloned and identified from South African clawed frog Xenopus laevis (designated XlGILT). The open reading frame (ORF) of XlGILT consists of 771 bases encoding a protein of 256 amino acids with an estimated molecular mass of 28.76kDa and a theoretical isoelectric point of 5.12. The N-terminus of the XlGILT was found to have a putative signal peptide with a cleavage site amino acid position at 15-16. SMART analysis showed that the XlGILT contained a GILT active-site C(69)GGC(72) motif and a GILT signature motif C(114)QHGKEECIGNLIETC(129). The expression levels of XlGILT mRNA were higher in spleen and peripheral blood mononuclear cells (PBMCs), moderate in liver, intestine, heart and kidney, and lower in lung. The XlGILT mRNA expression was significantly up-regulated in spleen in vivo and PBMCs in vitro after LPS stimulation. The soluble X. laevis GILT (XlsGILT) was inserted into a pET28a vector and expressed in BL21 (DE3) cells as a His-tag fusion enzyme. After purification, further study revealed that XlsGILT was capable of catalyzing the reduction of the interchain disulfide bonds intact IgG. These results will allow for further investigation to unravel the role of this key enzyme in class II MHC-restricted antigen processing and to use X. laevis as an in vivo model for related studies.

  4. Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius.

    PubMed

    Weyda, István; Lübeck, Mette; Ahring, Birgitte K; Lübeck, Peter S

    2014-04-01

    Aspergillus carbonarius accumulates xylitol when it grows on D-xylose. In fungi, D-xylose is reduced to xylitol by the NAD(P)H-dependent xylose reductase (XR). Xylitol is then further oxidized by the NAD(+)-dependent xylitol dehydrogenase (XDH). The cofactor impairment between the XR and XDH can lead to the accumulation of xylitol under oxygen-limiting conditions. Most of the XRs are NADPH dependent and contain a conserved Ile-Pro-Lys-Ser motif. The only known naturally occurring NADH-dependent XR (from Candida parapsilosis) carries an arginine residue instead of the lysine in this motif. In order to overcome xylitol accumulation in A. carbonarius a Lys-274 to Arg point mutation was introduced into the XR with the aim of changing the specificity toward NADH. The effect of the genetic engineering was examined in fermentation for citric acid production and xylitol accumulation by using D-xylose as the sole carbon source. Fermentation with the mutant strain showed a 2.8-fold reduction in xylitol accumulation and 4.5-fold increase in citric acid production compared to the wild-type strain. The fact that the mutant strain shows decreased xylitol levels is assumed to be associated with the capability of the mutated XR to use the NADH generated by the XDH, thus preventing the inhibition of XDH by the high levels of NADH and ensuring the flux of xylose through the pathway. This work shows that enhanced production of citric acid can be achieved using xylose as the sole carbon source by reducing accumulation of other by-products, such as xylitol.

  5. Identification of methyl coenzyme M reductase A(mcrA) genes associated with methane-oxidizing archaea

    SciTech Connect

    Hallam, Steven J.; Girguis, Peter R.; Preston, Christina M.; Richardson, Paul M.; DeLong, Edward F.

    2003-09-01

    Phylogenetic and stable-isotope analyses implicated two methanogen-like archaeal groups, ANME-1 and ANME-2, as key participants in the process of anaerobic methane oxidation. Although nothing is known about anaerobic methane oxidation at the molecular level, the evolutionary relationship between methane-oxidizing archaea (MOA) and methanogenic archaea raises the possibility that MOA have co-opted key elements of the methanogenic pathway, reversing many of its steps to oxidize methane anaerobically. In order to explore this hypothesis, the existence and genomic conservation of methyl coenzyme M reductase (MCR), the enzyme catalyzing the terminal step in methanogenesis, was studied in ANME-1 and ANME-2 archaea isolated from various marine environments. Clone libraries targeting a conserved region of the alpha subunit of MCR (mcrA) were generated and compared from environmental samples, laboratory-incubated microcosms, and fosmid libraries. Four out of five novel mcrA types identified from these sources were associated with ANME-1 orANME-2 group members. Assignment of mcrA types to specific phylogenetic groups was based on environmental clone recoveries, selective enrichment of specific MOA and mcrA types in a microcosm, phylogenetic congruence between mcrA and small-subunit rRNA tree topologies, and genomic context derived from fosmid sequences. Analysis of the ANME-1 and ANME-2 mcrA sequences suggested the potential for catalytic activity based on conservation of active-site amino acids. These results provide a basis for identifying methanotrophic archaea with mcrA sequences and define a functional genomic link between methanogenic and methanotrophic archaea.

  6. Structure of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12.

    PubMed Central

    Stewart, V; Parales, J; Merkel, S M

    1989-01-01

    narL and narX mediate nitrate induction of nitrate reductase synthesis and nitrate repression of fumarate reductase synthesis. We report here the nucleotide sequences of narL and narX. The deduced protein sequences aid in defining distinct subclasses of regulators and sensors in the family of two-component regulatory proteins. PMID:2649492

  7. Partial gene sequences for the A subunit of methyl-coenzyme M reductase (mcrI) as a phylogenetic tool for the family Methanosarcinaceae

    NASA Technical Reports Server (NTRS)

    Springer, E.; Sachs, M. S.; Woese, C. R.; Boone, D. R.

    1995-01-01

    Representatives of the family Methanosarcinaceae were analyzed phylogenetically by comparing partial sequences of their methyl-coenzyme M reductase (mcrI) genes. A 490-bp fragment from the A subunit of the gene was selected, amplified by the PCR, cloned, and sequenced for each of 25 strains belonging to the Methanosarcinaceae. The sequences obtained were aligned with the corresponding portions of five previously published sequences, and all of the sequences were compared to determine phylogenetic distances by Fitch distance matrix methods. We prepared analogous trees based on 16S rRNA sequences; these trees corresponded closely to the mcrI trees, although the mcrI sequences of pairs of organisms had 3.01 +/- 0.541 times more changes than the respective pairs of 16S rRNA sequences, suggesting that the mcrI fragment evolved about three times more rapidly than the 16S rRNA gene. The qualitative similarity of the mcrI and 16S rRNA trees suggests that transfer of genetic information between dissimilar organisms has not significantly affected these sequences, although we found inconsistencies between some mcrI distances that we measured and and previously published DNA reassociation data. It is unlikely that multiple mcrI isogenes were present in the organisms that we examined, because we found no major discrepancies in multiple determinations of mcrI sequences from the same organism. Our primers for the PCR also match analogous sites in the previously published mcrII sequences, but all of the sequences that we obtained from members of the Methanosarcinaceae were more closely related to mcrI sequences than to mcrII sequences, suggesting that members of the Methanosarcinaceae do not have distinct mcrII genes.

  8. Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

    PubMed Central

    Daimiel, Lidia A.; Fernández-Suárez, María E.; Rodríguez-Acebes, Sara; Crespo, Lorena; Lasunción, Miguel A.; Gómez-Coronado, Diego; Martínez-Botas, Javier

    2012-01-01

    DHCR24 (3β-hydroxysterol Δ24-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at −98/−90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5′-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression. PMID:23050906

  9. Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

    PubMed Central

    Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

    2015-01-01

    Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

  10. Molecular Cloning and Evidence for Osmoregulation of the Δ1-Pyrroline-5-Carboxylate Reductase (proC) Gene in Pea (Pisum sativum L.) 12

    PubMed Central

    Williamson, Cynthia L.; Slocum, Robert D.

    1992-01-01

    Several cDNA clones encoding Δ1-pyrroline-5-carboxylate reductase (P5CR, l-proline:NAD[P]+ 5-oxidoreductase, EC 1.5.1.2), which catalyzes the terminal step in proline biosynthesis, were isolated from a pea leaf library screened with a 32P-labeled Aval fragment of a soybean nodule P5CR cDNA (A.J. Delauney, D.P.S. Verma [1990] Mol Gen Genet 221: 299-305). DNA sequence analysis of one full-length 1.3-kb clone (pPPS3) indicated that the pea P5CR gene contains a single major open reading frame encoding a polypeptide of 28,242 Da. Genomic analysis suggested that two to three copies of the P5CR gene are present per haploid genome in pea. The primary structure of pea P5CR is 85% identical with that of soybean and exhibits significant homology to human, yeast, and Escherichia coli P5CR. The sequence of one of four highly conserved domains found in all prokaryotic and eukaryotic P5CRs is similar to the consensus sequence for the NAD(P)H-binding site of other enzymes. The pea P5CR cDNA hybridized to two transcripts, 1.3 and 1.1 kb in size, in polyadenylated RNA purified from leaf tissues of mature, light-grown plants (4 weeks old). Only the 1.3-kb transcript was detected in younger (1 week old) greened seedlings or in etiolated seedlings. In greened seedlings, steady-state levels of this 1.3-kb mRNA increased approximately 5-fold in root tissues within 6 h after plants were irrigated with 0.4 m NaCl, suggesting that expression of the P5CR gene is osmoregulated. Images Figure 3 Figure 4 Figure 5 PMID:11537868

  11. Phylogeography of Sulfate-Reducing Bacteria among Disturbed Sediments, Disclosed by Analysis of the Dissimilatory Sulfite Reductase Genes (dsrAB)

    PubMed Central

    Pérez-Jiménez, J. R.; Kerkhof, L. J.

    2005-01-01

    Sediment samples were collected worldwide from 16 locations on four continents (in New York, California, New Jersey, Virginia, Puerto Rico, Venezuela, Italy, Latvia, and South Korea) to assess the extent of the diversity and the distribution patterns of sulfate-reducing bacteria (SRB) in contaminated sediments. The SRB communities were examined by terminal restriction fragment (TRF) length polymorphism (TRFLP) analysis of the dissimilatory sulfite reductase genes (dsrAB) with NdeII digests. The fingerprints of dsrAB genes contained a total of 369 fluorescent TRFs, of which <20% were present in the GenBank database. The global sulfidogenic communities appeared to be significantly different among the anthropogenically impacted (petroleum-contaminated) sites, but nearly all were less diverse than pristine habitats, such as mangroves. A global SRB indicator species of petroleum pollution was not identified. However, several dsrAB gene sequences corresponding to hydrocarbon-degrading isolates or consortium members were detected in geographically widely separated polluted sites. Finally, a cluster analysis of the TRFLP fingerprints indicated that many SRB microbial communities were most similar on the basis of close geographic proximity (tens of kilometers). Yet, on larger scales (hundreds to thousands of kilometers) SRB communities could cluster with geographically widely separated sites and not necessarily with the site with the closest proximity. These data demonstrate that SRB populations do not adhere to a biogeographic distribution pattern similar to that of larger eukaryotic organisms, with the greatest species diversity radiating from the Indo-Pacific region. Rather, a patchy SRB distribution is encountered, implying an initially uniform SRB community that has differentiated over time. PMID:15691959

  12. Differential induction and suppression of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid.

    PubMed Central

    Choi, D; Ward, B L; Bostock, R M

    1992-01-01

    Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is essential for the biosynthesis of sesquiterpenoid phytoalexins and steroid derivatives in Solanaceous plants following stresses imposed by wounding and pathogen infection. To better understand this complex step in stress-responsive isoprenoid synthesis, we isolated three classes of cDNAS encoding HMGR (hmg1, hmg2, and hmg3) from a potato tuber library using a probe derived from an Arabidopsis HMGR cDNA. The potato cDNAs had extensive homology in portions of the protein coding regions but had low homology in the 3' untranslated regions. RNA gel blot analyses using gene-specific probes showed that hmg1 was strongly induced in tuber tissue by wounding, but the wound induction was strongly suppressed by treatment of the tissue with the fungal elicitor arachidonic acid or by inoculation with an incompatible or compatible race of the fungal pathogen Phytophtora infestans. The hmg2 and hmg3 mRNAs also accumulated in response to wounding, but in contrast to hmg1, these mRNAs were strongly enhanced by arachidonic acid or inoculation. Inoculation with a compatible race of P. infestans resulted in similar patterns in HMGR gene expression of hmg2 and hmg3 except that the magnitude and rate of the changes in mRNA levels were reduced relative to the incompatible interaction. The differential regulation of members of the HMGR gene family may explain in part the previously reported changes in HMGR enzyme activities following wounding and elicitor treatment. The suppression of hmg1 and the enhancement of hmg2 and hmg3 transcript levels following elicitor treatment or inoculation with the incompatible race parallel the suppression in steroid and stimulation of sesquiterpenoid accumulations observed in earlier investigations. The results are discussed in relation to the hypothesis that there are discrete organizational channels for sterol and sesquiterpene biosynthesis in potato and other Solanaceous species. PMID

  13. Different Alleles of a Gene Encoding Leucoanthocyanidin Reductase (PaLAR3) Influence Resistance against the Fungus Heterobasidion parviporum in Picea abies.

    PubMed

    Nemesio-Gorriz, Miguel; Hammerbacher, Almuth; Ihrmark, Katarina; Källman, Thomas; Olson, Åke; Lascoux, Martin; Stenlid, Jan; Gershenzon, Jonathan; Elfstrand, Malin

    2016-08-01

    Despite the fact that fungal diseases are a growing menace for conifers in modern silviculture, only a very limited number of molecular markers for pathogen resistance have been validated in conifer species. A previous genetic study indicated that the resistance of Norway spruce (Picea abies) to Heterobasidion annosum s.l., a pathogenic basidiomycete species complex, is linked to a quantitative trait loci that associates with differences in fungal growth in sapwood (FGS) that includes a gene, PaLAR3, which encodes a leucoanthocyanidin reductase. In this study, gene sequences showed the presence of two PaLAR3 allelic lineages in P. abies. Higher resistance was associated with the novel allele, which was found in low frequency in the four P. abies populations that we studied. Norway spruce plants carrying at least one copy of the novel allele showed a significant reduction in FGS after inoculation with Heterobasidion parviporum compared to their half-siblings carrying no copies, indicating dominance of this allele. The amount of (+) catechin, the enzymatic product of PaLAR3, was significantly higher in bark of trees homozygous for the novel allele. Although we observed that the in vitro activities of the enzymes encoded by the two alleles were similar, we could show that allele-specific transcript levels were significantly higher for the novel allele, indicating that regulation of gene expression is responsible for the observed effects in resistance, possibly caused by differences in cis-acting elements that we observe in the promoter region of the two alleles.

  14. Enhancing Heat Tolerance of the Little Dogwood Cornus canadensis L. f. with Introduction of a Superoxide Reductase Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus

    PubMed Central

    Geng, Xing-Min; Liu, Xiang; Ji, Mikyoung; Hoffmann, William A.; Grunden, Amy; Xiang, Qiu-Yun J.

    2016-01-01

    Production of reactive oxygen species (ROS) can be accelerated under various biotic and abiotic stresses causing lipid peroxidation, protein degradation, enzyme inactivation, and DNA damage. Superoxide reductase (SOR) is a novel antioxidant enzyme from Pyrococcus furiosus and is employed by this anaerobic hyperthermophilic archaeon for efficient detoxification of ROS. In this study, SOR was introduced into a flowering plant Cornus canadensis to enhance its heat tolerance and reduce heat induced damage. A fusion construct of the SOR gene and Green Fluorescent Protein gene (GFP) was introduced into C. canadensis using Agrobacterium-mediated transformation. Heat tolerance of the GFP-SOR expressing transgenic plants was investigated by observing morphological symptoms of heat injury and by examining changes in photosynthesis, malondialdehyde (MDA), and proline levels in the plants. Our results indicate that the expression of the P. furiosus SOR gene in the transgenic plants alleviated lipid peroxidation of cell membranes and photoinhibition of PS II, and decreased the accumulation of proline at 40°C. After a series of exposures to increasing temperatures, the SOR transgenic plants remained healthy and green whereas most of the non-transgenic plants dried up and were unable to recover. While it had previously been reported that expression of SOR in Arabidopsis enhanced heat tolerance, this is the first report of the successful demonstration of improved heat tolerance in a non-model plant resulting from the introduction of P. furiosus SOR. The study demonstrates the potential of SOR for crop improvement and that inherent limitations of plant heat tolerance can be ameliorated with P. furiosus SOR. PMID:26858741

  15. Identification of three new mutations in the NADH-cytochrome b5 reductase gene responsible for recessive congenital methemoglobinemia type II

    SciTech Connect

    Mota-Vieira, L.; Kaplan, J.C.; Kahn, A.; Leroux, A.

    1994-09-01

    Recessive congenital methemoglobinemia (RCM; McKusick N{degrees}25800) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases: in type I form, cyanosis is the only symptom and the enzyme is only defective in red blood cells; in type II form, cyanosis is associated with severe mental retardation and neurological impairment and the enzyme defect is systemic. We have identified three new molecular defects in two unrelated patients with type II RCM. A homozygous C{r_arrow}T transition in codon 218 (Arg) was detected in the cDNA of one patient, resulting in a premature stop codon (TGA) in exon 8. Restriction enzyme analysis of genomic DNA confirmed the homozygosity of the propositus and heterozygosity for an identical defect in both parents. The second patient was found to be a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation (T{r_arrow}C) with substitution of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele showed a 3 bp deletion of nucleotides 815-817 of the cDNA. The CTG ATG sequence at position 814-819 in exon 9 coding for Leu-271 and Met-272 was replaced by the CTG triplet, with conservation of the Leu-271 and loss of the Met-272. To our knowledge, these are the first examples of a homozygous nonsense mutation and of a compound heterozygous mutation detected in the cytb5r gene. This finding supports the diversity of genetic defects in the cytb5r gene leading to the severe form of the disease.

  16. A cluster of three genes (dapA, orf2, and dapB) of Brevibacterium lactofermentum encodes dihydrodipicolinate synthase, dihydrodipicolinate reductase, and a third polypeptide of unknown function.

    PubMed Central

    Pisabarro, A; Malumbres, M; Mateos, L M; Oguiza, J A; Martín, J F

    1993-01-01

    The dapA and dapB genes, encoding, respectively, dihydrodipicolinate synthase and dihydrodipicolinate reductase, the two first enzymes of the lysine branch of the aspartic amino acid family, were cloned from the DNA of the amino acid-producing bacterium Brevibacterium lactofermentum. The two genes were clustered in a 3.5-kb Sau3AI-BamHI fragment but were separated by an open reading frame of 750 nucleotides. The protein encoded by this open reading frame had little similarity to any protein in the data banks, and its function remains unknown. The three genes were translated in Escherichia coli, giving the corresponding polypeptides. Images PMID:8478336

  17. Comparative analysis of amino acid composition in the active site of nirk gene encoding copper-containing nitrite reductase (CuNiR) in bacterial spp.

    PubMed

    Adhikari, Utpal Kumar; Rahman, M Mizanur

    2017-04-01

    The nirk gene encoding the copper-containing nitrite reductase (CuNiR), a key catalytic enzyme in the environmental denitrification process that helps to produce nitric oxide from nitrite. The molecular mechanism of denitrification process is definitely complex and in this case a theoretical investigation has been conducted to know the sequence information and amino acid composition of the active site of CuNiR enzyme using various Bioinformatics tools. 10 Fasta formatted sequences were retrieved from the NCBI database and the domain and disordered regions identification and phylogenetic analyses were done on these sequences. The comparative modeling of protein was performed through Modeller 9v14 program and visualized by PyMOL tools. Validated protein models were deposited in the Protein Model Database (PMDB) (PMDB id: PM0080150 to PM0080159). Active sites of nirk encoding CuNiR enzyme were identified by Castp server. The PROCHECK showed significant scores for four protein models in the most favored regions of the Ramachandran plot. Active sites and cavities prediction exhibited that the amino acid, namely Glycine, Alanine, Histidine, Aspartic acid, Glutamic acid, Threonine, and Glutamine were common in four predicted protein models. The present in silico study anticipates that active site analyses result will pave the way for further research on the complex denitrification mechanism of the selected species in the experimental laboratory.

  18. Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    van Deenen, Nicole; Bachmann, Anne-Lena; Schmidt, Thomas; Schaller, Hubert; Sand, Jennifer; Prüfer, Dirk; Schulze Gronover, Christian

    2012-04-01

    Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products.

  19. Dominance of green sulfur bacteria in the chemocline of the meromictic Lake Suigetsu, Japan, as revealed by dissimilatory sulfite reductase gene analysis.

    PubMed

    Mori, Yumi; Kataoka, Takafumi; Okamura, Takahiko; Kondo, Ryuji

    2013-05-01

    This study investigated the spatiotemporal abundance and diversity of the α-subunit of the dissimilatory sulfite reductase gene (dsrA) in the meromictic Lake Suigetsu for assessing the sulfur-oxidizing bacterial community. The density of dsrA in the chemocline reached up to 3.1 × 10(6) copies ml(-1) in summer by means of quantitative real-time PCR and it was generally higher than deeper layers. Most of the dsrA clones sequenced were related to green sulfur bacteria such as Chlorobium phaeovibrioides, C. limicola, and C. luteolum. Below the chemocline of the lake, we also detected other dsrA clones related to the purple sulfur bacterium Halochromatium salexigens and some branching lineages of diverse sequences that were related to chemotrophic sulfur bacterial species such as Magnetospirillum gryphiswaldense, Candidatus Ruthia magnifica, and Candidatus Thiobios zoothamnicoli. The abundance and community compositions of sulfur-oxidizing bacteria changed depending on the water depth and season. This study indicated that the green sulfur bacteria dominated among sulfur-oxidizing bacterial population in the chemocline of Lake Suigetsu and that certain abiotic environmental variables were important factors that determined sulfur bacterial abundance and community structure.

  20. Methanogenic diversity studies within the rumen of Surti buffaloes based on methyl coenzyme M reductase A (mcrA) genes point to Methanobacteriales.

    PubMed

    Singh, Krishna M; Pandya, Paresh R; Parnerkar, Subhash; Tripathi, Ajai K; Ramani, Umed; Koringa, Prakash G; Rank, Dharamshi N; Joshi, Chaitanya G; Kothari, Ramesh K

    2010-01-01

    Methane emissions from ruminant livestock are considered to be one of the more potent forms of greenhouse gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, basic knowledge of the diversity of these microbes in breeds of buffalo is required. Therefore, the methanogenic community in the rumen of Surti buffaloes was analyzed by PCR amplification, cloning, and sequencing of methyl coenzyme M reductase (mcrA) gene. A total of 76 clones were identified, revealing 14 different sequences (phylotypes). All 14 sequences were similar to methanogens belonging to the order Methanobacteriales. Within Methanobacteriales, 12 clones (6 OTUs) were similar to Methanosphaera stadtmanae and the remaining 8 phylotypes (64 clones) were similar to unclassified Methanobacteriales. Overall, members of the Methanobacteriales dominated the mcrA clone library in the rumen of Surti buffalo. Further studies and effective strategies can be made to inhibit the growth of Methanobacteriales to reduce methane emission from the rumen which would help in preventing global warming.

  1. Increased incidence of cycloguanil resistance in malaria cases entering France from Africa, determined as point mutations in the parasites' dihydrofolate-reductase genes.

    PubMed

    Durand, R; di Piazza, J P; Longuet, C; Sécardin, Y; Clain, J; le Bras, J

    1999-01-01

    The incidence of cycloguanil resistance in 501 Plasmodium falciparum isolates from individuals entering France from Africa was estimated by a method based on PCR-restriction-fragment-length polymorphisms. None of the subjects had taken antifol prophylaxis. Annual incidence of the resistance, detected as a point mutation at codon 108 in the parasite's dihydrofolate-reductase gene, increased from 19.8% in 1995 to 43.6% in 1997 (P < 0.001). The proportion of isolates found to be susceptible (i.e. wild-type) among travellers returning from the African countries known as Group 2 in France (i.e. Burkina Faso, Côte d'Ivoire, Gambia, Ghana, Guinea, Liberia, Madagascar, Mali, Mauritania, Niger, Senegal, Sierra Leone, Tchad and Togo) was reasonably high (62.9%) and much higher than in the other subjects returning from other identifiable countries in Africa (35.3%). The antimalarial prophylaxis recommended in France to those travelling to Group-2 countries, chloroquine-proguanil, therefore still seems reasonable, although cycloguanil resistance may seriously undermine the efficacy of this drug combination in the future.

