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Sample records for synthase modulates cfa-induced

  1. Nitric oxide synthase modulates CFA-induced thermal hyperalgesia through cytokine regulation in mice.

    Science.gov (United States)

    Chen, Yong; Boettger, Michael K; Reif, Andreas; Schmitt, Angelika; Uçeyler, Nurcan; Sommer, Claudia

    2010-03-02

    Although it has been largely demonstrated that nitric oxide synthase (NOS), a key enzyme for nitric oxide (NO) production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Intraperitoneal (i.p.) pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor), aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor), L-N(G)-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor), but not L-N(5)-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor), significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1beta), and interleukin-10 (IL-10) gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1beta. The increase of the anti-inflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO) mice had lower gene expression of TNF, IL-1beta, and IL-10 following CFA, overall corroborating the inhibitor data. These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.

  2. Nitric oxide synthase modulates CFA-induced thermal hyperalgesia through cytokine regulation in mice

    Directory of Open Access Journals (Sweden)

    Üçeyler Nurcan

    2010-03-01

    Full Text Available Abstract Background Although it has been largely demonstrated that nitric oxide synthase (NOS, a key enzyme for nitric oxide (NO production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Results Intraperitoneal (i.p. pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor, aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor, L-N(G-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor, but not L-N(5-(1-iminoethyl-ornithine (L-NIO, a selective endothelial NOS inhibitor, significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl. injection of complete Freund's adjuvant (CFA. Real-time reverse transcription-polymerase chain reaction (RT-PCR revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF, interleukin-1 beta (IL-1β, and interleukin-10 (IL-10 gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1β. The increase of the anti-inflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO mice had lower gene expression of TNF, IL-1β, and IL-10 following CFA, overall corroborating the inhibitor data. Conclusion These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.

  3. Spinal translocator protein (TSPO) modulates pain behavior in rats with CFA-induced monoarthritis.

    Science.gov (United States)

    Hernstadt, Hayley; Wang, Shuxing; Lim, Grewo; Mao, Jianren

    2009-08-25

    Translocator protein 18 kDa (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is predominantly located in the mitochondrial outer membrane and plays an important role in steroidogenesis, immunomodulation, cell survival and proliferation. Previous studies have shown an increased expression of TSPO centrally in neuropathology, as well as in injured nerves. TSPO has also been implicated in modulation of nociception. In the present study, we examined the hypothesis that TSPO is involved in the initiation and maintenance of inflammatory pain using a rat model of Complete Freund's Adjuvant (CFA)-induced monoarthritis of the tibio-tarsal joint. Immunohistochemistry was performed using Iba-1 (microglia), NeuN (neurons), anti-Glial Fibrillary Acidic Protein, GFAP (astrocytes) and anti-PBR (TSPO) on Days 1, 7 and 14 after CFA-induced arthritis. Rats with CFA-induced monoarthritis showed mechanical allodynia and thermal hyperalgesia on the ipsilateral hindpaw, which correlated with the increased TSPO expression in ipsilateral laminae I-II on all experimental days. Iba-1 expression in the ipsilateral dorsal horn was also increased on Days 7 and 14. Moreover, TSPO was colocalized with Iba-1, GFAP and NeuN within the spinal cord dorsal horn. The TSPO agonist Ro5-4864, given intrathecally, dose-dependently retarded or prevented the development of mechanical allodynia and thermal hyperalgesia in rats with CFA-induced monoarthritis. These findings provide evidence that spinal TSPO is involved in the development and maintenance of inflammatory pain behaviors in rats. Thus, spinal TSPO may present a central target as a complementary therapy to reduce inflammatory pain.

  4. Ranolazine attenuation of CFA-induced mechanical hyperalgesia.

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    Casey, Gregory P; Roberts, Jomar S; Paul, Dennis; Diamond, Ivan; Gould, Harry J

    2010-01-01

    To determine whether ranolazine, a new anti-angina medication, could be an effective analgesic agent in complete Freund's adjuvant-induced inflammatory pain. Plantar injection of complete Freund's adjuvant (CFA) produces an extended period of hyperalgesia that is associated with a dramatic up-regulation of Na(v) 1.7 sodium channels in populations of large and small dorsal root ganglion neurons related to the injection site. Ranolazine appears to produce its anti-angina effect through blocking the late sodium current associated with the voltage-gated sodium channel, Na(v) 1.5. Because ranolazine also inhibits Na(v) 1.7, and 1.8, we sought to determine whether it could be an effective analgesic agent in CFA-induced inflammatory pain. Baseline determinations of withdrawal from thermal and mechanical stimulation were made in Sprague-Dawley rats ( approximately 300-350 x g). Following determination of baseline, one hindpaw in each group was injected with 0.1 mL of CFA. The contralateral paw received saline. Thermal and mechanical stimulation was repeated on the third day post-injection. Vehicle (0.9% isotonic saline; pH 3.0) or ranolazine was then administered in randomized and blinded doses either by intraperitoneal (ip) injection (0, 10, 20, and 50 mg/kg) or by oral gavage (po; 0, 20, 50, 100, and 200 mg/kg). Animals were again tested 30 minutes (ip) and 1 hour (po) after drug administration. Ranolazine produced dose-dependant analgesia on mechanical allodynia induced by CFA injection, but had no effect on thermal hyperalgesia. Ranolazine's potential as a new option for managing both angina and chronic inflammatory pain warrants further study.

  5. Pharmacokinetic/Pharmacodynamic Relationship of Gabapentin in a CFA-induced Inflammatory Hyperalgesia Rat Model

    DEFF Research Database (Denmark)

    Larsen, Malte Selch; Keizer, Ron; Munro, Gordon

    2016-01-01

    PURPOSE: Gabapentin displays non-linear drug disposition, which complicates dosing for optimal therapeutic effect. Thus, the current study was performed to elucidate the pharmacokinetic/pharmacodynamic (PKPD) relationship of gabapentin's effect on mechanical hypersensitivity in a rat model of CFA......-induced inflammatory hyperalgesia. METHODS: A semi-mechanistic population-based PKPD model was developed using nonlinear mixed-effects modelling, based on gabapentin plasma and brain extracellular fluid (ECF) time-concentration data and measurements of CFA-evoked mechanical hyperalgesia following administration...

  6. Modulation of hyaluronan synthase activity in cellular membrane fractions.

    Science.gov (United States)

    Vigetti, Davide; Genasetti, Anna; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Bartolini, Barbara; Moretto, Paola; De Luca, Giancarlo; Hascall, Vincent C; Passi, Alberto

    2009-10-30

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in eukaryotic cells and addressed the question of HAS activity during intracellular protein trafficking. We prepared three cellular fractions: plasma membrane, cytosol (containing membrane proteins mainly from the endoplasmic reticulum and Golgi), and nuclei. After incubation with UDP-sugar precursors, newly synthesized HA was quantified by polyacrylamide gel electrophoresis of fluorophore-labeled saccharides and high performance liquid chromatography. This new method measured HAS activity not only in the plasma membrane fraction but also in the cytosolic membranes. This new technique was used to evaluate the effects of 4-methylumbeliferone, phorbol 12-myristate 13-acetate, interleukin 1beta, platelet-derived growth factor BB, and tunicamycin on HAS activities. We found that HAS activity can be modulated by post-translational modification, such as phosphorylation and N-glycosylation. Interestingly, we detected a significant increase in HAS activity in the cytosolic membrane fraction after tunicamycin treatment. Since this compound is known to induce HA cable structures, this result links HAS activity alteration with the capability of the cell to promote HA cable formation.

  7. Differential modulation of nitric oxide synthases in aging: therapeutic opportunities

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    Stêfany Bruno De Assis Cau

    2012-06-01

    Full Text Available Vascular aging is the term that describes the structural and functional disturbances of the vasculature with advancing aging. The molecular mechanisms of aging-associated endothelial dysfunction are complex, but reduced nitric oxide (NO bioavailability and altered vascular expression and activity of NO synthase (NOS enzymes have been implicated as major players. Impaired vascular relaxation in aging has been attributed to reduced endothelial NOS (eNOS-derived NO, while increased inducible NOS (iNOS expression seems to account for nitrosative stress and disrupted vascular homeostasis. Although eNOS is considered the main source of NO in the vascular endothelium, neuronal NOS (nNOS also contributes to endothelial cells-derived NO, a mechanism that is reduced in aging. Pharmacological modulation of NO generation and expression/activity of NOS isoforms may represent a therapeutic alternative to prevent the progression of cardiovascular diseases. Accordingly, this review will focus on drugs that modulate NO bioavailability, such as nitrite anions and NO-releasing non-steroidal anti-inflammatory drugs, hormones (dehydroepiandrosterone and estrogen, statins, resveratrol and folic acid, since they may be useful to treat/to prevent aging-associated vascular dysfunction. The impact of these therapies on life quality in elderly and longevity will be discussed.

  8. Amelioratory effect of flavonoids rich Pergularia daemia extract against CFA induced arthritic rats.

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    Ananth, Devanesan Arul; Rameshkumar, Angappan; Jeyadevi, Ramachandran; Aseervatham, G Smilin Bell; Sripriya, Jaganathan; Bose, Prabaharan Chandra; Sivasudha, Thilagar

    2016-05-01

    Pergularia daemia Forsk. (Asclepiadaceae) is a traditionally reported medicinal herb used to treat joint pain and arthritis. However, there are no scientific reports about anti-arthritic activity of P. daemia methanolic extract on rats as animal model. This study identifies bioactive compounds present in the P. daemia methanolic extract and evaluates its anti-arthritic potential in CFA induced arthritic rats. Phytoconstituents of P. daemia extract were examined using LC-ESI/MS method. Anti-arthritic activity of P. daemia extract was determined by various biochemical experiments (RF, ESR and CRP), ultrasonography and histological analysis. LC-ESI/MS analysis resulted in the identification of major flavonoids compounds such as formononetin, qurecetin, chrysoeriol, taxifolin and naringenin. Serum biomarker analysis, after the treatment with PDME (500mg/kg b.w.) revealed that the hemoglobin (11.84±0.42g/dL) and RBC (8.38±0.67million/mm(3)) levels were significantly increased whereas WBC (8.91±0.38thousands/mm(3)), RF (17.94±0.45IU/mL), ESR (7.91±0.12mm/h) and CRP (22.56±0.26mg/L) levels were decreased when compared with the CFA induced arthritic control group. Histology results revealed that treatment with PDME has resulted in significant prevention against bony destruction by decreasing soft tissue swelling and narrowing of joint spaces (250 and 500mg/kg b.w.). Anti-arthritic effect of P. daemia might be due to the presence of these bioactive flavonoids. These findings lend pharmacological support to the reported folkloric use of P. daemia in the treatment and management of painful, arthritic inflammatory conditions. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. Modulation of hyaluronan synthase activity in cellular membrane fractions

    OpenAIRE

    Vigetti, Davide; Genasetti, A; Karousou, Evgenia; Viola, Manuela; Clerici, M; Bartolini, B; Moretto, Paola; DE LUCA, Giancarlo; Hascall, Vc; Passi, Alberto

    2009-01-01

    Hyaluronan (HA), the only non-sulfated glycosaminoglycan, is involved in morphogenesis, wound healing, inflammation, angiogenesis, and cancer. In mammals, HA is synthesized by three homologous HA synthases, HAS1, HAS2, and HAS3, that polymerize the HA chain using UDP-glucuronic acid and UDP-N-acetylglucosamine as precursors. Since the amount of HA is critical in several pathophysiological conditions, we developed a non-radioactive assay for measuring the activity of HA synthases (HASs) in euk...

  10. Protective Effects of Sinomenine on CFA-Induced Inflammatory Pain in Rats.

    Science.gov (United States)

    Yuan, Yan; Zhang, Yongjun; He, Xiaofeng; Fan, Shengdeng

    2018-04-05

    BACKGROUND The purpose of this study was to investigate the effects of sinomenine (SIN) on CFA-induced inflammatory pain in rats, and to explore the underlying molecular mechanisms. MATERIAL AND METHODS To determine the potential influences of SIN in the pathogenesis of inflammatory pain, an inflammatory pain (IP) mouse model was established and rats were treated with SIN (30 mg/kg). Behavioral tests were used to assess the MWT and TWL of the rats. ELISA assay was used to detect the level of inflammation cytokines. Western blotting and qRT-PCR were carried out to measure the related protein and mRNA expression level, respectively. RESULTS We found that the MWT and TWL of the CFA-treated rats were markedly lower than that of the control rats, and they were significantly increased by SIN administration. The results suggest that IP rats had higher levels of TNF-α, IL-1β and IL-6 compared with the control rats. SIN administration decreased the levels of TNF-α, IL-1β, and IL-6. In addition, we found that p-p65 and p-p38 expression notably decreased after SIN treatment in IP rats. Moreover, the results showed that SIN inhibited Cox-2 and PGE2 expression in IP rats. CONCLUSIONS The data indicate that SIN had a protective role in inflammatory pain through repressing inflammatory mediators via preventing the p38MAPK-NF-κB pathway.

  11. Hypothalamic GPR40 Signaling Activated by Free Long Chain Fatty Acids Suppresses CFA-Induced Inflammatory Chronic Pain

    OpenAIRE

    Nakamoto, Kazuo; Nishinaka, Takashi; Sato, Naoya; Mankura, Mitsumasa; Koyama, Yutaka; Kasuya, Fumiyo; Tokuyama, Shogo

    2013-01-01

    GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently inc...

  12. A time-dependent degeneration manner of condyle in rat CFA-induced inflamed TMJ.

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    Xu, Liqin; Guo, Huilin; Li, Cheng; Xu, Jie; Fang, Wei; Long, Xing

    2016-01-01

    Temporomandibular joint (TMJ) inflammation is a potential risk factor of osteoarthritis (OA) but the detailed degenerative changes in the inflamed TMJ remain unclear. In this study, we evaluated the changes of condylar cartilage and subchondral bone in rat inflamed TMJ induced by Freund's complete adjuvant (CFA). Articular cavity was injected with CFA and the TMJ samples were collected 1, 2, 3, and 4-week post-injection. Hematoxylin & Eosin (H&E) staining, toluidine blue (TB) staining, Safranin O (S.O) staining, Masson trichrome staining and micro-CT were used to assess TMJ degeneration during inflammation. Osteoclast and osteoblast activities were analyzed by tartrate-resistant acid phosphatase (TRAP) staining and osteocalcin (OCN) immunohistochemistry staining respectively. The expression of receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG) in condylar cartilage and subchondral bone was also evaluated through immunohistochemistry and RANKL/OPG ratio was evaluated. Reduced cartilage thickness, decreased number of chondrocytes, and down-regulated proteoglycan expression were observed in the condylar cartilage in the inflamed TMJ. Enhanced osteoclast activity, and expanded bone marrow cavity were reached the peak in the 2-week after CFA-injection. Meanwhile the RANKL/OPG ratio in the cartilage and subchondral bone also increased in the 2-week CFA-injection. Immature, unmineralized new bones with irregular trabecular bone structure, atypical condylar shape, up-regulated OCN expression, and decreased bone mineral density (BMD) were found in the inflamed TMJ. The time-dependent degeneration manner of TMJ cartilage and subchondral bone was found in CFA-induced arthritis rat model. The degeneration in the TMJ with inflammation might be a risk factor and should be concerned.

  13. Pharmacokinetic/Pharmacodynamic Relationship of Gabapentin in a CFA-induced Inflammatory Hyperalgesia Rat Model.

    Science.gov (United States)

    Larsen, Malte Selch; Keizer, Ron; Munro, Gordon; Mørk, Arne; Holm, René; Savic, Rada; Kreilgaard, Mads

    2016-05-01

    Gabapentin displays non-linear drug disposition, which complicates dosing for optimal therapeutic effect. Thus, the current study was performed to elucidate the pharmacokinetic/pharmacodynamic (PKPD) relationship of gabapentin's effect on mechanical hypersensitivity in a rat model of CFA-induced inflammatory hyperalgesia. A semi-mechanistic population-based PKPD model was developed using nonlinear mixed-effects modelling, based on gabapentin plasma and brain extracellular fluid (ECF) time-concentration data and measurements of CFA-evoked mechanical hyperalgesia following administration of a range of gabapentin doses (oral and intravenous). The plasma/brain ECF concentration-time profiles of gabapentin were adequately described with a two-compartment plasma model with saturable intestinal absorption rate (K m  = 44.1 mg/kg, V max  = 41.9 mg/h∙kg) and dose-dependent oral bioavailability linked to brain ECF concentration through a transit compartment. Brain ECF concentration was directly linked to a sigmoid E max function describing reversal of hyperalgesia (EC 50, plasma  = 16.7 μg/mL, EC 50, brain  = 3.3 μg/mL). The proposed semi-mechanistic population-based PKPD model provides further knowledge into the understanding of gabapentin's non-linear pharmacokinetics and the link between plasma/brain disposition and anti-hyperalgesic effects. The model suggests that intestinal absorption is the primary source of non-linearity and that the investigated rat model provides reasonable predictions of clinically effective plasma concentrations for gabapentin.

  14. Shp-1 dephosphorylates TRPV1 in dorsal root ganglion neurons and alleviates CFA-induced inflammatory pain in rats.

    Science.gov (United States)

    Xiao, Xing; Zhao, Xiao-Tao; Xu, Ling-Chi; Yue, Lu-Peng; Liu, Feng-Yu; Cai, Jie; Liao, Fei-Fei; Kong, Jin-Ge; Xing, Guo-Gang; Yi, Ming; Wan, You

    2015-04-01

    Transient receptor potential vanilloid 1 (TRPV1) receptors are expressed in nociceptive neurons of rat dorsal root ganglions (DRGs) and mediate inflammatory pain. Nonspecific inhibition of protein-tyrosine phosphatases (PTPs) increases the tyrosine phosphorylation of TRPV1 and sensitizes TRPV1. However, less is known about tyrosine phosphorylation's implication in inflammatory pain, compared with that of serine/threonine phosphorylation. Src homology 2 domain-containing tyrosine phosphatase 1 (Shp-1) is a key phosphatase dephosphorylating TRPV1. In this study, we reported that Shp-1 colocalized with and bound to TRPV1 in nociceptive DRG neurons. Shp-1 inhibitors, including sodium stibogluconate and PTP inhibitor III, sensitized TRPV1 in cultured DRG neurons. In naive rats, intrathecal injection of Shp-1 inhibitors increased both TRPV1 and tyrosine-phosphorylated TRPV1 in DRGs and induced thermal hyperalgesia, which was abolished by pretreatment with TRPV1 antagonists capsazepine, BCTC, or AMG9810. Complete Freund's adjuvant (CFA)-induced inflammatory pain in rats significantly increased the expression of Shp-1, TRPV1, and tyrosine-phosphorylated TRPV1, as well as the colocalization of Shp-1 and TRPV1 in DRGs. Intrathecal injection of sodium stibogluconate aggravated CFA-induced inflammatory pain, whereas Shp-1 overexpression in DRG neurons alleviated it. These results suggested that Shp-1 dephosphorylated and inhibited TRPV1 in DRG neurons, contributing to maintain thermal nociceptive thresholds in normal rats, and as a compensatory mechanism, Shp-1 increased in DRGs of rats with CFA-induced inflammatory pain, which was involved in protecting against excessive thermal hyperalgesia.

  15. Disruption of δ-opioid receptor phosphorylation at threonine 161 attenuates morphine tolerance in rats with CFA-induced inflammatory hypersensitivity.

    Science.gov (United States)

    Chen, Hai-Jing; Xie, Wei-Yan; Hu, Fang; Zhang, Ying; Wang, Jun; Wang, Yun

    2012-04-01

    Our previous study identified Threonine 161 (Thr-161), located in the second intracellular loop of the δ-opioid receptor (DOR), as the only consensus phosphorylation site for cyclin-dependent kinase 5 (Cdk5). The aim of this study was to assess the function of DOR phosphorylation by Cdk5 in complete Freund's adjuvant (CFA)-induced inflammatory pain and morphine tolerance. Dorsal root ganglion (DRG) neurons of rats with CFA-induced inflammatory pain were acutely dissociated and the biotinylation method was used to explore the membrane localization of phosphorylated DOR at Thr-161 (pThr-161-DOR), and paw withdrawal latency was measured after intrathecal delivery of drugs or Tat-peptide, using a radiant heat stimulator in rats with CFA-induced inflammatory pain. Both the total amount and the surface localization of pThr-161-DOR were significantly enhanced in the ipsilateral DRG following CFA injection. Intrathecal delivery of the engineered Tat fusion-interefering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L) increased inflammatory hypersensitivity, and inhibited DOR- but not µ-opioid receptor-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of Tat-DOR-2L postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain. Phosphorylation of DOR at Thr-161 by Cdk5 attenuates hypersensitivity and potentiates morphine tolerance in rats with CFA-induced inflammatory pain, while disruption of the phosphorylation of DOR at Thr-161 attenuates morphine tolerance.

  16. Pharmacological modulation of human platelet leukotriene C4-synthase.

    Science.gov (United States)

    Sala, A; Folco, G; Henson, P M; Murphy, R C

    1997-03-21

    The aim of this study was to test if human platelet leukotriene C4-synthase (LTC4-S) is pharmacologically different from cloned and expressed LTC4-S and, in light of the significant homologies between 5-lipoxygenase activating protein (FLAP) and LTC4-S, if different potencies of leukotriene synthesis inhibitors acting through binding with FLAP (FLAP inhibitors) reflect in different potencies as LTC4-S inhibitors. Leukotriene C4 (LTC4) synthesis by washed human platelets supplemented with synthetic leukotriene A4 (LTA4) was studied in the absence and presence of two different, structurally unrelated FLAP inhibitors (MK-886 and BAY-X1005) as well as a direct 5-lipoxygenase inhibitor (zileuton). LTC4 production was analyzed by RP-HPLC coupled to diode array detection. We report that human platelet LTC4-S was inhibited by MK-886 and BAY-X1005 (IC50 of 4.7 microM and 91.2 microM, respectively), but not by zileuton (inactive up to 300 microM); all 3 compounds were able to inhibit 5-lipoxygenase metabolite biosynthesis in intact human polymorphonuclear leukocytes (IC50 of 0.044 microM, 0.85 microM, and 1.5 microM, respectively). Platelet LTC4-S does not appear pharmacologically different from expression cloned LTC4-S. LTC4-S inhibition by FLAP inhibitors is in agreement with the significant homology reported for expression-cloned LTC4-S with FLAP, Furthermore, functional homology of the binding sites for inhibitors on LTC4-S and FLAP is suggested by the conservation of the relative potencies of MK-886 and BAY-X1005 vs FLAP-dependent 5-lipoxygenase activity and LTC4-S inhibition: MK-886 was 19.3-fold more potent than BAY-X1005 as FLAP inhibitor and 19.6-fold more potent than BAY-X1005 as LTC4-S inhibitor.

  17. Models of Inflammation: Carrageenan- or Complete Freund's Adjuvant (CFA)-Induced Edema and Hypersensitivity in the Rat.

    Science.gov (United States)

    McCarson, Kenneth E

    2015-09-01

    Animal models of inflammation are used to assess the production of inflammatory mediators at sites of inflammation, the processing of pain sensation at CNS sites, the anti-inflammatory properties of agents such as nonsteroidal anti-inflammatory drugs (NSAIDs), and the efficacy of putative analgesic compounds in reversing cutaneous hypersensitivity. Detailed in this unit are methods to elicit and measure carrageenan- and complete Freund's adjuvant (CFA)-induced cutaneous inflammation. Due to possible differences between the dorsal root sensory system and the trigeminal sensory system, injections into either the footpad or vibrissal pad are described. In this manner, cutaneous inflammation can be assessed in tissue innervated by the lumbar dorsal root ganglion neurons (footpad) or by the trigeminal ganglion neurons (vibrissal pad). Copyright © 2015 John Wiley & Sons, Inc.

  18. Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase.

    Science.gov (United States)

    Michel, J B; Feron, O; Sase, K; Prabhakar, P; Michel, T

    1997-10-10

    Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

  19. Anti-nociceptive effects of Tanshinone IIA (TIIA) in a rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain.

    Science.gov (United States)

    Sun, Shukai; Yin, Yue; Yin, Xin; Cao, Fale; Luo, Daoshu; Zhang, Ting; Li, Yunqing; Ni, Longxing

    2012-09-01

    Inflammatory pain is an important clinical symptom. The levels of extracellular signal-regulated kinases (ERKs) and the levels of cytokines such as interleukin 1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) play important roles in inflammatory pain. Tanshinone IIA (TIIA) is an important component of Danshen, a traditional Chinese medicine that has been commonly used to treat cardiovascular disease. In this study, we investigated the potential anti-inflammatory nociceptive effects of TIIA on complete Freund's adjuvant (CFA)-induced inflammation and inflammatory pain in rats. The effects of TIIA on CFA-induced thermal and mechanical hypersensitivity were investigated using behavioral tests. The levels of ERKs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transient receptor potential vanilloid 1 (TRPV1) in the fifth segment of the lumbar spinal cord (L5) ganglia were detected by Western blot, and the levels of mRNA and protein production of IL1-β, IL-6 and TNF-α were detected by real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immuno sorbent assay (ELISA). In this study, we found that TIIA attenuates the development of CFA-induced mechanical and thermal hypersensitivity. In addition, p-ERK and NF-κB expression levels were inhibited by TIIA, and the levels of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α were reduced. Finally, we found that the expression level of TRPV1 was significantly decreased after TIIA injection. This study demonstrated that TIIA has significant anti-nociceptive effects in a rat model of CFA-induced inflammatory pain. TIIA can inhibit the activation of ERK signaling pathways and the expression of pro-inflammatory cytokines. These results suggest that TIIA may be a potential anti-inflammatory and anti-nociceptive drug. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. In vivo anti-arthritic and anti-nociceptive effects of ethanol extract of Moringa oleifera leaves on complete Freund's adjuvant (CFA)-induced arthritis in rats.

    Science.gov (United States)

    Mahdi, Harith Jameel; Khan, Nurzalina Abdul Karim; Asmawi, Mohd Zaini Bin; Mahmud, Roziahanim; A/L Murugaiyah, Vikneswaran

    2018-03-01

    The medicinal uses of plants are in many cases based exclusively on traditional knowledge without enough scientific evidences. Different parts of Moringa oleifera were traditionally used for the treatment of wide variety of ailments including arthritis and joints pain. The present study had been designed to evaluate the anti-arthritic and anti-nociceptive activities of ethanol extract of Moringa leaves, this being the most abundant plant part suitable for commercial mass production of botanical medicinal products. Complete Freund's adjuvant (CFA)-induced arthritis in rats was used as disease model. CFA-induced inflammatory paw edema, body weight, arthritic index, X-ray radiography, hematological parameters, and walk track and locomotion analysis were all evaluated for the assessment of disease progression. In addition to that, anti-nociceptive activity was examined at different dose levels in both normal and arthritic-induced rats using Eddy's hot plate and tail flick thermal analgesia. The analysis of various arthritic assessment parameters used in this study revealed that Moringa extract has a considerable effect in preventing development or ameliorate arthritis disease severity. Moreover, the ethanol extract of Moringa leaves revealed significant anti-nociceptive activity at in both normal and CFA-induced arthritis rats in a dose-dependent manner. Ethanol extract of Moringa leaves appears to be a really promising as analgesic and arthritis medication, but a larger and more detailed preclinical and clinical studies especially in human is highly recommended.

  1. Transcutaneous electrical nerve stimulation on Yongquan acupoint reduces CFA-induced thermal hyperalgesia of rats via down-regulation of ERK2 phosphorylation and c-Fos expression.

    Science.gov (United States)

    Yang, Lin; Yang, Lianxue; Gao, Xiulai

    2010-07-01

    Activation of extracellular signal-regulated kinase-1/2 (ERK1/2) and its involvement in regulating gene expression in spinal dorsal horn, cortical and subcortical neurons by peripheral noxious stimulation contribute to pain hypersensitivity. Transcutaneous electrical nerve stimulation (TENS) is a treatment used in physiotherapy practice to promote analgesia in acute and chronic inflammatory conditions. In this study, a total number of 114 rats were used for three experiments. Effects of complete Freund's adjuvant (CFA)-induced inflammatory pain hypersensitivity and TENS analgesia on ERK1/2 phosphorylation and c-Fos protein expression were examined by using behavioral test, Western blot, and immunostaining methods. We found that CFA injection caused an area of localized swelling, erythema, hypersensitivity to thermal stimuli, the decreased response time of hind paw licking (HPL), as well as upregulation of c-Fos protein expression and ERK2 phosphorylation in the ipsilateral spinal dorsal horn and the contralateral primary somatosensory area of cortex and the amygdala of rats. TENS on Yongquan acupoint for 20 min produced obvious analgesic effects as demonstrated with increased HPL to thermal stimuli of CFA-treated rats. In addition, TENS application suppressed the CFA-induced ERK2 activation and c-Fos protein expression. These results suggest that down-regulation of ERK2 phosphorylation and c-Fos expression were involved in TENS inhibition on CFA-induced thermal hyperalgesia of rats.

  2. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase

    Science.gov (United States)

    Rajfer, R. A.; Kilic, A.; Neviaser, A. S.; Schulte, L. M.; Hlaing, S. M.; Landeros, J.; Ferrini, M. G.; Ebramzadeh, E.

    2017-01-01

    Objectives We investigated the effects on fracture healing of two up-regulators of inducible nitric oxide synthase (iNOS) in a rat model of an open femoral osteotomy: tadalafil, a phosphodiesterase inhibitor, and the recently reported nutraceutical, COMB-4 (consisting of L-citrulline, Paullinia cupana, ginger and muira puama), given orally for either 14 or 42 days. Materials and Methods Unilateral femoral osteotomies were created in 58 male rats and fixed with an intramedullary compression nail. Rats were treated daily either with vehicle, tadalafil or COMB-4. Biomechanical testing of the healed fracture was performed on day 42. The volume, mineral content and bone density of the callus were measured by quantitative CT on days 14 and 42. Expression of iNOS was measured by immunohistochemistry. Results When compared with the control group, the COMB-4 group exhibited 46% higher maximum strength (t-test, p = 0.029) and 92% higher stiffness (t-test, p = 0.023), but no significant changes were observed in the tadalafil group. At days 14 and 42, there was no significant difference between the three groups with respect to callus volume, mineral content and bone density. Expression of iNOS at day 14 was significantly higher in the COMB-4 group which, as expected, had returned to baseline levels at day 42. Conclusion This study demonstrates an enhancement in fracture healing by an oral natural product known to augment iNOS expression. Cite this article: R. A. Rajfer, A. Kilic, A. S. Neviaser, L. M. Schulte, S. M. Hlaing, J. Landeros, M. G. Ferrini, E. Ebramzadeh, S-H. Park. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase: Acceleration of fracture healing via inducible nitric oxide synthase. Bone Joint Res 2017:6:–97. DOI: 10.1302/2046-3758.62.BJR-2016-0164.R2. PMID:28188129

  3. Effect of new polyherbal formulations DF1911, DF2112 and DF2813 on CFA induced inflammation in rat model.

    Science.gov (United States)

    Nagarkar, Bhagyashri; Jagtap, Suresh

    2017-04-04

    Aim of the present study was to evaluate anti-inflammatory activity of newly developed polyherbal formulations DF1911, DF2112 and DF2813. These newly developed formulations are modifications of Dashamoola, a well known Ayurvedic formulation, along with addition of new plants. Complete Freund's adjuvant (CFA) induced inflammation in rat was used as an experimental model. Effects of the treatment in rats were monitored by physiological and biochemical parameters, histopathology and through gene expression studies. Diclofenac sodium showed maximum percentage inhibition (56.8 ± 3.5%) of paw edema followed by Dashamoola Kwatha (19.9 ± 1.8%). Among test formulations treated groups, DF1911 at 250 mg/kg bw (48.2 ± 5.4%, p CFA rats were normalized after treatment with test formulations. Results of serum markers and histopathological observations also supported the activity of formulations. Increased MDA levels in liver tissue of CFA injected animals significantly (p < 0.05) decreased by Diclofenac sodium and test formulation treated groups. DF1911, DF2112 and DF2813 showed down-regulation of IL1-β (~6.4-fold, ~5.2-fold and ~7.6-fold), IL-6 (~1.1-fold, ~1.6-fold and ~1.9-fold), TNF-α (~2.0-fold, ~4.6-fold and ~3.5-fold), and iNOS (~1.2-fold, ~1.8-fold and ~1.1-fold) in inflamed paw tissue compared to negative control group, respectively. The anti-inflammatory effects of DF1911 and DF2112 in rats were significantly higher than the Dashamoola Kwatha and are comparable to Diclofenac sodium.

  4. Hypothalamic GPR40 signaling activated by free long chain fatty acids suppresses CFA-induced inflammatory chronic pain.

    Directory of Open Access Journals (Sweden)

    Kazuo Nakamoto

    Full Text Available GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA. However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP, an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol, a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg and GW9508 (1.0 µg, a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg, a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long

  5. Hypothalamic GPR40 signaling activated by free long chain fatty acids suppresses CFA-induced inflammatory chronic pain.

    Science.gov (United States)

    Nakamoto, Kazuo; Nishinaka, Takashi; Sato, Naoya; Mankura, Mitsumasa; Koyama, Yutaka; Kasuya, Fumiyo; Tokuyama, Shogo

    2013-01-01

    GPR40 has been reported to be activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). However, reports studying functional role of GPR40 in the brain are lacking. The present study focused on the relationship between pain regulation and GPR40, investigating the functional roles of hypothalamic GPR40 during chronic pain caused using a complete Freund's adjuvant (CFA)-induced inflammatory chronic pain mouse model. GPR40 protein expression in the hypothalamus was transiently increased at day 7, but not at days 1, 3 and 14, after CFA injection. GPR40 was co-localized with NeuN, a neuron marker, but not with glial fibrillary acidic protein (GFAP), an astrocyte marker. At day 1 after CFA injection, GFAP protein expression was markedly increased in the hypothalamus. These increases were significantly inhibited by the intracerebroventricular injection of flavopiridol (15 nmol), a cyclin-dependent kinase inhibitor, depending on the decreases in both the increment of GPR40 protein expression and the induction of mechanical allodynia and thermal hyperalgesia at day 7 after CFA injection. Furthermore, the level of DHA in the hypothalamus tissue was significantly increased in a flavopiridol reversible manner at day 1, but not at day 7, after CFA injection. The intracerebroventricular injection of DHA (50 µg) and GW9508 (1.0 µg), a GPR40-selective agonist, significantly reduced mechanical allodynia and thermal hyperalgesia at day 7, but not at day 1, after CFA injection. These effects were inhibited by intracerebroventricular pretreatment with GW1100 (10 µg), a GPR40 antagonist. The protein expression of GPR40 was colocalized with that of β-endorphin and proopiomelanocortin, and a single intracerebroventricular injection of GW9508 (1.0 µg) significantly increased the number of neurons double-stained for c-Fos and proopiomelanocortin in the arcuate nucleus of the hypothalamus. Our findings suggest that hypothalamic GPR40 activated by free long chain fatty

  6. wALADin benzimidazoles differentially modulate the function of porphobilinogen synthase orthologs.

    Science.gov (United States)

    Lentz, Christian S; Halls, Victoria S; Hannam, Jeffrey S; Strassel, Silke; Lawrence, Sarah H; Jaffe, Eileen K; Famulok, Michael; Hoerauf, Achim; Pfarr, Kenneth M

    2014-03-27

    The heme biosynthesis enzyme porphobilinogen synthase (PBGS) is a potential drug target in several human pathogens. wALADin1 benzimidazoles have emerged as species-selective PBGS inhibitors against Wolbachia endobacteria of filarial worms. In the present study, we have systematically tested wALADins against PBGS orthologs from bacteria, protozoa, metazoa, and plants to elucidate the inhibitory spectrum. However, the effect of wALADin1 on different PBGS orthologs was not limited to inhibition: several orthologs were stimulated by wALADin1; others remained unaffected. We demonstrate that wALADins allosterically modulate the PBGS homooligomeric equilibrium with inhibition mediated by favoring low-activity oligomers, while 5-aminolevulinic acid, Mg(2+), or K(+) stabilized high-activity oligomers. Pseudomonas aeruginosa PBGS could be inhibited or stimulated by wALADin1 depending on these factors and pH. We have defined the wALADin chemotypes responsible for either inhibition or stimulation, facilitating the design of tailored PBGS modulators for potential application as antimicrobial agents, herbicides, or drugs for porphyric disorders.

  7. Upregulation of adrenomedullin in the spinal cord and dorsal root ganglia in the early phase of CFA-induced inflammation in rats.

    Science.gov (United States)

    Hong, Yanguo; Liu, Yushan; Chabot, Jean-Guy; Fournier, Alain; Quirion, Rémi

    2009-11-01

    Adrenomedullin (AM), a member of calcitonin gene-related peptide (CGRP) family, has been demonstrated to be a pronociceptive mediator [28]. This study was undertaken to investigate the role of AM in a model of complete Freund's adjuvant (CFA)-induced inflammatory pain. Injection of CFA, but not of saline, in the unilateral hindpaw produced an increase in the expression of AM-like immunoreactivity (AM-IR) in laminae I-II of the spinal cord as well as in small- and medium-sized dorsal root ganglion (DRG) neurons at 48 h. The content of AM in DRG on the side ipsilateral to CFA injection started to increase at 4 h and remained at high levels at 24 and 48 h. The selective antagonist of AM receptors, AM(22-52), administered intrathecally (i.t.) 24 h after CFA injection inhibited inflammation-associated hyperalgesia in a dose-dependent manner (2, 5 and 10 nmol). Impressively, this anti-hyperalgesic effect lasted for at least 24 h. I.t. administration of AM(22-52) (10 nmol) also reversed CFA-induced increase in AM-IR in the spinal dorsal horn and DRG. Furthermore, blockade of AM receptors abolished CFA-induced changes in the expression and content of CGRP-like immunoreactivity in these regions. Taken together, our results suggest that the upregulation of AM in DRG neurons contributes to the development of inflammatory pain, and this effect is mediated, at least in part, by enhancing the expression and release of CGRP. Blocking AM receptor downstream signaling effects using antagonists has the potential of relieving pain following the induction of inflammation.

  8. Involvement of PKA-dependent upregulation of nNOS-CGRP in adrenomedullin-initiated mechanistic pathway underlying CFA-induced response in rats.

    Science.gov (United States)

    Wang, Dongmei; Ruan, Liqin; Hong, Yanguo; Chabot, Jean-Guy; Quirion, Rémi

    2013-01-01

    We have previously shown that intrathecal administration of the adrenomedullin (AM) receptor antagonist AM(22-52) produces a long-lasting anti-hyperalgesia effect. This study examined the hypothesis that AM recruits other pronociceptive mediators in complete Freund's adjuvant (CFA)-induced inflammation. Injection of CFA in the hindpaw of rat produced an increase in the expression of nNOS in dorsal root ganglion (DRG) and the spinal dorsal horn. An intrathecal administration of AM(22-52), but not the CGRP antagonist BIBN4096BS, abolished the CFA-induced increase of nNOS. Moreover, AM-induced increase of CGRP was inhibited by the nNOS inhibitors L-NAME and 7-nitroindazole in cultured ganglion explants. Addition of AM to ganglion cultures induced an increase in nNOS protein, which was attenuated by the PKA inhibitor H-89. Treatment with AM also concentration-dependently increased cAMP content and pPKA protein level, but not its non-phosphorylated form, in cultured ganglia. In addition, nNOS was shown to be co-localized with the AM receptor components calcitonin receptor-like receptor and receptor activity-modifying protein 2- and 3 in DRG neurons. The present study suggests that the enhanced activity of nitric oxide (NO) mediates the biological action of AM at the spinal level and that AM recruits NO-CGRP via cAMP/PKA signaling in a mechanistic pathway underlying CFA-induced hyperalgesia. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Dynamic modulation of thymidylate synthase gene expression and fluorouracil sensitivity in human colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Kentaro Wakasa

    Full Text Available Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.

  10. Response of cardiac endothelial nitric oxide synthase to plasma viscosity modulation in acute isovolemic hemodilution

    Directory of Open Access Journals (Sweden)

    Kanyanatt Kanokwiroon

    2014-01-01

    Full Text Available Background: Endothelial nitric oxide synthase (eNOS is generally expressed in endocardial cells, vascular endothelial cells and ventricular myocytes. However, there is no experimental study elucidating the relationship between cardiac eNOS expression and elevated plasma viscosity in low oxygen delivery pathological conditions such as hemorrhagic shock-resuscitation and hemodilution. This study tested the hypothesis that elevated plasma viscosity increases cardiac eNOS expression in a hemodilution model, leading to positive effects on cardiac performance. Materials and Methods: Two groups of golden Syrian hamster underwent an acute isovolemic hemodilution where 40% of blood volume was exchanged with 2% (low-viscogenic plasma expander [LVPE] or 6% (high-viscogenic plasma expander [HVPE] of dextran 2000 kDa. In control group, experiment was performed without hemodilution. All groups were performed in awake condition. Experimental parameters, i.e., mean arterial blood pressure (MAP, heart rate, hematocrit, blood gas content and viscosity, were measured. The eNOS expression was evaluated by eNOS Western blot analysis. Results: After hemodilution, MAP decreased to 72% and 93% of baseline in the LVPE and HVPE, respectively. Furthermore, pO 2 in the LVPE group increased highest among the groups. Plasma viscosity in the HVPE group was significantly higher than that in control and LVPE groups. The expression of eNOS in the HVPE group showed higher intensity compared to other groups, especially compared with the control group. Conclusion: Our results demonstrated that cardiac eNOS has responded to plasma viscosity modulation with HVPE and LVPE. This particularly supports the previous studies that revealed the positive effects on cardiac function in animals hemodiluted with HVPE.

  11. Hyperglycemia adversely modulates endothelial nitric oxide synthase during anesthetic preconditioning through tetrahydrobiopterin- and heat shock protein 90-mediated mechanisms.

    Science.gov (United States)

    Amour, Julien; Brzezinska, Anna K; Jager, Zachary; Sullivan, Corbin; Weihrauch, Dorothee; Du, Jianhai; Vladic, Nikolina; Shi, Yang; Warltier, David C; Pratt, Phillip F; Kersten, Judy R

    2010-03-01

    Endothelial nitric oxide synthase activity is regulated by (6R-)5,6,7,8-tetrahydrobiopterin (BH4) and heat shock protein 90. The authors tested the hypothesis that hyperglycemia abolishes anesthetic preconditioning (APC) through BH4- and heat shock protein 90-dependent pathways. Myocardial infarct size was measured in rabbits in the absence or presence of APC (30 min of isoflurane), with or without hyperglycemia, and in the presence or absence of the BH4 precursor sepiapterin. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells cultured in normal (5.5 mm) or high (20 mm) glucose conditions, with or without sepiapterin (10 or 100 microm). APC decreased myocardial infarct size compared with control experiments (26 +/- 6% vs. 46 +/- 3%, respectively; P < 0.05), and this action was blocked by hyperglycemia (43 +/- 4%). Sepiapterin alone had no effect on infarct size (46 +/- 3%) but restored APC during hyperglycemia (21 +/- 3%). The beneficial actions of sepiapterin to restore APC were blocked by the nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl ester (47 +/- 2%) and the BH4 synthesis inhibitor N-acetylserotonin (46 +/- 3%). Isoflurane increased nitric oxide production to 177 +/- 13% of baseline, and this action was attenuated by high glucose concentrations (125 +/- 6%). Isoflurane increased, whereas high glucose attenuated intracellular BH4/7,8-dihydrobiopterin (BH2) (high performance liquid chromatography), heat shock protein 90-endothelial nitric oxide synthase colocalization (confocal microscopy) and endothelial nitric oxide synthase activation (immunoblotting). Sepiapterin increased BH4/BH2 and dose-dependently restored nitric oxide production during hyperglycemic conditions (149 +/- 12% and 175 +/- 9%; 10 and 100 microm, respectively). The results indicate that tetrahydrobiopterin and heat shock protein 90-regulated endothelial nitric oxide synthase activity play a central

  12. KYNA analogue SZR72 modifies CFA-induced dural inflammation- regarding expression of pERK1/2 and IL-1β in the rat trigeminal ganglion

    DEFF Research Database (Denmark)

    Lukács, M; Warfvinge, K; Kruse, L S

    2016-01-01

    modify the neurogenic inflammatory response in the trigeminal ganglion. METHODS: Inflammation in the trigeminal ganglion was induced by local dural application of Complete Freunds Adjuvant (CFA). Levels of phosphorylated MAP kinase pERK1/2 and IL-1β expression in V1 region of the trigeminal ganglion were...... investigated using immunohistochemistry and Western blot. FINDINGS: Pretreatment with one dose of SZR72 abolished the CFA-induced pERK1/2 and IL-1β activation in the trigeminal ganglion. No significant change was noted in case of repeated treatment with SZR72 as compared to a single dose. CONCLUSIONS......: This is the first study that demonstrates that one dose of KYNA analog before application of CFA can give anti-inflammatory response in a model of trigeminal activation, opening a new line for further investigations regarding possible effects of KYNA derivates....

  13. Modulation of Escherichia coli serine acetyltransferase catalytic activity in the cysteine synthase complex

    Czech Academy of Sciences Publication Activity Database

    Benoni, Roberto; De Bei, O.; Paredi, G.; Hayes, C. S.; Franko, N.; Mozzarelli, A.; Bettati, S.; Campanini, B.

    2017-01-01

    Roč. 591, č. 9 (2017), s. 1212-1224 ISSN 0014-5793 Institutional support: RVO:61388963 Keywords : cysteine synthase * protein - protein interaction * serine acetyltransferase Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.623, year: 2016

  14. Enhancement of fracture healing in the rat, modulated by compounds that stimulate inducible nitric oxide synthase

    OpenAIRE

    Rajfer, R. A.; Kilic, A.; Neviaser, A. S.; Schulte, L. M.; Hlaing, S. M.; Landeros, J.; Ferrini, M. G.; Ebramzadeh, E.; Park, S-H.

    2017-01-01

    Objectives We investigated the effects on fracture healing of two up-regulators of inducible nitric oxide synthase (iNOS) in a rat model of an open femoral osteotomy: tadalafil, a phosphodiesterase inhibitor, and the recently reported nutraceutical, COMB-4 (consisting of L-citrulline, Paullinia cupana, ginger and muira puama), given orally for either 14 or 42 days. Materials and Methods Unilateral femoral osteotomies were created in 58 male rats and fixed with an intramedullary compression na...

  15. KYNA analogue SZR72 modifies CFA-induced dural inflammation- regarding expression of pERK1/2 and IL-1β in the rat trigeminal ganglion.

    Science.gov (United States)

    Lukács, M; Warfvinge, K; Kruse, L S; Tajti, J; Fülöp, F; Toldi, J; Vécsei, L; Edvinsson, L

    2016-12-01

    Neurogenic inflammation has for decades been considered an important part of migraine pathophysiology. In the present study, we asked the question if administration of a novel kynurenic acid analogue (SZR72), precursor of an excitotoxin antagonist and anti-inflammatory substance, can modify the neurogenic inflammatory response in the trigeminal ganglion. Inflammation in the trigeminal ganglion was induced by local dural application of Complete Freunds Adjuvant (CFA). Levels of phosphorylated MAP kinase pERK1/2 and IL-1β expression in V1 region of the trigeminal ganglion were investigated using immunohistochemistry and Western blot. Pretreatment with one dose of SZR72 abolished the CFA-induced pERK1/2 and IL-1β activation in the trigeminal ganglion. No significant change was noted in case of repeated treatment with SZR72 as compared to a single dose. This is the first study that demonstrates that one dose of KYNA analog before application of CFA can give anti-inflammatory response in a model of trigeminal activation, opening a new line for further investigations regarding possible effects of KYNA derivates.

  16. Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

    Science.gov (United States)

    Toroser, D.; McMichael, R. Jr; Krause, K. P.; Kurreck, J.; Sonnewald, U.; Stitt, M.; Huber, S. C.; Davies, E. (Principal Investigator)

    1999-01-01

    Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

  17. Nicotianamine synthase overexpression positively modulates iron homeostasis-related genes in high iron rice

    Directory of Open Access Journals (Sweden)

    Meng eWang

    2013-05-01

    Full Text Available Nearly one-third of the world population, mostly women and children, suffer from iron malnutrition and its consequences, such as anemia or impaired mental development. Biofortification of rice, which is a staple crop for nearly half of the world’s population, can significantly contribute in alleviating iron deficiency. NFP rice (transgenic rice expressing nicotianamine synthase, ferritin and phytase genes has a more than six-fold increase in iron content in polished rice grains, resulting from the synergistic action of nicotianamine synthase (NAS and ferritin transgenes. We investigated iron homeostasis in NFP plants by analyzing the expression of 28 endogenous rice genes known to be involved in the homeostasis of iron and other metals, in iron-deficient and iron-sufficient conditions. RNA was collected from different tissues (roots, flag leaves, grains and at three developmental stages during grain filling. NFP plants showed increased sensitivity to iron-deficiency conditions and changes in the expression of endogenous genes involved in nicotianamine (NA metabolism, in comparison to their non-transgenic siblings. Elevated transcript levels were detected in NFP plants for several iron transporters. In contrast, expression of OsYSL2, which encodes a member of Yellow Stripe-like protein family, and a transporter of the NA-Fe(II complex was reduced in NFP plants under low iron conditions, indicating that expression of OsYSL2 is regulated by the endogenous iron status. Expression of the transgenes did not significantly affect overall iron homeostasis in NFP plants, which establishes the engineered push-pull mechanism as a suitable strategy to increase rice endosperm iron content.

  18. Increased serum IL-6 level time-dependently regulates hyperalgesia and spinal mu opioid receptor expression during CFA-induced arthritis.

    Science.gov (United States)

    Tekieh, E; Zaringhalam, Jalal; Manaheji, H; Maghsoudi, N; Alani, B; Zardooz, H

    2011-01-01

    Interleukin (IL)-6 is known to cause pro- and anti-inflammatory effects during different stages of inflammation. Recent therapeutic investigations have focused on treatment of various inflammatory disorders with anti-cytokine substances. As a result, the aim of this study was to further elucidate the influence of IL-6 in hyperalgesia and edema during different stages of Complete Freund's Adjuvant (CFA)-induced arthritis (AA) in male Wistar rats. AA was induced by a single subcutaneous injection of CFA into the rats' hindpaw. Anti-IL-6 was administered either daily or weekly during the 21 days of study. Spinal mu opioid receptor (mOR) expression was detected by Western blotting. Daily and weekly treatment with an anti-IL-6 antibody significantly decreased paw edema in the AA group compared to the AA control group. Additionally, daily and weekly anti-IL-6 administration significantly reduced hyperalgesia on day 7 in the AA group compared to the AA control group; however, there were significant increases in hyperalgesia in the antibody-treated group on days 14 and 21 compared to the AA control group. IL-6 antibody-induced increases in hyperalgesia on the 14 th and 21 st days after CFA injection correlated with a time-dependent, significant reduction in spinal mOR expression during anti-IL-6 treatment. Our study confirmed the important time-dependent relationship between serum IL-6 levels and hyperalgesia during AA. These results suggest that the stages of inflammation in AA must be considered for anti-hyperalgesic and anti-inflammatory interventions via anti-IL-6 antibody treatment.

  19. Intra-articular administration of an antibody against CSF-1 receptor reduces pain-related behaviors and inflammation in CFA-induced knee arthritis.