  2. Elevated total plasma homocysteine and 667C{r_arrow}T mutation of the 5,10-methylenetetrahydrofolate reductase gene in thrombotic vascular disease

    SciTech Connect

    De Franchis, R.; Sebastio, G.; Andria, G.

    1996-07-01

    Moderate elevation of total plasma homocysteine (tHcy) has been reported as an independent risk factor for thrombotic vascular disease, a well-known multifactorial disorder. Possible genetic causes of elevated tHcy include defects of the sulfur-containing amino acids metabolism due to deficiencies of cystathionine {Beta}-synthase, of 5,10-methylenetetrahydrofolate reductase (MTHFR), and of the enzymes of cobalamin metabolism. An impaired activity of MTHFR due to a thermolabile form of the enzyme has been observed in {le}28% of hyperhomocysteinemic patients with premature vascular disease. More recently, the molecular basis of such enzymatic thermolability has been related to a common mutation of the MTHFR gene, causing a C-to-T substitution at nt 677 (677C{r_arrow}T). This mutation was found in 38% of unselected chromosomes from 57 French Canadian individuals. The homozygous state for the mutation was present in 12% of these subjects and correlated with significantly elevated tHcy. Preliminary evidence indicates that the frequency of homozygotes for the 677C{r_arrow}T mutation may vary significantly in populations from different geographic areas. 5 refs., 2 tabs.

  3. A T-DNA Insertion Knockout of the Bifunctional Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase Gene Elevates Lysine Levels in Arabidopsis Seeds1

    PubMed Central

    Zhu, Xiaohong; Tang, Guiliang; Granier, Fabienne; Bouchez, David; Galili, Gad

    2001-01-01

    Plants possess both anabolic and catabolic pathways for the essential amino acid lysine (Lys). However, although the biosynthetic pathway was clearly shown to regulate Lys accumulation in plants, the functional significance of Lys catabolism has not been experimentally elucidated. To address this issue, we have isolated an Arabidopsis knockout mutant with a T-DNA inserted into exon 13 of the gene encoding Lys ketoglutarate reductase/saccharopine dehydrogenase. This bifunctional enzyme controls the first two steps of Lys catabolism. The phenotype of the LKR/SDH knockout was indistinguishable from wild-type plants under normal growth conditions, suggesting that Lys catabolism is not an essential pathway under standard growth conditions. However, mature seeds of the knockout mutant over-accumulated Lys compared with wild-type plants. This report provides the first direct evidence for the functional significance of Lys catabolism in regulating Lys accumulation in seeds. Such a knockout mutant may also provide new perspectives to improve the level of the essential amino acid Lys in plant seeds. PMID:11500552

  4. Molecular epidemiology of malaria in Cameroon. XI. Geographic distribution of Plasmodium falciparum isolates with dihydrofolate reductase gene mutations in southern and central Cameroon.

    PubMed

    Basco, Leonardo K; Ndounga, Mathieu; Tejiokem, Mathurin; Ngane, Vincent Foumane; Youmba, Jean-Christian; Ringwald, Pascal; Soula, Georges

    2002-10-01

    The DNA sequence of the dihydrofolate reductase (dhfr) gene, a molecular marker for pyrimethamine resistance, was determined for 178 field isolates of Plasmodium falciparum collected along the east-west axis in southern Cameroon. The proportion of isolates having the wild-type dhfr allele varied from 48.1% in the east (city of Bertoua) to 11.3-15.7% in central provinces (Yaounde and Eseka) and 0% in the littoral region (port city of Douala). Isolates with a single Asn-108 mutation or double mutations (Ile-51 or Arg-59 and Asn-108) constituted approximately 10% of the samples. Isolates with triple mutations (Ile-51, Arg-59, and Asn-108) were present in an equal proportion (48.1%) as the wild-type isolates in the east (Bertoua), while triple mutations predominated in Yaounde (62.3%), Eseka (62.7%), and Douala (78.9%). The distribution of triple dhfr mutations along the east-west axis in southern Cameroon suggests the presence of a decreasing gradient from the west coastal region to the central region and then to the east towards the interior of the country.

  5. Cloning of a novel aldo-keto reductase gene from Klebsiella sp. strain F51-1-2 and its functional expression in Escherichia coli.

    PubMed

    Jiang, Hong; Yang, Chao; Qu, Hong; Liu, Zheng; Fu, Q S; Qiao, Chuanling

    2007-08-01

    A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His(6)-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the K(m) and k(cat) values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 +/- 47.0 microM and 25.7 +/- 1.7 min(-1), respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.

  6. Co-expression of bacterial aspartate kinase and adenylylsulfate reductase genes substantially increases sulfur amino acid levels in transgenic alfalfa (Medicago sativa L.).

    PubMed

    Tong, Zongyong; Xie, Can; Ma, Lei; Liu, Liping; Jin, Yongsheng; Dong, Jiangli; Wang, Tao

    2014-01-01

    Alfalfa (Medicago sativa L.) is one of the most important forage crops used to feed livestock, such as cattle and sheep, and the sulfur amino acid (SAA) content of alfalfa is used as an index of its nutritional value. Aspartate kinase (AK) catalyzes the phosphorylation of aspartate to Asp-phosphate, the first step in the aspartate family biosynthesis pathway, and adenylylsulfate reductase (APR) catalyzes the conversion of activated sulfate to sulfite, providing reduced sulfur for the synthesis of cysteine, methionine, and other essential metabolites and secondary compounds. To reduce the feedback inhibition of other metabolites, we cloned bacterial AK and APR genes, modified AK, and introduced them into alfalfa. Compared to the wild-type alfalfa, the content of cysteine increased by 30% and that of methionine increased substantially by 60%. In addition, a substantial increase in the abundance of essential amino acids (EAAs), such as aspartate and lysine, was found. The results also indicated a close connection between amino acid metabolism and the tricarboxylic acid (TCA) cycle. The total amino acid content and the forage biomass tested showed no significant changes in the transgenic plants. This approach provides a new method for increasing SAAs and allows for the development of new genetically modified crops with enhanced nutritional value.

  7. Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant

    PubMed Central

    Chen, Fei; Yang, Yun; Yang, Lixia; Zhang, Guolin; Luo, Yinggang

    2015-01-01

    Camptothecin (CAM), a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR) encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR analysis showed that

  8. Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in Pacific northwest marine sediment communities

    SciTech Connect

    Braker, G.; Zhou, J.; Wu, L.; Devol, A.H.; Tiedje, J.M.

    2000-05-01

    Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that

  9. Methyltetrahydrofolate reductase C677T gene mutation and hyperhomocysteinemia as a novel risk factor for diabetic nephropathy.

    PubMed

    Ukinc, Kubilay; Ersoz, Halil Onder; Karahan, Caner; Erem, Cihangir; Eminagaoglu, Selcuk; Hacihasanoglu, Arif Bayram; Yilmaz, Mustafa; Kocak, Mustafa

    2009-10-01

    Hyperhomocysteinemia is a well-defined risk factor for endothelial dysfunction and atherosclerosis. A point mutation (677 C-T) of MTHFR gene results in a significant increase at plasma homocysteine levels. In this study we aimed to evaluate the effects of MTHFR gene mutation and consequent hyperhomocysteinemia on the development of diabetic microvascular complications in comparison with the other defined risk factors. Diabetic patients without a history of macrovascular complication or overt nephropathy enrolled into the study. The presence of MTHFR 677 C-T point mutation was evaluated by Real-Time PCR technique by using a LightCycler. MTHFR heterozygous mutation was present in 24 patients over 52. Patients with diabetes were divided into two groups according to the presence of MTHFR gene mutation. Both groups were well matched regarding age and diabetes duration. Metabolic parameters, plasma homocysteine, microalbuminuria, folic acid, and vitamin B12 levels were also studied. Presence of neuropathy and retinopathy were evaluated by specific tests. Duration of diabetes, BMI, systolic and diastolic blood pressure, plasma CRP, HbA1c, and lipid levels were not different between the two groups. Plasma homocysteine (12.89 +/- 1.74 and 8.98 +/- 1.91 micromol/l; P < 0.0001) and microalbuminuria levels (73.40 +/- 98.15 and 29.53 +/- 5.08 mg/day; P = 0.021) were significantly higher in the group with MTHFR gene mutation while creatinine clearance levels (101.1 +/- 42.6 and 136.21 +/- 51.50 ml/min; P = 0.008) were significantly lower. Sixteen over 22 (73%) of the patients with diabetic nephropathy had MTHFR gene mutation, while this was only 27% (8 over 30) in normoalbuminuric patients (P = 0.017). There was a significant correlation of plasma homocysteine level with microalbuminuria (r = 0.54; P = 0.031) in the patients with diabetic nephropathy who had C677T polymorphism. We did not find any specific association of MTHFR gene mutation and hyperhomocysteinemia with

  10. Adrenergic activation of steroid 5alpha-reductase gene expression in rat C6 glioma cells: involvement of cyclic amp/protein kinase A-mediated signaling pathway.

    PubMed

    Morita, Kyoji; Arimochi, Hideki; Tsuruo, Yoshihiro

    2004-01-01

    Steroid 5alpha-reductase (5alpha-R) is well known as the enzyme converting progesterone and other steroid hormones to their 5alpha-reduced metabolites and has been reported to be localized in both neuronal and glial cells in the brain. Previously, the enzyme activity in glial cells has been shown to be enhanced either by coculturing with neuronal cells or by adding the conditioned medium of neuronal cells, suggesting a possible implication of neuro-glial interactions in the regulation of neurosteroid metabolism in the brain. In the present studies, the effects of adrenergic agonists on 5alpha-R mRNA and protein levels in rat C6 glioma cells were examined as one of the model experiments for investigating the influence of neuronal activity on the expression of 5alpha-R gene in the glial cell. The direct challenge of beta-adrenergic agonists to glioma cells resulted in the rapid and transient elevation of 5alpha-R mRNA levels through the activation of the cyclic AMP (cAMP)/protein kinase A-mediated signaling pathway. Further studies showed that cAMP-induced 5alpha-R mRNA expression was completely abolished by pretreatment of cells with actinomycin D and also indicated that the elevation of 5alpha-R mRNA levels was accompanied by an increase in enzyme protein in the cells. These findings provide strong evidence that the stimulation of beta-adrenergic receptors might induce the transcriptional activation of 5alpha-R gene expression in glial cells, proposing the possibility that neuronal activity might be involved in the production of neuroactive 5alpha-reduced steroids in the brain.

  11. Lack of association between the methylenetetrahydropholate reductase gene A1298C polymorphism and neural tube defects in a Turkish study group.

    PubMed

    Yildiz, S H; Ozdemir Erdogan, M; Solak, M; Eser, O; Arıkan Terzi, E S; Eser, B; Kocabaş, V; Aslan, A

    2016-06-03

    The etiology underlying neural tube defects (NTDs) is not fully understood and is believed to involve a complex milieu of genetic and environmental factors. The A1298C polymorphism in the methylenetetrahydropholate reductase gene (MTHFR) has been associated with mild risk for NTDs. In this study, the genotype distribution of the MTHFR gene A1298C polymorphism and the levels of serum homocysteine, vitamin B12, and folate were evaluated in 33 children with NTDs, their mothers, and 46 healthy controls. Genotyping of the A1298C polymorphism was performed by real-time polymerase chain reaction. The A and C allele frequencies in children with NTDs and their mothers were similar to controls (P = 0.160). The 1298AA and 1298CC genotype frequencies (P = 0.551 and 0.062, respectively) in children with NTDs and their mothers did not differ from controls. On the other hand, the 1298AC genotype frequencies in children with NTDs and their mothers were significantly different from controls (P = 0.025). The genotype frequency of 1298AC was lower in children with NTDs than in controls. There was no significant association between clinical distribution of NTDs and 1298AA/AC/CC genotypes (P > 0.05). Serum vitamin B12 levels were higher in children with NTDs than their mothers and controls (P = 0.001). There were no differences among serum homocysteine and folate levels in all groups (P = 0.494 and 0.141, respectively). Both genetic and nutritional factors are important in the etiology of NTDs. Thus, the A1298C polymorphism cannot be regarded as a major risk factor for NTDs.

  12. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    PubMed

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections.

  13. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

    PubMed Central

    Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

    2002-01-01

    DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

  14. [Investigation of detoxification polymorphisms genes, methylenetetrahydrofolate-reductase (MTHFR) and P53 in the radiosensitive human cells].

    PubMed

    Shagirova, Zh M; Ushenkova, L N; Mikhaĭlov, V F; Kurbatova, L A; Kuz'mina, N S; Semiachkina, A N; Vasil'eva, I M; Mazurik, V K; Zasukhina, G D

    2010-01-01

    The genes of detoxication, MTHFR and p53 were studied in Down' and Ehlers-Danlos syndrome cells. The frequency GSTM1(0/0) genotype in Down syndrome patients was in 1.5 times higher than in control cells (p < 0.069). Opposite the frequency GSTM1(0/0) genotype in Ehlers-Danlos syndrome was 23.3% 2 times lower than in control cells (p < 0.034). This indication was in 2 times lower in women cells than in men cells and in 3 times lower than in control cells (p < 0.026). The mutations of p53 gene (7th exon) were detected in 4 from 11 Down patients (36.7%; in 2 cases af women and men), in Ehlers-Danlos patients--in 5 cases and only in men (29.4% among all the observed patients). The observations 24 healthy donors weren't revealed any mutations (p < 0.013-0.001). The hypothesis about the connection between gene polymorphisms which take a part in genome stability and radiosensitivity in Down and Ehlers-Danlos patients was developed.

  15. Detecting Nitrous Oxide Reductase (nosZ) Genes in Soil Metagenomes: Method Development and Implications for the Nitrogen Cycle

    PubMed Central

    Orellana, L. H.; Rodriguez-R, L. M.; Higgins, S.; Chee-Sanford, J. C.; Sanford, R. A.; Ritalahti, K. M.; Löffler, F. E.

    2014-01-01

    ABSTRACT Microbial activities in soils, such as (incomplete) denitrification, represent major sources of nitrous oxide (N2O), a potent greenhouse gas. The key enzyme for mitigating N2O emissions is NosZ, which catalyzes N2O reduction to N2. We recently described “atypical” functional NosZ proteins encoded by both denitrifiers and nondenitrifiers, which were missed in previous environmental surveys (R. A. Sanford et al., Proc. Natl. Acad. Sci. U. S. A. 109:19709–19714, 2012, doi:10.1073/pnas.1211238109). Here, we analyzed the abundance and diversity of both nosZ types in whole-genome shotgun metagenomes from sandy and silty loam agricultural soils that typify the U.S. Midwest corn belt. First, different search algorithms and parameters for detecting nosZ metagenomic reads were evaluated based on in silico-generated (mock) metagenomes. Using the derived cutoffs, 71 distinct alleles (95% amino acid identity level) encoding typical or atypical NosZ proteins were detected in both soil types. Remarkably, more than 70% of the total nosZ reads in both soils were classified as atypical, emphasizing that prior surveys underestimated nosZ abundance. Approximately 15% of the total nosZ reads were taxonomically related to Anaeromyxobacter, which was the most abundant genus encoding atypical NosZ-type proteins in both soil types. Further analyses revealed that atypical nosZ genes outnumbered typical nosZ genes in most publicly available soil metagenomes, underscoring their potential role in mediating N2O consumption in soils. Therefore, this study provides a bioinformatics strategy to reliably detect target genes in complex short-read metagenomes and suggests that the analysis of both typical and atypical nosZ sequences is required to understand and predict N2O flux in soils. PMID:24895307

  16. The FinR-regulated essential gene fprA, encoding ferredoxin NADP+ reductase: Roles in superoxide-mediated stress protection and virulence of Pseudomonas aeruginosa

    PubMed Central

    Boonma, Siriwan; Romsang, Adisak; Duang-nkern, Jintana; Atichartpongkul, Sopapan; Trinachartvanit, Wachareeporn; Vattanaviboon, Paiboon

    2017-01-01

    Pseudomonas aeruginosa has two genes encoding ferredoxin NADP(+) reductases, denoted fprA and fprB. We show here that P. aeruginosa fprA is an essential gene. However, the ΔfprA mutant could only be successfully constructed in PAO1 strains containing an extra copy of fprA on a mini-Tn7 vector integrated into the chromosome or carrying it on a temperature-sensitive plasmid. The strain containing an extra copy of the ferredoxin gene (fdx1) could suppress the essentiality of FprA. Other ferredoxin genes could not suppress the requirement for FprA, suggesting that Fdx1 mediates the essentiality of FprA. The expression of fprA was highly induced in response to treatments with a superoxide generator, paraquat, or sodium hypochlorite (NaOCl). The induction of fprA by these treatments depended on FinR, a LysR-family transcription regulator. In vivo and in vitro analysis suggested that oxidized FinR acted as a transcriptional activator of fprA expression by binding to its regulatory box, located 20 bases upstream of the fprA -35 promoter motif. This location of the FinR box also placed it between the -35 and -10 motifs of the finR promoter, where the reduced regulator functions as a repressor. Under uninduced conditions, binding of FinR repressed its own transcription but had no effect on fprA expression. Exposure to paraquat or NaOCl converted FinR to a transcriptional activator, leading to the expression of both fprA and finR. The ΔfinR mutant showed an increased paraquat sensitivity phenotype and attenuated virulence in the Drosophila melanogaster host model. These phenotypes could be complemented by high expression of fprA, indicating that the observed phenotypes of the ΔfinR mutant arose from the inability to up-regulate fprA expression. In addition, increased expression of fprB was unable to rescue essentiality of fprA or the superoxide-sensitive phenotype of the ΔfinR mutant, suggesting distinct mechanisms of the FprA and FprB enzymes. PMID:28187184

  17. Genetic Variation of Methylenetetrahydrofolate Reductase (MTHFR) and Thymidylate Synthase (TS) Genes Is Associated with Idiopathic Recurrent Implantation Failure

    PubMed Central

    Shim, Sung Han; Lee, Yubin; Kim, Ji Hyang; Jeon, Young Joo; Ko, Jung Jae; Lee, Woo Sik; Kim, Nam Keun

    2016-01-01

    The one-carbon metabolism pathway disorder was important role in successful pregnancy. The MTHFR and TS protein were crucial factor in one-carbon metabolism. To investigate the association between recurrent implantation failure (RIF) and enzymes in the one-carbon metabolism pathway. A total of 120 women diagnosed with RIF and 125 control subjects were genotyped for MTHFR 677C>T, 1298A>C, TSER 2R/3R and TS 1494del/ins by a polymerase chain reaction-restriction fragment length polymorphism assay. According to the gene-gene combination analysis, the MTHFR 677/MTHFR 1298 (TT/AA) and MTHFR 677/TS 1494 (TT/6bp6bp) genetic combinations were associated with relatively higher risks [adjusted odds ratio (AOR), 2.764; 95% CI, 1.065–7.174; P = 0.037 and AOR, 3.186; 95% CI, 1.241–8.178; P = 0.016] in RIF patients compared to the CC/AA (MTHFR 677/MTHFR 1298) and TT/6bp6bp (MTHFR 677/TS 1494) combinations, respectively. The results suggested that the combined MTHFR 677/MTHFR 1298 genotype might be associated with increased risk of RIF. To the best of our knowledge, this study is the first to elucidate the potential association of MTHFR, TS and TSER polymorphisms with RIF risk in Korean patients. PMID:27560137

  18. RNAi knock-downs support roles for the mucin-like (AeIMUC1) gene and short-chain dehydrogenase/reductase (SDR) gene in Aedes aegypti susceptibility to Plasmodium gallinaceum.

    PubMed

    Berois, M; Romero-Severson, J; Severson, D W

    2012-03-01

    The mosquito midgut represents the first barrier encountered by the Plasmodium parasite (Haemosporida: Plasmodiidae) when it is ingested in blood from an infected vertebrate. Previous studies identified the Aedes aegypti (L.) (Diptera: Culicidae) mucin-like (AeIMUC1) and short-chain dehydrogenase/reductase (SDR) genes as midgut-expressed candidate genes influencing susceptibility to infection by Plasmodium gallinaceum (Brumpt). We used RNA inference (RNAi) by double-stranded RNA (dsRNA) injections to examine ookinete survival to the oocyst stage following individual gene knock-downs. Double-stranded RNA gene knock-downs were performed 3 days prior to P. gallinaceum infection and oocyst development was evaluated at 7 days post-infection. Mean numbers of parasites developing to the oocyst stage were significantly reduced by 52.3% in dsAeIMUC1-injected females and by 36.5% in dsSDR-injected females compared with females injected with a dsβ-gal control. The prevalence of infection was significantly reduced in dsAeIMUC1- and dsSDR-injected females compared with females injected with dsβ-gal; these reductions resulted in a two- and three-fold increase in the number of uninfected individuals, respectively. Overall, these results suggest that both AeIMUC1 and SDR play a role in Ae. aegypti vector competence to P. gallinaceum.

  19. Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene Ala222Val and susceptibility to ovary cancer: a systematic review and meta-analysis.

    PubMed

    Pan, Xinwei; Lu, Yan; Long, Ying; Yao, Desheng

    2014-03-01

    Many studies have reported the role of methylenetetrahydrofolate reductase (MTHFR) gene Ala222Val polymorphism with ovary cancer risk, but the results remained controversial. To derive a more precise estimation of the relationship, a meta-analysis was performed. Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the association between MTHFR Ala222Val polymorphism and ovary cancer risk. A total of 8 studies including 3,723 cases and 4,001 controls were also involved in this meta-analysis. When all the eligible studies were pooled into this meta-analysis, no significant association between ovary cancer risk and MTHFR Ala222Val polymorphism was found in all genetic models [codominant model: OR = 0.980, 95% CI = 0.756-1.270, P h = 0.088, P = 0.877; dominant model: OR = 1.022, 95% CI = 0.864-1.208, P h = 0.033, P = 0.803; recessive model: OR = 1.050, 95% CI = 0.803-1.373, P h = 0.032, P = 0.723; allele comparison model: OR = 1.028, 95% CI = 0.898-1.178, P h = 0.012, P = 0.685]. In the stratified analysis by ethnicity, no evidence of any associations of this polymorphism with ovary cancer was found in the Caucasian populations. Our meta-analysis supports that the MTHFR Ala222Val polymorphism is not contributed to the risk of ovary cancer from currently available evidence.

  20. Spatiotemporal expression and transcriptional regulation of heme oxygenase and biliverdin reductase genes in zebrafish (Danio rerio) suggest novel roles during early developmental periods of heightened oxidative stress.

    PubMed

    Holowiecki, Andrew; O'Shields, Britton; Jenny, Matthew J

    2017-01-01

    Heme oxygenase 1 (HMOX1) degrades heme into biliverdin, which is subsequently converted to bilirubin by biliverdin reductase (BVRa or BVRb) in a manner analogous to the classic anti-oxidant glutathione-recycling pathway. To gain a better understanding of the potential antioxidant roles the BVR enzymes may play during development, the spatiotemporal expression and transcriptional regulation of zebrafish hmox1a, bvra and bvrb were characterized under basal conditions and in response to pro-oxidant exposure. All three genes displayed spatiotemporal expression patterns consistent with classic hematopoietic progenitors during development. Transient knockdown of Nrf2a did not attenuate the ability to detect bvra or bvrb by ISH, or alter spatial expression patterns in response to cadmium exposure. While hmox1a:mCherry fluorescence was documented within the intermediate cell mass, a transient location of primitive erythrocyte differentiation, expression was not fully attenuated in Nrf2a morphants, but real-time RT-PCR demonstrated a significant reduction in hmox1a expression. Furthermore, Gata-1 knockdown did not attenuate hmox1a:mCherry fluorescence. However, while there was a complete loss of detection of bvrb expression by ISH at 24hpf, bvra expression was greatly attenuated but still detectable in Gata-1 morphants. In contrast, 96 hpf Gata-1 morphants displayed increased bvra and bvrb expression within hematopoietic tissues. Finally, temporal expression patterns of enzymes involved in the generation and maintenance of NADPH were consistent with known changes in the cellular redox state during early zebrafish development. Together, these data suggest that Gata-1 and Nrf2a play differential roles in regulating the heme degradation enzymes during an early developmental period of heightened cellular stress.