    Science.gov (United States)

    Alvarado-Vazquez, P A; Morado-Urbina, C E; Castañeda-Corral, G; Acosta-Gonzalez, R I; Kitaura, H; Kimura, K; Takano-Yamamoto, T; Jiménez-Andrade, J M

    2015-01-01

    Several studies have shown that blockade of colony stimulating factor-1 (CSF-1) or its receptor (CSF-1R) inhibits disease progression in rodent models of rheumatoid arthritis (RA); however, the role of the CSF-1/CSF-1R pathway in RA-induced pain and functional deficits has not been studied. Thus, we examined the effect of chronic intra-articular administration of a monoclonal anti-CSF-1R antibody (AFS98) on spontaneous pain, knee edema and functional disabilities in mice with arthritis. Unilateral arthritis was produced by multiple injections of complete Freund's adjuvant (CFA) into the right knee joint of adult male ICR mice. CFA-injected mice were then treated twice weekly from day 10 until day 25 with anti-CSF-1R antibody (3 and 10 μg/5 μL per joint), isotype control (rat IgG 10 μg/5 μL per joint) or PBS (5 μl/joint). Knee edema, spontaneous flinching, vertical rearing and horizontal exploratory activity were assessed at different days. Additionally, counts of peripheral leukocytes and body weight were measured to evaluate general health status. Intra-articular treatment with anti-CSF-1R antibody significantly increased horizontal exploratory activity and vertical rearing as well as reduced spontaneous flinching behavior and knee edema as compared to CFA-induced arthritis mice treated with PBS. Treatment with this antibody neither significantly affect mouse body weight nor the number of peripheral leukocytes. These results suggest that blockade of CSF-1R at the initial injury site (joint) could represent a therapeutic alternative for improving the functional disabilities and attenuating pain and inflammation in patients with RA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  20. Effect of Mas-related gene (Mrg) receptors on hyperalgesia in rats with CFA-induced inflammation via direct and indirect mechanisms.

    Science.gov (United States)

    Jiang, Jianping; Wang, Dongmei; Zhou, Xiaolong; Huo, Yuping; Chen, Tingjun; Hu, Fenjuan; Quirion, Rémi; Hong, Yanguo

    2013-11-01

    Mas oncogene-related gene (Mrg) receptors are exclusively distributed in small-sized neurons in trigeminal and dorsal root ganglia (DRG). We investigated the effects of MrgC receptor activation on inflammatory hyperalgesia and its mechanisms. A selective MrgC receptor agonist, bovine adrenal medulla peptide 8-22 (BAM8-22) or melanocyte-stimulating hormone (MSH) or the μ-opioid receptor (MOR) antagonist CTAP was administered intrathecally (i.t.) in rats injected with complete Freund's adjuvant (CFA) in one hindpaw. Thermal and mechanical nociceptive responses were assessed. Neurochemicals were measured by immunocytochemistry, Western blot, ELISA and RT-PCR. CFA injection increased mRNA for MrgC receptors in lumbar DRG. BAM8-22 or MSH, given i.t., generated instant short and delayed long-lasting attenuations of CFA-induced thermal hyperalgesia, but not mechanical allodynia. These effects were associated with decreased up-regulation of neuronal NOS (nNOS), CGRP and c-Fos expression in the spinal dorsal horn and/or DRG. However, i.t. administration of CTAP blocked the induction by BAM8-22 of delayed anti-hyperalgesia and inhibition of nNOS and CGRP expression in DRG. BAM8-22 also increased mRNA for MORs and pro-opiomelanocortin, along with β-endorphin content in the lumbar spinal cord and/or DRG. MrgC receptors and nNOS were co-localized in DRG neurons. Activation of MrgC receptors suppressed up-regulation of pronociceptive mediators and consequently inhibited inflammatory pain, because of the activation of up-regulated MrgC receptors and subsequent endogenous activity at MORs. The uniquely distributed MrgC receptors could be a novel target for relieving inflammatory pain. © 2013 The British Pharmacological Society.

  1. Cardamonin (2',4'-dihydroxy-6'-methoxychalcone) isolated from Boesenbergia rotunda (L.) Mansf. inhibits CFA-induced rheumatoid arthritis in rats.

    Science.gov (United States)

    Voon, Fui-Ling; Sulaiman, Mohd Roslan; Akhtar, Muhammad Nadeem; Idris, Mohamad Fauzi; Akira, Ahmad; Perimal, Enoch Kumar; Israf, Daud Ahmad; Ming-Tatt, Lee

    2017-01-05

    Boesenbergia rotunda (L.) Mansf. had been traditionally used as herbs to treat pain and rheumatism. Cardamonin (2',4'-dihydroxy-6'-methoxychalcone) is a compound isolated from Boesenbergia rotunda (L.) Mansf.. Previous study had shown the potential of cardamonin in inhibiting the release of pro-inflammatory cytokines such as tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in vitro. Thus, the possible therapeutic effect of cardamonin in the rheumatoid arthritis (RA) joints is postulated. This study was performed to investigate the anti-arthritic properties of cardamonin in rat model of induced RA, particularly on the inflammatory and pain response of RA. Rheumatoid arthritis paw inflammation was induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA) in Sprague Dawley rats. Using four doses of cardamonin (0.625, 1.25, 2.5, and 5.0mg/kg), anti-arthritic activity was evaluated through the paw edema, mechanical allodynia and thermal hyperalgesia responses. Enzyme-linked immunosorbent assay (ELISA) was carried out to evaluate the plasma level of TNF-α, IL-1β, and IL-6. Histological slides were prepared from the harvested rat paws to observe the arthritic changes in the joints. Behavioral, biochemical, and histological studies showed that cardamonin demonstrated significant inhibition on RA-induced inflammatory and pain responses as well as progression of joint destruction in rats. ELISA results showed that there was significant inhibition in TNF-α, IL-1β, and IL-6 levels in plasma of the cardamonin-treated RA rats. Overall, cardamonin possesses potential anti-arthritic properties in CFA-induced RA rat model. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. C75, a fatty acid synthase inhibitor, modulates AMP-activated protein kinase to alter neuronal energy metabolism.

    Science.gov (United States)

    Landree, Leslie E; Hanlon, Andrea L; Strong, David W; Rumbaugh, Gavin; Miller, Ian M; Thupari, Jagan N; Connolly, Erin C; Huganir, Richard L; Richardson, Christine; Witters, Lee A; Kuhajda, Francis P; Ronnett, Gabriele V

    2004-01-30

    C75, a synthetic inhibitor of fatty acid synthase (FAS), is hypothesized to alter the metabolism of neurons in the hypothalamus that regulate feeding behavior to contribute to the decreased food intake and profound weight loss seen with C75 treatment. In the present study, we characterize the suitability of primary cultures of cortical neurons for studies designed to investigate the consequences of C75 treatment and the alteration of fatty acid metabolism in neurons. We demonstrate that in primary cortical neurons, C75 inhibits FAS activity and stimulates carnitine palmitoyltransferase-1 (CPT-1), consistent with its effects in peripheral tissues. C75 alters neuronal ATP levels and AMP-activated protein kinase (AMPK) activity. Neuronal ATP levels are affected in a biphasic manner with C75 treatment, decreasing initially, followed by a prolonged increase above control levels. Cerulenin, a FAS inhibitor, causes a similar biphasic change in ATP levels, although levels do not exceed control. C75 and cerulenin modulate AMPK phosphorylation and activity. TOFA, an inhibitor of acetyl-CoA carboxylase, increases ATP levels, but does not affect AMPK activity. Several downstream pathways are affected by C75 treatment, including glucose metabolism and acetyl-CoA carboxylase (ACC) phosphorylation. These data demonstrate that C75 modulates the levels of energy intermediates, thus, affecting the energy sensor AMPK. Similar effects in hypothalamic neurons could form the basis for the effects of C75 on feeding behavior.

  3. Modulating central gain in tinnitus: changes in nitric oxide synthase in the ventral cochlear nucleus.

    Science.gov (United States)

    Coomber, Ben; Kowalkowski, Victoria L; Berger, Joel I; Palmer, Alan Richard; Wallace, Mark Nelson

    2015-01-01

    A significant challenge in tinnitus research lies in explaining how acoustic insult leads to tinnitus in some individuals, but not others. One possibility is genetic variability in the expression and function of neuromodulators - components of neural signaling that alter the balance of excitation and inhibition in neural circuits. An example is nitric oxide (NO) - a free radical and potent neuromodulator in the mammalian brain - that regulates plasticity via both pre-synaptic and postsynaptic mechanisms. Changes in NO have previously been implicated in tinnitus generation, specifically in the ventral cochlear nucleus (VCN). Here, we examined nitric oxide synthase (NOS) - the enzyme responsible for NO production - in the guinea pig VCN following acoustic trauma. NOS was present in most cell types - including spherical and globular bushy cells, small, medium, and large multipolar cells, and octopus cells - spanning the entire extent of the VCN. The staining pattern was symmetrical in control animals. Unilateral acoustic over-exposure (AOE) resulted in marked asymmetries between ipsilateral and contralateral sides of the VCN in terms of the distribution of NOS across the cochlear nuclei in animals with behavioral evidence of tinnitus: fewer NOS-positive cells and a reduced level of NOS staining was present across the whole extent of the contralateral VCN, relative to the ipsilateral VCN. The asymmetric pattern of NOS-containing cells was observed as early as 1 day after AOE and was also present in some animals at 3, 7, and 21 days after AOE. However, it was not until 8 weeks after AOE, when tinnitus had developed, that asymmetries were significant overall, compared with control animals. Asymmetrical NOS expression was not correlated with shifts in the threshold hearing levels. Variability in NOS expression between animals may represent one underlying difference that can be linked to whether or not tinnitus develops after noise exposure.

  4. Modulating central gain in tinnitus: Changes in nitric oxide synthase in the ventral cochlear nucleus

    Directory of Open Access Journals (Sweden)

    Ben eCoomber

    2015-03-01

    Full Text Available A significant challenge in tinnitus research lies in explaining how acoustic insult leads to tinnitus in some individuals, but not others. One possibility is genetic variability in the expression and function of neuromodulators – components of neural signalling that alter the balance of excitation and inhibition in neural circuits. An example is nitric oxide (NO – a free radical and potent neuromodulator in the mammalian brain – that regulates plasticity via both presynaptic and postsynaptic mechanisms. Changes in NO have previously been implicated in tinnitus generation, specifically in the ventral cochlear nucleus (VCN. Here, we examined nitric oxide synthase (NOS – the enzyme responsible for NO production – in the guinea pig VCN following acoustic trauma. NOS was present in most cell types – including spherical and globular bushy cells, small, medium and large multipolar cells, and octopus cells – spanning the entire extent of the VCN. The staining pattern was symmetrical in control animals. Unilateral acoustic over-exposure (AOE resulted in marked asymmetries between ipsilateral and contralateral sides of the VCN in terms of the distribution of NOS across the cochlear nuclei in animals with behavioural evidence of tinnitus: fewer NOS-positive cells and a reduced level of NOS staining was present across the whole extent of the contralateral VCN, relative to the ipsilateral VCN. The asymmetric pattern of NOS-containing cells was observed as early as one day after AOE and was also present in some animals at 3, 7 and 21 days after AOE. However it was not until eight weeks after AOE, when tinnitus had developed, that asymmetries were significant overall, compared with control animals. Asymmetrical NOS expression was not correlated with shifts in the threshold hearing levels. Variability in NOS expression between animals may represent one underlying difference that can be linked to whether or not tinnitus develops after noise

  5. Transcutaneous electrical nerve stimulation attenuates CFA-induced hyperalgesia and inhibits spinal ERK1/2-COX-2 pathway activation in rats.

    Science.gov (United States)

    Fang, Jun-Fan; Liang, Yi; Du, Jun-Ying; Fang, Jian-Qiao

    2013-06-15

    Transcutaneous electrical nerve stimulation (TENS) is a non-pharmacologic treatment for pain relief. In previous animal studies, TENS effectively alleviated Complete Freund's Adjuvant (CFA)- or carrageenan-induced inflammatory pain. Although TENS is known to produce analgesia via opioid activation in the brain and at the spinal level, few reports have investigated the signal transduction pathways mediated by TENS. Prior studies have verified the importance of the activation of extracellular signal-regulated kinase (ERK) signal transduction pathway in the spinal cord dorsal horn (SCDH) in acute and persistent inflammatory pains. Here, by using CFA rat model, we tested the efficacy of TENS on inhibiting the expressions of p-ERK1/2 and of its downstream cyclooxygenase-2 (COX-2) and the level of prostaglandin E2 (PGE2) at spinal level. Rats were randomly divided into control, model and TENS groups, and injected subcutaneously with 100 μl CFA or saline in the plantar surface of right hind paw. Rats in the TENS group were treated with TENS (constant aquare wave, 2 Hz and 100 Hz alternating frequencies, intensities ranging from 1 to 2 mA, lasting for 30 min each time) at 5 h and 24 h after injection. Paw withdrawal thresholds (PWTs) were measured with dynamic plantar aesthesiometer at 3d before modeling and 5 h, 6 h, and 25 h after CFA injection. The ipsilateral sides of the lumbar spinal cord dosral horns were harvested for detecting the expressions of p-ERK1/2 and COX-2 by western blot analysis and qPCR, and PGE2 by ELISA. CFA-induced periphery inflammation decreased PWTs and increased paw volume of rats. TENS treatment significantly alleviated mechanical hyperalgesia caused by CFA. However, no anti-inflammatory effect of TENS was observed. Expression of p-ERK1/2 protein and COX-2 mRNA was significantly up-regualted at 5 h and 6 h after CFA injection, while COX-2 and PGE2 protein level only increased at 6 h after modeling. Furthermore, the high expression of p-ERK1

  6. A stable blue-light-derived signal modulates ultraviolet-light-induced activation of the chalcone-synthase gene in cultured parsley cells

    International Nuclear Information System (INIS)

    Ohl, S.; Hahlbrock, K.; Schäfer, E.

    1989-01-01

    Run-off transcription assays were used to demonstrate that both the ultraviolet (UV)-B and blue-light receptors control transcription rates for chalcone-synthase mRNA in the course of light-induced flavonoid synthesis in parsley (Petroselinum crispum Miller (A.W. Hill)) cell-suspension cultures. Blue and red light alone, presumably acting via a blue-light receptor and active phytochrome (far-red absorbing form) respectively, can induce accumulation of chalcone-synthase mRNA. The extent of the response is however considerably smaller than that obtained when these wavebands are applied in combination with UV light. A preirradiation with blue light strongly increases the response to a subsequent UV pulse and this modulating effect of blue light is stable for at least 20 h. The modulating effect is abolished by a UV induction but can be reestablished by a second irradiation with blue light. (author)

  7. The Class II trehalose 6-phosphate synthase gene PvTPS9 modulates trehalose metabolism in Phaseolus vulgaris nodules.

    Directory of Open Access Journals (Sweden)

    Aarón Barraza

    2016-11-01

    Full Text Available Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS of common bean (Phaseolus vulgaris, was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1% of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant.

  8. GNC and CGA1 Modulate Chlorophyll Biosynthesis and Glutamate Synthase (GLU1/Fd-GOGAT) Expression in Arabidopsis

    Science.gov (United States)

    Hudson, Darryl; Guevara, David; Yaish, Mahmoud W.; Hannam, Carol; Long, Nykoll; Clarke, Joseph D.; Bi, Yong-Mei; Rothstein, Steven J.

    2011-01-01

    Chloroplast development is an important determinant of plant productivity and is controlled by environmental factors including amounts of light and nitrogen as well as internal phytohormones including cytokinins and gibberellins (GA). The paralog GATA transcription factors GNC and CGA1/GNL up-regulated by light, nitrogen and cytokinin while also being repressed by GA signaling. Modifying the expression of these genes has previously been shown to influence chlorophyll content in Arabidopsis while also altering aspects of germination, elongation growth and flowering time. In this work, we also use transgenic lines to demonstrate that GNC and CGA1 exhibit a partially redundant control over chlorophyll biosynthesis. We provide novel evidence that GNC and CGA1 influence both chloroplast number and leaf starch in proportion to their transcript level. GNC and CGA1 were found to modify the expression of chloroplast localized GLUTAMATE SYNTHASE (GLU1/Fd-GOGAT), which is the primary factor controlling nitrogen assimilation in green tissue. Altering GNC and CGA1 expression was also found to modulate the expression of important chlorophyll biosynthesis genes (GUN4, HEMA1, PORB, and PORC). As previously demonstrated, the CGA1 transgenic plants demonstrated significantly altered timing to a number of developmental events including germination, leaf production, flowering time and senescence. In contrast, the GNC transgenic lines we analyzed maintain relatively normal growth phenotypes outside of differences in chloroplast development. Despite some evidence for partial divergence, results indicate that regulation of both GNC and CGA1 by light, nitrogen, cytokinin, and GA acts to modulate nitrogen assimilation, chloroplast development and starch production. Understanding the mechanisms controlling these processes is important for agricultural biotechnology. PMID:22102866

  9. Glycogen Synthase Kinase-3 Modulates Hyperosmotic-Induced Urea Transporter A1 Relocation in the Inner Medullary Collecting Duct Cells.

    Science.gov (United States)

    Li, Yong-Xia; Huang, Yun; Liu, Song; Mao, Yan; Yuan, Cheng-Yan; Yang, Xiao; Yao, Li-Jun

    2016-01-01

    Glycogen synthase kinase 3 (GSK3) regulates urine concentration by mediating the vasopressin-induced aquaporin 2 expression and water permeability, although it is unknown whether GSK3 also mediates the accumulation of the urea transporter A1 (UT-A1). The aim of this study is to investigate the effect of GSK3 on UT-A1 distribution. Mouse inner medullary collecting duct 3 cells were transfected with UT-A1-GFP construct. The stable transfected cells were cultured under hypertonic conditions, treated with GSK3 inhibitor lithium chloride, GSK3 activator, lysosome or proteasome inhibitor. The expression levels of UT-A1, GSK3, and phospho-GSK3 were analyzed using western blot. The interaction between UT-A1 and the Golgi apparatus was examined using confocal immunofluorescence microscope. The UT-A1 trafficking was examined using the biotinylation of surface membranes. UT-A1 dissociated away from the Golgi apparatus and translocated to the plasma membrane under hypertonic-NaCl and NaCl plus urea stimulation. This movement was accompanied by the increased phosphorylation of GSK3 and its localization on the cellular membrane. Moreover, these results were duplicated by treating the cells with the GSK3 inhibitor, and by contrast, were partially reversed by the GSK3 activator. Treating cells with a lysosome or proteasome inhibitor failed to attenuate the effects of hypertonic stimulus, indicating that the loss of UT-A1 from the Golgi was not due to degradation. Our results suggest that GSK3 may in part modulate the hypertonic-induced intracellular UT-A1 redistribution and its accumulation on the plasma membrane, which may constitute another mechanism by which GSK3 modulates urine concentration. © 2016 S. Karger AG, Basel.

  10. Regulation of Banana Phytoene Synthase (MaPSY) Expression, Characterization and Their Modulation under Various Abiotic Stress Conditions

    Science.gov (United States)

    Kaur, Navneet; Pandey, Ashutosh; Shivani; Kumar, Prateek; Pandey, Pankaj; Kesarwani, Atul K.; Mantri, Shrikant S.; Awasthi, Praveen; Tiwari, Siddharth

    2017-01-01

    Phytoene synthase (PSY) is a key regulatory enzyme of carotenoid biosynthesis pathway in plants. The present study examines the role of PSY in carotenogenesis and stress management in banana. Germplasm screening of 10 Indian cultivars showed that Nendran (3011.94 μg/100 g dry weight) and Rasthali (105.35 μg/100 g dry weight) contained the highest and lowest amounts of β-carotene, respectively in ripe fruit-pulp. Nendran ripe pulp also showed significantly higher antioxidant activity as compared to Rasthali. Meta-analysis of three banana PSY genes (MaPSY1, MaPSY2, and MaPSY3) was performed to identify their structural features, subcellular, and chromosomal localization in banana genome. The distinct expression patterns of MaPSY1, MaPSY2, and MaPSY3 genes were observed in various tissues, and fruit developmental stages of these two contrasting cultivars, suggesting differential regulation of the banana PSY genes. A positive correlation was observed between the expression of MaPSY1 and β-carotene accumulation in the ripe fruit-peel and pulp of Nendran. The presence of stress responsive cis-regulatory motifs in promoter region of MaPSY genes were correlated with the expression pattern during various stress (abscisic acid, methyl jasmonate, salicylic acid and dark) treatments. The positive modulation of MaPSY1 noticed under abiotic stresses suggested its role in plant physiological functions and defense response. The amino acid sequence analysis of the PSY proteins in contrasting cultivars revealed that all PSY comprises conserved domains related to enzyme activity. Bacterial complementation assay has validated the functional activity of six PSY proteins and among them PSY1 of Nendran (Nen-PSY1) gave the highest activity. These data provide new insights into the regulation of PSY expression in banana by developmental and stress related signals that can be explored in the banana improvement programs. PMID:28421096

  11. Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

    Directory of Open Access Journals (Sweden)

    Irene Mavelli

    2012-02-01

    Full Text Available Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis or “negative” (silencing mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1. Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

  12. Participation of hippocampal nitric oxide synthase and soluble guanylate cyclase in the modulation of behavioral responses elicited by the rat forced swimming test.

    Science.gov (United States)

    Sales, Amanda J; Hiroaki-Sato, Vinícius A; Joca, Sâmia R L

    2017-02-01

    Systemic or hippocampal administration of nitric oxide (NO) synthase inhibitors induces antidepressant-like effects in animals, implicating increased hippocampal levels of NO in the neurobiology of depression. However, the role played by different NO synthase in this process has not been clearly defined. As stress is able to induce neuroinflammatory mechanisms and trigger the expression of inducible nitric oxide synthase (iNOS) in the brain, as well as upregulate neuronal nitric oxide synthase (nNOS) activity, the aim of the present study was to investigate the possible differential contribution of hippocampal iNOS and nNOS in the modulation of the consequences of stress elicited by the forced swimming test. Male Wistar rats received intrahippocampal injections, immediately after the pretest or 1 h before the forced swimming test, of selective inhibitors of nNOS (N-propyl-L-arginine), iNOS (1400W), or sGC (ODQ), the main pharmacological target for NO. Stress exposure increased nNOS and phospho-nNOS levels at all time points, whereas iNOS expression was increased only 24 h after the pretest. All drugs induced an antidepressant-like effect. However, whereas the nNOS inhibitor was equally effective when injected at different times, the iNOS inhibitor was more effective 24 h after the pretest. These results suggest that hippocampal nNOS and iNOS contribute to increase in NO levels in response to stress, although with a differential time course after stress exposure.

  13. Engineering of Ion Sensing by the Cystathionine beta-Synthase Module of the ABC Transporter OpuA

    NARCIS (Netherlands)

    Mahmood, Nik A. B. N.; Biemans-Oldehinkel, Esther; Poolman, Bert

    2009-01-01

    We have previously shown that the C-terminal cystathionine beta-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (I(in)). Here, we show that a surface-exposed

  14. Imidazopyridine-Based Fatty Acid Synthase Inhibitors That Show Anti-HCV Activity and in Vivo Target Modulation.

    Science.gov (United States)

    Oslob, Johan D; Johnson, Russell J; Cai, Haiying; Feng, Shirley Q; Hu, Lily; Kosaka, Yuko; Lai, Julie; Sivaraja, Mohanram; Tep, Samnang; Yang, Hanbiao; Zaharia, Cristiana A; Evanchik, Marc J; McDowell, Robert S

    2013-01-10

    Potent imidazopyridine-based inhibitors of fatty acid synthase (FASN) are described. The compounds are shown to have antiviral (HCV replicon) activities that track with their biochemical activities. The most potent analogue (compound 19) also inhibits rat FASN and inhibits de novo palmitate synthesis in vitro (cell-based) as well as in vivo.

  15. Aspergillus fumigatus Trehalose-Regulatory Subunit Homolog Moonlights To Mediate Cell Wall Homeostasis through Modulation of Chitin Synthase Activity

    Directory of Open Access Journals (Sweden)

    Arsa Thammahong

    2017-04-01

    Full Text Available Trehalose biosynthesis is found in fungi but not humans. Proteins involved in trehalose biosynthesis are essential for fungal pathogen virulence in humans and plants through multiple mechanisms. Loss of canonical trehalose biosynthesis genes in the human pathogen Aspergillus fumigatus significantly alters cell wall structure and integrity, though the mechanistic link between these virulence-associated pathways remains enigmatic. Here we characterize genes, called tslA and tslB, which encode proteins that contain domains similar to those corresponding to trehalose-6-phosphate phosphatase but lack critical catalytic residues for phosphatase activity. Loss of tslA reduces trehalose content in both conidia and mycelia, impairs cell wall integrity, and significantly alters cell wall structure. To gain mechanistic insights into the role that TslA plays in cell wall homeostasis, immunoprecipitation assays coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS were used to reveal a direct interaction between TslA and CsmA, a type V chitin synthase enzyme. TslA regulates not only chitin synthase activity but also CsmA sub-cellular localization. Loss of TslA impacts the immunopathogenesis of murine invasive pulmonary aspergillosis through altering cytokine production and immune cell recruitment. In conclusion, our data provide a novel model whereby proteins in the trehalose pathway play a direct role in fungal cell wall homeostasis and consequently impact fungus-host interactions.

  16. Involvement of MrgprC in Electroacupuncture Analgesia for Attenuating CFA-Induced Thermal Hyperalgesia by Suppressing the TRPV1 Pathway

    Directory of Open Access Journals (Sweden)

    Ying-jun Liu

    2018-01-01

    Full Text Available Mas-related G-protein-coupled receptor C (MrgprC plays an important role in modulating chronic inflammatory pain. Electroacupuncture (EA has a satisfactory analgesic effect on chronic pain. This study aimed to investigate the involvement of MrgprC and its transient receptor potential vanilloid 1 (TRPV1 pathway in EA analgesia in chronic inflammatory pain. Chronic inflammatory pain was induced by subcutaneously injecting complete Freund’s adjuvant (CFA into the left hind paw. EA (2/100 Hz stimulation was administered. MrgprC siRNAs were intrathecally administered to inhibit MrgprC expression, and bovine adrenal medulla 8-22 (BAM8-22 was used to activate MrgprC. The mechanical allodynia was decreased by EA significantly since day 3. The piled analgesic effect of EA was partially blocked by 6 intrathecal administrations of MrgprC siRNA. Both EA and BAM8-22 could downregulate the expression of TRPV1 and PKC in both the DRG and the SCDH. Both EA and BAM8-22 could also decrease the TRPV1 translocation and p-TRPV1 level in both the DRG and the SCDH. The effects of EA on PKCε, TRPV1 translocation, and p-TRPV1 in both the DRG and the SCDH were reversed by MrgprC siRNA. The results indicated that MrgprC played crucial roles in chronic pain modulation and was involved in EA analgesia partially through the regulation of TRPV1 function at the DRG and SCDH levels.