  1. Ethnic variation of the C677T and A1298C polymorphisms in the methylenetetrahydrofolate-reductase (MTHFR) gene in southwestern Mexico.

    PubMed

    Antonio-Véjar, V; Del Moral-Hernández, O; Alarcón-Romero, L C; Flores-Alfaro, E; Leyva-Vázquez, M A; Hernández-Sotelo, D; Illades-Aguiar, B

    2014-09-29

    In this study, we examined the distribution of genotype and allele frequencies of the C677T and A1298C polymorphisms in the methylenetetrahydrofolate-reductase gene (MTHFR) in two ethnic groups in the State of Guerrero, Mexico, which were compared with those of the Mestizo population of the region. A comparative study was conducted on 455 women from two ethnic groups and a group of Mestizo women of the State of Guerrero, Mexico: 135 Nahuas, 124 Mixtecas, and 196 Mestizas. Genotyping of both polymorphisms were performed by using polymerase chain reaction-restriction fragment length polymorphism methods. We found that the 677TT genotype was more frequent in Nahua and Mixteca women compared to Mestiza women (P = 0.008), and the most prevalent genotype in both ethnic groups was the 1298AA genotype (P < 0.001). We also compared the 677T allele frequency obtained from the groups studied with the frequencies reported in other ethnic groups of Mexico (Huichol, Tarahumara, and Purepecha). There were significant differences between the three ethnic groups compared to Nahuas (Huicholes, P = 0.004; Tarahumaras, P < 0.001; Purepechas, P = 0.042). Our results indicated significant differences in the frequencies of the C677T and A1298C polymorphisms between the two ethnic groups and the Mestizo population of the State of Guerrero. In addition, we found strong differences with other ethnic groups in Mexico. These results could be useful for future studies investigating diseases related to folate metabolism, and could help the government to design specific nutrition programs for different ethnic groups.

  2. The C677T polymorphism of the methylenetetrahydrofolate reductase gene in Mexican mestizo neural-tube defect parents, control mestizo and native populations.

    PubMed

    Dávalos, I P; Olivares, N; Castillo, M T; Cantú, J M; Ibarra, B; Sandoval, L; Morán, M C; Gallegos, M P; Chakraborty, R; Rivas, F

    2000-01-01

    The C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene, associated with the thermolabile form of the enzyme, has reportedly been found to be increased in neural-tube defects (NTD), though this association is still unclear. A group of 107 mestizo parents of NTD children and five control populations: 101 mestizo (M), 50 Huichol (H), 38 Tarahumara (T), 21 Purepecha (P) and 20 Caucasian (C) individuals were typed for the MTHFR C677T variant by the PCR/RFLP (HinfI) method. Genotype frequencies were in agreement with the Hardy-Weinberg expectations in all six populations. Allele frequency (%) of the C677T variant was 45 in NTD, 44 in M, 56 in H, 36 in T, 57 in P, 35 in C. Pairwise inter-population comparisons of allele frequency disclosed a very similar distribution between NTD and M groups (exact test, P=0.92). Among controls, differences between M and individual native groups were NS (0.06

  3. Molecular structure and functional characterization of the gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in largemouth bass (Microptenus salmoides).

    PubMed

    Yang, Qian; Zhang, Jiaxin; Hu, Lingling; Lu, Jia; Sang, Ming; Zhang, Shuangquan

    2015-12-01

    The enzyme gamma-interferon-inducible lysosomal thiol reductase (GILT) plays a role in facilitating the processing and presentation of major histocompatibility complex (MHC) class II-restricted antigens and is also involved in MHC I-restricted antigens in adaptive immunity catalyzing disulfide bond reduction in mammals. In this study, we cloned a GILT gene homolog from largemouth bass (designated 'lbGILT'), a freshwater fish belonging to Perciformes and known for its nutritive value. We obtained the full-length cDNA of lbGILT by reverse transcription PCR and rapid amplification of cDNA ends. This cDNA is comprised of a 5'-untranslated region (UTR) of 87 bp, a 3'-UTR of 189 bp, and an open reading frame of 771 bp. It encodes a protein of 256 amino acids with a deduced molecular weight of 28.548 kDa and a predicted isoelectric point of 5.62. The deduced protein possesses the typical structural features of known GILTs, including an active site motif, two potential N-linked glycosylation sites, a GILT signature sequence, and six conserved cysteines. Tissue-specific expression of lbGILT was shown by real-time quantitative PCR. The expression of lbGILT mRNA was obviously up regulated in spleen and kidney after induction with lipopolysaccharide. Recombinant lbGILT was produced as an inclusion body with a His6 tag in ArcticExpress (DE3), and the protein was then washed, solubilized, and refolded. The refolded lbGILT showed reduction activity against an IgG substrate. These results suggest that lbGILT plays a role in innate immunity.

  4. Functional analysis of Plasmodium vivax dihydrofolate reductase-thymidylate synthase genes through stable transformation of Plasmodium falciparum.

    PubMed

    Auliff, Alyson M; Balu, Bharath; Chen, Nanhua; O'Neil, Michael T; Cheng, Qin; Adams, John H

    2012-01-01

    Mechanisms of drug resistance in Plasmodium vivax have been difficult to study partially because of the difficulties in culturing the parasite in vitro. This hampers monitoring drug resistance and research to develop or evaluate new drugs. There is an urgent need for a novel method to study mechanisms of P. vivax drug resistance. In this paper we report the development and application of the first Plasmodium falciparum expression system to stably express P. vivax dhfr-ts alleles. We used the piggyBac transposition system for the rapid integration of wild-type, single mutant (117N) and quadruple mutant (57L/58R/61M/117T) pvdhfr-ts alleles into the P. falciparum genome. The majority (81%) of the integrations occurred in non-coding regions of the genome; however, the levels of pvdhfr transcription driven by the P. falciparum dhfr promoter were not different between integrants of non-coding and coding regions. The integrated quadruple pvdhfr mutant allele was much less susceptible to antifolates than the wild-type and single mutant pvdhfr alleles. The resistance phenotype was stable without drug pressure. All the integrated clones were susceptible to the novel antifolate JPC-2067. Therefore, the piggyBac expression system provides a novel and important tool to investigate drug resistance mechanisms and gene functions in P. vivax.

  5. Quinone Reductase 2 Is a Catechol Quinone Reductase

    SciTech Connect

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao

    2008-09-05

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

  6. Identification of a 3-hydroxy-3-methylglutaryl-CoA reductase gene highly expressed in the root tissue of Taraxacum kok-saghyz

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Kazak dandelion (Taraxacum kok-saghyz, Tk) is a rubber-producing plant currently being investigated as a source of natural rubber for industrial applications. Like many other isoprenoids, rubber is a downstream product of the mevalonate pathway. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) en...

  7. The aldo-keto reductase superfamily homepage.

    PubMed

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  8. Cloning, sequencing, and growth phase-dependent transcription of the coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin reductase-encoding genes from Methanobacterium thermoautotrophicum delta H and Methanopyrus kandleri.

    PubMed Central

    Nölling, J; Pihl, T D; Reeve, J N

    1995-01-01

    The mer genes, which encode the coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin reductases (CH2 = H4MPT reductases), and their flanking regions have been cloned from Methanobacterium thermoautotrophicum delta H and Methanopyrus kandleri and sequenced. The mer genes have DNA sequences that are 57% identical and encode polypeptides with amino acid sequences that are 57% identical and 71% similar, with calculated molecular masses of 33.6 and 37.5 kDa, respectively. In M. thermoautotrophicum, mer transcription has been shown to initiate 10 bp upstream from the ATG translation initiating codon and to generate a monocistronic transcript approximately 1 kb in length. This transcript was synthesized at all stages of M. thermoautotrophicum delta H growth in batch cultures but was found to increase in abundance from the earliest stages of exponential growth, reaching a maximum level at the mid-exponential growth phase. For comparison, transcription of the ftr gene from M. thermoautotrophicum delta H that encodes the formylmethanofuran:tetrahydromethanopterin formyltransferase (A. A. DiMarco, K. A. Sment, J. Konisky, and R. S. Wolfe, J. Biol. Chem. 265:472-476, 1990) was included in this study. The ftr transcript was found similarly to be monocistronic and to be approximately 1 kb in length, but, in contrast to the mer transcript, the ftr transcript was present at maximum levels at both the early and the mid-exponential growth stages. PMID:8522533

  9. Inactivation of the lys7 gene, encoding saccharopine reductase in Penicillium chrysogenum, leads to accumulation of the secondary metabolite precursors piperideine-6-carboxylic acid and pipecolic acid from alpha-aminoadipic acid.

    PubMed

    Naranjo, Leopoldo; Martín de Valmaseda, Eva; Casqueiro, Javier; Ullán, Ricardo V; Lamas-Maceiras, Mónica; Bañuelos, Oscar; Martín, Juan F

    2004-02-01

    Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active alpha-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1-) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3'-end region. P. chrysogenum SR1- lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1- was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1- was grown with L-lysine as the sole nitrogen source and supplemented with DL-alpha-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1- with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from alpha-aminoadipic acid and not from L-lysine catabolism.

  10. New Rimocidin/CE-108 Derivatives Obtained by a Crotonyl-CoA Carboxylase/Reductase Gene Disruption in Streptomyces diastaticus var. 108: Substrates for the Polyene Carboxamide Synthase PcsA

    PubMed Central

    Escudero, Leticia; Al-Refai, Mahmoud; Nieto, Cristina; Laatsch, Hartmut; Malpartida, Francisco; Seco, Elena M.

    2015-01-01

    The rimJ gene, which codes for a crotonyl-CoA carboxylase/reductase, lies within the biosynthetic gene cluster for two polyketides belonging to the polyene macrolide group (CE-108 and rimocidin) produced by Streptomyces diastaticus var. 108. Disruption of rimJ by insertional inactivation gave rise to a recombinant strain overproducing new polyene derivatives besides the parental CE-108 (2a) and rimocidin (4a). The structure elucidation of one of them, CE-108D (3a), confirmed the incorporation of an alternative extender unit for elongation step 13. Other compounds were also overproduced in the fermentation broth of rimJ disruptant. The new compounds are in vivo substrates for the previously described polyene carboxamide synthase PcsA. The rimJ disruptant strain, constitutively expressing the pcsA gene, allowed the overproduction of CE-108E (3b), the corresponding carboxamide derivative of CE-108D (3a), with improved pharmacological properties. PMID:26284936

  11. A novel point mutation in a 3{prime} splice site of the NADH-cytochrome b{sub 5} reductase gene results in immunologically undetectable enzyme and impaired NADH-dependent ascorbate regeneration in cultured fibroblasts of a patient with type II hereditary methemoglobinemia

    SciTech Connect

    Shirabe, Komie; Takeshita, Masazumi; Landi, M.T.

    1995-08-01

    Hereditary methemoglobinemia with generalized deficiency of NADH-cytochrome b{sub 5} reductase (b{sub 5}R) (type II) is a rare disease characterized by severe developmental abnormalities, which often lead to premature death. Although the molecular relationship between the symptoms of this condition and the enzyme deficit are not understood, it is thought that an important cause is the loss of the lipid metabolizing activities of the endoplasmic reticulum-located reductase. However, the functions of the form located on outer mitochondrial membranes have not been considered previously. In this study, we have analyzed the gene of an Italian patient and identified a novel G{r_arrow}T transversion at the splice-acceptor site of the 9th exon, which results in the complete absence of immunologically detectable b{sub 5}R in blood cells and skin fibroblasts. In cultured fibroblasts of the patient, NADH-dependent cytochrome c reductase, ferricyanide reductase, and semidehydroascorbate reductase activities were severely reduced. The latter activity is known to be due to b{sub 5}R located on outer mitochondrial membranes. Thus, our results demonstrate that the reductase in its two membrane locations, endoplasmic reticulum and outer mitochondrial membranes, is the product of the same gene and suggest that a defect in ascorbate regeneration may contribute to the phenotype of hereditary methemoglobinemia of generalized type. 37 refs., 5 figs., 2 tabs.

  12. Mutations in the neverland gene turned Drosophila pachea into an obligate specialist species

    PubMed Central

    Lang, Michael; Murat, Sophie; Clark, Andrew G.; Gouppil, Géraldine; Blais, Catherine; Matzkin, Luciano M.; Guittard, Émilie; Yoshiyama−Yanagawa, Takuji; Kataoka, Hiroshi; Niwa, Ryusuke; Lafont, René; Dauphin−Villemant, Chantal; Orgogozo, Virginie

    2016-01-01

    Most living species exploit a limited range of resources. However, little is known on how tight links build up during evolution between specialist species and the hosts they utilize. We examined the dependence of Drosophila pachea on its single host, the senita cactus. Several amino acid changes in the Neverland oxygenase rendered D. pachea unable to transform cholesterol into 7-dehydrocholesterol (first reaction in the steroid hormone biosynthetic pathway in insects) and thus made D. pachea dependent on the uncommon sterols of its host plant. The neverland mutations increase survival on the cactus unusual sterols and are in a genomic region that faced recent positive selection. This study illustrates how relatively few genetic changes in a single gene may restrict the ecological niche of a species. PMID:23019649

  13. Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

    PubMed Central

    Kalb, Daniel; Lackner, Gerald

    2014-01-01

    A key step in fungal l-lysine biosynthesis is catalyzed by adenylate-forming l-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP+ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized l-α-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus l-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes. PMID:25085485

  14. Expression of 3-hydroxy-3-methylglutaryl-CoA reductase, p-hydroxybenzoate-m-geranyltransferase and genes of phenylpropanoid pathway exhibits positive correlation with shikonins content in arnebia [Arnebia euchroma (Royle) Johnston

    PubMed Central

    2010-01-01

    Background Geranyl pyrophosphate (GPP) and p-hydroxybenzoate (PHB) are the basic precursors involved in shikonins biosynthesis. GPP is derived from mevalonate (MVA) and/or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway(s), depending upon the metabolite and the plant system under consideration. PHB, however, is synthesized by only phenylpropanoid (PP) pathway. GPP and PHB are central moieties to yield shikonins through the synthesis of m-geranyl-p-hydroxybenzoate (GHB). Enzyme p-hydroxybenzoate-m-geranyltransferase (PGT) catalyses the coupling of GPP and PHB to yield GHB. The present research was carried out in shikonins yielding plant arnebia [Arnebia euchroma (Royle) Johnston], wherein no molecular work has been reported so far. The objective of the work was to identify the preferred GPP synthesizing pathway for shikonins biosynthesis, and to determine the regulatory genes involved in the biosynthesis of GPP, PHB and GHB. Results A cell suspension culture-based, low and high shikonins production systems were developed to facilitate pathway identification and finding the regulatory gene. Studies with mevinolin and fosmidomycin, inhibitors of MVA and MEP pathway, respectively suggested MVA as a preferred route of GPP supply for shikonins biosynthesis in arnebia. Accordingly, genes of MVA pathway (eight genes), PP pathway (three genes), and GHB biosynthesis were cloned. Expression studies showed down-regulation of all the genes in response to mevinolin treatment, whereas gene expression was not influenced by fosmidomycin. Expression of all the twelve genes vis-à-vis shikonins content in low and high shikonins production system, over a period of twelve days at frequent intervals, identified critical genes of shikonins biosynthesis in arnebia. Conclusion A positive correlation between shikonins content and expression of 3-hydroxy-3-methylglutaryl-CoA reductase (AeHMGR) and AePGT suggested critical role played by these genes in shikonins biosynthesis. Higher

  15. Involvement of de Novo Protein Synthesis, Protein Kinase, Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.).

    PubMed Central

    Choi, D.; Bostock, R. M.

    1994-01-01

    A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested. PMID:12232162

  16. 7-dehydrocholesterol efficiently supports Ret signaling in a mouse model of Smith-Opitz-Lemli syndrome.

    PubMed

    Gou-Fàbregas, Myriam; Macià, Anna; Anerillas, Carlos; Vaquero, Marta; Jové, Mariona; Jain, Sanjay; Ribera, Joan; Encinas, Mario

    2016-06-23

    Smith-Lemli-Opitz syndrome (SLOS) is a rare disorder of cholesterol synthesis. Affected individuals exhibit growth failure, intellectual disability and a broad spectrum of developmental malformations. Among them, renal agenesis or hypoplasia, decreased innervation of the gut, and ptosis are consistent with impaired Ret signaling. Ret is a receptor tyrosine kinase that achieves full activity when recruited to lipid rafts. Mice mutant for Ret are born with no kidneys and enteric neurons, and display sympathetic nervous system defects causing ptosis. Since cholesterol is a critical component of lipid rafts, here we tested the hypothesis of whether the cause of the above malformations found in SLOS is defective Ret signaling owing to improper lipid raft composition or function. No defects consistent with decreased Ret signaling were found in newborn Dhcr7(-/-) mice, or in Dhcr7(-/-) mice lacking one copy of Ret. Although kidneys from Dhcr7(-/-) mice showed a mild branching defect in vitro, GDNF was able to support survival and downstream signaling of sympathetic neurons. Consistently, GFRα1 correctly partitioned to lipid rafts in brain tissue. Finally, replacement experiments demonstrated that 7-DHC efficiently supports Ret signaling in vitro. Taken together, our findings do not support a role of Ret signaling in the pathogenesis of SLOS.

  17. 7-dehydrocholesterol efficiently supports Ret signaling in a mouse model of Smith-Opitz-Lemli syndrome

    PubMed Central

    Gou-Fàbregas, Myriam; Macià, Anna; Anerillas, Carlos; Vaquero, Marta; Jové, Mariona; Jain, Sanjay; Ribera, Joan; Encinas, Mario

    2016-01-01

    Smith-Lemli-Opitz syndrome (SLOS) is a rare disorder of cholesterol synthesis. Affected individuals exhibit growth failure, intellectual disability and a broad spectrum of developmental malformations. Among them, renal agenesis or hypoplasia, decreased innervation of the gut, and ptosis are consistent with impaired Ret signaling. Ret is a receptor tyrosine kinase that achieves full activity when recruited to lipid rafts. Mice mutant for Ret are born with no kidneys and enteric neurons, and display sympathetic nervous system defects causing ptosis. Since cholesterol is a critical component of lipid rafts, here we tested the hypothesis of whether the cause of the above malformations found in SLOS is defective Ret signaling owing to improper lipid raft composition or function. No defects consistent with decreased Ret signaling were found in newborn Dhcr7−/− mice, or in Dhcr7−/− mice lacking one copy of Ret. Although kidneys from Dhcr7−/− mice showed a mild branching defect in vitro, GDNF was able to support survival and downstream signaling of sympathetic neurons. Consistently, GFRα1 correctly partitioned to lipid rafts in brain tissue. Finally, replacement experiments demonstrated that 7-DHC efficiently supports Ret signaling in vitro. Taken together, our findings do not support a role of Ret signaling in the pathogenesis of SLOS. PMID:27334845

  18. Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes.

    PubMed

    Kim, Yeon-Bok; Kim, Sang-Min; Kang, Min-Kyoung; Kuzuyama, Tomohisa; Lee, Jong Kyu; Park, Seung-Chan; Shin, Sang-Chul; Kim, Soo-Un

    2009-05-01

    Pinus densiflora Siebold et Zucc. is the major green canopy species in the mountainous area of Korea. To assess the response of resin acid biosynthetic genes to mechanical and chemical stimuli, we cloned cDNAs of genes encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (1-deoxy-d-xylulose 5-phosphate synthase (PdDXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PdDXR) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (PdHDR)) by the rapid amplification of cDNA ends (RACE) technique. In addition, we cloned the gene encoding abietadiene synthase (PdABS) as a marker for the site of pine resin biosynthesis. PdHDR and PdDXS occurred as two gene families. In the phylogenetic trees, PdDXSs, PdDXR and PdHDRs each formed a separate clade from their respective angiosperm homologs. PdDXS2, PdHDR2 and PdDXR were most actively transcribed in stem wood, whereas PdABS was specifically transcribed. The abundance of PdDXS2 transcripts in wood in the resting state was generally 50-fold higher than the abundance of PdDXS1 transcripts, and PdHDR2 transcripts were more abundant by an order of magnitude in wood than in other tissues, with the ratio of PdHDR2 to PdHDR1 transcripts in wood being about 1. Application of 1 mM methyl jasmonate (MeJA) selectively enhanced the transcript levels of PdDXS2 and PdHDR2 in wood. The ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 reached 900 and 20, respectively, on the second day after MeJA treatment, whereas the transcript level of PdABS increased twofold by 3 days after MeJA treatment. Wounding of the stem differentially enhanced the transcript ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 to 300 and 70, respectively. The increase in the transcript levels of the MEP pathway genes in response to wounding was accompanied by two orders of magnitude increase in PdABS transcripts. These observations indicated that resin acid biosynthesis activity, represented by PdABS transcription, was correlated

  19. Expression of 3beta-HSD and P5betaR, genes respectively coding for Delta5-3beta-hydroxysteroid dehydrogenase and progesterone 5beta-reductase, in leaves and cell cultures of Digitalis lanata EHRH.

    PubMed

    Ernst, Mona; de Padua, Rodrigo Maia; Herl, Vanessa; Müller-Uri, Frieder; Kreis, Wolfgang

    2010-06-01

    Plants of the genus Digitalis produce 5 beta-cardenolides that are used in the therapy of cardiac insufficiency in humans. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5 beta-reductase (P5 betaR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC-MS methods. Expression of the respective genes, namely AY585867.1 (P5betaR gene) and DQ466890.1 (3beta-HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5betaR gene, encoding an enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in cardenolide-free tissues of cardenolide-containing plants.

  20. Stereoselective synthesis of (R)-phenylephrine using recombinant Escherichia coli cells expressing a novel short-chain dehydrogenase/reductase gene from Serratia marcescens BCRC 10948.

    PubMed

    Peng, Guan-Jhih; Kuan, Yi-Chia; Chou, Hsiao-Yi; Fu, Tze-Kai; Lin, Jia-Shin; Hsu, Wen-Hwei; Yang, Ming-Te

    2014-01-20

    (R)-Phenylephrine [(R)-PE] is an α1-adrenergic receptor agonist and is widely used as a nasal decongestant to treat the common cold without the side effects of other ephedrine adrenergic drugs. We identified a short-chain dehydrogenase/reductase (SM_SDR) from Serratia marcescens BCRC 10948 that was able to convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-PE. The SM_SDR used NADPH and NADH as cofactors with specific activities of 17.35±0.71 and 5.57±0.07mU/mg protein, respectively, at 30°C and pH 7.0, thereby indicating that this enzyme could be categorized as an NADPH-preferring short-chain dehydrogenase/reductase. Escherichia coli strain BL21 (DE3) expressing SM_SDR could convert HPMAE into (R)-PE with more than 99% enantiomeric excess. The productivity and conversion yield were 0.57mmolPE/lh and 51.06%, respectively, using 10mM HPMAE. Fructose was the most effective carbon source for the conversion of HPMAE to (R)-PE.

  1. A NADPH-dependent (S)-imine reductase (SIR) from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines: purification, characterization, gene cloning, and expression.

    PubMed

    Mitsukura, Koichi; Kuramoto, Tatsuya; Yoshida, Toyokazu; Kimoto, Norihiro; Yamamoto, Hiroaki; Nagasawa, Toru

    2013-09-01

    A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5 kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6 nmol min(-1) mg(-1), and their optical purities were 92.7 % ee, 96.4 % ee, and >99 % ee, respectively. Using a NADPH-regenerating system, 10 mM 2-MPN was converted to amine with 100 % conversion and 92 % ee after 24 h. The amino acid sequence analysis revealed that SIR showed about 60 % identity to 6-phosphogluconate dehydrogenase. However, it showed only 37 % identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.

  2. Role of polymorphisms in factor V (FV Leiden), prothrombin, plasminogen activator inhibitor type-1 (PAI-1), methylenetetrahydrofolate reductase (MTHFR) and cystathionine β-synthase (CBS) genes as risk factors for thrombophilias.