  17. Involvement of MrgprC in Electroacupuncture Analgesia for Attenuating CFA-Induced Thermal Hyperalgesia by Suppressing the TRPV1 Pathway.

    Science.gov (United States)

    Liu, Ying-Jun; Lin, Xiao-Xi; Fang, Jian-Qiao; Fang, Fang

    2018-01-01

    Mas-related G-protein-coupled receptor C (MrgprC) plays an important role in modulating chronic inflammatory pain. Electroacupuncture (EA) has a satisfactory analgesic effect on chronic pain. This study aimed to investigate the involvement of MrgprC and its transient receptor potential vanilloid 1 (TRPV1) pathway in EA analgesia in chronic inflammatory pain. Chronic inflammatory pain was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into the left hind paw. EA (2/100 Hz) stimulation was administered. MrgprC siRNAs were intrathecally administered to inhibit MrgprC expression, and bovine adrenal medulla 8-22 (BAM8-22) was used to activate MrgprC. The mechanical allodynia was decreased by EA significantly since day 3. The piled analgesic effect of EA was partially blocked by 6 intrathecal administrations of MrgprC siRNA. Both EA and BAM8-22 could downregulate the expression of TRPV1 and PKC in both the DRG and the SCDH. Both EA and BAM8-22 could also decrease the TRPV1 translocation and p-TRPV1 level in both the DRG and the SCDH. The effects of EA on PKC ε , TRPV1 translocation, and p-TRPV1 in both the DRG and the SCDH were reversed by MrgprC siRNA. The results indicated that MrgprC played crucial roles in chronic pain modulation and was involved in EA analgesia partially through the regulation of TRPV1 function at the DRG and SCDH levels.

  18. Platelet-derived growth factor-DD targeting arrests pathological angiogenesis by modulating glycogen synthase kinase-3beta phosphorylation.

    Science.gov (United States)

    Kumar, Anil; Hou, Xu; Lee, Chunsik; Li, Yang; Maminishkis, Arvydas; Tang, Zhongshu; Zhang, Fan; Langer, Harald F; Arjunan, Pachiappan; Dong, Lijin; Wu, Zhijian; Zhu, Linda Y; Wang, Lianchun; Min, Wang; Colosi, Peter; Chavakis, Triantafyllos; Li, Xuri

    2010-05-14

    Platelet-derived growth factor-DD (PDGF-DD) is a recently discovered member of the PDGF family. The role of PDGF-DD in pathological angiogenesis and the underlying cellular and molecular mechanisms remain largely unexplored. In this study, using different animal models, we showed that PDGF-DD expression was up-regulated during pathological angiogenesis, and inhibition of PDGF-DD suppressed both choroidal and retinal neovascularization. We also demonstrated a novel mechanism mediating the function of PDGF-DD. PDGF-DD induced glycogen synthase kinase-3beta (GSK3beta) Ser(9) phosphorylation and Tyr(216) dephosphorylation in vitro and in vivo, leading to increased cell survival. Consistently, GSK3beta activity was required for the antiangiogenic effect of PDGF-DD targeting. Moreover, PDGF-DD regulated the expression of GSK3beta and many other genes important for angiogenesis and apoptosis. Thus, we identified PDGF-DD as an important target gene for antiangiogenic therapy due to its pleiotropic effects on vascular and non-vascular cells. PDGF-DD inhibition may offer new therapeutic options to treat neovascular diseases.

  19. Modulation of inducible nitric oxide synthase gene expression in RAW 264.7 murine macrophages by Pacific ciguatoxin.

    Science.gov (United States)

    Kumar-Roiné, Shilpa; Matsui, Mariko; Chinain, Mireille; Laurent, Dominique; Pauillac, Serge

    2008-08-01

    To investigate the possible involvement of the nitric oxide radical (NO) in ciguatera fish poisoning (CFP), the in vitro effects of the main Pacific ciguatoxin (P-CTX-1B) and bacterial lipopolysaccharide (LPS) were comparatively studied on neuroblastoma Neuro-2a and on macrophage RAW 264.7 cell lines. NO accumulation was quantified by measuring nitrite levels in cellular supernatant using Griess reagent while the up-regulation of inducible nitric oxide synthase (iNOS) at the mRNA level was quantified via Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR). P-CTX-1B caused a concentration- and time-dependent induction of iNOS in RAW 264.7 cells but not in Neuro-2a cells. NO production was evidenced by increased nitrite levels in the 10 microM range after 48 h of RAW 264.7 cells exposure to LPS and P-CTX-1B (0.05 microg/ml and 6 nM, respectively). The expression of iNOS mRNA peaked at 8h for LPS then gradually decreased to low level at 48 h. In contrast, a sustained level was recorded with P-CTX-1B in the 8-48 h time interval. The addition of N(omega)-nitro-L-arginine methyl ester (L-NAME), a stereoselective NOS inhibitor, strongly diminished NO formation but had no effect on iNOS mRNA synthesis. The implication of NO in CFP paves the way for new therapies for both western and traditional medicines.

  20. Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1-dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

    Science.gov (United States)

    Dries, Eef; Santiago, Demetrio J; Johnson, Daniel M; Gilbert, Guillaume; Holemans, Patricia; Korte, Sanne M; Roderick, H Llewelyn; Sipido, Karin R

    2016-10-15

    The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca 2+ /calmodulin-dependent kinase II (CaMKII) activation during high-frequency stimulation. Sympathetic stimulation through β-adrenergic receptors activates an integrated signalling cascade, enhancing Ca 2+ cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during β-adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca 2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca 2+ transient are primarily protein kinase A-dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. In cardiac myocytes, β-adrenergic stimulation enhances Ca 2+ cycling through an integrated signalling cascade modulating L-type Ca 2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca 2+ /calmodulin-dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca 2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole-cell voltage-clamp and confocal line-scan imaging with Fluo-4 as a [Ca 2+ ] i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca 2+ sparks, which are assigned to coupled and non-coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca 2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces

  1. Efficacy and gastrointestinal tolerability of ML3403, a selective inhibitor of p38 MAP kinase and CBS-3595, a dual inhibitor of p38 MAP kinase and phosphodiesterase 4 in CFA-induced arthritis in rats.

    Science.gov (United States)

    Koch, Diana A; Silva, Rodrigo B M; de Souza, Alessandra H; Leite, Carlos E; Nicoletti, Natália F; Campos, Maria M; Laufer, Stefan; Morrone, Fernanda B

    2014-03-01

    Mitogen-activated protein kinase (MAPK) p38 inhibitors have entered the clinical phase, although many of them have failed due to high toxicity and lack of efficacy. In the present study we compared the effects of the selective p38 inhibitor ML3403 and the dual p38-PDE4 inhibitor CBS-3595, on inflammatory and nociceptive parameters in a model of polyarthritis in rats. Male Wistar rats (180-200 g) were used for the complete Freund's adjuvant (CFA)-induced arthritis model and they were evaluated at 14-21 days. We also analysed the effects of these pharmacological tools on liver and gastrointestinal toxicity and on cytokine levels. Repeated CBS-3595 (3 mg/kg) or ML3403 (10 mg/kg) administration produced significant anti-inflammatory actions in the chronic arthritis model induced by CFA. CBS-3595 and ML3403 treatment also markedly reduced the production of the proinflammatory cytokine IL-6 in the paw tissue, whereas it widely increased the levels of the anti-inflammatory cytokine IL-10. Moreover, CBS-3595 produced partial anti-allodynic effects in the CFA model at 4 and 8 days after treatment. Notably, ML3403 and CBS-3595 did not show marked signs of hepatoxicity, as supported by unaltered histological observations in the liver sections. Finally, both compounds were safe in the gastrointestinal tract, according to evaluation of intestinal biopsies. CBS-3595 displayed a superior profile regarding its anti-inflammatory effects. Thus p38 MAPK/PDE4 blocking might well constitute a relevant strategy for the treatment of RA.

  2. Benzalacetone Synthase

    Directory of Open Access Journals (Sweden)

    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  3. Deciphering the interplay between cysteine synthase and thiol cascade proteins in modulating Amphotericin B resistance and survival of Leishmania donovani under oxidative stress

    Directory of Open Access Journals (Sweden)

    Kuljit Singh

    2017-08-01

    Full Text Available Leishmania donovani is the causative organism of the neglected human disease known as visceral leishmaniasis which is often fatal, if left untreated. The cysteine biosynthesis pathway of Leishmania may serve as a potential drug target because it is different from human host and regulates downstream components of redox metabolism of the parasites; essential for their survival, pathogenicity and drug resistance. However, despite the apparent dependency of redox metabolism of cysteine biosynthesis pathway, the role of L. donovani cysteine synthase (LdCS in drug resistance and redox homeostasis has been unexplored. Herein, we report that over-expression of LdCS in Amphotericin B (Amp B sensitive strain (S1-OE modulates resistance towards oxidative stress and drug pressure. We observed that antioxidant enzyme activities were up-regulated in S1-OE parasites and these parasites alleviate intracellular reactive oxygen species (ROS efficiently by maintaining the reduced thiol pool. In contrast to S1-OE parasites, Amp B sensitive strain (S1 showed higher levels of ROS which was positively correlated with the protein carbonylation levels and negatively correlated with cell viability. Moreover, further investigations showed that LdCS over-expression also augments the ROS-primed induction of LdCS-GFP as well as endogenous LdCS and thiol pathway proteins (LdTryS, LdTryR and LdcTXN in L. donovani parasites; which probably aids in stress tolerance and drug resistance. In addition, the expression of LdCS was found to be up-regulated in Amp B resistant isolates and during infective stationary stages of growth and consistent with these observations, our ex vivo infectivity studies confirmed that LdCS over-expression enhances the infectivity of L. donovani parasites. Our results reveal a novel crosstalk between LdCS and thiol metabolic pathway proteins and demonstrate the crucial role of LdCS in drug resistance and redox homeostasis of Leishmania. Keywords

  4. Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity.

    Science.gov (United States)

    Gunasekera, Bhagya; Abou Diwan, Charbel; Altawallbeh, Ghaith; Kalil, Haitham; Maher, Shaimaa; Xu, Song; Bayachou, Mekki

    2018-03-07

    Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

  5. Modulation of fatty acid synthase degradation by concerted action of p38 MAP kinase, E3 ligase COP1, and SH2-tyrosine phosphatase Shp2.

    Science.gov (United States)

    Yu, Jianxiu; Deng, Rong; Zhu, Helen H; Zhang, Sharon S; Zhu, Changhong; Montminy, Marc; Davis, Roger; Feng, Gen-Sheng

    2013-02-08

    The Src-homology 2 (SH2) domain-containing tyrosine phosphatase Shp2 has been known to regulate various signaling pathways triggered by receptor and cytoplasmic tyrosine kinases. Here we describe a novel function of Shp2 in control of lipid metabolism by mediating degradation of fatty acid synthase (FASN). p38-phosphorylated COP1 accumulates in the cytoplasm and subsequently binds FASN through Shp2 here as an adapter, leading to FASN-Shp2-COP1 complex formation and FASN degradation mediated by ubiquitination pathway. By fasting p38 is activated and stimulates FASN protein degradation in mice. Consistently, the FASN protein levels are dramatically elevated in mouse liver and pancreas in which Shp2/Ptpn11 is selectively deleted. Thus, this study identifies a new activity for Shp2 in lipid metabolism.

  6. The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression.

    Science.gov (United States)

    Zhou, Fei; Sun, Tian-Hu; Zhao, Lei; Pan, Xi-Wu; Lu, Shan

    2015-01-01

    The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, -421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant.

  7. The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression

    Directory of Open Access Journals (Sweden)

    Fei eZhou

    2015-04-01

    Full Text Available The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, -421 bp from the translation initiation site which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA, we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD and constant dark (DD conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant.

  8. Modulation of Matrix Metalloproteinase 14, Tissue Inhibitor of Metalloproteinase 3, Tissue Inhibitor of Metalloproteinase 4, and Inducible Nitric Oxide Synthase in the Development of Periapical Lesions.

    Science.gov (United States)

    Cassanta, Lorena Teodoro de Castro; Rodrigues, Virmondes; Violatti-Filho, Jose Roberto; Teixeira Neto, Benedito Alves; Tavares, Vinícius Marques; Bernal, Eduarda Castelo Branco Araujo; Souza, Danila Malheiros; Araujo, Marcelo Sivieri; de Lima Pereira, Sanivia Aparecida; Rodrigues, Denise Bertulucci Rocha

    2017-07-01

    Periapical cysts and granulomas are chronic lesions caused by an inflammatory immune response against microbial challenge in the root canal. Different cell types, cytokines, and molecules have been associated with periapical lesion formation and expansion. Therefore, because of the chronic inflammatory state of these lesions, the aim of this study was to evaluate the in situ expression of matrix metalloproteinase (MMP)-14 and -19, tissue inhibitor of metalloproteinase (TIMP)-3 and -4, CD68, and inducible nitric oxide synthase (iNOS) in periapical cysts and granulomas. Sixteen cases of periapical cysts and 15 cases of periapical granulomas were analyzed. Ten normal dental pulps were used as the negative control. Immunohistochemistry was performed with anti-MMP-19, anti-MMP-14, anti-TIMP-3, anti-TIMP-4, anti-iNOS, and anti-CD68 antibodies. The expression of TIMP-3, TIMP-4, iNOS, and CD68 was significantly higher in both the cyst and granuloma groups than in the control group. TIMP-4 was also significantly higher in cases of chronic apical abscess. There was also a significant difference in the expression of MMP-14 between the cyst and control groups. However, there were no differences in the expression of MMP-19 between the 3 groups. Our data suggest that the expression of MMP-14, TIMP-3, and TIMP-4 is associated with the development of periapical lesions. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  9. Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response.

    Science.gov (United States)

    Zhang, Conggang; Liu, Zeyu; Bunker, Eric; Ramirez, Adrian; Lee, Schuyler; Peng, Yinghua; Tan, Aik-Choon; Eckhardt, S Gail; Chapnick, Douglas A; Liu, Xuedong

    2017-09-08

    Sorafenib (Nexavar) is a broad-spectrum multikinase inhibitor that proves effective in treating advanced renal-cell carcinoma and liver cancer. Despite its well-characterized mechanism of action on several established cancer-related protein kinases, sorafenib causes variable responses among human tumors, although the cause for this variation is unknown. In an unbiased screening of an oncology drug library, we found that sorafenib activates recruitment of the ubiquitin E3 ligase Parkin to damaged mitochondria. We show that sorafenib inhibits the activity of both complex II/III of the electron transport chain and ATP synthase. Dual inhibition of these complexes, but not inhibition of each individual complex, stabilizes the serine-threonine protein kinase PINK1 on the mitochondrial outer membrane and activates Parkin. Unlike the protonophore carbonyl cyanide m -chlorophenylhydrazone, which activates the mitophagy response, sorafenib treatment triggers PINK1/Parkin-dependent cellular apoptosis, which is attenuated upon Bcl-2 overexpression. In summary, our results reveal a new mechanism of action for sorafenib as a mitocan and suggest that high Parkin activity levels could make tumor cells more sensitive to sorafenib's actions, providing one possible explanation why Parkin may be a tumor suppressor gene. These insights could be useful in developing new rationally designed combination therapies with sorafenib. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Mutations in the FMN domain modulate MCD spectra of the heme site in the oxygenase domain of inducible nitric oxide synthase.

    Science.gov (United States)

    Sempombe, Joseph; Elmore, Bradley O; Sun, Xi; Dupont, Andrea; Ghosh, Dipak K; Guillemette, J Guy; Kirk, Martin L; Feng, Changjian

    2009-05-27

    The nitric oxide synthase (NOS) output state for NO production is a complex of the flavin mononucleotide (FMN)-binding domain and the heme domain, and thereby it facilitates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN-heme interactions are important in the formation of the output state because they guide the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bidomain oxygenase/FMN construct have been observed by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins clearly indicates that a properly docked FMN domain contributes to the observed L-Arg perturbation of the heme MCD spectrum in the wild-type protein and that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.

  11. Sequence of the gamma-subunit of Spirulina platensis : a new principle of thiol modulation of F0F1 ATP synthase?

    NARCIS (Netherlands)

    Steinemann, D.; Lill, H

    1995-01-01

    The gene encoding the gamma subunit of Spirulina platensis F0F1, the relative of the chloroplast F1 subunit responsible for thiol activation, has been cloned and sequenced. As in other cyanobacteria, a specific couple of cysteines like those involved in thiol modulation of the chloroplast enzyme was

  12. Extracellular Protein Kinase A Modulates Intracellular Calcium/Calmodulin-Dependent Protein Kinase II, Nitric Oxide Synthase, and the Glutamate-Nitric Oxide-cGMP Pathway in Cerebellum. Differential Effects in Hyperammonemia.

    Science.gov (United States)

    Cabrera-Pastor, Andrea; Llansola, Marta; Felipo, Vicente

    2016-12-21

    Extracellular protein kinases, including cAMP-dependent protein kinase (PKA), modulate neuronal functions including N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation. NMDA receptor activation increases calcium, which binds to calmodulin and activates nitric oxide synthase (NOS), increasing nitric oxide (NO), which activates guanylate cyclase, increasing cGMP, which is released to the extracellular fluid, allowing analysis of this glutamate-NO-cGMP pathway in vivo by microdialysis. The function of this pathway is impaired in hyperammonemic rats. The aims of this work were to assess (1) whether the glutamate-NO-cGMP pathway is modulated in cerebellum in vivo by an extracellular PKA, (2) the role of phosphorylation and activity of calcium/calmodulin-dependent protein kinase II (CaMKII) and NOS in the pathway modulation by extracellular PKA, and (3) whether the effects are different in hyperammonemic and control rats. The pathway was analyzed by in vivo microdialysis. The role of extracellular PKA was analyzed by inhibiting it with a membrane-impermeable inhibitor. The mechanisms involved were analyzed in freshly isolated cerebellar slices from control and hyperammonemic rats. In control rats, inhibiting extracellular PKA reduces the glutamate-NO-cGMP pathway function in vivo. This is due to reduction of CaMKII phosphorylation and activity, which reduces NOS phosphorylation at Ser1417 and NOS activity, resulting in reduced guanylate cyclase activation and cGMP formation. In hyperammonemic rats, under basal conditions, CaMKII phosphorylation and activity are increased, increasing NOS phosphorylation at Ser847, which reduces NOS activity, guanylate cyclase activation, and cGMP. Inhibiting extracellular PKA in hyperammonemic rats normalizes CaMKII phosphorylation and activity, NOS phosphorylation, NOS activity, and cGMP, restoring normal function of the pathway.

  13. Glyoxalase I reduces glycative and oxidative stress and prevents age-related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation.

    Science.gov (United States)

    Jo-Watanabe, Airi; Ohse, Takamoto; Nishimatsu, Hiroaki; Takahashi, Masao; Ikeda, Yoichiro; Wada, Takehiko; Shirakawa, Jun-ichi; Nagai, Ryoji; Miyata, Toshio; Nagano, Tetsuo; Hirata, Yasunobu; Inagi, Reiko; Nangaku, Masaomi

    2014-06-01

    Endothelial dysfunction is a major contributor to cardiovascular disease (CVD), particularly in elderly people. Studies have demonstrated the role of glycation in endothelial dysfunction in nonphysiological models, but the physiological role of glycation in age-related endothelial dysfunction has been poorly addressed. Here, to investigate how vascular glycation affects age-related endothelial function, we employed rats systemically overexpressing glyoxalase I (GLO1), which detoxifies methylglyoxal (MG), a representative precursor of glycation. Four groups of rats were examined, namely young (13 weeks old), mid-age (53 weeks old) wild-type, and GLO1 transgenic (WT/GLO1 Tg) rats. Age-related acceleration in glycation was attenuated in GLO1 Tg rats, together with lower aortic carboxymethyllysine (CML) and urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Age-related impairment of endothelium-dependent vasorelaxation was attenuated in GLO1 Tg rats, whereas endothelium-independent vasorelaxation was not different between WT and GLO1 Tg rats. Nitric oxide (NO) production was decreased in mid-age WT rats, but not in mid-age GLO1 Tg rats. Age-related inactivation of endothelial NO synthase (eNOS) due to phosphorylation of eNOS on Thr495 and dephosphorylation on Ser1177 was ameliorated in GLO1 Tg rats. In vitro, MG increased phosphorylation of eNOS (Thr495) in primary human aortic endothelial cells (HAECs), and overexpression of GLO1 decreased glycative stress and phosphorylation of eNOS (Thr495). Together, GLO1 reduced age-related endothelial glycative and oxidative stress, altered phohphorylation of eNOS, and attenuated endothelial dysfunction. As a molecular mechanism, GLO1 lessened inhibitory phosphorylation of eNOS (Thr495) by reducing glycative stress. Our study demonstrates that blunting glycative stress prevents the long-term impact of endothelial dysfunction on vascular aging. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons

  14. Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase.

    Science.gov (United States)

    Talwar, Pooja Saigal; Gupta, Ravi; Maurya, Arun Kumar; Deswal, Renu

    2012-11-01

    Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 μM) and l-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (∼13%) and almost two fold increase in V(max). PMA (438 nM), a kinase activator, activated BjNOSla ∼1.9 fold while its inactive analog 4αPDD was ineffective. Calcium and PMA activated the enzyme to ∼3 folds. Interestingly, 1,2-DG6 (2.5 μM) and PS (1 μM) with calcium activated the enzyme activity to ∼7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (∼90%) and calphostin-C (∼40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  15. Nitric Oxide Synthase 1 Modulates Basal and β-Adrenergic-Stimulated Contractility by Rapid and Reversible Redox-Dependent S-Nitrosylation of the Heart

    Science.gov (United States)

    Vielma, Alejandra Z.; León, Luisa; Fernández, Ignacio C.; González, Daniel R.

    2016-01-01

    S-nitrosylation of several Ca2+ regulating proteins in response to β-adrenergic stimulation was recently described in the heart; however the specific nitric oxide synthase (NOS) isoform and signaling pathways responsible for this modification have not been elucidated. NOS-1 activity increases inotropism, therefore, we tested whether β-adrenergic stimulation induces NOS-1-dependent S-nitrosylation of total proteins, the ryanodine receptor (RyR2), SERCA2 and the L-Type Ca2+ channel (LTCC). In the isolated rat heart, isoproterenol (10 nM, 3-min) increased S-nitrosylation of total cardiac proteins (+46±14%) and RyR2 (+146±77%), without affecting S-nitrosylation of SERCA2 and LTCC. Selective NOS-1 blockade with S-methyl-L-thiocitrulline (SMTC) and Nω-propyl-l-arginine decreased basal contractility and relaxation (−25–30%) and basal S-nitrosylation of total proteins (−25–60%), RyR2, SERCA2 and LTCC (−60–75%). NOS-1 inhibition reduced (−25–40%) the inotropic response and protein S-nitrosylation induced by isoproterenol, particularly that of RyR2 (−85±7%). Tempol, a superoxide scavenger, mimicked the effects of NOS-1 inhibition on inotropism and protein S-nitrosylation; whereas selective NOS-3 inhibitor L-N5-(1-Iminoethyl)ornithine had no effect. Inhibition of NOS-1 did not affect phospholamban phosphorylation, but reduced its oligomerization. Attenuation of contractility was abolished by PKA blockade and unaffected by guanylate cyclase inhibition. Additionally, in isolated mouse cardiomyocytes, NOS-1 inhibition or removal reduced the Ca2+-transient amplitude and sarcomere shortening induced by isoproterenol or by direct PKA activation. We conclude that 1) normal cardiac performance requires basal NOS-1 activity and S-nitrosylation of the calcium-cycling machinery; 2) β-adrenergic stimulation induces rapid and reversible NOS-1 dependent, PKA and ROS-dependent, S-nitrosylation of RyR2 and other proteins, accounting for about one third of its

  16. Hybrid polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Hagen, Andrew; Katz, Leonard; Keasling, Jay D.; Poust, Sean; Zhang, Jingwei; Zotchev, Sergey

    2016-05-10

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an even-chain or odd-chain diacid or lactam or diamine. The present invention also provides for a host cell comprising the PKS and when cultured produces the even-chain diacid, odd-chain diacid, or KAPA. The present invention also provides for a host cell comprising the PKS capable of synthesizing a pimelic acid or KAPA, and when cultured produces biotin.

  17. An Arabidopsis callose synthase

    DEFF Research Database (Denmark)

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole

    2002-01-01

    in the Arabidopsis mpk4 mutant which exhibits systemic acquired resistance (SAR), elevated beta-1,3-glucan synthase activity, and increased callose levels. In addition, AtGsl5 is a likely target of salicylic acid (SA)-dependent SAR, since AtGsl5 mRNA accumulation is induced by SA in wild-type plants, while...... expression of the nahG salicylate hydroxylase reduces AtGsl5 mRNA levels in the mpk4 mutant. These results indicate that AtGsl5 is likely involved in callose synthesis in flowering tissues and in the mpk4 mutant....

  18. Monoterpene synthases from common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wise, Mitchell Lynn (Pullman, WA); Katahira, Eva Joy (Pullman, WA); Savage, Thomas Jonathan (Christchurch 5, NZ)

    1999-01-01

    cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

  19. ATP Synthase, a Target for Dementia and Aging?

    Science.gov (United States)

    Larrick, James W; Larrick, Jasmine W; Mendelsohn, Andrew R

    2018-02-01

    Advancing age is the biggest risk factor for development for the major life-threatening diseases in industrialized nations accounting for >90% of deaths. Alzheimer's dementia (AD) is among the most devastating. Currently approved therapies fail to slow progression of the disease, providing only modest improvements in memory. Recently reported work describes mechanistic studies of J147, a promising therapeutic molecule previously shown to rescue the severe cognitive deficits exhibited by aged, transgenic AD mice. Apparently, J147 targets the mitochondrial alpha-F1-ATP synthase (ATP5A). Modest inhibition of the ATP synthase modulates intracellular calcium to activate AMP-activated protein kinase to inhibit mammalian target of rapamycin, a known mechanism of lifespan extension from worms to mammals.

  20. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    OpenAIRE

    Karuppiah, Vijaykumar; Ranaghan, Kara E.; Leferink, Nicole G. H.; Johannissen, Linus O.; Shanmugam, Muralidharan; Ní Cheallaigh, Aisling; Bennett, Nathan J.; Kearsey, Lewis J.; Takano, Eriko; Gardiner, John M.; van der Kamp, Marc W.; Hay, Sam; Mulholland, Adrian J.; Leys, David; Scrutton, Nigel S.

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS)...

  1. Producing biofuels using polyketide synthases

    Science.gov (United States)

    Katz, Leonard; Fortman, Jeffrey L; Keasling, Jay D

    2013-04-16

    The present invention provides for a non-naturally occurring polyketide synthase (PKS) capable of synthesizing a carboxylic acid or a lactone, and a composition such that a carboxylic acid or lactone is included. The carboxylic acid or lactone, or derivative thereof, is useful as a biofuel. The present invention also provides for a recombinant nucleic acid or vector that encodes such a PKS, and host cells which also have such a recombinant nucleic acid or vector. The present invention also provides for a method of producing such carboxylic acids or lactones using such a PKS.

  2. Longevity in vivo of primary cell wall cellulose synthases.

    Science.gov (United States)

    Hill, Joseph Lee; Josephs, Cooper; Barnes, William J; Anderson, Charles T; Tien, Ming

    2018-02-01

    Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell. Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

  3. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  4. Threonine phosphorylation of rat liver glycogen synthase

    International Nuclear Information System (INIS)

    Arino, J.; Arro, M.; Guinovart, J.J.

    1985-01-01

    32 P-labeled glycogen synthase specifically immunoprecipitated from 32 P-phosphate incubated rat hepatocytes contains, in addition to [ 32 P] phosphoserine, significant levels of [ 32 P] phosphothreonine. When the 32 P-immunoprecipitate was cleaved with CNBr, the [ 32 P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32 P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

  5. Synthetic biology of polyketide synthases

    DEFF Research Database (Denmark)

    Yuzawa, Satoshi; Backman, Tyler W.H.; Keasling, Jay D.

    2018-01-01

    ). The modules are composed of enzymatic domains that share sequence and functional similarity across all known PKSs. We have used the nomenclature of synthetic biology to classify the enzymatic domains and modules as parts and devices, respectively, and have generated detailed lists of both. In addition, we...... realize the potential that synthetic biology approaches bring to this class of molecules....

  6. Effects of acetoacetyl-CoA synthase expression on production of farnesene in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Tippmann, Stefan; Ferreira, Raphael; Siewers, Verena

    2017-01-01

    to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible...... functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain...

  7. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    cells were quantitated using enzyme immunometric assays. The activity of GSK-3β (serine-9-phosphorylated GSK-3β/total GSK-3β) was lower at baseline compared with follow-up. No significant mean change over time was observed in levels of total GSK-3β and serine-9-phosphorylated GSK-3β. Exploratory......Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...... with emotional lability, subjective mood fluctuations and cognitive function in healthy individuals. Thirty-seven healthy subjects were evaluated with neuropsychological tests and blood samples at baseline and 12-week follow-up. Total GSK-3β and serine-9-phosphorylated GSK-3β in peripheral blood mononuclear...

  8. Crystal structure of riboflavin synthase

    Energy Technology Data Exchange (ETDEWEB)

    Liao, D.-I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B. (DuPont); (NWU)

    2010-03-05

    Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. The first three-dimensional structure of the enzyme was determined at 2.0 {angstrom} resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar {beta} barrels and a C-terminal {alpha} helix. The similar {beta} barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The {beta} barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the {beta} barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

  9. The cellulose synthase companion proteins act non-redundantly with CELLULOSE SYNTHASE INTERACTING1/POM2 and CELLULOSE SYNTHASE 6

    OpenAIRE

    Endler, Anne; Schneider, Rene; Kesten, Christopher; Lampugnani, Edwin R.; Persson, Staffan

    2016-01-01

    Cellulose is a cell wall constituent that is essential for plant growth and development, and an important raw material for a range of industrial applications. Cellulose is synthesized at the plasma membrane by massive cellulose synthase (CesA) complexes that track along cortical microtubules in elongating cells of Arabidopsis through the activity of the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1). In a recent study we identified another family of proteins that also are associated with the ...

  10. Sphingomyelin synthases regulate protein trafficking and secretion.

    Directory of Open Access Journals (Sweden)

    Marimuthu Subathra

    Full Text Available Sphingomyelin synthases (SMS1 and 2 represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG. SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD, to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN, the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2 are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi.

  11. Cyclophilin D Promotes Brain Mitochondrial F1FO ATP Synthase Dysfunction in Aging Mice.

    Science.gov (United States)

    Gauba, Esha; Guo, Lan; Du, Heng

    2017-01-01

    Brain aging is the known strongest risk factor for Alzheimer's disease (AD). In recent years, mitochondrial deficits have been proposed to be a common mechanism linking brain aging to AD. Therefore, to elucidate the causative mechanisms of mitochondrial dysfunction in aging brains is of paramount importance for our understanding of the pathogenesis of AD, in particular its sporadic form. Cyclophilin D (CypD) is a specific mitochondrial protein. Recent studies have shown that F1FO ATP synthase oligomycin sensitivity conferring protein (OSCP) is a binding partner of CypD. The interaction of CypD with OSCP modulates F1FO ATP synthase function and mediates mitochondrial permeability transition pore (mPTP) opening. Here, we have found that increased CypD expression, enhanced CypD/OSCP interaction, and selective loss of OSCP are prominent brain mitochondrial changes in aging mice. Along with these changes, brain mitochondria from the aging mice demonstrated decreased F1FO ATP synthase activity and defective F1FO complex coupling. In contrast, CypD deficient mice exhibited substantially mitigated brain mitochondrial F1FO ATP synthase dysfunction with relatively preserved mitochondrial function during aging. Interestingly, the aging-related OSCP loss was also dramatically attenuated by CypD depletion. Therefore, the simplest interpretation of this study is that CypD promotes F1FO ATP synthase dysfunction and the resultant mitochondrial deficits in aging brains. In addition, in view of CypD and F1FO ATP synthase alterations seen in AD brains, the results further suggest that CypD-mediated F1FO ATP synthase deregulation is a shared mechanism linking mitochondrial deficits in brain aging and AD.

  12. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-05-24

    May 24, 2010 ... chronic periodontitis (CP), 31 with gingivitis (G) and 50 healthy controls. Probing depth ..... Periodontal disease in pregnancy I. Prevalence and severity. ... endothelial nitric oxide synthase gene in premenopausal women with.

  13. High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A. [Instituto Leloir, Buenos Aires (Argentina); Braden, B.C. [Bowie State Univ., Maryland (United States)

    2004-07-01

    The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

  14. High order quaternary arrangement confers increased structural stability to Brucella Spp. lumazine synthase

    International Nuclear Information System (INIS)

    Zylberman, V.; Craig, P.O.; Klinke, S.; Cauerhff, A.; Goldbaum, F.A.; Braden, B.C.

    2004-01-01

    The penultimate step in the pathway of riboflavin biosynthesis is catalyzed by the enzyme lumazine synthase (LS). One of the most distinctive characteristics of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from practically the same structural monomeric unit. The pentameric structure is formed by five 18 kDa monomers, each extensively contacting neighboring monomers. The icosahedral structure consists of 60 LS monomers arranged as twelve pentamers giving rise to a capsid exhibiting icosahedral 532 symmetry. In all lumazine synthases studied, the topologically equivalent active sites are located at the interfaces between adjacent subunits in the pentameric modules. The Brucella spp. lumazine synthase (BLS) sequence clearly diverges from pentameric and icosahedral enzymes. This unusual divergence prompted to further investigate on its quaternary arrangement. In the present work, we demonstrate by means of solution Light Scattering and X-ray structural analyses that BLS assembles as a very stable dimer of pentamers representing a third category of quaternary assembly for lumazine synthases. We also describe by spectroscopic studies the thermodynamic stability of this oligomeric protein, and postulate a mechanism for dissociation/unfolding of this macromolecular assembly. The higher molecular order of BLS increases its stability 20 deg C compared to pentameric lumazine synthases. The decameric arrangement described in this work highlights the importance of quaternary interactions in the stabilization of proteins. (author)

  15. Regulation of galactan synthase expression to modify galactan content in plants

    Science.gov (United States)

    None

    2017-08-22

    The disclosure provides methods of engineering plants to modulate galactan content. Specifically, the disclosure provides methods for engineering a plant to increase the galactan content in a plant tissue by inducing expression of beta-1,4-galactan synthase (GALS), modulated by a heterologous promoter. Further disclosed are the methods of modulating expression level of GALS under the regulation of a transcription factor, as well as overexpression of UDP-galactose epimerse in the same plant tissue. Tissue specific promoters and transcription factors can be used in the methods are also provided.

  16. Terpene synthases from Cannabis sativa.

    Directory of Open Access Journals (Sweden)

    Judith K Booth

    Full Text Available Cannabis (Cannabis sativa plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E-β-ocimene, (--limonene, (+-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  17. Terpene synthases from Cannabis sativa.

    Science.gov (United States)

    Booth, Judith K; Page, Jonathan E; Bohlmann, Jörg

    2017-01-01

    Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

  18. Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in Arabidopsis

    DEFF Research Database (Denmark)

    Bernal Giraldo, Adriana Jimena; Jensen, Jacob Krüger; Harholt, Jesper

    2007-01-01

    Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs...... are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module...

  19. Neuronal nitric oxide synthase is dislocated in type I fibers of myalgic muscle but can recover with physical exercise training

    DEFF Research Database (Denmark)

    Jensen, L; Andersen, L L; Schrøder, H D

    2015-01-01

    Trapezius myalgia is the most common type of chronic neck pain. While physical exercise reduces pain and improves muscle function, the underlying mechanisms remain unclear. Nitric oxide (NO) signaling is important in modulating cellular function, and a dysfunctional neuronal NO synthase (nNOS) ma...

  20. Enhanced growth and improved vascular function in offspring from successive pregnancies in endothelial nitric oxide synthase knockout mice

    NARCIS (Netherlands)

    Longo, M; Jain, [No Value; Langenveld, J; Vedernikov, YP; Garfield, RE; Hankins, GDV; Anderson, GD; Saade, GR

    2004-01-01

    Objective: Transgenic mice that lack endothelial nitric oxide synthase have offspring with growth deficiency and abnormal vascular reactivity in later life. Our objective was to evaluate the role of parity in the modulation of the fetal programming of growth and vascular responses in these

  1. Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

  2. The effects of pressure on arthritic knees in a rat model of CFA-induced arthritis.

    Science.gov (United States)

    Koo, Sung Tae; Lee, Chang-Hyung; Choi, Hyeunseok; Shin, Yong Il; Ha, Ki Tae; Ye, Hanna; Shim, Hyun Bo

    2013-01-01

    Pain is influenced by weather changes under certain circumstances, and inflammatory pain in animal models is ameliorated by pressure, but the underlying mechanism of atmospheric pressure has not been clearly elucidated. To examine the effect of pressure on pain in an arthritic animal model. Controlled animal study. Laboratory animal study. Following an injection of complete Freund's adjuvant (CFA) into one side of a knee joint, 32 rats were assigned randomly to 2 groups and either placed under 1 or 2.5 atmospheres absolute (ATA) in a hyperbaric chamber for 5 hours. The pain levels were assessed daily for up to 2 weeks post-injection to determine the changes in weight bearing (WB) of the affected limbs. In addition, the levels of gelatinase, MMP-2, and MMP-9 expression in the synovial fluids of the knees were analyzed. After arthritis induction, the rats in the 1 ATA group showed reduced WB of the affected limbs (CFA injection in the 1 ATA group. However, repetitive exposure to 2.5 ATA significantly reduced this ratio in the 2.5 ATA group. Although a sufficient number of samples were used to support the hypothesis that high atmospheric pressure improves a painful condition in this study, an additional larger-scale study will be needed to confirm these findings. Exposure to elevated pressures appears to relieve arthritic pain for extended periods by reducing the inflammatory process and should be considered as a possible alternative pain-reducing therapy.

  3. Acquiring the optimal time for hyperbaric therapy in the rat model of CFA induced arthritis.

    Science.gov (United States)

    Koo, Sung Tae; Lee, Chang-Hyung; Shin, Yong Il; Ko, Hyun Yoon; Lee, Da Gyo; Jeong, Han-Sol

    2014-01-01

    We previously published an article about the pressure effect using a rheumatoid animal model. Hyperbaric therapy appears to be beneficial in treating rheumatoid arthritis (RA) by reducing the inflammatory process in an animal model. In this sense, acquiring the optimal pressure-treatment time parameter for RA is important and no optimal hyperbaric therapy time has been suggested up to now. The purpose of our study was to acquire the optimal time for hyperbaric therapy in the RA rat model. Controlled animal study. Following injection of complete Freund's adjuvant (CFA) into one side of the knee joint, 32 rats were randomly assigned to 3 different time groups (1, 3, 5 hours a day) under 1.5 atmospheres absolute (ATA) hyperbaric chamber for 12 days. The pain levels were assessed daily for 2 weeks by weight bearing force (WBF) of the affected limb. In addition, the levels of gelatinase, MMP-2, and MMP-9 expression in the synovial fluids of the knees were analyzed. The reduction of WBF was high at 2 days after injection and then it was spontaneously increased up to 14 days in all 3 groups. There were significant differences of WBF between 5 hours and control during the third through twelfth days, between 3 hours and control during the third through fifth and tenth through twelfth days, and between 3 hours and 5 hours during the third through seventh days (P CFA injection in all groups compared to the initial findings, however, the 3 hour group showed a smaller MMP-9/MMP-2 ratio than the control group. Although enough samples were used for the study to support our hypothesis, more samples will be needed to raise the validity and reliability. The effect of hyperbaric treatment appears to be dependent upon the elevated therapy time under 1.5 ATA pressure for a short period of time; however, the long-term effects were similar in all pressure groups. Further study will be needed to acquire the optimal pressure-treatment parameter relationship in various conditions for clinical application.

  4. Cloning and expression of pineapple sucrosephosphate synthase ...

    African Journals Online (AJOL)

    A 1132-base pairs (bp) polymerase-chain-reaction product of sucrose-phosphate synthase (SPS) (EC 2.3.1.14) from pineapple (Ananas comosus cv. Comte de paris) fruit was cloned and nominated as Ac- SPS1. The sequence encodes a putative 377 amino acids protein containing two serine conserved features that had ...

  5. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  6. HAEM SYNTHASE AND COBALT PORPHYRIN SYNTHASE IN VARIOUS MICRO-ORGANISMS.

    Science.gov (United States)

    PORRA, R J; ROSS, B D

    1965-03-01

    1. The preparation of a crude extract of Clostridium tetanomorphum containing cobalt porphyrin synthase but little haem-synthase activity is described. 2. The properties of cobalt porphyrin synthase in the clostridial extracts is compared with the properties of a haem synthase present in crude extracts of the yeast Torulopsis utilis. 3. Cobalt porphyrin synthase in extracts of C. tetanomorphum inserts Co(2+) ions into the following dicarboxylic porphyrins in descending order of rate of insertion: meso-, deutero- and proto-porphyrins. Esterification renders meso- and deutero-porphyrins inactive as substrates. Neither the tetracarboxylic (coproporphyrin III) nor the octacarboxylic (uroporphyrin III) compounds are converted into cobalt porphyrins by the extract, but the non-enzymic incorporation of Co(2+) ions into these two porphyrins is rapid. These extracts are unable to insert Mn(2+), Zn(2+), Mg(2+) or Cu(2+) ions into mesoporphyrin. 4. Crude extracts of T. utilis readily insert both Co(2+) and Fe(2+) ions into deutero-, meso, and proto-porphyrins. Unlike the extracts of C. tetanomorphum, these preparations catalyse the insertion of Co(2+) ions into deuteroporphyrin more rapidly than into mesoporphyrin. This parallels the formation of haems by the T. utilis extract. 5. Cobalt porphyrin synthase is present in the particulate fraction of the extracts of C. tetanomorphum but requires a heat-stable factor present in the soluble fraction. This soluble factor can be replaced by GSH. 6. Cobalt porphyrin synthase in the clostridial extract is inhibited by iodoacetamide and to a smaller extent by p-chloromercuribenzoate and N-ethylmaleimide. The haem synthases of T. utilis and Micrococcus denitrificans are also inhibited by various thiol reagents.

  7. The biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase.

    Science.gov (United States)

    Demissie, Zerihun A; Erland, Lauren A E; Rheault, Mark R; Mahmoud, Soheil S

    2013-03-01

    Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 μm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.

  8. Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

    Science.gov (United States)

    Maldonado, Rodrigo; Mancilla, Héctor; Villarroel-Espíndola, Franz; Slebe, Felipe; Slebe, Juan Carlos; Méndez, Raúl; Guinovart, Joan J; Concha, Ilona I

    2016-11-01

    Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated with RNA-related molecules. J. Cell. Biochem. 117: 2597-2607, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. Nitric Oxide Synthase and Cyclooxygenase Pathways: A Complex Interplay in Cellular Signaling.

    Science.gov (United States)

    Sorokin, Andrey

    2016-01-01

    The cellular reaction to external challenges is a tightly regulated process consisting of integrated processes mediated by a variety of signaling molecules, generated as a result of modulation of corresponding biosynthetic systems. Both, nitric oxide synthase (NOS) and cyclooxygenase (COX) systems, consist of constitutive forms (NOS1, NOS3 and COX-1), which are mostly involved in housekeeping tasks, and inducible forms (NOS2 and COX-2), which shape the cellular response to stress and variety of bioactive agents. The complex interplay between NOS and COX pathways can be observed at least at three levels. Firstly, products of NOS and Cox systems can mediate the regulation and the expression of inducible forms (NOS2 and COX-2) in response of similar and dissimilar stimulus. Secondly, the reciprocal modulation of cyclooxygenase activity by nitric oxide and NOS activity by prostaglandins at the posttranslational level has been shown to occur. Mechanisms by which nitric oxide can modulate prostaglandin synthesis include direct S-nitrosylation of COX and inactivation of prostaglandin I synthase by peroxynitrite, product of superoxide reaction with nitric oxide. Prostaglandins, conversely, can promote an increased association of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase) with NOS1, thereby reducing its activity. The third level of interplay is provided by intracellular crosstalk of signaling pathways stimulated by products of NOS and COX which contributes significantly to the complexity of cellular signaling. Since modulation of COX and NOS pathways was shown to be principally involved in a variety of pathological conditions, the dissection of their complex relationship is needed for better understanding of possible therapeutic strategies. This review focuses on implications of interplay between NOS and COX for cellular function and signal integration.

  10. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    International Nuclear Information System (INIS)

    Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

    2014-01-01

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus

  11. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  12. Clinical significance of Phosphatidyl Inositol Synthase overexpression in oral cancer

    International Nuclear Information System (INIS)

    Kaur, Jatinder; Sawhney, Meenakshi; DattaGupta, Siddartha; Shukla, Nootan K; Srivastava, Anurag; Ralhan, Ranju

    2010-01-01

    We reported increased levels of Phosphatidyl Inositol synthase (PI synthase), (enzyme that catalyses phosphatidyl inositol (PI) synthesis-implicated in intracellular signaling and regulation of cell growth) in smokeless tobacco (ST) exposed oral cell cultures by differential display. This study determined the clinical significance of PI synthase overexpression in oral squamous cell carcinoma (OSCC) and premalignant lesions (leukoplakia), and identified the downstream signaling proteins in PI synthase pathway that are perturbed by smokeless tobacco (ST) exposure. Tissue microarray (TMA) Immunohistochemistry, Western blotting, Confocal laser scan microscopy, RT-PCR were performed to define the expression of PI synthase in clinical samples and in oral cell culture systems. Significant increase in PI synthase immunoreactivity was observed in premalignant lesions and OSCCs as compared to oral normal tissues (p = 0.000). Further, PI synthase expression was significantly associated with de-differentiation of OSCCs, (p = 0.005) and tobacco consumption (p = 0.03, OR = 9.0). Exposure of oral cell systems to smokeless tobacco (ST) in vitro confirmed increase in PI synthase, Phosphatidylinositol 3-kinase (PI3K) and cyclin D1 levels. Collectively, increased PI synthase expression was found to be an early event in oral cancer and a target for smokeless tobacco

  13. Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Berta Alquézar

    2017-08-01

    Full Text Available Citrus aroma and flavor, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers, and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defense against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical, and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck genome sequence allowed us to characterize for the first time the terpene synthase (TPS family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional TPSs. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2, and 5 putative full length genes each. Additionally, sweet orange β-farnesene synthase, (Z-β-cubebene/α-copaene synthase, two β-caryophyllene synthases, and three multiproduct enzymes yielding β-cadinene/α-copaene, β-elemene, and β-cadinene/ledene/allo-aromandendrene as major products were identified, and functionally characterized via in vivo recombinant Escherichia coli assays.

  14. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity

    DEFF Research Database (Denmark)

    Yang, Ting; Gao, Liping; Hu, Hao

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first path-way-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1′-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate...

  15. Constitutive nitric oxide synthase (cNOS activity in Langerhans islets from streptozotocin diabetic rats

    Directory of Open Access Journals (Sweden)

    Fonovich de Schroeder T.M.

    1998-01-01

    Full Text Available Nitric oxide synthase activity was measured in Langerhans islets isolated from control and streptozotocin diabetic rats. The activity of the enzyme was linear up to 150 µg of protein from control rats and was optimal at 0.1 µM calcium, when it was measured after 45 min of incubation at 37oC in the presence of 200 µM arginine. Specific activity of the enzyme was 25 x 10-4 nmol [3H]citrulline 45 min-1 mg protein-1. Streptozotocin diabetic rats exhibited less enzyme activity both in total pancreas homogenate and in isolated Langerhans islets when compared to control animals. Nitric oxide synthase activity measured in control and diabetic rats 15 days after the last streptozotocin injection in the second group of animals corresponded only to a constitutive enzyme since it was not inhibited by aminoguanidine in any of the mentioned groups. Hyperglycemia in diabetic rats may be the consequence of impaired insulin release caused at least in part by reduced positive modulation mediated by constitutive nitric oxide synthase activity, which was dramatically reduced in islets severely damaged after streptozotocin treatment.

  16. Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA)

    Science.gov (United States)

    2011-01-01

    Background Staphylococcus aureus is a human pathogen that produces extracellular adenosine to evade clearance by the host immune system, an activity attributed to the 5'-nucleotidase activity of adenosine synthase (AdsA). In mammals, conversion of adenosine triphosphate to adenosine is catalyzed in a two-step process: ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTDPases) hydrolyze ATP and ADP to AMP, whereas 5'-nucleotidases hydrolyze AMP to adenosine. NTPDases harbor apyrase conserved regions (ACRs) that are critical for activity. Results NTPDase ACR motifs are absent in AdsA, yet we report here that recombinant AdsA hydrolyzes ADP and ATP in addition to AMP. Competition assays suggest that hydrolysis occurs following binding of all three substrates at a unique site. Alanine substitution of two amino acids, aspartic acid 127 and histidine 196 within the 5'-nucleotidase signature sequence, leads to reduced AMP or ADP hydrolysis but does not affect the binding of these substrates. Conclusion Collectively, these results provide insight into the unique ability of AdsA to produce adenosine through the consecutive hydrolysis of ATP, ADP and AMP, thereby endowing S. aureus with the ability to modulate host immune responses. PMID:22035583

  17. Oxide Synthase Expression by p38 MAP Kinase

    Directory of Open Access Journals (Sweden)

    Tuija Turpeinen

    2011-01-01

    Full Text Available The role of dual specificity phosphatase 1 (DUSP1 in inducible nitric oxide synthase (iNOS expression in A549 human pulmonary epithelial cells, J774 mouse macrophages and primary mouse bone marrow-derived macrophages (BMMs was investigated. iNOS expression was induced by a cytokine mixture (TNF, IFNγ and IL-1β in A549 cells and by LPS in J774 cells, and it was inhibited by p38 MAPK inhibitors SB202190 and BIRB 796. Stimulation with cytokine mixture or LPS enhanced also DUSP1 expression. Down-regulation of DUSP1 by siRNA increased p38 MAPK phosphorylation and iNOS expression in A549 and J774 cells. In addition, LPS-induced iNOS expression was enhanced in BMMs from DUSP1(−/− mice as compared to that in BMMs from wild-type mice. The results indicate that DUSP1 suppresses iNOS expression by limiting p38 MAPK activity in human and mouse cells. Compounds that enhance DUSP1 expression or modulate its function may be beneficial in diseases complicated with increased iNOS-mediated NO production.

  18. Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity.

    Science.gov (United States)

    Gasanov, Sardar E; Kim, Aleksandr A; Yaguzhinsky, Lev S; Dagda, Ruben K

    2018-02-01

    Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1 H NMR and 31 P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F 0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F 0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F 0 sector, and thereby increase ATP synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Sequence analysis of cereal sucrose synthase genes and isolation ...

    African Journals Online (AJOL)

    SERVER

    2007-10-18

    Oct 18, 2007 ... sequencing of sucrose synthase gene fragment from sor- ghum using primers designed at their conserved exons. MATERIALS AND METHODS. Multiple sequence alignment. Sucrose synthase gene sequences of various cereals like rice, maize, and barley were accessed from NCBI Genbank database.

  20. Prostaglandin H synthase immunoreactivity in human gut. An immunohistochemical study

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Rumessen, J J; Qvortrup, Klaus

    1991-01-01

    Prostaglandins exhibit a variety of actions on intestinal smooth muscle depending upon the type, dose and muscle layer studied. As the cellular origin of prostaglandin H (PGH) synthase has not been established with certainty in the human gut wall, we studied the localization of PGH synthase...