    PubMed

    Miranda-Vilela, A L

    2012-09-01

    Thrombophilias are defined as a predisposition to thrombosis due to hematological changes which induce blood hypercoagulability; they can be inherited or acquired. They are individually characterized by a large phenotypic variability, even when they occur within the same family. Hereditary thrombophilias are, in most cases, due to changes related to physiological coagulation inhibitors or mutations in the genes of coagulation factors. High levels of plasma homocysteine may also be responsible for vaso-occlusive episodes and may have acquired (nutritional deficiencies of folate and vitamins B6 and B12) and/or genetic causes (mutations in the genes responsible for expression of enzymes involved in the intracellular metabolism of homocysteine). Considering that: (1) thromboses are events of multigenic and multifactorial etiopathology; (2) the presence of mutations in several genes significantly increases the risk of their occurrence; (3) the vascular territory (venous and/or arterial) affected involves different pathophysiological mechanisms and treatments, knowledge of genetic variants that may contribute to the risk and variability of the phenotypic manifestations of these diseases is extremely important. This understanding may provide support for a more individualized and therefore more effective treatment for thrombophilia carriers. Thus, this mini-review aims to address a comprehensive summary of thrombophilias and thrombosis, and discuss the role of polymorphisms in Factor V (FV Leiden), Prothrombin, Plasminogen activator inhibitor type-1 (PAI-1), Methylenetetrahydrofolate reductase (MTHFR) and Cystathionine β-synthase (CBS) genes as risk factors for thrombophilias.

  3. Regulation of the Lactobacillus Strains on HMGCoA Reductase Gene Transcription in Human HepG2 Cells via Nuclear Factor-κB.

    PubMed

    Chen, Kun; Li, Shaocong; Chen, Fang; Li, Jun; Luo, Xuegang

    2016-02-01

    Lactic acid bacteria have been identified to be effective in reducing cholesterol levels. Most of the mechanistic studies were focused on the bile salt deconjugation ability of bile salt hydrolase in lactic acid bacteria. However, the mechanism by which Lactobacillus decreases cholesterol levels has not been thoroughly studied in intact primate cells. 3-Hydroxy-3- methyl-glutaryl-coenzyme A reductase (HMGCR) is the vital enzyme in cholesterol synthesis. To confirm the effect of probiotic Lactobacillus strains on HMGCR level, in the present study, human hepatoma HepG2 cells were treated with Lactobacillus strains, and then the HMGCR level was illustrated by luciferase reporter assay and RT-PCR. The results showed that the level of HMGCR was suppressed after being treated with the live Lactobacillus strains. These works might set a foundation for the following study of the antihyperlipidemic effects of L. acidophilus, and contribute to the development of functional foods or drugs that benefit patients suffering from hyperlipidemia diseases.

  4. Engineering class I cytochrome P450 by gene fusion with NADPH-dependent reductase and S. avermitilis host development for daidzein biotransformation.

    PubMed

    Choi, Kwon-Young; Jung, EunOk; Yun, Hyungdon; Yang, Yung-Hun; Kim, Byung-Gee

    2014-10-01

    Daidzein C6 hydroxylase (6-DH, nfa12130), which is a class I type of cytochrome P450 enzyme, catalyzes a hydroxylation reaction at the C6-position of the daidzein A-ring and requires auxiliary electron transfer proteins. Current utilization of cytochrome P450 (CYP) enzymes is limited by low coupling efficiency, which necessitates extramolecular electron transfers, and low driving forces, which derive electron flows from tightly regulated NADPH redox balances into the heterogeneous CYP catalytic cycle. To overcome such limitations, the heme domain of the 6-DH enzyme was genetically fused with the NADPH-reductase domain of self-sufficient CYP102D1 to enhance electron transfer efficiencies through intramolecular electron transfer and switching cofactor preference from NADH into NADPH. 6-DH-reductase fusion enzyme displayed distinct spectral properties of both flavoprotein and heme proteins and catalyzed daidzein hydroxylation more efficiently with a k cat/K m value of 120.3 ± 11.5 [10(3) M(-1) s(-1)], which was about three times higher than that of the 6-DH-FdxC-FdrA reconstituted system. Moreover, to obtain a higher redox driving force, a Streptomyces avermitilis host system was developed for heterologous expression of fusion 6-DH enzyme and whole cell biotransformation of daidzein. The whole cell reaction using the final recombinant strain, S. avermitilisΔcyp105D7::fusion 6-DH (nfa12130), resulted in 8.3 ± 1.4 % of 6-OHD yield from 25.4 mg/L of daidzein.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  7. Characterization of erythrose reductases from filamentous fungi

    PubMed Central

    2013-01-01

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

  8. Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.

    PubMed

    Kavanagh, K L; Jörnvall, H; Persson, B; Oppermann, U

    2008-12-01

    Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an alpha/beta folding pattern with a central beta sheet flanked by 2 - 3 alpha-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

  9. Methylenetetrahydrofolate reductase gene germ-line C677T and A1298C SNPs are associated with colorectal cancer risk in the Turkish population.

    PubMed

    Ozen, Filiz; Sen, Metin; Ozdemir, Ozturk

    2014-01-01

    Colorectal cancer (CRC) is the third most common cause of death due to cancer in the worldwide and the incidence is also increasing in Turkey. Our present aim was to investigate any association between germ-line methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms and CRC risk in Turkey. A total of 86 CRC cases and 212 control individuals of the same ethnicity were included in the current study. Peripheral blood-DNA samples were used for genotyping by StripAssay technique, based on the reverse- hybridization principle and real-time PCR methods. Results were compared in Pearson Chi-square and multiple logistic regression models. The MTHFR 677TT (homozygous) genotype was found in 20.9% and the T allele frequency 4.2-fold increased in CRC when compared with the control group.The second SNP MTHFR 1298CC (homozygous) genotype was found in 14.0% and the C allele frequency 1.4-fold elevated in the CRC group. The current data suggest strong associations between both SNPs of germ-line MTHFR 677 C>T and 1298 A>C genotypes and CRC susceptibility in the Turkish population. Now the results need to be confirmed with a larger sample size.

  10. Overexpression and Suppression of Artemisia annua 4-Hydroxy-3-Methylbut-2-enyl Diphosphate Reductase 1 Gene (AaHDR1) Differentially Regulate Artemisinin and Terpenoid Biosynthesis

    PubMed Central

    Ma, Dongming; Li, Gui; Zhu, Yue; Xie, De-Yu

    2017-01-01

    4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) catalyzes the last step of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP). To date, little is known regarding effects of an increase or a decrease of a HDR expression on terpenoid and other metabolite profiles in plants. In our study, an Artemisia annua HDR cDNA (namely AaHDR1) was cloned from leaves. Expression profiling showed that it was highly expressed in leaves, roots, stems, and flowers with different levels. Green florescence protein fusion and confocal microscope analyses showed that AaHDR1 was localized in chloroplasts. The overexpression of AaHDR1 increased contents of artemisinin, arteannuin B and other sesquiterpenes, and multiple monoterpenes. By contrast, the suppression of AaHDR1 by anti-sense led to opposite results. In addition, an untargeted metabolic profiling showed that the overexpression and suppression altered non-polar metabolite profiles. In conclusion, the overexpression and suppression of AaHDR1 protein level in plastids differentially affect artemisinin and other terpenoid biosynthesis, and alter non-polar metabolite profiles of A. annua. Particularly, its overexpression leading to the increase of artemisinin production is informative to future metabolic engineering of this antimalarial medicine. PMID:28197158

  11. Identification and characterization of the LysR-type transcriptional regulator HsdR for steroid-inducible expression of the 3α-hydroxysteroid dehydrogenase/carbonyl reductase gene in Comamonas testosteroni.

    PubMed

    Gong, Wenjie; Xiong, Guangming; Maser, Edmund

    2012-02-01

    3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni is a key enzyme in steroid degradation in soil and water. 3α-HSD/CR gene (hsdA) expression can be induced by steroids like testosterone and progesterone. Previously, we have shown that the induction of hsdA expression by steroids is a derepression where steroidal inducers bind to two repressors, RepA and RepB, thereby preventing the blocking of hsdA transcription and translation, respectively (G. Xiong and E. Maser, J. Biol. Chem. 276:9961-9970, 2001; G. Xiong, H. J. Martin, and E. Maser, J. Biol. Chem. 278:47400-47407, 2003). In the present study, a new LysR-type transcriptional factor, HsdR, for 3α-HSD/CR expression in C. testosteroni has been identified. The hsdR gene is located 2.58 kb downstream from hsdA on the C. testosteroni ATCC 11996 chromosome with an orientation opposite that of hsdA. The hsdR gene was cloned and recombinant HsdR protein was produced, as was anti-HsdR polyclonal antibodies. While heterologous transformation systems revealed that HsdR activates the expression of the hsdA gene, electrophoresis mobility shift assays showed that HsdR specifically binds to the hsdA promoter region. Interestingly, the activity of HsdR is dependent on decreased repression by RepA. Furthermore, in vitro binding assays indicated that HsdR can come into contact with RNA polymerase. As expected, an hsdR knockout mutant expressed low levels of 3α-HSD/CR compared to that of wild-type C. testosteroni after testosterone induction. In conclusion, HsdR is a positive transcription factor for the hsdA gene and promotes the induction of 3α-HSD/CR expression in C. testosteroni.

  12. Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase.

    PubMed

    Zumft, W G; Dreusch, A; Löchelt, S; Cuypers, H; Friedrich, B; Schneider, B

    1992-08-15

    The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri [Viebrock, A. & Zumft, W. G. (1988) J. Bacteriol. 170, 4658-4668] and the subunit II of cytochrome-c oxidase (COII). Both types of enzymes possess the CuA binding site. The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22-kb PstI fragment (NS220) of nosZ from P. stutzeri. The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A. eutrophus) and 70695 Da (P. aeruginosa). The N-terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport. A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences. Only three out of nine cysteine residues of the NosZ protein (P. stutzeri) are invariant. Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus. Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes. The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences. Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination. Of 23 histidine residues in NosZ (P. stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences. Conserved tryptophan residues are located close to several potential copper ligands. Trp615 may contribute to the observed quenching of

  13. Evidence for chemoautotrophic symbiosis in a Mediterranean cold seep clam (Bivalvia: Lucinidae): comparative sequence analysis of bacterial 16S rRNA, APS reductase and RubisCO genes.

    PubMed

    Duperron, Sébastien; Fiala-Médioni, Aline; Caprais, Jean-Claude; Olu, Karine; Sibuet, Myriam

    2007-01-01

    Symbioses between lucinid clams (Bivalvia: Lucinidae) and autotrophic sulphide-oxidizing bacteria have mainly been studied in shallow coastal species, and information regarding deep-sea species is scarce. Here we study the symbiosis of a clam, resembling Lucinoma kazani, which was recently collected in sediment cores from new cold-seep sites in the vicinity of the Nile deep-sea fan, eastern Mediterranean, at depths ranging from 507 to 1691 m. A dominant bacterial phylotype, related to the sulphide-oxidizing symbiont of Lucinoma aequizonata, was identified in gill tissue by comparative 16S rRNA gene sequence analysis. A second phylotype, related to spirochete sequences, was identified twice in a library of 94 clones. Comparative analyses of gene sequences encoding the APS reductase alpha subunit and ribulose-1,5-bisphosphate carboxylase oxygenase support the hypothesis that the dominant symbiont can perform sulphide oxidation and autotrophy. Transmission electron micrographs of gills confirmed the dominance of sulphide-oxidizing bacteria, which display typical vacuoles, and delta(13)C values measured in gill and foot tissue further support the hypothesis for a chemoautotrophic-sourced host carbon nutrition.

  14. A new and improved method based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the determination of A1298C mutation in the methylenetetrahydrofolate reductase (MTHFR) gene.

    PubMed

    Machnik, Grzegorz; Zapala, Malgorzata; Pelc, Ewa; Gasecka-Czapla, Monika; Kaczmarczyk, Grzegorz; Okopien, Boguslaw

    2013-01-01

    Intracellular folate homeostasis and metabolism is regulated by numerous genes. Among them, 5,10-methylenetetrahydrofolate reductase (MTHFR) is of special interest because of its involvement in regulation of the homocysteine level in the body as a result of folate metabolism. Moreover, some studies demonstrated that the homocysteine plasma level in individuals may be influenced by polymorphisms present in the MTHFR gene. Two common, clinically relevant mutations have been described: MTHFR C677T and MTHFR A1298C. Although several laboratory techniques allow genotyping of both polymorphisms, PCR-RFLP analysis is simple to perform, relatively cheap, and thus one of the most utilized. In the case of A1298C, the PCR-RFLP technique that utilizes MboII endonuclease class II requires an acrylamide gel electrophoresis, since agarose gel electrophoresis is unable to resolve short deoxyribonucleic acid (DNA) fragments after restriction digestion. Agarose gel electrophoresis is commonly preferred over that of acrylamide. To resolve this inconvenience, a novel PCR-RFLP, AjuI-based method to genotype A1298C alleles has been developed that can be performed on standard agarose gel.

  15. Carbon-carbon double-bond reductases in nature.

    PubMed

    Huang, Minmin; Hu, Haihong; Ma, Li; Zhou, Quan; Yu, Lushan; Zeng, Su

    2014-08-01

    Reduction of C = C bonds by reductases, found in a variety of microorganisms (e.g. yeasts, bacteria, and lower fungi), animals, and plants has applications in the production of metabolites that include pharmacologically active drugs and other chemicals. Therefore, the reductase enzymes that mediate this transformation have become important therapeutic targets and biotechnological tools. These reductases are broad-spectrum, in that, they can act on isolation/conjugation C = C-bond compounds, α,β-unsaturated carbonyl compounds, carboxylic acids, acid derivatives, and nitro compounds. In addition, several mutations in the reductase gene have been identified, some associated with diseases. Several of these reductases have been cloned and/or purified, and studies to further characterize them and determine their structure in order to identify potential industrial biocatalysts are still in progress. In this study, crucial reductases for bioreduction of C = C bonds have been reviewed with emphasis on their principal substrates and effective inhibitors, their distribution, genetic polymorphisms, and implications in human disease and treatment.

  16. Meta-analysis of Methylenetetrahydrofolate reductase maternal gene in Down syndrome: increased susceptibility in women carriers of the MTHFR 677T allele.

    PubMed

    Victorino, D B; Godoy, M F; Goloni-Bertollo, E M; Pavarino, E C

    2014-08-01

    Because a number of data studies include some controversial results about Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and Down syndrome (DS), we performed a meta-analysis to determine a more precise estimation of this association. Studies were searched on PubMed, EMBASE and Lilacs-Scielo, up to April 2013, and they were eligible if they included case mothers (DSM) that have gave birth to children with DS, and controls mothers (CM) that have gave birth to healthy children without chromosomal abnormality, syndrome or malformation. The combined odds ratio with 95% confidence intervals was calculated by fixed or random effects models to assess the strength of associations. Potential sources of heterogeneity between studies were evaluated using Q test and the I(2). Publication bias was estimated using Begg's test and Egger's linear regression test. Sensitivity analyses were performed by using allelic, dominant, recessive and codominant genetic models, Hardy-Weinberg equilibrium (HWE) and ethnicity. Twenty-two studies with 2,223 DSM and 2,807 CM were included for MTHFR C677T and 15 studies with 1,601 DSM and 1,849 CM were included for MTHFR A1298C. Overall analysis suggests an association of the MTHFR C677T polymorphism with maternal risk for DS. Moreover, no association between the MTHFR A1298C polymorphism and maternal risk for DS was found. There is also evidence of higher heterogeneity, with I(2) test values ranging from 8 to 89%. No evidence of publication bias was found. Taken together, our meta-analysis implied that the T allele carriers might carry an increased maternal risk for DS.

  17. Down-regulation of Leucaena leucocephala cinnamoyl CoA reductase (LlCCR) gene induces significant changes in phenotype, soluble phenolic pools and lignin in transgenic tobacco.

    PubMed

    Prashant, S; Srilakshmi Sunita, M; Pramod, S; Gupta, Ranadheer K; Anil Kumar, S; Rao Karumanchi, S; Rawal, S K; Kavi Kishor, P B

    2011-12-01

    cDNA and genomic clones of cinnamoyl CoA reductase measuring 1011 and 2992 bp were isolated from a leguminous pulpwood tree Leucaena leucocephala, named as LlCCR. The cDNA exhibited 80-85% homology both at the nucleotide and amino acid levels with other known sequences. The genomic sequence contained five exons and four introns. Sense and antisense constructs of LlCCR were introduced in tobacco plants to up and down-regulate this key enzyme of lignification. The primary transformants showed a good correlation between CCR transcript levels and its activity. Most of the CCR down-regulated lines displayed stunted growth and development, wrinkled leaves and delayed senescence. These lines accumulated unusual phenolics like ferulic and sinapic acids in cell wall. Histochemical staining suggested reduction in aldehyde units and increased syringyl over guaiacyl (S/G) ratio of lignin. Anatomical studies showed thin walled, elongated xylem fibres, collapsed vessels with drastic reduction of secondary xylem. The transmission electron microscopic studies revealed modification of ultrastructure and topochemical distribution of wall polysaccharides and lignin in the xylem fibres. CCR down-regulated lines showed increased thickness of secondary wall layers and poor lignification of S2 and S3 wall layers. The severely down-regulated line AS17 exhibited 24.7% reduction of Klason lignin with an increase of 15% holocellulose content. Contrarily, the CCR up-regulated lines exhibited robust growth, development and significant increase in lignin content. The altered lignin profiles observed in transgenic tobacco lines support a role for CCR down-regulation in improving wood properties of L. leucocephala exclusively used in the pulp and paper industry of India.

  18. Denitrifying alphaproteobacteria from the Arabian Sea that express nosZ, the gene encoding nitrous oxide reductase, in oxic and suboxic waters.

    PubMed

    Wyman, Michael; Hodgson, Sylvia; Bird, Clare

    2013-04-01

    Marine ecosystems are significant sources of the powerful greenhouse gas nitrous oxide (N2O). A by-product of nitrification and an intermediate in the denitrification pathway, N2O is formed primarily in oxygen-deficient waters and sediments. We describe the isolation of a group of alphaproteobacteria from the suboxic waters of the Arabian Sea that are phylogenetically affiliated with Labrenzia spp. and other denitrifiers. Quantitative PCR assays revealed that these organisms were very broadly distributed in this semienclosed ocean basin. Their biogeographical range extended from the productive, upwelling region off the Omani shelf to the clear, oligotrophic waters that are found much further south and also included the mesotrophic waters overlying the oxygen minimum zone (OMZ) in the northeastern sector of the Arabian Sea. These organisms actively expressed NosZ (N2O reductase, the terminal step in the denitrification pathway) within the OMZ, an established region of pelagic denitrification. They were found in greatest numbers outside the OMZ, however, and nosZ mRNAs were also readily detected near the base of the upper mixed layer in nutrient-poor, oxic regions. Our findings provide firm molecular evidence of a potential sink for N2O within well-ventilated, oceanic surface waters in this biogeochemically important region. We show that the Labrenzia-like denitrifiers and their close relatives are habitual colonizers of the pseudobenthic environment provided by Trichodesmium spp. We develop the conjecture that the O2-depleted microzones that occur within the colonies of these filamentous, diazotrophic cyanobacteria might provide unexpected niches for the reduction of nitrogen oxides in tropical and subtropical surface waters.

  19. Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae).

    PubMed

    Liu, Su; Liang, Qing-Mei; Huang, Yuan-Jie; Yuan, Xin; Zhou, Wen-Wu; Qiao, Fei; Cheng, Jiaan; Gurr, Geoff M; Zhu, Zeng-Rong

    2013-01-01

    NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems.

  20. Potential Application of the Oryza sativa Monodehydroascorbate Reductase Gene (OsMDHAR) to Improve the Stress Tolerance and Fermentative Capacity of Saccharomyces cerevisiae

    PubMed Central

    Kim, Yul-Ho; Park, Ae-Kyung; Kim, Han-Woo; Lee, Jun-Hyuk; Yoon, Ho-Sung

    2016-01-01

    Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is an important enzyme for ascorbate recycling. To examine whether heterologous expression of MDHAR from Oryza sativa (OsMDHAR) can prevent the deleterious effects of unfavorable growth conditions, we constructed a transgenic yeast strain harboring a recombinant plasmid carrying OsMDHAR (p426GPD::OsMDHAR). OsMDHAR-expressing yeast cells displayed enhanced tolerance to hydrogen peroxide by maintaining redox homoeostasis, proteostasis, and the ascorbate (AsA)-like pool following the accumulation of antioxidant enzymes and molecules, metabolic enzymes, and molecular chaperones and their cofactors, compared to wild-type (WT) cells carrying vector alone. The addition of exogenous AsA or its analogue isoascorbic acid increased the viability of WT and ara2Δ cells under oxidative stress. Furthermore, the survival of OsMDHAR-expressing cells was greater than that of WT cells when cells at mid-log growth phase were exposed to high concentrations of ethanol. High OsMDHAR expression also improved the fermentative capacity of the yeast during glucose-based batch fermentation at a standard cultivation temperature (30°C). The alcohol yield of OsMDHAR-expressing transgenic yeast during fermentation was approximately 25% (0.18 g·g-1) higher than that of WT yeast. Accordingly, OsMDHAR-expressing transgenic yeast showed prolonged survival during the environmental stresses produced during fermentation. These results suggest that heterologous OsMDHAR expression increases tolerance to reactive oxygen species-induced oxidative stress by improving cellular redox homeostasis and improves survival during fermentation, which enhances fermentative capacity. PMID:27392090

  1. Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

    PubMed Central

    Hoffmann, Tamara; Frankenberg, Nicole; Marino, Marco; Jahn, Dieter

    1998-01-01

    During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation. PMID:9422613

  2. Nitrate and periplasmic nitrate reductases

    PubMed Central

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

    2014-01-01

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

  3. Analysis of Dissimilatory Sulfite Reductase and 16S rRNA Gene Fragments from Deep-Sea Hydrothermal Sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific

    PubMed Central

    Nakagawa, Tatsunori; Ishibashi, Jun-Ichiro; Maruyama, Akihiko; Yamanaka, Toshiro; Morimoto, Yusuke; Kimura, Hiroyuki; Urabe, Tetsuro; Fukui, Manabu

    2004-01-01

    This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5°C and natural vent fluids at 7°C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and γ- and ɛ-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with “Nanoarchaeota.” The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300°C were affiliated with the δ-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4°C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments. PMID:14711668

  4. Genetic susceptibility of methylenetetrahydrofolate reductase (MTHFR) gene C677T, A1298C, and G1793A polymorphisms with risk for bladder transitional cell carcinoma in men.

    PubMed

    Safarinejad, Mohammad Reza; Shafiei, Nayyer; Safarinejad, Shiva

    2011-12-01

    We performed a case-control study of 158 bladder transitional cell carcinoma (TCC) cases and 316 controls to investigate the association between methylenetetrahydrofolate reductase (MTHFR) C677T, A1298G, and G1793A polymorphisms and bladder cancer susceptibility by polymerase chain reaction restriction fragment length polymorphism (PCR-RLFP) technique. The controls were frequency-matched to the cases by age (± 5 years), ethnicity, and smoking status. We also measured serum levels of total homocysteine (tHcy), folate, and vitamin B12. It was found that the 1298AC (odds ratio, OR = 3.74; 95% confidence interval, CI = 2.34-5.47; P = 0.001) and 1298CC (OR = 3.46, 95% CI = 2.37-5.52; P = 0.001) genotypes of MTHFR A1298C were significantly associated with increased risk of bladder TCC. The MTHFR C677T and G1793A polymorphisms were not associated with bladder TCC. After stratification for grade and stage, we observed that the 677TT (OR = 4.47, 95% CI = 2.74-6.72; P = 0.001) and MTHFR 1298CC (OR = 4.78, 95% CI = 2.82-6.89; P = 0.001) genotypes of MTHFR were associated with increased risk of muscle-invasive bladder TCC. We also found that the MTHFR 677CT+1298AA genotypes were associated with an approximately 70% reduction in risk of bladder cancer (OR = 0.31; 95% CI = 0.15-0.68) compared to the combined referent genotype. There were 8 haplotypes and 16 haplotype genotypes based on these three variants. When we used the haplotypes and assumed that the 677T, 1298C, and 1793G alleles were risk alleles, the adjusted odds ratios increased as the number of risk alleles increased: 1.00 for 0-1 variant, 1.88 (1.4-2.7) for any two risk alleles and 2.07 (1.6-2.8) for any three risk alleles. Serum tHcy levels were significantly higher in carriers of the 677T, 1298C, and 1793G alleles compared to noncarriers (all P < 0.01). There was no significant correlation between serum levels of tHcy and folate and bladder cancer risk. Further studies in larger samples size and different

  5. Comparison of the expression of human equilibrative nucleotide transporter 1 (hENT1) and ribonucleotide reductase subunit M1 (RRM1) genes in seven non-Hodgkin lymphoma cell lines.