  1. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...

  2. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    Directory of Open Access Journals (Sweden)

    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  3. Predicted cycloartenol synthase protein from Kandelia obovata and Rhizophora stylosa using online software of Phyre2 and Swiss-model

    Science.gov (United States)

    Basyuni, M.; Sulistiyono, N.; Wati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

    2018-03-01

    Cloning of Kandelia obovata KcCAS gene (previously known as Kandelia candel) and Rhizophora stylosa RsCAS have already have been reported and encoded cycloartenol synthases. In this study, the predicted KcCAS and RsCAS protein were analyzed using online software of Phyre2 and Swiss-model. The protein modelling for KcCAS and RsCAS cycloartenol synthases was determined using Pyre2 had similar results with slightly different in sequence identity. By contrast, the Swiss-model for KcCAS slightly had higher sequence identity (47.31%) and Qmean (0.70) compared to RsCAS. No difference of ligands binding site which is considered as modulators for both cycloartenol synthases. The range of predicted protein derived from 91-757 amino acid residues with coverage sequence similarities 0.86, respectively from template model of lanosterol synthase from the human. Homology modelling revealed that 706 residues (93% of the amino acid sequence) had been modelled with 100.0% confidence by the single highest scoring template for both KcCAS and RsCAS using Phyre2. This coverage was more elevated than swiss-model predicted (86%). The present study suggested that both genes are responsible for the genesis of cycloartenol in these mangrove plants.

  4. Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

    Science.gov (United States)

    Ober, Dietrich; Hartmann, Thomas

    1999-01-01

    Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids. PMID:10611289

  5. Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase

    OpenAIRE

    Andrews, Rachel E.; Galileo, Deni S.; Martin-DeLeon, Patricia A.

    2015-01-01

    Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca2+ efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca2+, and that these fertility-modulating proteins are present...

  6. Nitric oxide synthase gene G298 allele

    International Nuclear Information System (INIS)

    Nagib El-Kilany, Galal E.; Nayel, Ehab; Hazzaa, Sahar

    2004-01-01

    Background: Nitric oxide (NO) has an important effect on blood pressure, arterial wall, and the basal release of endothelial NO in hypertension (HPN) may be reduced. Until now, there is no solid data revealing the potential role of the polymorphism of the nitric oxide synthase gene (NOS) in patients with HPN and microvascular angina. Aim: The aim of the present study is to investigate the gene of endothelial nitric oxide synthase (eNOS), as the polymorphism of this gene may be a putative candidate for HPN and initiate the process of atherosclerosis. Methods: Sixty participants were recruited for this study; 50 were hypertensive patients complaining of chest pain [30 of them have electrocardiogram (EKG) changes of ischemia], 20 had isolated HPN, and 10 healthy volunteers served as control. All patients underwent stress myocardial perfusion imaging (MPI) and coronary angiography. Genotyping of eNOS for all patients and controls was performed. The linkages between HPN, microvascular angina and eNOS gene polymorphism were investigated. Results: MPI and coronary angiography revealed that 15 patients had chest pain with true ischemia and reversible myocardial perfusion defects (multiple and mild) but normal epicardial coronary arteries (microvascular angina), while 15 patients had significant coronary artery disease (CAD), and 20 hypertensive patients showed normal perfusion scan and coronary angiography. The prevalence of the NOS G 298 allele was higher in the hypertensive group with microvascular angina (documented by MPI) than it was among the control participants (P<.005). The eNOS allele was significantly higher in the hypertensive group than in the control participants, but there was no significant difference in homozygote mutants among hypertensive participants, x-syndrome and patients with CAD. Conclusion: eNOS gene polymorphism is proved to be an important etiology in microvascular angina (x-syndrome) among hypertensive patients. In addition, the eNOS mutant

  7. Glycogen synthase activation by sugars in isolated hepatocytes.

    Science.gov (United States)

    Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

    1988-07-01

    We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

  8. Generation and Functional Evaluation of Designer Monoterpene Synthases.

    Science.gov (United States)

    Srividya, N; Lange, I; Lange, B M

    2016-01-01

    Monoterpene synthases are highly versatile enzymes that catalyze the first committed step in the pathways toward terpenoids, the structurally most diverse class of plant natural products. Recent advancements in our understanding of the reaction mechanism have enabled engineering approaches to develop mutant monoterpene synthases that produce specific monoterpenes. In this chapter, we are describing protocols to introduce targeted mutations, express mutant enzyme catalysts in heterologous hosts, and assess their catalytic properties. Mutant monoterpene synthases have the potential to contribute significantly to synthetic biology efforts aimed at producing larger amounts of commercially attractive monoterpenes. © 2016 Elsevier Inc. All rights reserved.

  9. Geranylfarnesyl diphosphate synthase from Methanosarcina mazei: Different role, different evolution

    International Nuclear Information System (INIS)

    Ogawa, Takuya; Yoshimura, Tohru; Hemmi, Hisashi

    2010-01-01

    The gene of (all-E) geranylfarnesyl diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Goe1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) prenyl diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl diphosphate synthase from a thermophilic archaeon.

  10. Glutamylation of the DNA sensor cGAS regulates its binding and synthase activity in antiviral immunity.

    Science.gov (United States)

    Xia, Pengyan; Ye, Buqing; Wang, Shuo; Zhu, Xiaoxiao; Du, Ying; Xiong, Zhen; Tian, Yong; Fan, Zusen

    2016-04-01

    Cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA during viral infection and catalyzes synthesis of the dinucleotide cGAMP, which activates the adaptor STING to initiate antiviral responses. Here we found that deficiency in the carboxypeptidase CCP5 or CCP6 led to susceptibility to DNA viruses. CCP5 and CCP6 were required for activation of the transcription factor IRF3 and interferons. Polyglutamylation of cGAS by the enzyme TTLL6 impeded its DNA-binding ability, whereas TTLL4-mediated monoglutamylation of cGAS blocked its synthase activity. Conversely, CCP6 removed the polyglutamylation of cGAS, whereas CCP5 hydrolyzed the monoglutamylation of cGAS, which together led to the activation of cGAS. Therefore, glutamylation and deglutamylation of cGAS tightly modulate immune responses to infection with DNA viruses.

  11. Isolation of developing secondary xylem specific cellulose synthase ...

    Indian Academy of Sciences (India)

    The present study aimed at identifying developing secondary xylem specific cellulose synthase genes from .... the First strand cDNA synthesis kit (Fermentas, Pittsburgh,. USA). .... ing height of the rooted cutting, girth of the stem, leaf area.

  12. Cloning and expression of pineapple sucrose- phosphate synthase ...

    African Journals Online (AJOL)

    hope&shola

    2010-12-06

    Dec 6, 2010 ... phosphate; EDTA, ethylene diamine tetraacetic acid; Ivr, invertase; SS .... phenolics, tannins and artifacts due to differences of tissue composition ..... Banana sucrose-phosphate synthase gene expression during fruit ripening.

  13. Inhibitors of Fatty Acid Synthase for Prostate Cancer. Revision

    Science.gov (United States)

    2013-05-01

    acetyl- cholinesterase inhibitors have been developed, many with femtomolar binding affinities (7). This body of literature also confirms that the...AD_________________ Award Number: W81XWH-09-1-0204 TITLE: Inhibitors of Fatty Acid Synthase for...May 2013 2. REPORT TYPE Revised Final 3. DATES COVERED 01 May 2009-30 Apr 2013 4. TITLE AND SUBTITLE Inhibitors of Fatty Acid Synthase for

  14. Inhibitors of Fatty Acid Synthase for Prostate Cancer

    Science.gov (United States)

    2012-05-01

    compounds. For example, numerous classes of acetyl- cholinesterase inhibitors have been developed, m any with fe mtomolar binding affinities (7). This...AD_________________ Award Number: W81XWH-09-1-0204 TITLE: Inhibitors of Fatty Acid Synthase for...CONTRACT NUMBER Inhibitors of Fatty Acid Synthase for Prostate Cancer 5b. GRANT NUMBER W81XWH-09-1-0204 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR

  15. Homocystinuria due to cystathionine beta synthase deficiency

    Directory of Open Access Journals (Sweden)

    Rao T

    2008-01-01

    Full Text Available A two year-old male child presented with cutis marmorata congenita universalis, brittle hair, mild mental retardation, and finger spasms. Biochemical findings include increased levels of homocysteine in the blood-106.62 µmol/L (normal levels: 5.90-16µmol/L. Biochemical tests such as the silver nitroprusside and nitroprusside tests were positive suggesting homocystinuria. The patient was treated with oral pyridoxine therapy for three months. The child responded well to this therapy and the muscle spasms as well as skin manifestations such as cutis marmorata subsided. The treatment is being continued; the case is reported here because of its rarity. Homocysteinuria arising due to cystathionine beta-synthase (CBS deficiency is an autosomal recessive disorder of methionine metabolism that produces increased levels of urinary homocysteine and methionine It manifests itself in vascular, central nervous system, cutaneous, and connective tissue disturbances and phenotypically resembles Marfan′s syndrome. Skin manifestations include malar flush, thin hair, and cutis reticulata / marmorata.

  16. The Eucalyptus terpene synthase gene family.

    Science.gov (United States)

    Külheim, Carsten; Padovan, Amanda; Hefer, Charles; Krause, Sandra T; Köllner, Tobias G; Myburg, Alexander A; Degenhardt, Jörg; Foley, William J

    2015-06-11

    Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts. The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus. Our data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.

  17. Converting S-limonene synthase to pinene or phellandrene synthases reveals the plasticity of the active site.

    Science.gov (United States)

    Xu, Jinkun; Ai, Ying; Wang, Jianhui; Xu, Jingwei; Zhang, Yongkang; Yang, Dong

    2017-05-01

    S-limonene synthase is a model monoterpene synthase that cyclizes geranyl pyrophosphate (GPP) to form S-limonene. It is a relatively specific enzyme as the majority of its products are composed of limonene. In this study, we converted it to pinene or phellandrene synthases after introducing N345A/L423A/S454A or N345I mutations. Further studies on N345 suggest the polarity of this residue plays a critical role in limonene production by stabilizing the terpinyl cation intermediate. If it is mutated to a non-polar residue, further cyclization or hydride shifts occurs so the carbocation migrates towards the pyrophosphate, leading to the production of pinene or phellandrene. On the other hand, mutant enzymes that still possess a polar residue at this position produce limonene as the major product. N345 is not the only polar residue that may stabilize the terpinyl cation because it is not strictly conserved among limonene synthases across species and there are also several other polar residues in this area. These residues could form a "polar pocket" that may collectively play this stabilizing role. Our study provides important insights into the catalytic mechanism of limonene synthases. Furthermore, it also has wider implications on the evolution of terpene synthases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. The Tomato Terpene Synthase Gene Family1[W][OA

    Science.gov (United States)

    Falara, Vasiliki; Akhtar, Tariq A.; Nguyen, Thuong T.H.; Spyropoulou, Eleni A.; Bleeker, Petra M.; Schauvinhold, Ines; Matsuba, Yuki; Bonini, Megan E.; Schilmiller, Anthony L.; Last, Robert L.; Schuurink, Robert C.; Pichersky, Eran

    2011-01-01

    Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far. PMID:21813655

  19. Characterization of the human gene (TBXAS1) encoding thromboxane synthase.

    Science.gov (United States)

    Miyata, A; Yokoyama, C; Ihara, H; Bandoh, S; Takeda, O; Takahashi, E; Tanabe, T

    1994-09-01

    The gene encoding human thromboxane synthase (TBXAS1) was isolated from a human EMBL3 genomic library using human platelet thromboxane synthase cDNA as a probe. Nucleotide sequencing revealed that the human thromboxane synthase gene spans more than 75 kb and consists of 13 exons and 12 introns, of which the splice donor and acceptor sites conform to the GT/AG rule. The exon-intron boundaries of the thromboxane synthase gene were similar to those of the human cytochrome P450 nifedipine oxidase gene (CYP3A4) except for introns 9 and 10, although the primary sequences of these enzymes exhibited 35.8% identity each other. The 1.2-kb of the 5'-flanking region sequence contained potential binding sites for several transcription factors (AP-1, AP-2, GATA-1, CCAAT box, xenobiotic-response element, PEA-3, LF-A1, myb, basic transcription element and cAMP-response element). Primer-extension analysis indicated the multiple transcription-start sites, and the major start site was identified as an adenine residue located 142 bases upstream of the translation-initiation site. However, neither a typical TATA box nor a typical CAAT box is found within the 100-b upstream of the translation-initiation site. Southern-blot analysis revealed the presence of one copy of the thromboxane synthase gene per haploid genome. Furthermore, a fluorescence in situ hybridization study revealed that the human gene for thromboxane synthase is localized to band q33-q34 of the long arm of chromosome 7. A tissue-distribution study demonstrated that thromboxane synthase mRNA is widely expressed in human tissues and is particularly abundant in peripheral blood leukocyte, spleen, lung and liver. The low but significant levels of mRNA were observed in kidney, placenta and thymus.

  20. Nitrite reductase activity and inhibition of H₂S biogenesis by human cystathionine ß-synthase.

    Directory of Open Access Journals (Sweden)

    Carmen Gherasim

    Full Text Available Nitrite was recognized as a potent vasodilator >130 years and has more recently emerged as an endogenous signaling molecule and modulator of gene expression. Understanding the molecular mechanisms that regulate nitrite metabolism is essential for its use as a potential diagnostic marker as well as therapeutic agent for cardiovascular diseases. In this study, we have identified human cystathionine ß-synthase (CBS as a new player in nitrite reduction with implications for the nitrite-dependent control of H₂S production. This novel activity of CBS exploits the catalytic property of its unusual heme cofactor to reduce nitrite and generate NO. Evidence for the possible physiological relevance of this reaction is provided by the formation of ferrous-nitrosyl (Fe(II-NO CBS in the presence of NADPH, the human diflavin methionine synthase reductase (MSR and nitrite. Formation of Fe(II-NO CBS via its nitrite reductase activity inhibits CBS, providing an avenue for regulating biogenesis of H₂S and cysteine, the limiting reagent for synthesis of glutathione, a major antioxidant. Our results also suggest a possible role for CBS in intracellular NO biogenesis particularly under hypoxic conditions. The participation of a regulatory heme cofactor in CBS in nitrite reduction is unexpected and expands the repertoire of proteins that can liberate NO from the intracellular nitrite pool. Our results reveal a potential molecular mechanism for cross-talk between nitrite, NO and H₂S biology.

  1. Highly divergent mitochondrial ATP synthase complexes in Tetrahymena thermophila.

    Directory of Open Access Journals (Sweden)

    Praveen Balabaskaran Nina

    2010-07-01

    Full Text Available The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1 sector catalyzes ATP synthesis, whereas the F(o sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1 and F(o sectors are highly conserved across prokaryotes and eukaryotes. Therefore, it was a surprise that genes encoding the a and b subunits as well as other components of the F(o sector were undetectable in the sequenced genomes of a variety of apicomplexan parasites. While the parasitic existence of these organisms could explain the apparent incomplete nature of ATP synthase in Apicomplexa, genes for these essential components were absent even in Tetrahymena thermophila, a free-living ciliate belonging to a sister clade of Apicomplexa, which demonstrates robust oxidative phosphorylation. This observation raises the possibility that the entire clade of Alveolata may have invented novel means to operate ATP synthase complexes. To assess this remarkable possibility, we have carried out an investigation of the ATP synthase from T. thermophila. Blue native polyacrylamide gel electrophoresis (BN-PAGE revealed the ATP synthase to be present as a large complex. Structural study based on single particle electron microscopy analysis suggested the complex to be a dimer with several unique structures including an unusually large domain on the intermembrane side of the ATP synthase and novel domains flanking the c subunit rings. The two monomers were in a parallel configuration rather than the angled configuration previously observed in other organisms. Proteomic analyses of well-resolved ATP synthase complexes from 2-D BN/BN-PAGE identified orthologs of seven canonical ATP synthase subunits, and at least 13 novel proteins that constitute subunits apparently limited to the ciliate lineage. A mitochondrially encoded protein, Ymf66, with predicted eight transmembrane domains could be a

  2. Sucrose Phosphate Synthase and Sucrose Accumulation at Low Temperature 1

    Science.gov (United States)

    Guy, Charles L.; Huber, Joan L. A.; Huber, Steven C.

    1992-01-01

    The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance. Images Figure 1 Figure 2 Figure 3 PMID:16652990

  3. Wounding stimulates ALLENE OXIDE SYNTHASE gene and increases the level of jasmonic acid in Ipomoea nil cotyledons

    Directory of Open Access Journals (Sweden)

    Emilia Wilmowicz

    2016-03-01

    Full Text Available Allene oxide synthase (AOS encodes the first enzyme in the lipoxygenase pathway, which is responsible for jasmonic acid (JA formation. In this study we report the molecular cloning and characterization of InAOS from Ipomoea nil. The full-length gene is composed of 1662 bp and encodes for 519 amino acids. The predicted InAOS contains PLN02648 motif, which is evolutionarily conserved and characteristic for functional enzymatic proteins. We have shown that wounding led to a strong stimulation of the examined gene activity in cotyledons and an increase in JA level, which suggest that this compound may be a modulator of stress responses in I. nil.

  4. Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Cortés-Vieyra Ricarda

    2012-06-01

    Full Text Available Abstract Glycogen synthase kinase 3β (GSK3β plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

  5. Glycogen synthase kinase 3 alpha phosphorylates and regulates the osteogenic activity of Osterix.

    Science.gov (United States)

    Li, Hongyan; Jeong, Hyung Min; Choi, You Hee; Lee, Sung Ho; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl

    2013-05-10

    Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens.

    Science.gov (United States)

    Cortés-Vieyra, Ricarda; Bravo-Patiño, Alejandro; Valdez-Alarcón, Juan J; Juárez, Marcos Cajero; Finlay, B Brett; Baizabal-Aguirre, Víctor M

    2012-06-12

    Glycogen synthase kinase 3β (GSK3β) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3β may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3β and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

  7. Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

    Science.gov (United States)

    Nordström, Viola; Willershäuser, Monja; Herzer, Silke; Rozman, Jan; von Bohlen Und Halbach, Oliver; Meldner, Sascha; Rothermel, Ulrike; Kaden, Sylvia; Roth, Fabian C; Waldeck, Clemens; Gretz, Norbert; de Angelis, Martin Hrabě; Draguhn, Andreas; Klingenspor, Martin; Gröne, Hermann-Josef; Jennemann, Richard

    2013-01-01

    Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.

  8. Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

    Science.gov (United States)

    Despinasse, Yolande; Fiorucci, Sébastien; Antonczak, Serge; Moja, Sandrine; Bony, Aurélie; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis; Jullien, Frédéric

    2017-05-01

    Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221. Copyright © 2017. Published by Elsevier Ltd.

  9. Sesquiterpene Synthase-3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Fusion Protein Responsible for Hirsutene Biosynthesis in Stereum hirsutum.

    Science.gov (United States)

    Flynn, Christopher M; Schmidt-Dannert, Claudia

    2018-06-01

    The wood-rotting mushroom Stereum hirsutum is a known producer of a large number of namesake hirsutenoids, many with important bioactivities. Hirsutenoids form a structurally diverse and distinct class of sesquiterpenoids. No genes involved in hirsutenoid biosynthesis have yet been identified or their enzymes characterized. Here, we describe the cloning and functional characterization of a hirsutene synthase as an unexpected fusion protein of a sesquiterpene synthase (STS) with a C-terminal 3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-CoA) synthase (HMGS) domain. Both the full-length fusion protein and truncated STS domain are highly product-specific 1,11-cyclizing STS enzymes with kinetic properties typical of STSs. Complementation studies in Saccharomyces cerevisiae confirmed that the HMGS domain is also functional in vivo Phylogenetic analysis shows that the hirsutene synthase domain does not form a clade with other previously characterized sesquiterpene synthases from Basidiomycota. Comparative gene structure analysis of this hirsutene synthase with characterized fungal enzymes reveals a significantly higher intron density, suggesting that this enzyme may be acquired by horizontal gene transfer. In contrast, the HMGS domain is clearly related to other fungal homologs. This STS-HMGS fusion protein is part of a biosynthetic gene cluster that includes P450s and oxidases that are expressed and could be cloned from cDNA. Finally, this unusual fusion of a terpene synthase to an HMGS domain, which is not generally recognized as a key regulatory enzyme of the mevalonate isoprenoid precursor pathway, led to the identification of additional HMGS duplications in many fungal genomes, including the localization of HMGSs in other predicted sesquiterpenoid biosynthetic gene clusters. IMPORTANCE Hirsutenoids represent a structurally diverse class of bioactive sesquiterpenoids isolated from fungi. Identification of their biosynthetic pathways will provide

  10. Impaired glycogen synthase activity and mitochondrial dysfunction in skeletal muscle

    DEFF Research Database (Denmark)

    Højlund, Kurt; Beck-Nielsen, Henning

    2006-01-01

    Insulin resistance in skeletal muscle is a major hallmark of type 2 diabetes and an early detectable abnormality in the development of this disease. The cellular mechanisms of insulin resistance include impaired insulin-mediated muscle glycogen synthesis and increased intramyocellular lipid content......, whereas impaired insulin activation of muscle glycogen synthase represents a consistent, molecular defect found in both type 2 diabetic and high-risk individuals. Despite several studies of the insulin signaling pathway believed to mediate dephosphorylation and hence activation of glycogen synthase......, the molecular mechanisms responsible for this defect remain unknown. Recently, the use of phospho-specific antibodies in human diabetic muscle has revealed hyperphosphorylation of glycogen synthase at sites not regulated by the classical insulin signaling pathway. In addition, novel approaches such as gene...

  11. Class II recombinant phosphoribosyl diphosphate synthase from spinach

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    to other PRPP synthases the activity of spinach PRPP synthase isozyme 3 is independent of P(i), and the enzyme is inhibited by ribonucleoside diphosphates in a purely competitive manner, which indicates a lack of allosteric inhibition by these compounds. In addition spinach PRPP synthase isozyme 3 shows...... an unusual low specificity toward diphosphoryl donors by accepting dATP, GTP, CTP, and UTP in addition to ATP. The kinetic mechanism of the enzyme is an ordered steady state Bi Bi mechanism with K(ATP) and K(Rib-5-P) values of 170 and 110 micrometer, respectively, and a V(max) value of 13.1 micromol (min x...... mg of protein)(-1). The enzyme has an absolute requirement for magnesium ions, and maximal activity is obtained at 40 degrees C at pH 7.6....

  12. Intra-articular injection of Botulinum toxin A reduces neurogenic inflammation in CFA-induced arthritic rat model.

    Science.gov (United States)

    Wang, Lin; Wang, Kaile; Chu, Xiao; Li, Tieshan; Shen, Nana; Fan, Chenglei; Niu, Zhenyuan; Zhang, Xiaochen; Hu, Luoman

    2017-02-01

    Currently, administration of Botulinum toxin Type A (BoNT/A) to treat arthritic pain has promising efficacy in clinical research. However, the mechanisms underlying anti-neurogenic inflammation mediated by BoNT/A remains unclear. The aim of this study was to demonstrate the effectiveness in macro and micro levels and to explore the causal mechanism of BoNT/A. Wistar rats (n = 60) were injected with 50ul complete Freund's adjuvant (CFA) in the left ankle joint capsule to establish a model of chronic monoarthritis. Pain behaviour (Evoked pain assessment) and infrared thermal imaging testing were performed at the macroscopic level to assess the effectiveness of analgesia and anti-inflammation. Western blotting and immunofluorescence staining were used at the microscopic level in an attempt to determine the mechanisms of anti-nociceptive or anti-inflammatory effects of BoNT/A. Additionally, hematoxylin-eosin staining was also used to visualise the cartilage and the synovial degenerative conditions of arthritis. By comparing the outcome of the evoked pain test and immunofluorescence staining, there was a significant improvement in BoNT/A compared with the normal saline (NS) injected control group. In addition, thermal variations showed that the temperature of ipsilateral ankle joint increased between 1 and 2 weeks following injection of CFA, but decreased after 3 weeks (still above the contralateral side). However, the temperature showed no difference between the BoNT/A group and NS group after treatment. The expression of IL-1β or TNF-α in the ankle synovial tissue was significantly decreased in the BoNT/A group compared to the NS group (p < 0.05). Based on the HE assessment, cartilage degeneration and infiltration of inflammatory cells in the BoNT/A group was alleviated compared to the NS group after treatment. In conclusion, we proposed the hypothesis that intra-articular BoNT/A administration does play an important role in anti-neurogenic inflammation. The possible mechanisms might be that BoNT/A prevents the release of nociceptive nerve peptides at the injection site and then suppresses the expression of inflammatory cytokines. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Variable transcriptional responsiveness of the P2X3 receptor gene during CFA-induced inflammatory hyperalgesia.

    Science.gov (United States)

    Nuñez-Badinez, Paulina; Sepúlveda, Hugo; Diaz, Emilio; Greffrath, Wolfgang; Treede, Rolf-Detlef; Stehberg, Jimmy; Montecino, Martin; van Zundert, Brigitte

    2018-05-01

    The purinergic receptor P2X3 (P2X3-R) plays important roles in molecular pathways of pain, and reduction of its activity or expression effectively reduces chronic inflammatory and neuropathic pain sensation. Inflammation, nerve injury, and cancer-induced pain can increase P2X3-R mRNA and/or protein levels in dorsal root ganglia (DRG). However, P2X3-R expression is unaltered or even reduced in other pain studies. The reasons for these discrepancies are unknown and might depend on the applied traumatic intervention or on intrinsic factors such as age, gender, genetic background, and/or epigenetics. In this study, we sought to get insights into the molecular mechanisms responsible for inflammatory hyperalgesia by determining P2X3-R expression in DRG neurons of juvenile male rats that received a Complete Freund's Adjuvant (CFA) bilateral paw injection. We demonstrate that all CFA-treated rats showed inflammatory hyperalgesia, however, only a fraction (14-20%) displayed increased P2X3-R mRNA levels, reproducible across both sides. Immunostaining assays did not reveal significant increases in the percentage of P2X3-positive neurons, indicating that increased P2X3-R at DRG somas is not critical for inducing inflammatory hyperalgesia in CFA-treated rats. Chromatin immunoprecipitation (ChIP) assays showed a correlated (R 2  = 0.671) enrichment of the transcription factor Runx1 and the epigenetic active mark histone H3 acetylation (H3Ac) at the P2X3-R gene promoter in a fraction of the CFA-treated rats. These results suggest that animal-specific increases in P2X3-R mRNA levels are likely associated with the genetic/epigenetic context of the P2X3-R locus that controls P2X3-R gene transcription by recruiting Runx1 and epigenetic co-regulators that mediate histone acetylation. © 2017 Wiley Periodicals, Inc.