    PubMed

    Zhao, H B; Zhang, X F; Shi, F; Zhang, M Z; Xue, W L

    2016-05-06

    We investigated the variability in the expression of human equilibrative nucleoside transporter 1 (hENT1) and ribonucleotide reductase subunit M1 (RRM1) in non-Hodgkin lymphoma cell lines. hENT1 and RRM1 mRNA expression levels in natural killer (NK) cells and seven non-Hodgkin lymphoma cell lines (YTS, SNK-6, Jeko-1, ly-1, Raji, Karpas, and Jurket) were studied using reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) and the results were compared using the Student t-test. mRNA expression of hENT1 was detectable in YTS, SNK-6, Jeko-1, ly-1, Raji, Karpas, Jurket, and NK cells, which revealed variability in gene expression. There were significant differences in the mRNA expression values of hENT1 (P = 0.021) and RRM1 (P = 0.002) compared to those in NK cells. mRNA expression of both hENT1 and RRM1 was closely associated with non-Hodgkin lymphoma cell proliferation. Differential expression analysis of hENT1 and RRM1 in non-Hodgkin lymphoma cell lines may provide novel drug leads for precision medicine.

  6. Multiple types of 8-vinyl reductases for (bacterio)chlorophyll biosynthesis occur in many green sulfur bacteria.

    PubMed

    Liu, Zhenfeng; Bryant, Donald A

    2011-09-01

    Two 8-vinyl reductases, BciA and BciB, have been identified in chlorophototrophs. The bciA gene of Chlorobaculum tepidum was replaced with genes similar to bciB from other green sulfur bacteria. Pigment analyses of the complemented strains showed that the bciB homologs encode 8-vinyl reductases similar to those of cyanobacteria.

  7. Association of sequence variations in vitamin K epoxide reductase and gamma-glutamyl carboxylas genes with biochemical measures of vitamin K status

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic factors, specifically the VKORC1 and GGCX genes, have been shown to contribute to the interindividual variability in response to the vitamin K-antagonist, warfarin, which influences the dose required to achieve the desired anticoagulation response. These differences in warfarin sensitivity ...

  8. Senescence-induced ectopic expression of the A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate influx, and nitrate reductase activity, but does not affect grain yield.

    PubMed

    Sykorová, Blanka; Kuresová, Gabriela; Daskalova, Sasha; Trcková, Marie; Hoyerová, Klára; Raimanová, Ivana; Motyka, Václav; Trávnícková, Alena; Elliott, Malcolm C; Kamínek, Miroslav

    2008-01-01

    The manipulation of cytokinin levels by senescence-regulated expression of the Agrobacterium tumefaciens ipt gene through its control by the Arabidopsis SAG12 (senescence-associated gene 12) promoter is an efficient tool for the prolongation of leaf photosynthetic activity which potentially can affect plant productivity. In the present study, the efficiency of this approach was tested on wheat (Triticum aestivum L.)-a monocarpic plant characterized by a fast switch from vegetative to reproductive growth, and rapid translocation of metabolites from leaves to developing grains after anthesis. When compared with the wild-type (WT) control plants, the SAG12::ipt wheat plants exhibited delayed chlorophyll degradation only when grown under limited nitrogen (N) supply. Ten days after anthesis the content of chlorophyll and bioactive cytokinins of the first (flag) leaf of the transgenic plants was 32% and 65% higher, respectively, than that of the control. There was a progressive increase in nitrate influx and nitrate reductase activity. However, the SAG12::ipt and the WT plants did not show differences in yield-related parameters including number of grains and grain weight. These results suggest that the delay of leaf senescence in wheat also delays the translocation of metabolites from leaves to developing grains, as indicated by higher accumulation of ((15)N-labelled) N in spikes of control compared with transgenic plants prior to anthesis. This delay interferes with the wheat reproductive strategy that is based on a fast programmed translocation of metabolites from the senescing leaves to the reproductive sinks shortly after anthesis.

  9. Expression analysis of cinnamoyl-CoA reductase (CCR) gene in developing seedlings of Leucaena leucocephala: a pulp yielding tree species.

    PubMed

    Srivastava, Sameer; Gupta, Ranadheer K; Arha, Manish; Vishwakarma, Rishi K; Rawal, Shuban K; Kavi Kishor, P B; Khan, Bashir M

    2011-02-01

    Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as Ll-CCR; the sequence analysis revealed an Open Reading Frame (ORF) of 1005 bp. Phylogenetic analysis showed that Ll-CCR sequence is highly homologous to CCRs from other dicot plants. The 2992 bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L. leucocephala contains two copies as revealed by DNA blot hybridization. Ll-CCR gene was over-expressed in Escherichia coli, which showed a molecular mass of approximately 38 kDa. Protein blot analysis revealed that Ll-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15 d in case of roots and stem as revealed by QRT-PCR studies in 0-15 d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5, 10 and 15 d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry.

  10. Bacterial morphinone reductase is related to Old Yellow Enzyme.

    PubMed Central

    French, C E; Bruce, N C

    1995-01-01

    Morphinone reductase, produced by Pseudomonas putida M10, catalyses the NADH-dependent saturation of the carbon-carbon double bond of morphinone and codeinone, and is believed to be involved in the metabolism of morphine and codeine. The structural gene encoding morphinone reductase, designated morB, was cloned from Ps. putida M10 genomic DNA by the use of degenerate oligonucleotide probes based on elements of the amino acid sequence of the purified enzyme. Sequence analysis and structural characteristics indicated that morphinone reductase is related to the flavoprotein alpha/beta-barrel oxidoreductases, and is particularly similar to Old Yellow Enzyme of Saccharomyces spp. and the related oestrogen-binding protein of Candida albicans. Expressed sequence tags from several plant species show high homology to these enzymes, suggesting the presence of a family of enzymes conserved in plants and fungi. Although related bacterial proteins are known, morphinone reductase appears to be more similar to the eukaryotic proteins. Morphinone reductase was overexpressed in Escherichia coli, and has potential applications for the industrial preparation of semisynthetic opiates. Images Figure 1 Figure 5 PMID:8554504

  11. A detoxifying oxygen reductase in the anaerobic protozoan Entamoeba histolytica.

    PubMed

    Vicente, João B; Tran, Vy; Pinto, Liliana; Teixeira, Miguel; Singh, Upinder

    2012-09-01

    We report the characterization of a bacterial-type oxygen reductase abundant in the cytoplasm of the anaerobic protozoan parasite Entamoeba histolytica. Upon host infection, E. histolytica is confronted with various oxygen tensions in the host intestine, as well as increased reactive oxygen and nitrogen species at the site of local tissue inflammation. Resistance to oxygen-derived stress thus plays an important role in the pathogenic potential of E. histolytica. The genome of E. histolytica has four genes that encode flavodiiron proteins, which are bacterial-type oxygen or nitric oxide reductases and were likely acquired by lateral gene transfer from prokaryotes. The EhFdp1 gene has higher expression in virulent than in nonvirulent Entamoeba strains and species, hinting that the response to oxidative stress may be one correlate of virulence potential. We demonstrate that EhFdp1 is abundantly expressed in the cytoplasm of E. histolytica and that the protein levels are markedly increased (up to ~5-fold) upon oxygen exposure. Additionally, we produced fully functional recombinant EhFdp1 and demonstrated that this enzyme is a specific and robust oxygen reductase but has poor nitric oxide reductase activity. This observation represents a new mechanism of oxygen resistance in the anaerobic protozoan pathogen E. histolytica.

  12. Zeatin reductase in Phaseolus embryos

    SciTech Connect

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

    1989-04-01

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

  13. The C677T mutation of the 5,10-methylenetetrahydrofolate reductase gene is a moderate risk factor for spina bifida in Italy.

    PubMed Central

    de Franchis, R; Buoninconti, A; Mandato, C; Pepe, A; Sperandeo, M P; Del Gado, R; Capra, V; Salvaggio, E; Andria, G; Mastroiacovo, P

    1998-01-01

    OBJECTIVE: To estimate the risk for spina bifida associated with the common mutation C677T of the MTHFR gene in a country with a relatively low prevalence of NTDs. DESIGN: Case-control study. SUBJECTS: Cases: 203 living patients affected with spina bifida (173 myelomeningocele and 30 lipomeningocele); controls: 583 subjects (306 young adults and 277 unselected newborns) from northern and central-southern Italy. SETTING: Cases: three spina bifida centres; young adult controls: DNA banks; newborn controls: regional neonatal screening centres. MAIN OUTCOME MEASURES: Prevalence of the C677T genotypes in cases and controls by place of birth; odds ratios for spina bifida and estimated attributable fraction. RESULTS: The prevalence of T/T, T/C, and C/C genotype was 16.6%, 53.7%, and 29.7% in controls and 25.6%, 43.8%, and 30.6% in cases, respectively. We found no differences between type of defect or place of birth. The odds ratio for spina bifida associated with the T/T genotype v C/C plus T/C was 1.73 (95% CI 1.15, 2.59) and the corresponding attributable fraction was 10.8%. No increased risk was found for heterozygous patients (OR=0.79, 95% CI 0.53-1.18). CONCLUSION: This study, as well as the meta-analysis we updated, shows that homozygosity for the MTHFR C677T mutation is a moderate risk factor in Europe, and even in Italy where there is a relatively low prevalence of spina bifida. The estimated attributable fraction associated with this risk factor explains only a small proportion of cases preventable by periconceptional folic acid supplementation. Thus, other genes involved in folate-homocysteine metabolism, their interaction, and the interaction between genetic and environmental factors should be investigated further. PMID:9863598

  14. Diversity of Dissimilatory Sulfite Reductase Genes (dsrAB) in a Salt Marsh Impacted by Long-Term Acid Mine Drainage▿ †

    PubMed Central

    Moreau, John W.; Zierenberg, Robert A.; Banfield, Jillian F.

    2010-01-01

    Sulfate-reducing bacteria (SRB) play a major role in the coupled biogeochemical cycling of sulfur and chalcophilic metal(loid)s. By implication, they can exert a strong influence on the speciation and mobility of multiple metal(loid) contaminants. In this study, we combined DsrAB gene sequencing and sulfur isotopic profiling to identify the phylogeny and distribution of SRB and to assess their metabolic activity in salt marsh sediments exposed to acid mine drainage (AMD) for over 100 years. Recovered dsrAB sequences from three sites sampled along an AMD flow path indicated the dominance of a single Desulfovibrio species. Other major sequence clades were related most closely to Desulfosarcina, Desulfococcus, Desulfobulbus, and Desulfosporosinus species. The presence of metal sulfides with low δ34S values relative to δ34S values of pore water sulfate showed that sediment SRB populations were actively reducing sulfate under ambient conditions (pH of ∼2), although possibly within less acidic microenvironments. Interestingly, δ34S values for pore water sulfate were lower than those for sulfate delivered during tidal inundation of marsh sediments. 16S rRNA gene sequence data from sediments and sulfur isotope data confirmed that sulfur-oxidizing bacteria drove the reoxidation of biogenic sulfide coupled to oxygen or nitrate reduction over a timescale of hours. Collectively, these findings imply a highly dynamic microbially mediated cycling of sulfate and sulfide, and thus the speciation and mobility of chalcophilic contaminant metal(loid)s, in AMD-impacted marsh sediments. PMID:20472728

  15. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

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

    2013-04-23

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

  16. Association of the I264T variant in the sulfide quinone reductase-like (SQRDL) gene with osteoporosis in Korean postmenopausal women.

    PubMed

    Jin, Hyun-Seok; Kim, Jeonghyun; Park, Sangwook; Park, Eunkuk; Kim, Bo-Young; Choi, Vit-Na; Yoo, Young-Hyun; Kim, Bom-Taeck; Jeong, Seon-Yong

    2015-01-01

    To identify novel susceptibility variants for osteoporosis in Korean postmenopausal women, we performed a genome-wide association analysis of 1180 nonsynonymous single nucleotide polymorphisms (nsSNPs) in 405 individuals with osteoporosis and 722 normal controls of the Korean Association Resource cohort. A logistic regression analysis revealed 72 nsSNPs that showed a significant association with osteoporosis (p<0.05). The top 10 nsSNPs showing the lowest p-values (p = 5.2×10-4-8.5×10-3) were further studied to investigate their effects at the protein level. Based on the results of an in silico prediction of the protein's functional effect based on amino acid alterations and a sequence conservation evaluation of the amino acid residues at the positions of the nsSNPs among orthologues, we selected one nsSNP in the SQRDL gene (rs1044032, SQRDL I264T) as a meaningful genetic variant associated with postmenopausal osteoporosis. To assess whether the SQRDL I264T variant played a functional role in the pathogenesis of osteoporosis, we examined the in vitro effect of the nsSNP on bone remodeling. Overexpression of the SQRDL I264T variant in the preosteoblast MC3T3-E1 cells significantly increased alkaline phosphatase activity, mineralization, and the mRNA expression of osteoblastogenesis markers, Runx2, Sp7, and Bglap genes, whereas the SQRDL wild type had no effect or a negative effect on osteoblast differentiation. Overexpression of the SQRDL I264T variant did not affect osteoclast differentiation of the primary-cultured monocytes. The known effects of hydrogen sulfide (H2S) on bone remodeling may explain the findings of the current study, which demonstrated the functional role of the H2S-catalyzing enzyme SQRDL I264T variant in osteoblast differentiation. In conclusion, the results of the statistical and experimental analyses indicate that the SQRDL I264T nsSNP may be a significant susceptibility variant for osteoporosis in Korean postmenopausal women that is involved

  17. Association of the I264T Variant in the Sulfide Quinone Reductase-Like (SQRDL) Gene with Osteoporosis in Korean Postmenopausal Women

    PubMed Central

    Park, Eunkuk; Kim, Bo-Young; Choi, Vit-Na; Yoo, Young-Hyun; Kim, Bom-Taeck; Jeong, Seon-Yong

    2015-01-01

    To identify novel susceptibility variants for osteoporosis in Korean postmenopausal women, we performed a genome-wide association analysis of 1180 nonsynonymous single nucleotide polymorphisms (nsSNPs) in 405 individuals with osteoporosis and 722 normal controls of the Korean Association Resource cohort. A logistic regression analysis revealed 72 nsSNPs that showed a significant association with osteoporosis (p<0.05). The top 10 nsSNPs showing the lowest p-values (p = 5.2×10-4–8.5×10-3) were further studied to investigate their effects at the protein level. Based on the results of an in silico prediction of the protein’s functional effect based on amino acid alterations and a sequence conservation evaluation of the amino acid residues at the positions of the nsSNPs among orthologues, we selected one nsSNP in the SQRDL gene (rs1044032, SQRDL I264T) as a meaningful genetic variant associated with postmenopausal osteoporosis. To assess whether the SQRDL I264T variant played a functional role in the pathogenesis of osteoporosis, we examined the in vitro effect of the nsSNP on bone remodeling. Overexpression of the SQRDL I264T variant in the preosteoblast MC3T3-E1 cells significantly increased alkaline phosphatase activity, mineralization, and the mRNA expression of osteoblastogenesis markers, Runx2, Sp7, and Bglap genes, whereas the SQRDL wild type had no effect or a negative effect on osteoblast differentiation. Overexpression of the SQRDL I264T variant did not affect osteoclast differentiation of the primary-cultured monocytes. The known effects of hydrogen sulfide (H2S) on bone remodeling may explain the findings of the current study, which demonstrated the functional role of the H2S-catalyzing enzyme SQRDL I264T variant in osteoblast differentiation. In conclusion, the results of the statistical and experimental analyses indicate that the SQRDL I264T nsSNP may be a significant susceptibility variant for osteoporosis in Korean postmenopausal women that is

  18. Effect of the methylenetetrahydrofolate reductase gene polymorphisms on homocysteine, folate and vitamin B12 in patients with bipolar disorder and relatives.

    PubMed

    Ozbek, Zeynep; Kucukali, Cem Ismail; Ozkok, Elif; Orhan, Nurcan; Aydin, Makbule; Kilic, Gamze; Sazci, Ali; Kara, Ihsan

    2008-07-01

    We investigated the effect of polymorphic variants of c.1298A>C (Glu429Ala) and c.677C>T (Ala222Val) in methylenetetrahydrofolate (MTHFR) gene on the total homocysteine (tHcy), folate and B12 levels in patients with bipolar disorder, first-degree relatives of patients, and controls. The c.677C>T and c.1298A>C polymorphisms in MTHFR were determined by polymerase chain reaction-restriction fragment length polymorphism in 197 bipolar patients, 278 relatives and 238 controls. tHcy and folate and vitamin B12 levels were measured by Fluorescence Polarization Immunoassay and Electrochemiluminescence, respectively. The tHcy was significantly increased in patients and relatives. In contrast, folate and B12 were significantly lower in patients and relatives. Gender was not considered as a significant determinant in the multivariate analysis. Genotypes of c.1298A>C and c.677C>T were correlated with tHcy, folate and B12. Patients and relatives carrying TT and/or AA and AC genotypes had elevated tHcy and reduced folate and B12 levels. High tHcy but low folate and vitamin B12 levels may be a risk factor for development of bipolar disorder.

  19. Increased production of wax esters in transgenic tobacco plants by expression of a fatty acid reductase:wax synthase gene fusion.

    PubMed

    Aslan, Selcuk; Hofvander, Per; Dutta, Paresh; Sun, Chuanxin; Sitbon, Folke

    2015-12-01

    Wax esters are hydrophobic lipids consisting of a fatty acid moiety linked to a fatty alcohol with an ester bond. Plant-derived wax esters are today of particular concern for their potential as cost-effective and sustainable sources of lubricants. However, this aspect is hampered by the fact that the level of wax esters in plants generally is too low to allow commercial exploitation. To investigate whether wax ester biosynthesis can be increased in plants using transgenic approaches, we have here exploited a fusion between two bacterial genes together encoding a single wax ester-forming enzyme, and targeted the resulting protein to chloroplasts in stably transformed tobacco (Nicotiana benthamiana) plants. Compared to wild-type controls, transgenic plants showed both in leaves and stems a significant increase in the total level of wax esters, being eight-fold at the whole plant level. The profiles of fatty acid methyl ester and fatty alcohol in wax esters were related, and C16 and C18 molecules constituted predominant forms. Strong transformants displayed certain developmental aberrations, such as stunted growth and chlorotic leaves and stems. These negative effects were associated with an accumulation of fatty alcohols, suggesting that an adequate balance between formation and esterification of fatty alcohols is crucial for a high wax ester production. The results show that wax ester engineering in transgenic plants is feasible, and suggest that higher yields may become achieved in the near future.

  20. Characterization and expression of gamma-interferon-inducible lysosomal thiol reductase (GILT) gene in amphioxus Branchiostoma belcheri with implications for GILT in innate immune response.

    PubMed

    Liu, Naiguo; Zhang, Shicui; Liu, Zhenhui; Gaowa, Saren; Wang, Yongjun

    2007-04-01

    An amphioxus cDNA, AmphiGILT, encoding GILT protein was isolated from the gut cDNA library of Branchiostoma belcheri. It codes for a deduced protein of 254 amino acids, which has all the main features typical of GILT proteins including the signature sequence CQHGX(2)CX(2)NX(4)C, CXXC motif and 11 conserved cysteines. Phylogenetic analysis showed that AmphiGILT and sea urchin GILT clubbed together and positioned at the base of vertebrate GILT clade, suggesting that both AmphiGILT and sea urchin GILT might share some characteristics of the archetype of vertebrate GILT proteins. The genomic DNA sequence of B. floridae contains seven exons and six introns, which is similar to vertebrate GILT exon-intron organization. AmphiGILT was expressed in a tissue-specific manner with the most abundant mRNA in the digestive system including hepatic caecum and hind-gut. It was also found that mammalian IFN-gamma only exerted a slight effect on the expression of GILT gene in amphioxus, forming a contrast to the marked induction of human and mouse GILT expression by IFN-gamma. Taken the absence of the adaptive immune system including MHC class II molecules and lymphocytes into consideration, these results suggest that AmphiGILT is highly likely to play a role in the innate immune responses in amphioxus.

  1. Cloning and Sequence Analysis of Two Pseudomonas Flavoprotein Xenobiotic Reductases

    PubMed Central

    Blehert, David S.; Fox, Brian G.; Chambliss, Glenn H.

    1999-01-01

    The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from Pseudomonas putida II-B and P. fluorescens I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The P. putida gene, xenA, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that removes either the terminal or central nitro groups from NG and that reduces 2-cyclohexen-1-one but did not readily reduce 2,4,6-trinitrotoluene (TNT). The P. fluorescens gene, xenB, encodes a 37,441-Da monomeric, NAD(P)H-dependent flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily react with 2-cyclohexen-1-one. Heterologous expression of xenA and xenB was demonstrated in Escherichia coli DH5α. The transcription initiation sites of both xenA and xenB were identified by primer extension analysis. BLAST analyses conducted with the P. putida xenA and the P. fluorescens xenB sequences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryotic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serve a detoxification role in antioxidant defense systems. PMID:10515912

  2. Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aldehyde reductase gene ARI1 is a recently characterized member of intermediate subfamily under SDR (short-chain dehydrogenase/reductase) superfamily that revealed mechanisms of in situ detoxification of furfural and HMF for tolerance of Saccharomyces cerevisiae. Uncharacterized open reading frames ...

  3. Steroid 5β-Reductase from Leaves of Vitis vinifera: Molecular Cloning, Expression, and Modeling.

    PubMed

    Ernst, Mona; Munkert, Jennifer; Campa, Manuela; Malnoy, Mickael; Martens, Stefan; Müller-Uri, Frieder

    2015-11-25

    A steroid 5β-reductase gene corresponding to the hypothetical protein LOC100247199 from leaves of Vitis vinifera (var. 'Chardonnay') was cloned and overexpressed in Escherichia coli. The recombinant protein showed 5β-reductase activity when progesterone was used as a substrate. The reaction was stereoselective, producing only 5β-products such as 5β-pregnane-3,20-dione. Other small substrates (terpenoids and enones) were also accepted as substrates, indicating the highly promiscuous character of the enzyme class. Our results show that the steroid 5β-reductase gene, encoding an orthologous enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in leaves of the cardenolide-free plant V. vinifera. We emphasize the fact that, on some occasions, different reductases (e.g., progesterone 5β-reductase and monoterpenoid reductase) can also use molecules that are similar to the final products as a substrate. Therefore, in planta, the different reductases may contribute to the immense number of diverse small natural products finally leading to the flavor of wine.

  4. Nitrate reductase from Rhodopseudomonas sphaeroides.

    PubMed Central

    Kerber, N L; Cardenas, J

    1982-01-01

    The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed. PMID:6978883

  5. Identification and characterization of 2-naphthoyl-coenzyme A reductase, the prototype of a novel class of dearomatizing reductases.

    PubMed

    Eberlein, Christian; Estelmann, Sebastian; Seifert, Jana; von Bergen, Martin; Müller, Michael; Meckenstock, Rainer U; Boll, Matthias

    2013-06-01

    The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl-coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl-CoA by ATP-dependent or -independent benzoyl-CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2-naphthoyl-CoA reductase was purified from extracts of the naphthalene-degrading, sulphidogenic enrichment culture N47. The oxygen-tolerant enzyme dearomatized the non-activated ring of 2-naphthoyl-CoA by a four-electron reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin-containing 'old yellow enzyme' family. NCR contained FAD, FMN, and an iron-sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2-naphthoyl-CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl-CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl-CoA reductases.

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  7. Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter.

    PubMed Central

    Snape, J R; Walkley, N A; Morby, A P; Nicklin, S; White, G F

    1997-01-01

    Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km = 23 microM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35 degrees C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the alpha/beta barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively. PMID:9401040

  8. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network.

    PubMed

    Couto, Narciso; Wood, Jennifer; Barber, Jill

    2016-06-01

    In this review article we examine the role of glutathione reductase in the regulation, modulation and maintenance of cellular redox homoeostasis. Glutathione reductase is responsible for maintaining the supply of reduced glutathione; one of the most abundant reducing thiols in the majority of cells. In its reduced form, glutathione plays key roles in the cellular control of reactive oxygen species. Reactive oxygen species act as intracellular and extracellular signalling molecules and complex cross talk between levels of reactive oxygen species, levels of oxidised and reduced glutathione and other thiols, and antioxidant enzymes such as glutathione reductase determine the most suitable conditions for redox control within a cell or for activation of programmed cell death. Additionally, we discuss the translation and expression of glutathione reductase in a number of organisms including yeast and humans. In yeast and human cells, a single gene expresses more than one form of glutathione reductase, destined for residence in the cytoplasm or for translocation to different organelles; in plants, however, two genes encoding this protein have been described. In general, insects and kinetoplastids (a group of protozoa, including Plasmodia and Trypanosoma) do not express glutathione reductase or glutathione biosynthetic enzymes. Instead, they express either the thioredoxin system or the trypanothione system. The thioredoxin system is also present in organisms that have the glutathione system and there may be overlapping functions with cross-talk between the two systems. Finally we evaluate therapeutic targets to overcome oxidative stress associated cellular disorders.

  9. Methylenetetrahydrofolate Reductase C677T: Hypoplastic Left Heart and Thrombosis.

    PubMed

    Spronk, Kimberly J; Olivero, Anthony D; Haw, Marcus P; Vettukattil, Joseph J

    2015-10-01

    The incidence of congenital heart defects is higher in infants with mutation of methylenetetrahydrofolate reductase (MTHFR) gene. The MTHFR C677T gene decreases the bioavailability of folate and increases plasma homocysteine, a risk factor for thrombosis. There have been no reported cases in the literature on the clinical implications of this procoagulable state in the setting of cyanotic heart disease, which itself has prothrombotic predisposition. Two patients with hypoplastic left heart syndrome developed postoperative thrombotic complications, both were homozygous for MTHFR C677T. We present these cases and highlight the implications of MTHFR mutation in the management of complex congenital heart disease.

  10. The respiratory arsenate reductase from Bacillus selenitireducens strain MLS10

    USGS Publications Warehouse

    Afkar, E.; Lisak, J.; Saltikov, C.; Basu, P.; Oremland, R.S.; Stolz, J.F.

    2003-01-01

    The respiratory arsenate reductase from the Gram-positive, haloalkaliphile, Bacillus selenitireducens strain MLS10 was purified and characterized. It is a membrane bound heterodimer (150 kDa) composed of two subunits ArrA (110 kDa) and ArrB (34 kDa), with an apparent Km for arsenate of 34 ??M and Vmax of 2.5 ??mol min-1 mg-1. Optimal activity occurred at pH 9.5 and 150 g l-1 of NaCl. Metal analysis (inductively coupled plasma mass spectrometry) of the holoenzyme and sequence analysis of the catalytic subunit (ArrA; the gene for which was cloned and sequenced) indicate it is a member of the DMSO reductase family of molybdoproteins. ?? 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

  11. Two fatty acyl reductases involved in moth pheromone biosynthesis

    PubMed Central

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

    2016-01-01

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

  12. Cysteine-286 as the site of acylation of the Lux-specific fatty acyl-CoA reductase.

    PubMed

    Lee, C Y; Meighen, E A

    1997-04-04

    The channelling of fatty acids into the fatty aldehyde substrate for the bacterial bioluminescence reaction is catalyzed by a fatty acid reductase multienzyme complex, which channels fatty acids through the thioesterase (LuxD), synthetase (LuxE) and reductase (LuxC) components. Although all three components can be readily acylated in extracts of different luminescent bacteria, this complex has been successfully purified only from Photobacterium phosphoreum and the sites of acylation identified on LuxD and LuxE. To identify the acylation site on LuxC, the nucleotide sequence of P. phosphoreum luxC has been determined and the gene expressed in a mutant Escherichia coli strain. Even in crude extracts, the acylated reductase intermediate as well as acyl-CoA reductase activity could be readily detected, providing the basis for analysis of mutant reductases. Comparison of the amino-acid sequences of LuxC from P. phosphoreum, P. leiognathi and other luminescent bacteria, showed that only three cysteine residues (C171, C279, and C286) were conserved. As a cysteine residue on LuxC has been implicated in fatty acyl transfer, each of the conserved cysteine residues of the P. phosphoreum and P. leiognathi reductases was converted to a serine residue, and the properties of the mutant proteins examined. Only mutation of C286-blocked reductase activity and prevented formation of the acylated reductase intermediate, showing that C286 is the site of acylation on LuxC.

  13. Neuroprotective role for carbonyl reductase?

    PubMed

    Maser, Edmund

    2006-02-24

    Oxidative stress is increasingly implicated in neurodegenerative disorders including Alzheimer's, Parkinson's, Huntington's, and Creutzfeld-Jakob diseases or amyotrophic lateral sclerosis. Reactive oxygen species seem to play a significant role in neuronal cell death in that they generate reactive aldehydes from membrane lipid peroxidation. Several neuronal diseases are associated with increased accumulation of abnormal protein adducts of reactive aldehydes, which mediate oxidative stress-linked pathological events, including cellular growth inhibition and apoptosis induction. Combining findings on neurodegeneration and oxidative stress in Drosophila with studies on the metabolic characteristics of the human enzyme carbonyl reductase (CR), it is clear now that CR has a potential physiological role for neuroprotection in humans. Several lines of evidence suggest that CR represents a significant pathway for the detoxification of reactive aldehydes derived from lipid peroxidation and that CR in humans is essential for neuronal cell survival and to confer protection against oxidative stress-induced brain degeneration.

  14. Androgen Regulation of 5α-Reductase Isoenzymes in Prostate Cancer: Implications for Prostate Cancer Prevention

    PubMed Central

    Li, Jin; Ding, Zhiyong; Wang, Zhengxin; Lu, Jing-Fang; Maity, Sankar N.; Navone, Nora M.; Logothetis, Christopher J.; Mills, Gordon B.; Kim, Jeri

    2011-01-01

    The enzyme 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), performs key functions in the androgen receptor (AR) signaling pathway. The three isoenzymes of 5α-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of 5α-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type–specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE) on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of 5α-reductase isoenzymes may confer response or resistance to 5α-reductase inhibitors and thus may have importance in prostate cancer prevention. PMID:22194926

  15. Aldose reductases influence prostaglandin F2α levels and adipocyte differentiation in male mouse and human species.

    PubMed

    Pastel, Emilie; Pointud, Jean-Christophe; Loubeau, Gaëlle; Dani, Christian; Slim, Karem; Martin, Gwenaëlle; Volat, Fanny; Sahut-Barnola, Isabelle; Val, Pierre; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2015-05-01

    Aldose reductases (AKR1B) are widely expressed oxidoreductases whose physiological function remains elusive. Some isoforms are genuine prostaglandin F2α (PGF2α) synthases, suggesting they might influence adipose homeostasis because PGF2α inhibits adipogenesis. This was shown by Akr1b7 gene ablation in the mouse, which resulted in increased adiposity related to a lower PGF2α content in fat. Yet humans have no ortholog gene for Akr1b7, so the role of aldose reductases in human adipose homeostasis remains to be explored. We analyzed expression of genes encoding human and mouse aldose reductase isoforms in adipose tissues and differentiating adipocytes to assess conserved mechanisms regulating PGF2α synthesis and adipogenesis. The Akr1b3 gene encoded the most abundant isoform in mouse adipose tissue, whereas Akr1b7 encoded the only isoform enriched in the stromal vascular fraction. Most mouse aldose reductase gene expression peaked in early adipogenesis of 3T3-L1 cells and diminished with differentiation. In contrast with its mouse ortholog Akr1b3, AKR1B1 expression increased throughout differentiation of human multipotent adipose-derived stem cells, paralleling PGF2α release, whereas PGF2α receptor (FP) levels collapsed in early differentiation. Pharmacological inhibition of aldose reductase using Statil altered PGF2α production and enhanced human multipotent adipose-derived stem adipocyte differentiation. As expected, the adipogenic effects of Statil were counteracted by an FP agonist (cloprostenol). Thus, in both species aldose reductase-dependent PGF2α production could be important in early differentiation to restrict adipogenesis. PGF2α antiadipogenic signaling could then be toned down through the FP receptor or aldose reductases down-regulation in human and mouse cells, respectively. Our data suggest that aldose reductase inhibitors could have obesogenic potential.

  16. Polymorphisms of methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), and thymidylate synthase (TYMS) in multiple myeloma risk.

    PubMed

    Lima, Carmen S P; Ortega, Manoela M; Ozelo, Margareth C; Araujo, Renato C; De Souza, Cármino A; Lorand-Metze, Irene; Annichino-Bizzacchi, Joyce M; Costa, Fernando F

    2008-03-01

    We tested whether the polymorphisms of the methylenetetrahydrofolate reductase gene, MTHFR C677T and A1298C, the methionine synthase gene, MTR A2756G, the methionine synthase reductase gene, MTRR A66G, and the thymidylate synthase gene, TYMS 2R-->3R, involved in folate and methionine metabolism, altered the risk for multiple myeloma (MM). Genomic DNA from 123MM patients and 188 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. The frequency of the MTR 2756 AG plus GG genotype was higher in patients than in controls (39.8% versus 23.4%, P=0.001). Individual carriers of the variant allele G had a 2.31 (95% CI: 1.38-3.87)-fold increased risk for MM compared with others. In contrast, similar frequencies of the MTHFR, the MTRR and the TYMS genotypes were seen in patients and controls. These results suggest, for the first time, a role for the MTR A2756G polymorphism in MM risk in our country, but should be confirmed by large-scale epidemiological studies with patients and controls age matched.

  17. Rnr4p, a novel ribonucleotide reductase small-subunit protein.

    PubMed Central

    Wang, P J; Chabes, A; Casagrande, R; Tian, X C; Thelander, L; Huffaker, T C

    1997-01-01

    Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are alpha2beta2 tetramers; each of the larger, alpha subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, beta subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode alpha subunits and one gene, RNR2, that encodes a beta subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the beta-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex. PMID:9315671

  18. Genetics Home Reference: 5-alpha reductase deficiency

    MedlinePlus

    ... About half of these individuals adopt a male gender role in adolescence or early adulthood. Related Information ... 1730-5. Citation on PubMed Cohen-Kettenis PT. Gender change in 46,XY persons with 5alpha-reductase- ...

  19. Mutational Spectrum of Smith-Lemli-Opitz Syndrome Patients in Hungary

    PubMed Central

    Balogh, I.; Koczok, K.; Szabó, G.P.; Török, O.; Hadzsiev, K.; Csábi, G.; Balogh, L.; Dzsudzsák, E.; Ajzner, É.; Szabó, L.; Csákváry, V.; Oláh, A.V.

    2012-01-01

    Smith-Lemli-Opitz (SLO) syndrome is an autosomal recessive disorder characterized by multiple congenital abnormalities and mental retardation. The condition is caused by the deficiency of 7-dehydrocholesterol reductase (DHCR7) which catalyzes the final step in cholesterol biosynthesis. Biochemical diagnosis is based on increased concentration of 7-dehydrocholesterol (7-DHC) in the patient serum. Both life expectancy and quality of life are severely affected by the disease. The estimated prevalence of SLO syndrome ranges between 1:20,000 and 1:40,000 among Caucasians. Although the mutational spectrum of the disease is wide, approximately 10 mutations are responsible for more than 80% of the cases. These mutations show a large interethnic variability. There are no mutation distribution data from Hungary to date. Thirteen patients were diagnosed with SLO syndrome in our laboratory. As first-line tests, serum 7-DHC and total cholesterol were measured and, in positive cases, molecular genetic analysis of the DHCR7 gene was performed. Complete genetic background of the disease could be identified in 12 cases. In 1 case only 1 mutation was detected in a heterozygote form. One patient was homozygous for the common splice site mutation c.964–1G>C, while all other patients were compound heterozygotes. One novel missense mutation, c.374A>G (p.Tyr125Cys) was identified. PMID:23293579

  20. A dissimilatory nitrite reductase in Paracoccus halodenitrificans

    NASA Technical Reports Server (NTRS)

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

    1984-01-01

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

  1. Characterization of thyroidal glutathione reductase

    SciTech Connect

    Raasch, R.J.

    1989-01-01

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

  2. Use of a Simple, Colorimetric Assay to Demonstrate Conditions for Induction of Nitrate Reductase in Plants.

    ERIC Educational Resources Information Center

    Harley, Suzanne M.

    1993-01-01

    Nitrate assimilation by plants provides an excellent system for demonstrating control of gene expression in a eukaryotic organism. Describes an assay method that allows students to complete experiments designed around the measurement of nitrate reductase within a three-hour laboratory experiment. (PR)

  3. Chicken muscle aldose reductase: purification, properties and relationship to other chicken aldo/keto reductases.

    PubMed

    Murphy, D G; Davidson, W S

    1986-01-01

    An enzyme that catalyzes the NADPH-dependent reduction of a wide range of aromatic and hydroxy-aliphatic aldehydes was purified from chicken breast muscle. This enzyme shares many properties with mammalian aldose reductases including molecular weight, relative substrate specificity, Michaelis constants, an inhibitor specificity. Therefore, it seems appropriate to call this enzyme an aldose reductase (EC 1.1.1.21). Chicken muscle aldose reductase appears to be kinetically identical to an aldose reductase that has been purified from chicken kidney (Hara et al., Eur. J. Biochem. 133, 207-214) and to hen muscle L-glycol dehydrogenase (Bernado et al., Biochim. biophys. Acta 659, 189-198). The association of this aldose reductase with muscular dystrophy in the chick is discussed.

  4. Properties of the arsenate reductase of plasmid R773.

    PubMed

    Gladysheva, T B; Oden, K L; Rosen, B P

    1994-06-14

    Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest.

  5. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

    PubMed

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-07-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

  6. Organization of dimethyl sulfoxide reductase in the plasma membrane of Escherichia coli.

    PubMed Central

    Sambasivarao, D; Scraba, D G; Trieber, C; Weiner, J H

    1990-01-01

    Dimethyl sulfoxide reductase is a trimeric, membrane-bound, iron-sulfur molybdoenzyme induced in Escherichia coli under anaerobic growth conditions. The enzyme catalyzes the reduction of dimethyl sulfoxide, trimethylamine N-oxide, and a variety of S- and N-oxide compounds. The topology of dimethyl sulfoxide reductase subunits was probed by a combination of techniques. Immunoblot analysis of the periplasmic proteins from the osmotic shock and chloroform wash fluids indicated that the subunits were not free in the periplasm. The reductase was susceptible to proteases in everted membrane vesicles, but the enzyme in outer membrane-permeabilized cells became protease sensitive only after detergent solubilization of the E. coli plasma membrane. Lactoperoxidase catalyzed the iodination of each of the three subunits in an everted membrane vesicle preparation. Antibodies to dimethyl sulfoxide reductase and fumarate reductase specifically agglutinated the everted membrane vesicles. No TnphoA fusions could be found in the dmsA or -B genes, indicating that these subunits were not translocated to the periplasm. Immunogold electron microscopy of everted membrane vesicles and thin sections by using antibodies to the DmsABC, DmsA, DmsB subunits resulted in specific labeling of the cytoplasmic surface of the inner membrane. These results show that the DmsA (catalytic subunit) and DmsB (electron transfer subunit) are membrane-extrinsic subunits facing the cytoplasmic side of the plasma membrane. Images PMID:2170332

  7. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

    PubMed Central

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-01-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control. PMID:10861207

  8. Regulation of 5alpha-reductase isoforms by oxytocin in the rat ventral prostate.

    PubMed

    Assinder, S J; Johnson, C; King, K; Nicholson, H D

    2004-12-01

    Oxytocin (OT) is present in the male reproductive tract, where it is known to modulate contractility, cell growth, and steroidogenesis. Little is known about how OT regulates these processes. This study describes the localization of OT receptor in the rat ventral prostate and investigates if OT regulates gene expression and/or activity of 5alpha-reductase isoforms I and II. The ventral prostates of adult male Wistar rats were collected following daily sc administration of saline (control), OT, a specific OT antagonist or both OT plus antagonist for 3 d. Expression of the OT receptor was identified in the ventral prostate by RT-PCR and Western blot, and confirmed to be a single active binding site by radioreceptor assay. Immunohistochemistry localized the receptor to the epithelium of prostatic acini and to the stromal tissue. Real-time RT-PCR determined that OT treatment significantly reduced expression of 5alpha-reductase I but significantly increased 5alpha-reductase II expression in the ventral prostate. Activity of both isoforms of 5alpha-reductase was significantly increased by OT, resulting in increased concentration of prostatic dihydrotestosterone. In conclusion, OT is involved in regulating conversion of testosterone to the biologically active dihydrotestosterone in the rat ventral prostate. It does so by differential regulation of 5alpha-reductase isoforms I and II.

  9. Respiratory arsenate reductase as a bidirectional enzyme

    USGS Publications Warehouse

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

    2009-01-01

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

  10. Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases.

    PubMed

    Sattler, Steven A; Walker, Alexander M; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee

    2017-02-01

    Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species.

  11. The tyrosyl free radical in ribonucleotide reductase.

    PubMed Central

    Gräslund, A; Sahlin, M; Sjöberg, B M

    1985-01-01

    The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins B1 and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of iron-radical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase. The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative. In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis. Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical. PMID:3007085

  12. Cloning, molecular characterization and functional analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza Bge. f. alba.

    PubMed

    Hao, Gangping; Shi, Renjiu; Tao, Ru; Fang, Qian; Jiang, Xingyu; Ji, Haiwei; Feng, Lei; Huang, Luqi

    2013-09-01

    The enzyme 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) is a terminal-acting enzyme in the plastid MEP pathway, which produce isoprenoid precursors. The full-length cDNA of HDR, designated SmHDR1 (Genbank Accession No. JX516088), was isolated for the first time from Salvia miltiorrhiza Bge. f. alba. SmHDR1 contains a 1389-bp open reading frame encoding 463 amino acids. The deduced SmHDR1 protein, which shows high identity to HDRs of other plant species, is predicted to possess a chloroplast transit peptide at the N-terminus and four conserved cysteine residues. Transcription pattern analysis revealed that SmHDR1 has high levels of transcription in leaves and low levels of transcription in roots and stems. The expression of SmHDR1 was induced by 0.1 mM methyl-jasmonate (MeJA) and salicylic acid (SA), but not by 0.1 mM abscisic acid (ABA), in the hairy roots of S. miltiorrhiza Bge. f. alba. Complementation of SmHDR1 in the Escherichia coli HDR mutant MG1655 ara < > ispH demonstrated the function of this enzyme. A functional color assay in E. coli showed that SmHDR1 accelerates the biosynthesis of β-carotene, indicating that SmHDR1 encodes a functional protein. Overexpression of SmHDR1 enhanced the production of tanshinones in cultured hairy roots of S. miltiorrhiza Bge. f. alba. These results indicate that SmHDR1 is a novel and important enzyme involved in the biosynthesis of diterpenoid tanshinones in S. miltiorrhiza Bge. f. alba.

  13. Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, intakes of folate and related B vitamins and colorectal cancer: a case-control study in a population with relatively low folate intake.

    PubMed

    Sharp, Linda; Little, Julian; Brockton, Nigel T; Cotton, Seonaidh C; Masson, Lindsey F; Haites, Neva E; Cassidy, Jim

    2008-02-01

    Folate is key in one-carbon metabolism, disruption of which can interfere with DNA synthesis, repair, and methylation. Efficient one-carbon metabolism requires other B vitamins and the optimal activity of enzymes including 5,10-methylenetetrahydrofolate reductase (MTHFR). We report a population-based case-control study of folate intake, related dietary factors and MTHFR polymorphisms (C677T, A1298C) and colorectal cancer in a population with relatively high colorectal cancer incidence and relatively low folate intake. A total of 264 cases with histologically confirmed incident colorectal cancer and 408 controls participated. There was no clear trend in risk with reported intakes of total, or dietary, folate, riboflavin, vitamin B12 or vitamin B6, nor were there interactions between folate intake and the other B vitamins or alcohol. For C677T, risk decreased with increasing variant alleles (multivariate OR for CT v. CC = 0.77 (95 % CI 0.52, 1.16); OR for TT v. CC = 0.62 (95 % CI 0.31, 1.24)), which, although not statistically significant, was consistent with previous studies. For A1298C, compared with AA subjects, CC subjects had modest, non-significant, reduced risk (multivariate OR = 0.81 (95 % CI 0.45, 1.49)). There were significant interactions between total folate and C677T (P = 0.029) and A1298C (P = 0.025), and total vitamin B6 and both polymorphisms (C677T, P = 0.016; A1298C, P = 0.033), although the patterns observed differed from previous studies. Seen against the setting of low folate intake, the results suggest that the role of folate metabolism in colorectal cancer aetiology may be more complex than previously thought. Investigation of particular folate vitamers (for example, tetrahydrofolate, 5,10-methylenetetrahydrofolate) may help clarify carcinogenesis pathways.

  14. The polymorphisms in methylenetetrahydrofolate reductase, methionine synthase, methionine synthase reductase, and the risk of colorectal cancer.

    PubMed

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results.

  15. The Polymorphisms in Methylenetetrahydrofolate Reductase, Methionine Synthase, Methionine Synthase Reductase, and the Risk of Colorectal Cancer

    PubMed Central

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results. PMID:22719222

  16. Evaluation of nitrate reductase activity in Rhizobium japonicum

    SciTech Connect

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

    1983-08-01

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

  17. Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

    PubMed

    Barber, M J; Quinn, G B

    2001-11-01

    A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities

  18. Nitrate metabolism in tobacco leaves overexpressing Arabidopsis nitrite reductase.

    PubMed

    Davenport, Susie; Le Lay, Pascaline; Sanchez-Tamburrrino, Juan Pablo

    2015-12-01

    Primary nitrogen assimilation in plants includes the reduction of nitrite to ammonium in the chloroplasts by the enzyme nitrite reductase (NiR EC:1.7.7.1) or in the plastids of non-photosynthetic organs. Here we report on a study overexpressing the Arabidopsis thaliana NiR (AtNiR) gene in tobacco plants under the control of a constitutive promoter (CERV - Carnation Etched Ring Virus). The aim was to overexpress AtNiR in an attempt to alter the level of residual nitrite in the leaf which can act as precursor to the formation of nitrosamines. The impact of increasing the activity of AtNiR produced an increase in leaf protein and a stay-green phenotype in the primary transformed AtNiR population. Investigation of the T1 homozygous population demonstrated elevated nitrate reductase (NR) activity, reductions in leaf nitrite and nitrate and the amino acids proline, glutamine and glutamate. Chlorophyl content of the transgenic lines was increased, as evidenced by the stay-green phenotype. This reveals the importance of NiR in primary nitrogen assimilation and how modification of this key enzyme affects both the nitrogen and carbon metabolism of tobacco plants.

  19. Post-translational Regulation of Nitrate Reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. Fumarate Reductase Activity of Streptococcus faecalis

    PubMed Central

    Aue, B. J.; Diebel, R. H.