  14. Topical dura mater application of CFA induces enhanced expression of c-fos and glutamate in rat trigeminal nucleus caudalis

    DEFF Research Database (Denmark)

    Lukács, M; Warfvinge, K; Tajti, J

    2017-01-01

    BACKGROUND: Migraine is a debilitating neurological disorder where trigeminovascular activation plays a key role. We have previously reported that local application of Complete Freund's Adjuvant (CFA) onto the dura mater caused activation in rat trigeminal ganglion (TG) which was abolished......) was achieved by application of CFA onto the dural parietal surface. SZR72 was given intraperitoneally (i.p.), one dose prior CFA deposition and repeatedly daily for 7 days. Immunohistochemical studies were performed for mapping glutamate, c-fos, PACAP, substance P, IL-6, IL-1β and TNFα in the TNC/Sp5 and other...... regions of the brainstem and at the C1-C2 regions of the spinal cord. RESULTS: We found that CFA increased c-fos and glutamate immunoreactivity in TNC and C1-C2 neurons. This effect was mitigated by SZR72. PACAP positive fibers were detected in the fasciculus cuneatus and gracilis. Substance P, TNFα, IL-6...

  15. Chronic inhibition of nitric oxide synthase augments the ACTH response to exercise.

    Science.gov (United States)

    Jankord, Ryan; McAllister, Richard M; Ganjam, Venkataseshu K; Laughlin, M Harold

    2009-03-01

    Exercise can activate the hypothalamo-pituitary-adrenocortical (HPA) axis, and regular exercise training can impact how the HPA axis responds to stress. The mechanism by which acute exercise induces HPA activity is unclear. Therefore, the purpose of this study was to test the hypothesis that nitric oxide modulates the neuroendocrine component of the HPA axis during exercise. Female Yucatan miniature swine were treated with N-nitro-l-arginine methyl ester (l-NAME) to test the effect of chronic nitric oxide synthase (NOS) inhibition on the ACTH response to exercise. In addition, we tested the effect of NOS inhibition on blood flow to tissues of the HPA axis and report the effects of handling and treadmill exercise on the plasma concentrations of ACTH and cortisol. Chronic NOS inhibition decreased plasma NO(x) levels by 44%, increased mean arterial blood pressure by 46%, and increased expression of neuronal NOS in carotid arteries. Vascular conductance was decreased in the frontal cortex, the hypothalamus, and the adrenal gland. Chronic NOS inhibition exaggerated the ACTH response to exercise. In contrast, chronic NOS inhibition decreased the ACTH response to restraint, suggesting that the role of NO in modulating HPA activity is stressor dependent. These results demonstrate that NOS activity modulates the response of the neuroendocrine component of the HPA axis during exercise stress.

  16. Virtual Screening of Novel Glucosamine-6-Phosphate Synthase Inhibitors.

    Science.gov (United States)

    Lather, Amit; Sharma, Sunil; Khatkar, Anurag

    2018-01-01

    Infections caused by microorganisms are the major cause of death today. The tremendous and improper use of antimicrobial agents leads to antimicrobial resistance. Various currently available antimicrobial drugs are inadequate to control the infections and lead to various adverse drug reactions. Efforts based on computer-aided drug design (CADD) can excavate a large number of databases to generate new, potent hits and minimize the requirement of time as well as money for the discovery of newer antimicrobials. Pharmaceutical sciences also have made development with advances in drug designing concepts. The current research article focuses on the study of various G-6-P synthase inhibitors from literature cited molecular database. Docking analysis was conducted and ADMET data of various molecules was evaluated by Schrodinger Glide and PreADMET software, respectively. Here, the results presented efficacy of various inhibitors towards enzyme G-6-P synthase. Docking scores, binding energy and ADMET data of various molecules showed good inhibitory potential toward G-6-P synthase as compared to standard antibiotics. This novel antimicrobial drug target G-6-P synthase has not so extensively been explored for its application in antimicrobial therapy, so the work done so far proved highly essential. This article has helped the drug researchers and scientists to intensively explore about this wonderful antimicrobial drug target. The Schrodinger, Inc. (New York, USA) software was utilized to carry out the computational calculations and docking studies. The hardware configuration was Intel® core (TM) i5-4210U CPU @ 2.40GHz, RAM memory 4.0 GB under 64-bit window operating system. The ADMET data was calculated by using the PreADMET tool (PreADMET ver. 2.0). All the computational work was completed in the Laboratory for Enzyme Inhibition Studies, Department of Pharmaceutical Sciences, M.D. University, Rohtak, INDIA. Molecular docking studies were carried out to identify the binding

  17. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  18. Highly Divergent Mitochondrial ATP Synthase Complexes in Tetrahymena thermophila

    NARCIS (Netherlands)

    Nina, Praveen Balabaskaran; Dudkina, Natalya V.; Kane, Lesley A.; van Eyk, Jennifer E.; Boekema, Egbert J.; Mather, Michael W.; Vaidya, Akhil B.; Eisen, Jonathan A.

    The F-type ATP synthase complex is a rotary nano-motor driven by proton motive force to synthesize ATP. Its F(1) sector catalyzes ATP synthesis, whereas the F(o) sector conducts the protons and provides a stator for the rotary action of the complex. Components of both F(1) and F(o) sectors are

  19. Predicting the catalytic sites of isopenicillin N synthase (IPNS ...

    African Journals Online (AJOL)

    Isopenicillin N synthase (IPNS) related Non-haem iron-dependent oxygenases and oxidases (NHIDOX) demonstrated a striking structural conservativeness, even with low protein sequence homology. It is evident that these enzymes have an architecturally similar catalytic centre with active ligands lining the reactive pocket.

  20. Studies on the Active Site of Deacetoxycephalosporin C Synthase

    NARCIS (Netherlands)

    Lloyd, Matthew D.; Lee, Hwei-Jen; Harlos, Karl; Zhang, Zhi-Hong; Baldwin, Jack E.; Schofield, Christopher J.; Charnock, John M.; Garner, C. David; Hara, Takane; Terwisscha van Scheltinga, Anke C.; Valegård, Karin; Viklund, Jenny A.C.; Hajdu, Janos; Andersson, Inger; Danielsson, Åke; Bhikhabhai, Rama

    1999-01-01

    The Fe(II) and 2-oxoglutarate-dependent dioxygenase deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was expressed at ca 25% of total soluble protein in Escherichia coli and purified by an efficient large-scale procedure. Purified protein catalysed the conversions of

  1. Beta-Glucan Synthase Gene Expression in Pleurotus sp

    International Nuclear Information System (INIS)

    Azhar Mohamad; Nie, H.J.

    2016-01-01

    Pleurotus sp. is a popular edible mushroom, containing various functional component, in particular, Beta-glucan. Beta-glucans is a part of glucan family of polysaccharides and supposedly contribute to medicinal and nutritional value of Pleurotus.sp. In order to understand the distribution of Beta-glucan in Pleurotus.sp, the Beta-glucan synthase gene expression was determined and compared in different part of Pleurotus, namely mycelium, stripe and cap. The Pleurotus.sp RNA was extracted using commercial kit, employing Tissuelyser ll (Qiagen, USA) to disrupt the cell walls. Then the RNA was quantified by Nano drop (Thermo Fisher, USA) and visualized using denaturing agarose gel. RNA with good OD 260.280 reading (∼2.0) was chosen and converted to cDNA. Using Laccase synthase gene as home keeping gene, Beta-glucan synthase gene expression was quantified using CFX 96 Real Time PCR detection system (Biorad, USA). Preliminary result shows that Beta-glucan synthase was relatively expressed the most in stripe, followed by mycelium and barely in cap. (author)

  2. Isolation and expression of the Pneumocystis carinii thymidylate synthase gene

    DEFF Research Database (Denmark)

    Edman, U; Edman, J C; Lundgren, B

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar...

  3. Insight into Biochemical Characterization of Plant Sesquiterpene Synthases

    DEFF Research Database (Denmark)

    Manczak, Tom; Simonsen, Henrik Toft

    2016-01-01

    A fast and reproducible protocol was established for enzymatic characterization of plant sesquiterpene synthases that can incorporate radioactivity in their products. The method utilizes the 96-well format in conjunction with cluster tubes and enables processing of >200 samples a day. Along...... with reduced reagent usage, it allows further reduction in the use of radioactive isotopes and flammable organic solvents. The sesquiterpene synthases previously characterized were expressed in yeast, and the plant-derived Thapsia garganica kunzeaol synthase TgTPS2 was tested in this method. KM for TgTPS2...... was found to be 0.55 μM; the turnover number, kcat, was found to be 0.29 s-1, kcat for TgTPS2 is in agreement with that of terpene synthases of other plants, and kcat/KM was found to be 0.53 s-1 μM-1 for TgTPS2. The kinetic parameters were in agreement with previously published data....

  4. Endothelial nitric oxide synthase polymorphism G298T in ...

    Indian Academy of Sciences (India)

    Supplementary data: Endothelial nitric oxide synthase polymorphism G298T in association with oxidative DNA damage in coronary atherosclerosis. Rajesh G. Kumar, Mrudula K. Spurthi, Kishore G. Kumar, Sanjib K. Sahu and Surekha H. Rani. J. Genet. 91, 349–352. Table 1. The demographic and clinical data of the CHD ...

  5. ATP synthase--a marvellous rotary engine of the cell.

    Science.gov (United States)

    Yoshida, M; Muneyuki, E; Hisabori, T

    2001-09-01

    ATP synthase can be thought of as a complex of two motors--the ATP-driven F1 motor and the proton-driven Fo motor--that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

  6. Contribution of granule bound starch synthase in kernel modification ...

    African Journals Online (AJOL)

    The role of gbssI and gbssII genes, encoding granule bound starch synthase enzyme I and II, respectively, in quality protein maize (QPM) were studied at different days after pollination (DAP). Total RNA was used for first strand cDNA synthesis using the ImpromIISriptTM reverse transcriptase. No detectable levels of gbssI ...

  7. Dihydropteroate synthase gene mutations in Pneumocystis and sulfa resistance

    DEFF Research Database (Denmark)

    Huang, Laurence; Crothers, Kristina; Atzori, Chiara

    2004-01-01

    in the dihydropteroate synthase (DHPS) gene. Similar mutations have been observed in P. jirovecii. Studies have consistently demonstrated a significant association between the use of sulfa drugs for PCP prophylaxis and DHPS gene mutations. Whether these mutations confer resistance to TMP-SMX or dapsone plus trimethoprim...

  8. Analysis of genetic variation of inducible nitric oxide synthase and ...

    African Journals Online (AJOL)

    The genetic diversity of 100 Malaysian native chickens was investigated using polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) for two candidate genes: inducible nitric oxide synthase (INOS) and natural resistance-associated macrophage protein 1 (NRAMP1). The two genes were selected ...

  9. Characterising the cellulose synthase complexes of cell walls

    NARCIS (Netherlands)

    Mansoori Zangir, N.

    2012-01-01

    One of the characteristics of the plant kingdom is the presence of a structural cell wall. Cellulose is a major component in both the primary and secondary cell walls of plants. In higher plants cellulose is synthesized by so called rosette protein complexes with cellulose synthases (CESAs) as

  10. Molecular cloning and characterization of strictosidine synthase, a ...

    African Journals Online (AJOL)

    Mitragynine is one of the most dominant indole alkaloids present in the leaves of Mitragyna speciosa, a species of Rubiaceae. This alkaloid is believed to be synthesized via condensation of the amino acid derivative, tryptamine and secologanine by the action of strictosidine synthase (STR). The cDNA clone encoding STR ...

  11. Functional Characterization of Sesquiterpene Synthase from Polygonum minus

    Directory of Open Access Journals (Sweden)

    Su-Fang Ee

    2014-01-01

    Full Text Available Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS has a complete open reading frame (ORF of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of β-sesquiphellandrene.

  12. Identifying the catalytic components of cellulose synthase and the maize mixed-linkage beta-glucan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Nicholas C Carpita

    2009-04-20

    Five specific objectives of this project are to develop strategies to identify the genes that encode the catalytic components of "mixed-linkage" (1→3),(1→4)-beta-D-glucans in grasses, to determine the protein components of the synthase complex, and determine the biochemical mechanism of synthesis. We have used proteomic approaches to define intrinsic and extrinsic polypeptides of Golgi membranes that are associated with polysaccharide synthesis and trafficking. We were successful in producing recombinant catalytic domains of cellulose synthase genes and discovered that they dimerize upon concentration, indicating that two CesA proteins form the catalytic unit. We characterized a brittle stalk2 mutant as a defect in a COBRA-like protein that results in compromised lignin-cellulose interactions that decrease tissue flexibility. We used virus-induced gene silencing of barley cell wall polysaccharide synthesis by BSMV in an attempt to silence specific members of the cellulose synthase-like gene family. However, we unexpectedly found that regardless of the specificity of the target gene, whole gene interaction networks were silenced. We discovered the cause to be an antisense transcript of the cellulose synthase gene initiated small interfering RNAs that spread silencing to related genes.

  13. Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia.

    Science.gov (United States)

    Jullien, Frédéric; Moja, Sandrine; Bony, Aurélie; Legrand, Sylvain; Petit, Cécile; Benabdelkader, Tarek; Poirot, Kévin; Fiorucci, Sébastien; Guitton, Yann; Nicolè, Florence; Baudino, Sylvie; Magnard, Jean-Louis

    2014-01-01

    In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a (E)-β-caryophyllene (LaCARS) and a τ-cadinol synthase (LaCADS). τ-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum γ-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum δ-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of τ-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and (E)-β-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender.

  14. Evolutionary and mechanistic insights from the reconstruction of α-humulene synthases from a modern (+)-germacrene A synthase.

    Science.gov (United States)

    Gonzalez, Veronica; Touchet, Sabrina; Grundy, Daniel J; Faraldos, Juan A; Allemann, Rudolf K

    2014-10-15

    Germacrene A synthase (GAS) from Solidago canadensis catalyzes the conversion of farnesyl diphosphate (FDP) to the plant sesquiterpene (+)-germacrene A. After diphosphate expulsion, farnesyl cation reacts with the distal 10,11-double bond to afford germacrene A (>96%) and <2% α-humulene, which arises from 1,11-cyclization of FDP. The origin of the 1,11-activity of GAS was investigated by amino acid sequence alignments of 1,10- and 1,11-synthases and comparisons of X-ray crystal structures with the homology model of GAS; a triad [Thr 401-Gly 402-Gly 403] that might be responsible for the predominant 1,10-cyclization activity of GAS was identified. Replacement of Gly 402 with residues of increasing size led to a progressive increase of 1,11-cyclization. The catalytic robustness of these 1,10- /1,11-GAS variants point to Gly 402 as a functional switch of evolutionary significance and suggests that enzymes with strict functionalities have evolved from less specific ancestors through a small number of substitutions. Similar results were obtained with germacrene D synthase (GDS) upon replacement of the homologous active-site residue Gly 404: GDS-G404V generated approximately 20% bicyclogermacrene, a hydrocarbon with a cyclopropane ring that underlines the dual 1,10-/1,11-cyclization activity of this mutant. This suggests that the reaction pathways to germacrenes and humulenes might be connected through a bridged 1,10,11-carbocation intermediate or transition state that resembles bicyclogermacrene. Mechanistic studies using [1-(3)H1]-10-fluorofarnesyl diphosphate and deuterium-labeling experiments with [12,13-(2)H6]-FDP support a germacrene-humulene rearrangement linking 1,10- and 1,11-pathways. These results support the bioinformatics proposal that modern 1,10-synthases could have evolved from promiscuous 1,11-sesquiterpene synthases.

  15. Construction of ivermectin producer by domain swaps of avermectin polyketide synthase in Streptomyces avermitilis.

    Science.gov (United States)

    Zhang, Xiaolin; Chen, Zhi; Li, Meng; Wen, Ying; Song, Yuan; Li, Jilun

    2006-10-01

    Ivermectin, 22, 23-dihydroavermectin B1, is commercially important in human, veterinary medicine, and pesticides. It is currently synthesized by chemical reduction of the double bond between C22 and C23 of avermectins B1, which are a mixture of B1a (>80%) and B1b (produced by fermentation of Streptomyces avermitilis. The cost of ivermectin is much higher than that of avermectins B1 owing to the necessity of region-specific hydrogenation at C22-C23 of avermectins B1 with rhodium chloride as the catalyst for producing ivermectin. Here we report that ivermectin can be produced directly by fermentation of recombinant strains constructed through targeted genetic engineering of the avermectin polyketide synthase (PKS) in S. avermitilis Olm73-12, which produces only avermectins B and not avermectins A and oligomycin. The DNA region encoding the dehydratase (DH) and ketoreductase (KR) domains of module 2 from the avermectin PKS in S. avermitilis Olm73-12 was replaced by the DNA fragment encoding the DH, enoylreductase, and KR domains from module 4 of the pikromycin PKS of Streptomyces venezuelae ATCC 15439 using a gene replacement vector pXL211. Twenty-seven of mutants were found to produce a small amount of 22, 23-dihydroavermectin B1a and avermectin B1a and B2a by high performance liquid chromatography and liquid chromatography mass spectrometry analysis. This study might provide a route to the low-cost production of ivermectin by fermentation.

  16. Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid

    Energy Technology Data Exchange (ETDEWEB)

    Hagen, A; Poust, S; De Rond, T; Fortman, JL; Katz, L; Petzold, CJ; Keasling, JD

    2015-10-26

    Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design–build–test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS’ first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to “debug” PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.

  17. Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

    Directory of Open Access Journals (Sweden)

    Lu Frances Fangjia

    2012-11-01

    Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  18. Thalidomide ameliorates portal hypertension via nitric oxide synthase independent reduced systolic blood pressure.

    Science.gov (United States)

    Theodorakis, Nicholas G; Wang, Yining N; Korshunov, Vyacheslav A; Maluccio, Mary A; Skill, Nicholas J

    2015-04-14

    Portal hypertension is a common complication of liver cirrhosis and significantly increases mortality and morbidity. Previous reports have suggested that the compound thalidomide attenuates portal hypertension (PHT). However, the mechanism for this action is not fully elucidated. One hypothesis is that thalidomide destabilizes tumor necrosis factor α (TNFα) mRNA and therefore diminishes TNFα induction of nitric oxide synthase (NOS) and the production of nitric oxide (NO). To examine this hypothesis, we utilized the murine partial portal vein ligation (PVL) PHT model in combination with endothelial or inducible NOS isoform gene knockout mice. Wild type, inducible nitric oxide synthase (iNOS)(-/-) and endothelial nitric oxide synthase (eNOS)(-/-) mice received either PVL or sham surgery and were given either thalidomide or vehicle. Serum nitrate (total nitrate, NOx) was measured daily for 7 d as a surrogate of NO synthesis. Serum TNFα level was quantified by enzyme-linked immunosorbent assay. TNFα mRNA was quantified in liver and aorta tissue by reverse transcription-polymerase chain reaction. PHT was determined by recording splenic pulp pressure (SPP) and abdominal aortic flow after 0-7 d. Response to thalidomide was determined by measurement of SPP and mean arterial pressure (MAP). SPP, abdominal aortic flow (Qao) and plasma NOx were increased in wild type and iNOS(-/-) PVL mice when compared to sham operated control mice. In contrast, SPP, Qao and plasma NOx were not increased in eNOS(-/-) PVL mice when compared to sham controls. Serum TNFα level in both sham and PVL mice was below the detection limit of the commercial ELISA used. Therefore, the effect of thalidomide on serum TNFα levels was undetermined in wild type, eNOS(-/-) or iNOS(-/-) mice. Thalidomide acutely increased plasma NOx in wild type and eNOS(-/-) mice but not iNOS(-/-) mice. Moreover, thalidomide temporarily (0-90 min) decreased mean arterial pressure, SPP and Qao in wild type, e

  19. Air pollution alters brain and pituitary endothelin-1 and inducible nitric oxide synthase gene expression.

    Science.gov (United States)

    Thomson, Errol M; Kumarathasan, Prem; Calderón-Garcidueñas, Lilian; Vincent, Renaud

    2007-10-01

    Recent work suggests that air pollution is a risk factor for cerebrovascular and neurodegenerative disease. Effects of inhaled pollutants on the production of vasoactive factors such as endothelin (ET) and nitric oxide (NO) in the brain may be relevant to disease pathogenesis. Inhaled pollutants increase circulating levels of ET-1 and ET-3, and the pituitary is a potential source of plasma ET, but the effects of pollutants on the expression of ET and NO synthase genes in the brain and pituitary are not known. In the present study, Fischer-344 rats were exposed by nose-only inhalation to particles (0, 5, 50mg/m3 EHC-93), ozone (0, 0.4, 0.8 ppm), or combinations of particles and ozone for 4 h. Real-time reverse transcription polymerase chain reaction was used to measure mRNA levels in the cerebral hemisphere and pituitary 0 and 24 h post-exposure. Ozone inhalation significantly increased preproET-1 but decreased preproET-3 mRNAs in the cerebral hemisphere, while increasing mRNA levels of preproET-1, preproET-3, and the ET-converting enzyme (ECE)-1 in the pituitary. Inducible NO synthase (iNOS) was initially decreased in the cerebral hemisphere after ozone inhalation, but increased 24 h post-exposure. Particles decreased tumour necrosis factor (TNF)-alpha mRNA in the cerebral hemisphere, and both particles and ozone decreased TNF-alpha mRNA in the pituitary. Our results show that ozone and particulate matter rapidly modulate the expression of genes involved in key vasoregulatory pathways in the brain and pituitary, substantiating the notion that inhaled pollutants induce cerebrovascular effects.

  20. Regulation of C. elegans fat uptake and storage by acyl-CoA synthase-3 is dependent on NR5A family nuclear hormone receptor nhr-25

    DEFF Research Database (Denmark)

    Mullaney, Brendan C; Blind, Raymond D; Lemieux, George A

    2010-01-01

    Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3...... mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine...... program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans....

  1. In vitro biochemical characterization of all barley endosperm starch synthases

    DEFF Research Database (Denmark)

    Cuesta-Seijo, Jose A.; Nielsen, Morten M.; Ruzanski, Christian

    2016-01-01

    Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs). While the overall starch synthase (SS) reaction is known, the functional differences between the five SS....... Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results...... define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis...

  2. Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum).

    Science.gov (United States)

    Yang, Chang-Qing; Wu, Xiu-Ming; Ruan, Ju-Xin; Hu, Wen-Li; Mao, Yin-Bo; Chen, Xiao-Ya; Wang, Ling-Jian

    2013-12-01

    Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Nitric oxide synthase isoforms in spontaneous and salt hypertension

    Czech Academy of Sciences Publication Activity Database

    Hojná, Silvie; Kuneš, Jaroslav; Zicha, Josef

    2007-01-01

    Roč. 25, Suppl. 2 (2007), S 338-S 338 ISSN 0263-6352. [European Meeting on Hypertension /17./. 15.06.2007-19.06.2007, Milan] R&D Projects: GA MŠk(CZ) 1M0510 Institutional research plan: CEZ:AV0Z50110509 Keywords : nitric oxide synthase isoforms * spontaneous and salt hypertension Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

  4. Use of linalool synthase in genetic engineering of scent production

    Science.gov (United States)

    Pichersky, Eran

    1998-01-01

    A purified S-linalool synthase polypeptide from Clarkia breweri is disclosed as is the recombinant polypeptide and nucleic acid sequences encoding the polypeptide. Also disclosed are antibodies immunoreactive with the purified peptide and with recombinant versions of the polypeptide. Methods of using the nucleic acid sequences, as well as methods of enhancing the smell and the flavor of plants expressing the nucleic acid sequences are also disclosed.

  5. Trypanosoma brucei solanesyl-diphosphate synthase localizes to the mitochondrion

    Czech Academy of Sciences Publication Activity Database

    Lai, D.-H.; Bontempi, E. J.; Lukeš, Julius

    2012-01-01

    Roč. 183, č. 2 (2012), s. 189-192 ISSN 0166-6851 R&D Projects: GA ČR(CZ) GAP305/11/2179 Institutional support: RVO:60077344 Keywords : Trypanosoma brucei * Sleeping sickness * Ubiquinone * Solanesyl-diphosphate synthase * Digitonin permeabilization * In situ tagging Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.734, year: 2012 http://www.sciencedirect.com/science/article/pii/S0166685112000539

  6. Multi-substrate terpene synthases: their occurrence and physiological significance

    Directory of Open Access Journals (Sweden)

    Leila Pazouki

    2016-07-01

    Full Text Available Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15, and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5, mono- (C10 and diterpenes (C20. Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles.

  7. From bacterial to human dihydrouridine synthase: automated structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Whelan, Fiona, E-mail: fiona.whelan@york.ac.uk; Jenkins, Huw T., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom); Griffiths, Samuel C. [University of Oxford, Headington, Oxford OX3 7BN (United Kingdom); Byrne, Robert T. [Ludwig-Maximilians-University Munich, Feodor-Lynen-Strasse 25, 81377 Munich (Germany); Dodson, Eleanor J.; Antson, Alfred A., E-mail: fiona.whelan@york.ac.uk [The University of York, Heslington, York YO10 5DD (United Kingdom)

    2015-06-30

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer.

  8. From bacterial to human dihydrouridine synthase: automated structure determination

    International Nuclear Information System (INIS)

    Whelan, Fiona; Jenkins, Huw T.; Griffiths, Samuel C.; Byrne, Robert T.; Dodson, Eleanor J.; Antson, Alfred A.