    1967-01-01

    Some characteristics of a fumarate reductase from Streptococcus faecalis are described. The enzyme had a pH optimum of 7.4; optimal activity was observed when the ionic strength of the phosphate buffer was adjusted to 0.088. The Km value of the enzyme for reduced flavin mononucleotide was 2 × 10−4 m as determined with a 26-fold preparation. In addition to fumarate, the enzyme reduced maleate and mesaconate. No succinate dehydrogenase activity was detected, but succinate did act as an inhibitor of the fumarate reductase activity. Other inhibitors were malonate, citraconate, and trans-, trans-muconate. Metal-chelating agents did not inhibit the enzyme. A limited inhibition by sulfhydryl-binding agents was observed, and the preparations were sensitive to air oxidation and storage. Glycine, alanine, histidine, and possibly lysine stimulated fumarate reductase activity in the cell-free extracts. However, growth in media supplemented with glycine did not enhance fumarate reductase activity. The enzymatic activity appears to be constitutive. PMID:4960892

  1. Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri.

    PubMed

    Arese, Marzia; Zumft, Walter G; Cutruzzolà, Francesca

    2003-01-01

    Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.

  2. Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica

    PubMed Central

    Mock, Johanna; Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

    2014-01-01

    Moorella thermoacetica can grow with H2 and CO2, forming acetic acid from 2 CO2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown. PMID:25002540

  3. Cloning, expression and characterization of a putative 2,5-diketo-D-gluconic acid reductase in Comamonas testosteroni.

    PubMed

    Chen, Yuanan; Ji, Wei; Zhang, Hao; Zhang, Xiao; Yu, Yuanhua

    2015-06-05

    Aldo-keto reductases (AKRs) are a superfamily of soluble NAD(P)(H) oxidoreductases. The function of the enzymes is to reduce aldehydes and ketones into primary and secondary alcohols. We have cloned a 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene from Comamonas testosteroni (C. testosteroni) ATCC11996 (a Gram-negative bacterium which can use steroids as carbon and energy source) into plasmid pET-15b and over expressed in Escherichia coli BL21 (DE3). The protein was purified by His-tag Metal chelating affinity chromatography column. The 2,5-diketo-D-gluconic acid reductase (2,5DKGR) gene contains 1062 bp and could be translated into a protein of 353 amino acid residues. Three consensus sequences of the AKR superfamily are found as GxxxxDxAxxY, LxxxGxxxPxxGxG and LxxxxxxxxxDxxxxH. GxxxxDxAxxY is the active site, LxxxGxxxPxxGxG is the Cofactor-binding site for NAD(P)(H), LxxxxxxxxxDxxxxH is used for supporting the 3D structure. 2,5-diketo-D-gluconic acid reductase gene of C. testosteroni was knocked out and a mutant M-AKR was obtained. Compared to wild type C. testosteroni, degradations of testosterone, estradiol, oestrone and methyltestosterone in mutant M-AKR were decreased. Therefore, 2,5-diketo-D-gluconic acid reductase in C. testosteroni is involved in steroid degradation.

  4. Physiological Roles for Two Periplasmic Nitrate Reductases in Rhodobacter sphaeroides 2.4.3 (ATCC 17025)▿

    PubMed Central

    Hartsock, Angela; Shapleigh, James P.

    2011-01-01

    The metabolically versatile purple bacterium Rhodobacter sphaeroides 2.4.3 is a denitrifier whose genome contains two periplasmic nitrate reductase-encoding gene clusters. This work demonstrates nonredundant physiological roles for these two enzymes. One cluster is expressed aerobically and repressed under low oxygen while the second is maximally expressed under low oxygen. Insertional inactivation of the aerobically expressed nitrate reductase eliminated aerobic nitrate reduction, but cells of this strain could still respire nitrate anaerobically. In contrast, when the anaerobic nitrate reductase was absent, aerobic nitrate reduction was detectable, but anaerobic nitrate reduction was impaired. The aerobic nitrate reductase was expressed but not utilized in liquid culture but was utilized during growth on solid medium. Growth on a variety of carbon sources, with the exception of malate, the most oxidized substrate used, resulted in nitrite production on solid medium. This is consistent with a role for the aerobic nitrate reductase in redox homeostasis. These results show that one of the nitrate reductases is specific for respiration and denitrification while the other likely plays a role in redox homeostasis during aerobic growth. PMID:21949073

  5. The Saccharomyces cerevisiae aldose reductase is implied in the metabolism of methylglyoxal in response to stress conditions.

    PubMed

    Aguilera, J; Prieto, J A

    2001-07-01

    The enzyme aldose reductase plays an important role in the osmo-protection mechanism of diverse organisms. Here, we show that yeast aldose reductase is encoded by the GRE3 gene. Expression of GRE3 is carbon-source independent and up-regulated by different stress conditions, such as NaCl, H2O2, 39 degrees C and carbon starvation. Measurements of enzyme activity and intracellular sorbitol in wild-type cells also indicate that yeast aldose reductase is stress-regulated. Overexpression of GRE3 increases methylglyoxal tolerance in Saccharomyces cerevisiae. Furthermore, high expression of GRE3 complements the deficiency of the glyoxalase system of a glo1delta mutant strain. Consistent with this, in vitro and in vivo assays of yeast aldose reductase activity indicate that methylglyoxal is an endogenous substrate of aldose reductase. Furthermore, addition of NaCl or H2O2 to exponential-phase cells triggers an initial transient increase in the intracellular level of methylglyoxal, which is dependent on the Gre3p and Glo1p function. These observations indicate that the metabolism of methylglyoxal is stimulated under stress conditions; and they support a methylglyoxal degradative pathway, in which this compound is metabolised by the action of aldose reductase.

  6. The Smith-Lemli-Opitz syndrome

    PubMed Central

    Kelley, R.; Hennekam, R.

    2000-01-01

    The Smith-Lemli-Opitz syndrome (SLOS) is one of the archetypical multiple congenital malformation syndromes. The recent discovery of the biochemical cause of SLOS and the subsequent redefinition of SLOS as an inborn error of cholesterol metabolism have led to important new treatment possibilities for affected patients. Moreover, the recent recognition of the important role of cholesterol in vertebrate embryogenesis, especially with regard to the hedgehog embryonic signalling pathway and its effects on the expression of homeobox genes, has provided an explanation for the abnormal morphogenesis in the syndrome. The well known role of cholesterol in the formation of steroid hormones has also provided a possible explanation for the abnormal behavioural characteristics of SLOS.


Keywords: Smith-Lemli-Opitz syndrome; cholesterol metabolism; 7-dehydrocholesterol reductase; clinical history; management PMID:10807690

  7. Augmentation of CFTR maturation by S-nitrosoglutathione reductase

    PubMed Central

    Sawczak, Victoria; Zaidi, Atiya; Butler, Maya; Bennett, Deric; Getsy, Paulina; Zeinomar, Maryam; Greenberg, Zivi; Forbes, Michael; Rehman, Shagufta; Jyothikumar, Vinod; DeRonde, Kim; Sattar, Abdus; Smith, Laura; Corey, Deborah; Straub, Adam; Sun, Fei; Palmer, Lisa; Periasamy, Ammasi; Randell, Scott; Kelley, Thomas J.; Lewis, Stephen J.

    2015-01-01

    S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o−) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o− cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions. PMID:26637637

  8. Molecular cloning and characterization of a novel Dehydrogenase/reductase (SDR family) member 1 genea from human fetal brain.

    PubMed

    Wu, Q; Xu, M; Cheng, C; Zhou, Z; Huang, Y; Zhao, W; Zeng, L; Xu, J; Fu, X; Ying, K; Xie, Y; Mao, Y

    2001-01-01

    Short-chain dehydrogenases/reductases (SDR) constitute a large protein family of NAD(P)(H)-dependent oxidoreductase. They are defined by distinct, common sequence motifs and show a wide range of substrate specialisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human SDR-type dehydrogenase/reductase gene named Dehydrogenase/reductase (SDR family) member 1 (DHRS1). The DHRS1 cDNA is 1411 base pair in length, encoding a 314-amino-acid polypeptide which has a SDR motif. Northern blot reveals two bands, of about 0.9 and 1.4 kb in size. These two forms are expressed in many tissues. The DHRS1 gene is localized on chromosome 14q21.3. It has 9 exons and spans 9.2 kb of the genomic DNA.

  9. B-vitamins, methylenetetrahydrofolate reductase (MTHFR) and hypertension.

    PubMed

    Ward, Mary; Wilson, Carol P; Strain, J J; Horigan, Geraldine; Scott, John M; McNulty, Helene

    2011-07-01

    Hypertension is a leading risk factor for cardiovascular disease (CVD) and stroke. A common polymorphism in the gene encoding the enzyme methylenetetrahydrofolate reductase (MTHFR), previously identified as the main genetic determinant of elevated homocysteine concentration and also recognized as a risk factor for CVD, appears to be independently associated with hypertension. The B-vitamin riboflavin is required as a cofactor by MTHFR and recent evidence suggests it may have a role in modulating blood pressure, specifically in those with the homozygous mutant MTHFR 677 TT genotype. If studies confirm that this genetic predisposition to hypertension is correctable by low-dose riboflavin, the findings could have important implications for the management of hypertension given that the frequency of this polymorphism ranges from 3 to 32 % worldwide.

  10. [Molecular characterizations of two dehydroascorbate reductases from Selaginella moellendorffii].

    PubMed

    Cheng, Zishuo; Lan, Ting; Li, Di; Yang, Hailing; Zeng, Qingyin

    2011-01-01

    Plant dehydroascorbate reductase (DHAR) is a physiologically important reducing enzyme in the ascorbate-glutathione recycling reaction. In this study, two DHARs genes (SmDHAR1 and SmDHAR2) were isolated from Selaginella moellendorffii. The SmDHAR1 and SmDHAR2 genes encode two proteins of 218 and 241 amino acid residues, with a calculated molecular mass of 23.97 kDa and 27.33 kDa, respectively. The genomic sequence analysis showed SmDHAR1 and SmDHAR2 contained five and six introns, respectively. Reverse transcription PCR revealed that the SmDHAR1 and SmDHAR2 were constitutive expression genes in S. moellendorffii. The recombinant SmDHAR1 and SmDHAR2 proteins were overexpressed in E. coli, and were purified by Ni-affinity chromatography. The recombinant SmDHAR1 showed 116-fold higher enzymatic activity towards the substrate dehydroascorbate than recombinant SmDHAR2. The recombinant SmDHAR1 showed higher thermal stability than recombinant SmDHAR2. These results indicated obvious functional divergence between the duplicate genes SmDHAR1 and SmDHAR2.

  11. Molecular Cloning of Complementary DNA Encoding Maize Nitrite Reductase

    PubMed Central

    Lahners, Kristine; Kramer, Vance; Back, Eduard; Privalle, Laura; Rothstein, Steven

    1988-01-01

    Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize. Images Fig. 3 Fig. 4 Fig. 5 PMID:16666376

  12. FRUCTOSE-6-PHOSPHATE REDUCTASE FROM SALMONELLA GALLINARUM

    PubMed Central

    Zancan, Glaci T.; Bacila, Metry

    1964-01-01

    Zancan, Glaci T. (Universidade do Paraná, Curitiba, Paraná, Brazil), and Metry Bacila. Fructose-6-phosphate reductase from Salmonella gallinarum. J. Bacteriol. 87:614–618. 1964.—A fructose-6-phosphate reductase present in cell-free extracts of Salmonella gallinarum was purified approximately 42 times. The optimal pH for this enzyme is 8.0. The enzyme is specific for fructose-6-phosphate and reduced nicotinamide adenine dinucleotide (NADH). The dissociation constants are 1.78 × 10−4m for fructose-6-phosphate and 8.3 × 10−5m for NADH. The Q10, reaction order, and equilibrium constant were determined. The enzyme is sensitive to p-chloromercuribenzoic acid, but not to o-iodosobenzoic acid nor to N-ethylmaleimide. PMID:14127579

  13. Type I and type II fatty acid biosynthesis in Eimeria tenella: enoyl reductase activity and structure.

    PubMed

    Lu, J Z; Muench, S P; Allary, M; Campbell, S; Roberts, C W; Mui, E; McLeod, R L; Rice, D W; Prigge, S T

    2007-12-01

    Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

  14. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

    PubMed Central

    Dimster-Denk, D; Thorsness, M K; Rine, J

    1994-01-01

    In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation. Images PMID:7949422

  15. Structural characterization and functional validation of aldose reductase from the resurrection plant Xerophyta viscosa.

    PubMed

    Singh, Preeti; Sarin, Neera Bhalla

    2014-11-01

    Aldose reductases are key enzymes in the detoxification of reactive aldehyde compounds like methylglyoxal (MG) and malondialdehyde. The present study describes for first time the preliminary biochemical and structural characterization of the aldose reductase (ALDRXV4) enzyme from the resurrection plant Xerophyta viscosa. The ALDRXV4 cDNA was expressed in E. coli using pET28a expression vector, and the protein was purified using affinity chromatography. The recombinant protein showed a molecular mass of ~36 kDa. The K M (1.2 mM) and k cat (14.5 s(-1)) of the protein determined using MG as substrate was found to be comparable with other reported homologs. Three-dimensional structure prediction based on homology modeling suggested several similarities with the other aldose reductases reported from plants. Circular dichroism spectroscopy results supported the bioinformatic prediction of alpha-beta helix nature of aldose reductase proteins. Subcellular localization studies revealed that the ALDRXV4-GFP fusion protein was localized both in the nucleus and the cytoplasm. The E. coli cells overexpressing ALDRXV4 exhibited improved growth and showed tolerance against diverse abiotic stresses induced by high salt (500 mM NaCl), osmoticum (10 % PEG 6000), heavy metal (20 mM CdCl2), and MG (5 mM). Based on these results, we propose that ALDRXV4 gene from X. viscosa could be a potential candidate for developing stress-tolerant crop plants.

  16. Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones

    PubMed Central

    Nie, Yao; Xiao, Rong; Xu, Yan; Montelione, Gaetano T.

    2014-01-01

    Application of biocatalysis in the synthesis of chiral molecules is one of the greenest technologies for the replacement of chemical routes due to its environmentally benign reaction conditions and unparalleled chemo-, regio-and stereoselectivities. We have been interested in searching for carbonyl reductase enzymes and assessing their substrate specificity and stereoselectivity. We now report a gene cluster identified in Candida parapsilosis that consists of four open reading frames including three putative stereospecific carbonyl reductases (scr1, scr2, and scr3) and an alcohol dehydrogenase (cpadh). These newly identified three stereospecific carbonyl reductases (SCRs) showed high catalytic activities for producing (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone with NADPH as the coenzyme. Together with CPADH, all four enzymes from this cluster are carbonyl reductases with novel anti-Prelog stereoselectivity. SCR1 and SCR3 exhibited distinct specificities to acetophenone derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities to ethyl 4-chloro-3-oxobutyrate, a β-ketoester with important pharmaceutical potentials. Our study also showed that genomic mining is a powerful tool for the discovery of new enzymes. PMID:21505708

  17. Novel anti-Prelog stereospecific carbonyl reductases from Candida parapsilosis for asymmetric reduction of prochiral ketones.

    PubMed

    Nie, Yao; Xiao, Rong; Xu, Yan; Montelione, Gaetano T

    2011-06-07

    The application of biocatalysis to the synthesis of chiral molecules is one of the greenest technologies for the replacement of chemical routes due to its environmentally benign reaction conditions and unparalleled chemo-, regio- and stereoselectivities. We have been interested in searching for carbonyl reductase enzymes and assessing their substrate specificity and stereoselectivity. We now report a gene cluster identified in Candida parapsilosis that consists of four open reading frames including three putative stereospecific carbonyl reductases (scr1, scr2, and scr3) and an alcohol dehydrogenase (cpadh). These newly identified three stereospecific carbonyl reductases (SCRs) showed high catalytic activities for producing (S)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone with NADPH as the coenzyme. Together with CPADH, all four enzymes from this cluster are carbonyl reductases with novel anti-Prelog stereoselectivity. SCR1 and SCR3 exhibited distinct specificities to acetophenone derivatives and chloro-substituted 2-hydroxyacetophenones, and especially very high activities towards ethyl 4-chloro-3-oxobutyrate, a β-ketoester with important pharmaceutical potential. Our study also showed that genomic mining is a powerful tool for the discovery of new enzymes.

  18. Nitrate reduction in Haloferax alexandrinus: the case of assimilatory nitrate reductase.

    PubMed

    Kilic, Volkan; Kilic, Gözde Aydoğan; Kutlu, Hatice Mehtap; Martínez-Espinosa, Rosa María

    2017-03-21

    Haloferax alexandrinus Strain TM JCM 10717(T) = IFO 16590(T) is an extreme halophilic archaeon able to produce significant amounts of canthaxanthin. Its genome sequence has been analysed in this work using bioinformatics tools available at Expasy in order to look for genes encoding nitrate reductase-like proteins: respiratory nitrate reductase (Nar) and/or assimilatory nitrate reductase (Nas). The ability of the cells to reduce nitrate under aerobic conditions was tested. The enzyme in charge of nitrate reduction under aerobic conditions (Nas) has been purified and characterised. It is a monomeric enzyme (72 ± 1.8 kDa) that requires high salt concentration for stability and activity. The optimum pH value for activity was 9.5. Effectiveness of different substrates, electron donors, cofactors and inhibitors was also reported. High nitrite concentrations were detected within the culture media during aerobic/microaerobic cells growth. The main conclusion from the results is that this haloarchaeon reduces nitrate aerobically thanks to Nas and may induce denitrification under anaerobic/microaerobic conditions using nitrate as electron acceptor. The study sheds light on the role played by haloarchaea in the biogeochemical cycle of nitrogen, paying special attention to nitrate reduction processes. Besides, it provides useful information for future attempts on microecological and biotechnological implications of haloarchaeal nitrate reductases.

  19. Phenotypic classification of male pseudohermaphroditism due to steroid 5{alpha}-reductase 2 deficiency

    SciTech Connect

    Sinnecker, G.H.G; Hiort, O.; Kruse, K.; Dibbelt, L.

    1996-05-03

    Conversion of testosterone (T) to dihydrotestosterone (DHT) in genital tissue is catalysed by the enzyme 5{alpha}-reductase 2, which is encoded by the SRD5A2 gene. The potent androgen DHT is required for full masculinization of the external genitalia. Mutations of the SRD5A2 gene inhibit enzyme activity, diminish DHT formation, and hence cause masculinization defects of varying degree. The classical syndrome, formerly described as pseudovaginal perineoscrotal hypospadias, is characterized by a predominantly female phenotype at birth and significant virilization without gynecomastia at puberty. We investigated nine patients with steroid 5{alpha}-reductase 2 deficiency (SRD). T/DHT-ratios were highly increased in the classical syndrome, but variable in the less severe affected patients. Mutations in the SRD5A2 gene had been characterized using PCR-SSCP analysis and direct DNA sequencing. A small deletion was encountered in two patients, while all other patients had single base mutations which result in amino acid substitutions. We conclude that phenotypes may vary widely in patients with SRD5A2 gene mutations spanning the whole range from completely female to normal male without distinctive clinical signs of the disease. Hence, steroid 5{alpha}-reductase deficiency should be considered not only in sex reversed patients with female or ambiguous phenotypes, but also in those with mild symptoms of undermasculinization as encountered in patients with hypospadias and/or micropenis. A classification based on the severity of the masculinization defect may be used for correlation of phenotypes with enzyme activities and genotypes, and for comparisons of phenotypes between different patients as the basis for clinical decisions to be made in patients with pseudohermaphroditism due to steroid 5{alpha}-reductase 2 deficiency. 22 refs., 2 figs., 2 tabs.

  20. A Ferredoxin Disulfide Reductase Delivers Electrons to the Methanosarcina barkeri Class III Ribonucleotide Reductase

    PubMed Central

    2015-01-01

    Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRs reflects the diversity of electron carriers used in anaerobic metabolism. PMID:26536144

  1. Evidence for a Ustilago maydis steroid 5alpha-reductase by functional expression in Arabidopsis det2-1 mutants.

    PubMed

    Basse, Christoph W; Kerschbamer, Christine; Brustmann, Markus; Altmann, Thomas; Kahmann, Regine

    2002-06-01

    We have identified a gene (udh1) in the basidiomycete Ustilago maydis that is induced during the parasitic interaction with its host plant maize (Zea mays). udh1 encodes a protein with high similarity to mammalian and plant 5alpha-steroid reductases. Udh1 differs from those of known 5alpha-steroid reductases by six additional domains, partially predicted to be membrane-spanning. A fusion protein of Udh1 and the green fluorescent protein provided evidence for endoplasmic reticulum localization in U. maydis. The function of the Udh1 protein was demonstrated by complementing Arabidopsis det2-1 mutants, which display a dwarf phenotype due to a mutation in the 5alpha-steroid reductase encoding DET2 gene. det2-1 mutant plants expressing either the udh1 or the DET2 gene controlled by the cauliflower mosaic virus 35S promoter differed from wild-type Columbia plants by accelerated stem growth, flower and seed development and a reduction in size and number of rosette leaves. The accelerated growth phenotype of udh1 transgenic plants was stably inherited and was favored under reduced light conditions. Truncation of the N-terminal 70 amino acids of the Udh1 protein abolished the ability to restore growth in det2-1 plants. Our results demonstrate the existence of a 5alpha-steroid reductase encoding gene in fungi and suggest a common ancestor between fungal, plant, and mammalian proteins.

  2. Design and synthesis of 2-pyridones as novel inhibitors of the Bacillus anthracis enoyl-ACP reductase.

    PubMed

    Tipparaju, Suresh K; Joyasawal, Sipak; Forrester, Sara; Mulhearn, Debbie C; Pegan, Scott; Johnson, Michael E; Mesecar, Andrew D; Kozikowski, Alan P

    2008-06-15

    Enoyl-ACP reductase (ENR), the product of the FabI gene, from Bacillus anthracis (BaENR) is responsible for catalyzing the final step of bacterial fatty acid biosynthesis. A number of novel 2-pyridone derivatives were synthesized and shown to be potent inhibitors of BaENR.

  3. Cytochrome P450 3A, NADPH cytochrome P450 reductase and cytochrome b5 in the upper airways in horse.

    PubMed

    Tydén, E; Olsén, L; Tallkvist, J; Tjälve, H; Larsson, P

    2008-08-01

    Gene and protein expression as well as catalytic activity of cytochrome P450 (CYP) 3A were studied in the nasal olfactory and respiratory mucosa and the tracheal mucosa of the horse. We also examined the activity of NADPH cytochrome P450 reductase (NADPH P450 reductase), the amount of cytochrome b(5) and the total CYP content in these tissues. Comparative values for the above were obtained using liver as a control. The CYP3A related catalytic activity in the tissues of the upper airways was considerably higher than in the liver. The CYP3A gene and protein expression, on the other hand, was higher in the liver than in the upper airway tissues. Thus, the pattern of CYP3A metabolic activity does not correlate with the CYP3A gene and protein expression. Our results showed that the activity of NADPH P450 reductase and the level of cytochrome b(5) in the relation to the gene and protein expression of CYP3A were higher in the tissues of the upper airways than in the liver. It is concluded that CYP3A related metabolism in horse is not solely dependent on the expression of the enzyme but also on adequate levels of NADPH P450 reductase and cytochrome b(5).

  4. Genotypic variation in sulfur assimilation and metabolism of onion (Allium cepa L.) III. Characterization of sulfite reductase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic and cDNA sequences corresponding to a ferredoxin-sulfite reductase (SiR) have been cloned from bulb onion (Allium cepa L.) and the expression of the gene and activity of the enzyme characterised with respect to sulfur (S) supply. Cloning, mapping and expression studies revealed that onion ha...

  5. Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil

    PubMed Central

    Mueller, L.; Hauser, P. M.; Gauye, F.

    2017-01-01

    ABSTRACT Lausannevirus belongs to the family Marseilleviridae within the group of nucleocytoplasmic large DNA viruses (NCLDVs). These giant viruses exhibit unique features, including a large genome, ranging from 100 kb to 2.5 Mb and including from 150 to more than 2,500 genes, as well as the presence of genes coding for proteins involved in transcription and translation. The large majority of Lausannevirus open reading frames have unknown functions. Interestingly, a bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is encoded in the Lausannevirus genome. The enzyme plays central roles in DNA precursor biosynthesis. DHFR is the pharmacological target of antifolates, such as trimethoprim, pyrimethamine, and proguanil. First, the functionality of Lausannevirus DHFR-TS was demonstrated by the successful complementation of a DHFR-deficient Saccharomyces cerevisiae strain with a plasmid expressing the heterologous gene. Additionally, using this heterologous expression system, we demonstrated the in vitro susceptibility of Lausannevirus DHFR-TS to proguanil and its resistance to pyrimethamine and trimethoprim. Proguanil may provide a unique and useful treatment if Lausannevirus proves to be a human pathogen. To our knowledge, this is the first time that a DHFR-TS has been described and characterized in an NCLDV. PMID:28137801

  6. Methionine sulfoxide reductase contributes to meeting dietary methionine requirements

    PubMed Central

    Zhao, Hang; Kim, Geumsoo; Levine, Rodney L.

    2012-01-01

    Methionine sulfoxide reductases are present in all aerobic organisms. They contribute to antioxidant defenses by reducing methionine sulfoxide in proteins back to methionine. However, the actual in vivo roles of these reductases are not well defined. Since methionine is an essential amino acid in mammals, we hypothesized that methionine sulfoxide reductases may provide a portion of the dietary methionine requirement by recycling methionine sulfoxide. We used a classical bioassay, the growth of weanling mice fed diets varying in methionine, and applied it to mice genetically engineered to alter the levels of methionine sulfoxide reductase A or B1. Mice of all genotypes were growth retarded when raised on chow containing 0.10% methionine instead of the standard 0.45% methionine. Retardation was significantly greater in knockout mice lacking both reductases. We conclude that the methionine sulfoxide reductases can provide methionine for growth in mice with limited intake of methionine, such as may occur in the wild. PMID:22521563

  7. Acrolein-Induced Dyslipidemia and Acute Phase Response Independenly of HMG-CoA Reductase

    PubMed Central

    Conklin, Daniel J.; Prough, Russell A.; Juvan, Peter; Rezen, Tadeja; Rozman, Damjana; Haberzettl, Petra; Srivastava, Sanjay; Bhatnagar, Aruni

    2012-01-01

    Scope Aldehydes are ubiquitous natural constituents of foods, water and beverages. Dietary intake represents the greatest source of exposure to acrolein and related aldehydes. Oral acrolein induces dyslipidemia acutely and chronically increases atherosclerosis in mice, yet the mechanisms are unknown. Because lipid synthesis and trafficking are largely under hepatic control, we examined hepatic genes in murine models of acute and chronic oral acrolein exposure. Methods and results Changes in hepatic gene expression were examined using a Steroltalk microarray. Acute acrolein feeding modified plasma and hepatic proteins and increased plasma triglycerides within 15 min. By 6h, acrolein altered hepatic gene expression including Insig1, Insig2 and Hmgcr genes and stimulated an acute phase response (APR) with up-regulation of serum amyloid A genes (Saa) and systemic hypoalbuminemia. To test if decreased HMG-CoA reductase activity could modify acrolein-induced dyslipidemia or the APR, mice were pretreated with simvastatin. Statin treatment, however, did not alter acrolein-induced dyslipidemia or hypoalbuminemia associated with an APR. Few hepatic genes were dysregulated by chronic acrolein feeding in apoE-null mice. These studies confirmed that acute acrolein exposure altered expression of hepatic genes involved with lipid synthesis and trafficking and APR, and thus, indicated a hepatic locus of acrolein-induced dyslipidemia and APR that was independent of HMG CoA-reductase. Conclusion Dietary intake of acrolein could contribute to cardiovascular disease risk by disturbing hepatic function. PMID:21812109

  8. Structural Elucidation of Chalcone Reductase and Implications for Deoxychalcone Biosynthesis

    PubMed Central

    Bomati, Erin K.; Austin, Michael B.; Bowman, Marianne E.; Dixon, Richard A.; Noel, Joseph P.

    2010-01-01

    4,2′,4′,6′-tetrahydroxychalcone (chalcone) and 4,2′,4′-trihydroxychalcone (deoxychalcone) serve as precursors of ecologically important flavonoids and isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone reductase; however, the identity of the chalcone reductase substrate out of the possible substrates formed during the multistep reaction catalyzed by chalcone synthase remains experimentally elusive. We report here the three-dimensional structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose the identity and binding mode of its substrate, namely the non-aromatized coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of the fully extended coumaryl-tetraketide thioester intermediate. In the absence of a ternary complex, the quality of the refined NADP+-bound chalcone reductase structure serves as a template for computer-assisted docking to evaluate the likelihood of possible substrates. Interestingly, chalcone reductase adopts the three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto reductase fold is structurally distinct from all known ketoreductases of fatty acid biosynthesis, which instead belong to the short-chain dehydrogenase/reductase superfamily. The results presented here provide structural support for convergent functional evolution of these two ketoreductases that share similar roles in the biosynthesis of fatty acids/polyketides. In addition, the chalcone reductase structure represents the first protein structure of a member of the aldo/ketoreductase 4 family. Therefore, the chalcone reductase structure serves as a template for the homology modeling of other aldo/ketoreductase 4 family members, including the reductase involved in morphine biosynthesis, namely codeinone reductase. PMID:15970585

  9. Adventitious Arsenate Reductase Activity of the Catalytic Domain of the Human Cdc25B and Cdc25C Phosphatases†

    PubMed Central

    Bhattacharjee, Hiranmoy; Sheng, Ju; Ajees, A. Abdul; Mukhopadhyay, Rita; Rosen, Barry P.

    2013-01-01

    A number of eukaryotic enzymes that function as arsenate reductases are homologues of the catalytic domain of the human Cdc25 phosphatase. For example, the Leishmania major enzyme LmACR2 is both a phosphatase and an arsenate reductase, and its structure bears similarity to the structure of the catalytic domain of human Cdc25 phosphatase. These reductases contain an active site C-X5-R signature motif, where C is the catalytic cysteine, the five X residues form a phosphate binding loop, and R is a highly conserved arginine, which is also present in human Cdc25 phosphatases. We therefore investigated the possibility that the three human Cdc25 isoforms might have adventitious arsenate reductase activity. The sequences for the catalytic domains of Cdc25A, -B, and -C were cloned individually into a prokaryotic expression vector, and their gene products were purified from a bacterial host using nickel affinity chromatography. While each of the three Cdc25 catalytic domains exhibited phosphatase activity, arsenate reductase activity was observed only with Cdc25B and -C. These two enzymes reduced inorganic arsenate but not methylated pentavalent arsenicals. Alteration of either the cysteine and arginine residues of the Cys-X5-Arg motif led to the loss of both reductase and phosphatase activities. Our observations suggest that Cdc25B and -C may adventitiously reduce arsenate to the more toxic arsenite and may also provide a framework for identifying other human protein tyrosine phosphatases containing the active site Cys-X5-Arg loop that might moonlight as arsenate reductases. PMID:20025242

  10. Purification, Characterization, and Overexpression of Flavin Reductase Involved in Dibenzothiophene Desulfurization by Rhodococcus erythropolis D-1

    PubMed Central

    Matsubara, Toshiyuki; Ohshiro, Takashi; Nishina, Yoshihiro; Izumi, Yoshikazu

    2001-01-01

    The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the Km values for NADH and FMN were 208 and 10.8 μM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35°C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80°C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705–1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain. PMID:11229908

  11. Mutation and repair induced by the carcinogen 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in the dihydrofolate reductase gene of Chinese hamster ovary cells and conformational modeling of the dG-C8-PhIP adduct in DNA.

    PubMed

    Carothers, A M; Yuan, W; Hingerty, B E; Broyde, S; Grunberger, D; Snyderwine, E G

    1994-01-01

    Three experiments using 20 microM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C-->T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a-1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by 32P-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 x 10(-6), the adduct levels after a 1-h exposure of UA21 cells to 20 microM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural

  12. Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice.

    PubMed

    Windahl, Sara H; Andersson, Niklas; Börjesson, Anna E; Swanson, Charlotte; Svensson, Johan; Movérare-Skrtic, Sofia; Sjögren, Klara; Shao, Ruijin; Lagerquist, Marie K; Ohlsson, Claes

    2011-01-01

    Androgens are important regulators of bone mass but the relative importance of testosterone (T) versus dihydrotestosterone (DHT) for the activation of the androgen receptor (AR) in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2), encoded by separate genes (Srd5a1 and Srd5a2). 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1⁻/⁻ mice. Four-month-old male Srd5a1⁻/⁻ mice had reduced trabecular bone mineral density (-36%, p<0.05) and cortical bone mineral content (-15%, p<0.05) but unchanged serum androgen levels compared with wild type (WT) mice. The cortical bone dimensions were reduced in the male Srd5a1⁻/⁻ mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05) in orchidectomized WT mice but not in orchidectomized Srd5a1⁻/⁻ mice. Male Srd5a1⁻/⁻ mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05). Female Srd5a1⁻/⁻ mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1⁻/⁻ mice, is an indirect effect mediated by elevated circulating androgen levels.

  13. Limited proteolysis of the nitrate reductase from spinach leaves.

    PubMed

    Kubo, Y; Ogura, N; Nakagawa, H

    1988-12-25

    The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.

  14. Serenoa repens (Permixon) inhibits the 5alpha-reductase activity of human prostate cancer cell lines without interfering with PSA expression.

    PubMed

    Habib, Fouad K; Ross, Margaret; Ho, Clement K H; Lyons, Valerie; Chapman, Karen

    2005-03-20

    The phytotherapeutic agent Serenoa repens is an effective dual inhibitor of 5alpha-reductase isoenzyme activity in the prostate. Unlike other 5alpha-reductase inhibitors, Serenoa repens induces its effects without interfering with the cellular capacity to secrete PSA. Here, we focussed on the possible pathways that might differentiate the action of Permixon from that of synthetic 5alpha-reductase inhibitors. We demonstrate that Serenoa repens, unlike other 5alpha-reductase inhibitors, does not inhibit binding between activated AR and the steroid receptor-binding consensus in the promoter region of the PSA gene. This was shown by a combination of techniques: assessment of the effect of Permixon on androgen action in the LNCaP prostate cancer cell line revealed no suppression of AR and maintenance of PSA protein expression at control levels. This was consistent with reporter gene experiments showing that Permixon failed to interfere with AR-mediated transcriptional activation of PSA and that both testosterone and DHT were equally effective at maintaining this activity. Our results demonstrate that despite Serenoa repens effective inhibition of 5alpha-reductase activity in the prostate, it did not suppress PSA secretion. Therefore, we confirm the therapeutic advantage of Serenoa repens over other 5alpha-reductase inhibitors as treatment with the phytotherapeutic agent will permit the continuous use of PSA measurements as a useful biomarker for prostate cancer screening and for evaluating tumour progression.

  15. Functional analysis of a nitrite reductase promoter from birch in transgenic tobacco.

    PubMed

    Warning; Hachtel

    2000-06-29

    Nitrate assimilation is a highly regulated process in higher plants, and the regulatory cues governing gene expression in this pathway include both external and internal factors. In birch (Betula pendula Roth) the expression of nitrate reductase (NR) and nitrite reductase (NiR) genes is co-regulated by light and nitrate at the transcriptional level. In order to identify cis-acting DNA-elements involved in light and nitrate induction of the birch NiR gene, a 0.9 kb 5' flanking region of the NiR gene was isolated, analysed on the DNA level, and the transcription start site was determined. Deletion analysis of the birch NiR promoter region fused to the GUS reporter gene (uidA) in transgenic tobacco (Nicotiana tabacum) revealed the presence of light- and nitrate-responsive promoter fragments. The responsive fragments showed different activities in leaves and roots. Further, gel mobility shift assays using nuclear proteins from leaves detected a specific DNA-binding activity to the sequence between -146 and -267 bp that was induced in darkness and disappeared in the light. The deletion analysis has shown that this region is critical for light inducibility of the birch NiR gene in leaves.

  16. Molybdate-dependent expression of the periplasmic nitrate reductase in Bradyrhizobium japonicum.

    PubMed

    Bonnard, N; Tresierra-Ayala, A; Bedmar, E J; Delgado, M J

    2005-02-01

    The napEDABC genes of Bradyrhizobium japonicum encode the periplasmic nitrate reductase, an Mo-containing enzyme which catalyses the reduction of nitrate to nitrite when oxygen concentrations are limiting. In this bacterium, another set of genes, modABC, code for a high affinity ABC-type Mo transport system. A B. japonicum modA mutant has been obtained that is not capable of growing anaerobically with nitrate and lacks nitrate reductase activity. Under nitrate respiring conditions, when Mo concentrations are limiting, the B. japonicum modA mutant lacked both the 90 kDa protein corresponding to the NapA component of the periplasmic nitrate reductase, and the membrane-bound 25 kDa c-type cytochrome NapC. Regulatory studies using a napE-lacZ fusion indicated that napE expression was highly reduced in the modA mutant background when the cells were incubated anaerobically with nitrate under Mo-deficient conditions.

  17. Evolution of the Ferric Reductase Domain (FRD) Superfamily: Modularity, Functional Diversification, and Signature Motifs

    PubMed Central

    Zhang, Xuezhi; Krause, Karl-Heinz; Xenarios, Ioannis; Soldati, Thierry; Boeckmann, Brigitte

    2013-01-01

    A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria. PMID:23505460

  18. Trichomonas vaginalis flavin reductase 1 and its role in metronidazole resistance.

    PubMed

    Leitsch, David; Janssen, Brian D; Kolarich, Daniel; Johnson, Patricia J; Duchêne, Michael

    2014-01-01

    The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defence in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis.

  19. Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

    PubMed Central

    Booker, S; Stubbe, J

    1993-01-01

    Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statistically significant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. leichmannii. PMID:8397403

  20. A qualitative and quantitative cytochemical assay of dihydrofolate reductase in erythroid cells.

    PubMed

    Nano, R; Gerzeli, G; Invernizzi, R; Supino, R

    1989-01-01

    The distribution and intensity of dihydrofolate reductase (DHFR) cytochemically demonstrable was studied in erythroid cells. Cells of normal human bone marrow, of human erythroleukaemia (M6), and cells of the Friend (MEL) clone 745A murine erythroleukaemia (also after differentiation with dimethylsulphoxide, DMSO) were stained according to Gerzeli and de Piceis Polver (1969) technique; quantification of the reaction product was made using a Vickers M86 microdensitometer. The enzyme activity progressively decreased during the normal differentiation of the erythropoietic series while persisted at high levels in erythroleukaemia cells. It can be suggested that in the 1st case, the cytochemical pattern of dihydrofolate reductase may be a useful added tool for studying the erythroid differentiation. In the 2nd case, the increased level of this enzyme may be related to an amplification of the gene of DHFR in the malignant transformation.

  1. Methylenetetrahydrofolate reductase (MTHFR) deficiency enhances resistance against cytomegalovirus infection.

    PubMed

    Fodil-Cornu, N; Kozij, N; Wu, Q; Rozen, R; Vidal, S M

    2009-10-01

    Folates provide one-carbon units for nucleotide synthesis and methylation reactions. A common polymorphism in the MTHFR gene (677C --> T) results in reduced enzymatic activity, and is associated with an increased risk for neural tube defects and cardiovascular disease. The high prevalence of this polymorphism suggests that it may have experienced a selective advantage under environmental pressure, possibly an infectious agent. To test the hypothesis that methylenetetrahydrofolate reductase (MTHFR) genotype influences the outcome of infectious disease, we examined the response of Mthfr-deficient mice against mouse cytomegalovirus (MCMV) infection. Acute MCMV infection of Mthfr(-/-) mice resulted in early control of cytokine secretion, decreased viral titer and preservation of spleen immune cells, in contrast to Mthfr wild-type littermates. The phenotype was abolished in MTHFR transgenic mice carrying an extra copy of the gene. Infection of primary fibroblasts with MCMV showed a decrease in viral replication and in the number of productively infected cells in Mthfr(+/-) fibroblasts compared with wild-type cells. These results indicate that Mthfr deficiency protects against MCMV infection in vivo and in vitro, suggesting that human genetic variants may provide an advantage in the host response against certain pathogens.

  2. [Inborn error of cholesterol biosynthesis: Smith-Lemli-Opitz syndrome].

    PubMed

    Koczok, Katalin; V Oláh, Anna; P Szabó, Gabriella; Oláh, Éva; Török, Olga; Balogh, István

    2015-10-18

    Smith-Lemli-Opitz syndrome is an autosomal recessive mental retardation and multiple malformation syndrome caused by deficiency of the 7-dehydrocholesterol reductase, the enzyme catalyzing the last step in cholesterol biosynthesis. The authors summarize the pathophysiology, epidemiology, clinical picture, diagnostics and therapy of the disease based on a review of the international literature. Since 2004, fourteen patients have been diagnosed with Smith-Lemli-Opitz syndrome in Hungary, which suggests an underdiagnosis of the disease based upon estimated incidence data. Due to deficiency of the 7-dehydrocholesterol reductase, serum cholesterol concentration is low and 7-dehydrocholesterol concentration is elevated in blood and tissues; the latter being highly specific for the syndrome. Detection of disease-causing mutations makes the prenatal diagnosis possible. The clinical spectrum is wide, the most common symptom is syndactyly of the second and third toes. Standard therapy is cholesterol supplementation. Recent publications suggest that oxidative compounds of 7-dehydrocholesterol may play a role in the pathophysiology of the disease as well.

  3. Smith-Lemli-Opitz syndrome produced in rats with AY 9944 treated by intravenous injection of lipoprotein cholesterol.

    PubMed

    Chambers, C M; McLean, M P; Ness, G C

    1997-01-31

    A limitation to treating Smith-Lemli-Opitz infants by giving dietary cholesterol is their impaired ability to absorb cholesterol due to a deficiency of bile acids. Since intravenously administered lipoprotein cholesterol should not require bile acids for uptake into tissues, we tested the effects of this form of cholesterol on tissue cholesterol and 7-dehydrocholesterol levels in an animal model of SLO, created by feeding rats 0.02% AY 9944. Intravenous administration of 15 mg of bovine cholesterol supertrate twice daily increased serum cholesterol levels from 11 to over 250 mg/dl. This treatment increased liver cholesterol levels from 309 to over 900 micrograms/g and lowered hepatic 7-dehydrocholesterol levels from 1546 to 909 micrograms/g. A combination of iv cholesterol and 2% dietary cholesterol was most effective as it raised hepatic cholesterol levels to 1950 micrograms/g, which is 50% above normal. 7-Dehydrocholesterol levels were decreased to 760 micrograms/g. Similar responses were seen for heart, lung, kidney, and testes. Brain sterol levels were not significantly affected. AY 9944 caused a modest increase in hepatic HMG-CoA reductase activity. Administration of dietary cholesterol together with iv cholesterol lowered hepatic HMG-CoA reductase activity to barely detectable levels. The data indicate that the combination of iv and dietary cholesterol was most effective in raising cholesterol levels, lowering 7-dehydrocholesterol levels, and inhibiting de novo cholesterol biosynthesis.

  4. Enzyme toolbox: novel enantiocomplementary imine reductases.

    PubMed

    Scheller, Philipp N; Fademrecht, Silvia; Hofelzer, Sebastian; Pleiss, Jürgen; Leipold, Friedemann; Turner, Nicholas J; Nestl, Bettina M; Hauer, Bernhard

    2014-10-13

    Reducing reactions are among the most useful transformations for the generation of chiral compounds in the fine-chemical industry. Because of their exquisite selectivities, enzymatic approaches have emerged as the method of choice for the reduction of C=O and activated C=C bonds. However, stereoselective enzymatic reduction of C=N bonds is still in its infancy-it was only recently described after the discovery of enzymes capable of imine reduction. In our work, we increased the spectrum of imine-reducing enzymes by database analysis. By combining the currently available knowledge about the function of imine reductases with the experimentally uncharacterized diversity stored in protein sequence databases, three novel imine reductases with complementary enantiopreference were identified along with amino acids important for catalysis. Furthermore, their reducing capability was demonstrated by the reduction of the pharmaceutically relevant prochiral imine 2-methylpyrroline. These novel enzymes exhibited comparable to higher catalytic efficiencies than previously described enzymes, and their biosynthetic potential is highlighted by the full conversion of 2-methylpyrroline in whole cells with excellent selectivities.

  5. Soluble ascorbate free radical reductase in the human lens.

    PubMed

    Bando, M; Obazawa, H

    1994-01-01

    A major and a minor ascorbate free radical (AFR) reductase were separated from the soluble fraction in the human lens cortex by DEAE-cellulose ion-exchange column chromatography. These AFR reductases also exhibited diaphorase activity using dichlorophenolindophenol and ferricyanide as electron acceptors. The major AFR reductase was partially purified by 5'AMP-Sepharose 4B affinity column chromatography. This partially purified AFR reductase showed a single band of diaphorase activity in native polyacrylamide disc gel electrophoresis. This activity band corresponded to the major protein observed in protein staining by Coomassie Brilliant Blue. However, the protein staining by Coomassie Brilliant Blue showed this activity band surrounded by diffused staining. Molecular weight of the partially purified AFR reductase was determined to be 32 kDa by gel filtration, and the apparent Km value for AFR was about 15 microM. This major lens AFR reductase could be distinguished from soluble Neurospora, Euglena and cucumber AFR reductases, and from two ubiquitous enzymes with reduction activity of AFR and/or foreign compounds, ie, NADH-cytochrome b5 reductase and DT-diaphorase, by their molecular weights, Km values and/or ion-exchange chromatographic behaviors.

  6. Prokaryotic arsenate reductase enhances arsenate resistance in Mammalian cells.

    PubMed

    Wu, Dan; Tao, Xuanyu; Wu, Gaofeng; Li, Xiangkai; Liu, Pu

    2014-01-01

    Arsenic is a well-known heavy metal toxicant in the environment. Bioremediation of heavy metals has been proposed as a low-cost and eco-friendly method. This article described some of recent patents on transgenic plants with enhanced heavy metal resistance. Further, to test whether genetic modification of mammalian cells could render higher arsenic resistance, a prokaryotic arsenic reductase gene arsC was transfected into human liver cancer cell HepG2. In the stably transfected cells, the expression level of arsC gene was determined by quantitative real-time PCR. Results showed that arsC was expressed in HepG2 cells and the expression was upregulated by 3 folds upon arsenate induction. To further test whether arsC has function in HepG2 cells, the viability of HepG2-pCI-ArsC cells exposed to arsenite or arsenate was compared to that of HepG2-pCI cells without arsC gene. The results indicated that arsC increased the viability of HepG2 cells by 25% in arsenate, but not in arsenite. And the test of reducing ability of stably transfected cells revealed that the concentration of accumulated trivalent arsenic increased by 25% in HepG2-pCI-ArsC cells. To determine the intracellular localization of ArsC, a fusion vector with fluorescent marker pEGFP-N1-ArsC was constructed and transfected into.HepG2. Laser confocal microscopy showed that EGFP-ArsC fusion protein was distributed throughout the cells. Taken together, these results demonstrated that prokaryotic arsenic resistant gene arsC integrated successfully into HepG2 genome and enhanced arsenate resistance of HepG2, which brought new insights of arsenic detoxification in mammalian cells.

  7. The Effect of Small Molecules on Sterol Homeostasis: Measuring 7-Dehydrocholesterol in Dhcr7-Deficient Neuro2a Cells and Human Fibroblasts.

    PubMed

    Korade, Zeljka; Kim, Hye-Young H; Tallman, Keri A; Liu, Wei; Koczok, Katalin; Balogh, Istvan; Xu, Libin; Mirnics, Karoly; Porter, Ned A

    2016-02-11

    Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith-Lemli-Opitz syndrome (SLOS) patients and linked to specific trans