    2015-01-01

    The crystal structure of a human dihydrouridine synthase, an enzyme associated with lung cancer, with 18% sequence identity to a T. maritima enzyme, has been determined at 1.9 Å resolution by molecular replacement after extensive molecular remodelling of the template. The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1–340) determined at 1.9 Å resolution is presented. It is shown that the structure can be determined automatically by phenix.mr-rosetta starting from a bacterial Dus enzyme with only 18% sequence identity and a significantly divergent structure. The overall fold of the human Dus2 is similar to that of bacterial enzymes, but has a larger recognition domain and a unique three-stranded antiparallel β-sheet insertion into the catalytic domain that packs next to the recognition domain, contributing to domain–domain interactions. The structure may inform the development of novel therapeutic approaches in the fight against lung cancer

  9. Dynamics of meso and thermo citrate synthases with implicit solvation

    Science.gov (United States)

    Cordeiro, J. M. M.

    The dynamics of hydration of meso and thermo citrate synthases has been investigated using the EEF1 methodology implemented with the CHARMM program. The native enzymes are composed of two identical subunits, each divided into a small and large domain. The dynamics behavior of both enzymes at 30°C and 60°C has been compared. The results of simulations show that during the hydration process, each subunit follows a different pathway of hydration, in spite of the identical sequence. The hydrated structures were compared with the crystalline structure, and the root mean square deviation (RMSD) of each residue along the trajectory was calculated. The regions with larger and smaller mobility were identified. In particular, helices belonging to the small domain are more mobile than those of the large domain. In contrast, the residues that constitute the active site show a much lower displacement compared with the crystalline structure. Hydration free energy calculations point out that Thermoplasma acidophilum citrate synthase (TCS) is more stable than chicken citrate synthase (CCS), at high temperatures. Such result has been ascribed to the higher number of superficial charges in the thermophilic homologue, which stabilizes the enzyme, while the mesophilic homologue denatures. These results are in accord with the experimental found that TCS keeps activity at temperatures farther apart from the catalysis regular temperature than the CCS.

  10. Mechanism of Action and Inhibition of dehydrosqualene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    F Lin; C Liu; Y Liu; Y Zhang; K Wang; W Jeng; T Ko; R Cao; A Wang; E Oldfield

    2011-12-31

    'Head-to-head' terpene synthases catalyze the first committed steps in sterol and carotenoid biosynthesis: the condensation of two isoprenoid diphosphates to form cyclopropylcarbinyl diphosphates, followed by ring opening. Here, we report the structures of Staphylococcus aureus dehydrosqualene synthase (CrtM) complexed with its reaction intermediate, presqualene diphosphate (PSPP), the dehydrosqualene (DHS) product, as well as a series of inhibitors. The results indicate that, on initial diphosphate loss, the primary carbocation so formed bends down into the interior of the protein to react with C2,3 double bond in the prenyl acceptor to form PSPP, with the lower two-thirds of both PSPP chains occupying essentially the same positions as found in the two farnesyl chains in the substrates. The second-half reaction is then initiated by the PSPP diphosphate returning back to the Mg{sup 2+} cluster for ionization, with the resultant DHS so formed being trapped in a surface pocket. This mechanism is supported by the observation that cationic inhibitors (of interest as antiinfectives) bind with their positive charge located in the same region as the cyclopropyl carbinyl group; that S-thiolo-diphosphates only inhibit when in the allylic site; activity results on 11 mutants show that both DXXXD conserved domains are essential for PSPP ionization; and the observation that head-to-tail isoprenoid synthases as well as terpene cyclases have ionization and alkene-donor sites which spatially overlap those found in CrtM.

  11. Phytochelatin synthase activity as a marker of metal pollution

    Energy Technology Data Exchange (ETDEWEB)

    Zitka, Ondrej; Krystofova, Olga; Sobrova, Pavlina [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Adam, Vojtech [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Zehnalek, Josef; Beklova, Miroslava [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Kizek, Rene, E-mail: kizek@sci.muni.cz [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic)

    2011-08-30

    Highlights: {yields} New tool for determination of phytochelatin synthase activity. {yields} The optimization of experimental condition for determination of the enzyme activity. {yields} First evaluation of K{sub m} for the enzyme. {yields} The effects of cadmium (II) not only on the activity of the enzyme but also on K{sub m}. -- Abstract: The synthesis of phytochelatins is catalyzed by {gamma}-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO{sub 3}){sub 2} for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35 {sup o}C for 30 min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270 fkat) in treated cells was more than seven times higher in comparison to control ones. K{sub m} for PCS was estimated as 2.3 mM.

  12. Phytochelatin synthase activity as a marker of metal pollution

    International Nuclear Information System (INIS)

    Zitka, Ondrej; Krystofova, Olga; Sobrova, Pavlina; Adam, Vojtech; Zehnalek, Josef; Beklova, Miroslava; Kizek, Rene

    2011-01-01

    Highlights: → New tool for determination of phytochelatin synthase activity. → The optimization of experimental condition for determination of the enzyme activity. → First evaluation of K m for the enzyme. → The effects of cadmium (II) not only on the activity of the enzyme but also on K m . -- Abstract: The synthesis of phytochelatins is catalyzed by γ-Glu-Cys dipeptidyl transpeptidase called phytochelatin synthase (PCS). Aim of this study was to suggest a new tool for determination of phytochelatin synthase activity in the tobacco BY-2 cells treated with different concentrations of the Cd(II). After the optimization steps, an experiment on BY-2 cells exposed to different concentrations of Cd(NO 3 ) 2 for 3 days was performed. At the end of the experiment, cells were harvested and homogenized. Reduced glutathione and cadmium (II) ions were added to the cell suspension supernatant. These mixtures were incubated at 35 o C for 30 min and analysed using high performance liquid chromatography coupled with electrochemical detector (HPLC-ED). The results revealed that PCS activity rises markedly with increasing concentration of cadmium (II) ions. The lowest concentration of the toxic metal ions caused almost three fold increase in PCS activity as compared to control samples. The activity of PCS (270 fkat) in treated cells was more than seven times higher in comparison to control ones. K m for PCS was estimated as 2.3 mM.

  13. The primary defect in glycogen synthase activity is not based on increased glycogen synthase kinase-3a activity in diabetic myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael; Brusgaard, Klaus; Handberg, Aa.

    2004-01-01

    The mechanism responsible for the diminished activation of glycogen synthase (GS) in diabetic myotubes remains unclear, but may involve increased activity and/or expression of glycogen synthase kinase-3 (GSK-3). In myotubes established from type 2 diabetic and healthy control subjects we determined...

  14. Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases.

    Directory of Open Access Journals (Sweden)

    Yi Tang

    2004-02-01

    Full Text Available Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs. The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group, some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii the minimal PKS controls polyketide chain length by counting the number of atoms

  15. Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues

    Science.gov (United States)

    Koo, Hyun Jo; Gang, David R.

    2012-01-01

    The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (−)-caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+)-α-turmerone and (+)-β-turmerone, are produced from (−)-α-zingiberene and (−)-β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase. PMID:23272109

  16. [BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

    Science.gov (United States)

    Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Podvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

    2015-01-01

    A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research.

  17. Suites of terpene synthases explain differential terpenoid production in ginger and turmeric tissues.

    Directory of Open Access Journals (Sweden)

    Hyun Jo Koo

    Full Text Available The essential oils of ginger (Zingiber officinale and turmeric (Curcuma longa contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+-germacrene D synthase and (S-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (--caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+-α-turmerone and (+-β-turmerone, are produced from (--α-zingiberene and (--β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase.

  18. Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Kragelund, Birthe B; von Wettstein-Knowles, Penny

    2007-01-01

    Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual...... activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high...... degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic...

  19. Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.

    Science.gov (United States)

    Jin, Zhehao; Kim, Jin-Hee; Park, Sang Un; Kim, Soo-Un

    2016-12-01

    Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

  20. Irreducible Specht modules are signed Young modules

    OpenAIRE

    Hemmer, David J.

    2005-01-01

    Recently Donkin defined signed Young modules as a simultaneous generalization of Young and twisted Young modules for the symmetric group. We show that in odd characteristic, if a Specht module $S^\\lambda$ is irreducible, then $S^\\lambda$ is a signed Young module. Thus the set of irreducible Specht modules coincides with the set of irreducible signed Young modules. This provides evidence for our conjecture that the signed Young modules are precisely the class of indecomposable self-dual module...

  1. New procedures to measure synthase and phosphatase activities of bis-phosphoglycerate mutase. Interest for development of therapeutic drugs

    International Nuclear Information System (INIS)

    Ravel, P.; Garel, M.C.; Toullec, D.

    1997-01-01

    In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentrations is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bis-phosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allows us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phospho-glycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM. (author)

  2. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants.

    Science.gov (United States)

    Degenhardt, Jörg; Köllner, Tobias G; Gershenzon, Jonathan

    2009-01-01

    The multitude of terpene carbon skeletons in plants is formed by enzymes known as terpene synthases. This review covers the monoterpene and sesquiterpene synthases presenting an up-to-date list of enzymes reported and evidence for their ability to form multiple products. The reaction mechanisms of these enzyme classes are described, and information on how terpene synthase proteins mediate catalysis is summarized. Correlations between specific amino acid motifs and terpene synthase function are described, including an analysis of the relationships between active site sequence and cyclization type and a discussion of whether specific protein features might facilitate multiple product formation.

  3. Enhancement of vascular targeting by inhibitors of nitric oxide synthase

    International Nuclear Information System (INIS)

    Davis, Peter D.; Tozer, Gillian M.; Naylor, Matthew A.; Thomson, Peter; Lewis, Gemma; Hill, Sally A.

    2002-01-01

    Purpose: This study investigates the enhancement of the vascular targeting activity of the tubulin-binding agent combretastatin A4 phosphate (CA4P) by various inhibitors of nitric oxide synthases. Methods and Materials: The syngeneic tumors CaNT and SaS growing in CBA mice were used for this study. Reduction in perfused vascular volume was measured by injection of Hoechst 33342 24 h after drug administration. Necrosis (hematoxylin and eosin stain) was assessed also at 24 h after treatment. Combretastatin A4 phosphate was synthesized by a modification of the published procedure and the nitric oxide synthase inhibitors L-NNA, L-NMMA, L-NIO, L-NIL, S-MTC, S-EIT, AMP, AMT, and L-TC, obtained from commercial sources. Results: A statistically significant augmentation of the reduction in perfused vascular volume by CA4P in the CaNT tumor was observed with L-NNA, AMP, and AMT. An increase in CA4P-induced necrosis in the same tumor achieved significance with L-NNA, L-NMMA, L-NIL, and AMT. CA4P induced little necrosis in the SaS tumor, but combination with the inhibitors L-NNA, L-NMMA, L-NIO, S-EIT, and L-TC was effective. Conclusions: Augmentation of CA4P activity by nitric oxide synthase inhibitors of different structural classes supports a nitric oxide-related mechanism for this effect. L-NNA was the most effective inhibitor studied

  4. Incorporation of phosphate into glycogen by glycogen synthase.

    Science.gov (United States)

    Contreras, Christopher J; Segvich, Dyann M; Mahalingan, Krishna; Chikwana, Vimbai M; Kirley, Terence L; Hurley, Thomas D; DePaoli-Roach, Anna A; Roach, Peter J

    2016-05-01

    The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of (32)P from [β-(32)P]UDP-glucose to glycogen by glycogen synthase. The (32)P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The (32)P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-(32)P]UDP generated in the reaction. Furthermore, [(32)P]UDP did not bind non-covalently to glycogen. The (32)P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Signed Young Modules and Simple Specht Modules

    OpenAIRE

    Danz, Susanne; Lim, Kay Jin

    2015-01-01

    By a result of Hemmer, every simple Specht module of a finite symmetric group over a field of odd characteristic is a signed Young module. While Specht modules are parametrized by partitions, indecomposable signed Young modules are parametrized by certain pairs of partitions. The main result of this article establishes the signed Young module labels of simple Specht modules. Along the way we prove a number of results concerning indecomposable signed Young modules that are of independent inter...

  6. Microsomal prostaglandin E synthase-1 in rheumatic diseases

    Directory of Open Access Journals (Sweden)

    Marina eKorotkova

    2011-01-01

    Full Text Available Microsomal prostaglandin E synthase-1 (mPGES-1 is a well recognized target for the development of novel anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases and other inflammatory conditions. In this review, we focus on mPGES-1 in rheumatic diseases with the aim to cover the most recent advances in the understanding of mPGES-1 in rheumatoid arthritis, osteoarthritis and inflammatory myopathies. Novel findings regarding regulation of mPGES1 cell expression as well as enzyme inhibitors are also summarized.

  7. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal......NOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age...

  8. Fatty acid synthase inhibitors isolated from Punica granatum L

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, He-Zhong [School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, (China); Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing, E-mail: zhaoyx1011@163.com [Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou (China); Fan, Hui-Jin; Ma, Xiao-Feng, E-mail: maxiaofeng@gucas.ac.cn [College of Life Sciences, Graduate University of Chinese Academy of Sciences, Beijing (China)

    2012-05-15

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC{sub 50} value of 10.3 {mu}mol L{sup -1}. (author)

  9. Fatty acid synthase inhibitors isolated from Punica granatum L

    International Nuclear Information System (INIS)

    Jiang, He-Zhong; Ma, Qing-Yun; Liang, Wen-Juan; Huang, Sheng-Zhuo; Dai, Hao-Fu; Wang, Peng-Cheng; Zhao, You-Xing; Fan, Hui-Jin; Ma, Xiao-Feng

    2012-01-01

    The aim of this work is the isolation of fatty acid synthase (FAS) inhibitors from the ethyl acetate extracts of fruit peels of Punica granatum L. Bioassay-guided chemical investigation of the fruit peels resulted in the isolation of seventeen compounds mainly including triterpenoids and phenolic compounds, from which one new oleanane-type triterpene (punicaone) along with fourteen known compounds were isolated for the first time from this plant. Seven isolates were evaluated for inhibitory activities of FAS and two compounds showed to be active. Particularly, flavogallonic acid exhibited strong FAS inhibitory activity with IC 50 value of 10.3 μmol L -1 . (author)

  10. Memory Modulation

    NARCIS (Netherlands)

    Roozendaal, Benno; McGaugh, James L.

    2011-01-01

    Our memories are not all created equally strong: Some experiences are well remembered while others are remembered poorly, if at all. Research on memory modulation investigates the neurobiological processes and systems that contribute to such differences in the strength of our memories. Extensive

  11. Module descriptor

    DEFF Research Database (Denmark)

    Vincenti, Gordon; Klausen, Bodil; Kjær Jensen, Jesper

    2016-01-01

    The Module Descriptor including a Teacher’s Guide explains and describes how to work innovatively and co-creatively with wicked problems and young people. The descriptor shows how interested educators and lecturers in Europe can copy the lessons of the Erasmus+ project HIP when teaching their own...

  12. Role of Modular Polyketide Synthases in the Production of Polyether Ladder Compounds in Ciguatoxin-Producing Gambierdiscus polynesiensis and G. excentricus (Dinophyceae).

    Science.gov (United States)

    Kohli, Gurjeet S; Campbell, Katrina; John, Uwe; Smith, Kirsty F; Fraga, Santiago; Rhodes, Lesley L; Murray, Shauna A

    2017-09-01

    Gambierdiscus, a benthic dinoflagellate, produces ciguatoxins that cause the human illness Ciguatera. Ciguatoxins are polyether ladder compounds that have a polyketide origin, indicating that polyketide synthases (PKS) are involved in their production. We sequenced transcriptomes of Gambierdiscus excentricus and Gambierdiscus polynesiensis and found 264 contigs encoding single domain ketoacyl synthases (KS; G. excentricus: 106, G. polynesiensis: 143) and ketoreductases (KR; G. excentricus: 7, G. polynesiensis: 8) with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 24 contigs (G. excentricus: 3, G. polynesiensis: 21) encoding multiple PKS domains (forming typical type I PKSs modules) were found. The proposed structure produced by one of these megasynthases resembles a partial carbon backbone of a polyether ladder compound. Seventeen contigs encoding single domain KS, KR, s-malonyltransacylase, dehydratase and enoyl reductase with sequence similarity to type II fatty acid synthases (FAS) in plants were found. Type I PKS and type II FAS genes were distinguished based on the arrangement of domains on the contigs and their sequence similarity and phylogenetic clustering with known PKS/FAS genes in other organisms. This differentiation of PKS and FAS pathways in Gambierdiscus is important, as it will facilitate approaches to investigating toxin biosynthesis pathways in dinoflagellates. © 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.

  13. Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I modular polyketide synthases: opportunities and challenges.

    Science.gov (United States)

    Yuzawa, Satoshi; Keasling, Jay D; Katz, Leonard

    2017-04-01

    Complex polyketides comprise a large number of natural products that have broad application in medicine and agriculture. They are produced in bacteria and fungi from large enzyme complexes named type I modular polyketide synthases (PKSs) that are composed of multifunctional polypeptides containing discrete enzymatic domains organized into modules. The modular nature of PKSs has enabled a multitude of efforts to engineer the PKS genes to produce novel polyketides of predicted structure. We have repurposed PKSs to produce a number of short-chain mono- and di-carboxylic acids and ketones that could have applications as fuels or industrial chemicals.

  14. Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I modular polyketide synthases: opportunities and challenges

    Energy Technology Data Exchange (ETDEWEB)

    Yuzawa, Satoshi [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Keasling, Jay D. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). QB3 Inst.; Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering; Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering; Technical Univ. of Denmark, Horsholm (Denmark). Novo Nordisk Foundation Center for Biosustainability; Katz, Leonard [Univ. of California, Berkeley, CA (United States). QB3 Inst.

    2016-11-16

    Complex polyketides comprise a large number of natural products that have broad application in medicine and agriculture. They are produced in bacteria and fungi from large enzyme complexes named type I modular polyketide synthases (PKSs) that are composed of multifunctional polypeptides containing discrete enzymatic domains organized into modules. The modular nature of PKSs has enabled a multitude of efforts to engineer the PKS genes to produce novel polyketides of predicted structure. Finally, we have repurposed PKSs to produce a number of short-chain mono- and di-carboxylic acids and ketones that could have applications as fuels or industrial chemicals.

  15. Overexpression of the Anthocyanidin Synthase Gene in Strawberry Enhances Antioxidant Capacity and Cytotoxic Effects on Human Hepatic Cancer Cells.

    Science.gov (United States)

    Giampieri, Francesca; Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Mazzoni, Luca; Capocasa, Franco; Sabbadini, Silvia; Alvarez-Suarez, Josè M; Afrin, Sadia; Rosati, Carlo; Pandolfini, Tiziana; Molesini, Barbara; Sánchez-Sevilla, José F; Amaya, Iraida; Mezzetti, Bruno; Battino, Maurizio

    2018-01-24

    Food fortification through the increase and/or modulation of bioactive compounds has become a major goal for preventing several diseases, including cancer. Here, strawberry lines of cv. Calypso transformed with a construct containing an anthocyanidin synthase (ANS) gene were produced to study the effects on anthocyanin biosynthesis, metabolism, and transcriptome. Three strawberry ANS transgenic lines (ANS L5, ANS L15, and ANS L18) were analyzed for phytochemical composition and total antioxidant capacity (TAC), and their fruit extracts were assessed for cytotoxic effects on hepatocellular carcinoma. ANS L18 fruits had the highest levels of total phenolics and flavonoids, while those of ANS L15 had the highest anthocyanin concentration; TAC positively correlated with total polyphenol content. Fruit transcriptome was also specifically affected in the polyphenol biosynthesis and in other related metabolic pathways. Fruit extracts of all lines exerted cytotoxic effects in a dose/time-dependent manner, increasing cellular apoptosis and free radical levels and impairing mitochondrial functionality.

  16. Effects of various nitric oxide synthase inhibitors on AlCl3-induced neuronal injury in rats

    Directory of Open Access Journals (Sweden)

    IVANA STEVANOVIĆ

    2009-05-01

    Full Text Available The present study was aimed at determining the effectiveness of nitric oxide synthase (NOS inhibitors: N-nitro-L-arginine methyl ester, 7-nitroindazole and aminoguanidine in modulating the toxicity of AlCl3 on superoxide production and the malondialdehyde concentration of Wistar rats. The animals were sacrificed 10 min and 3 days after the treatment and the forebrain cortex was removed. The results show that AlCl3 exposure promotes oxidative stress in different neural areas. The biochemical changes observed in the neuronal tissues show that aluminum acts as pro-oxidant, while NOS inhibitors exert an anti-oxidant action in AlCl3-treated animals.

  17. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

    International Nuclear Information System (INIS)

    Takabe, Piia; Bart, Geneviève; Ropponen, Antti; Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna

    2015-01-01

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma

  18. Towards Precision Engineering of Canonical Polyketide Synthase Domains: Recent Advances and Future Prospects

    Directory of Open Access Journals (Sweden)

    Carmen L. Bayly

    2017-02-01

    Full Text Available Modular polyketide synthases (mPKSs build functionalized polymeric chains, some of which have become blockbuster therapeutics. Organized into repeating clusters (modules of independently-folding domains, these assembly-line-like megasynthases can be engineered by introducing non-native components. However, poor introduction points and incompatible domain combinations can cause both unintended products and dramatically reduced activity. This limits the engineering and combinatorial potential of mPKSs, precluding access to further potential therapeutics. Different regions on a given mPKS domain determine how it interacts both with its substrate and with other domains. Within the assembly line, these interactions are crucial to the proper ordering of reactions and efficient polyketide construction. Achieving control over these domain functions, through precision engineering at key regions, would greatly expand our catalogue of accessible polyketide products. Canonical mPKS domains, given that they are among the most well-characterized, are excellent candidates for such fine-tuning. The current minireview summarizes recent advances in the mechanistic understanding and subsequent precision engineering of canonical mPKS domains, focusing largely on developments in the past year.

  19. Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption

    Science.gov (United States)

    van't Hof, R. J.; Armour, K. J.; Smith, L. M.; Armour, K. E.; Wei, X. Q.; Liew, F. Y.; Ralston, S. H.

    2000-01-01

    Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study, we investigated the role that the iNOS pathway plays in mediating the bone-resorbing effects of IL-1 by studying mice with targeted disruption of the iNOS gene. Studies in vitro and in vivo showed that iNOS-deficient mice exhibited profound defects of IL-1-induced osteoclastic bone resorption but responded normally to calciotropic hormones such as 1,25 dihydroxyvitamin D3 and parathyroid hormone. Immunohistochemical studies and electrophoretic mobility shift assays performed on bone marrow cocultures from iNOS-deficient mice showed abnormalities in IL-1-induced nuclear translocation of the p65 component of NFκB and in NFκB-DNA binding, which were reversed by treatment with the NO donor S-nitroso-acetyl penicillamine. These results show that the iNOS pathway is essential for IL-1-induced bone resorption and suggest that the effects of NO may be mediated by modulating IL-1-induced nuclear activation of NFκB in osteoclast precursors. PMID:10869429

  20. Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica).

    Science.gov (United States)

    Dare, Andrew P; Tomes, Sumathi; Jones, Midori; McGhie, Tony K; Stevenson, David E; Johnson, Ross A; Greenwood, David R; Hellens, Roger P

    2013-05-01

    We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down-regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down-regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS-silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS-silenced line was smaller than the 'Royal Gala' controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS-silenced line compared with the 'Royal Gala' control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

  1. Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Masahiro Myojo

    Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

  2. Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption.

    Science.gov (United States)

    van't Hof, R J; Armour, K J; Smith, L M; Armour, K E; Wei, X Q; Liew, F Y; Ralston, S H

    2000-07-05

    Nitric oxide has been suggested to be involved in the regulation of bone turnover, especially in pathological conditions characterized by release of bone-resorbing cytokines. The cytokine IL-1 is thought to act as a mediator of periarticular bone loss and tissue damage in inflammatory diseases such as rheumatoid arthritis. IL-1 is a potent stimulator of both osteoclastic bone resorption and expression of inducible nitric oxide synthase (iNOS) in bone cells and other cell types. In this study, we investigated the role that the iNOS pathway plays in mediating the bone-resorbing effects of IL-1 by studying mice with targeted disruption of the iNOS gene. Studies in vitro and in vivo showed that iNOS-deficient mice exhibited profound defects of IL-1-induced osteoclastic bone resorption but responded normally to calciotropic hormones such as 1,25 dihydroxyvitamin D3 and parathyroid hormone. Immunohistochemical studies and electrophoretic mobility shift assays performed on bone marrow cocultures from iNOS-deficient mice showed abnormalities in IL-1-induced nuclear translocation of the p65 component of NFkappaB and in NFkappaB-DNA binding, which were reversed by treatment with the NO donor S-nitroso-acetyl penicillamine. These results show that the iNOS pathway is essential for IL-1-induced bone resorption and suggest that the effects of NO may be mediated by modulating IL-1-induced nuclear activation of NFkappaB in osteoclast precursors.

  3. Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Takabe, Piia, E-mail: piia.takabe@uef.fi [University of Eastern Finland, Institute of Biomedicine, 70211 Kuopio (Finland); Bart, Geneviève [University of Eastern Finland, Institute of Biomedicine, 70211 Kuopio (Finland); Ropponen, Antti [University of Eastern Finland, Institute of Clinical Medicine, 70211 Kuopio (Finland); Rilla, Kirsi; Tammi, Markku; Tammi, Raija; Pasonen-Seppänen, Sanna [University of Eastern Finland, Institute of Biomedicine, 70211 Kuopio (Finland)

    2015-09-10

    Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.

  4. Regulation of mouse brain glycogen synthase kinase-3 by atypical antipsychotics.

    Science.gov (United States)

    Li, Xiaohua; Rosborough, Kelley M; Friedman, Ari B; Zhu, Wawa; Roth, Kevin A

    2007-02-01

    Glycogen synthase kinase-3 (GSK3) has been recognized as an important enzyme that modulates many aspects of neuronal function. Accumulating evidence implicates abnormal activity of GSK3 in mood disorders and schizophrenia, and GSK3 is a potential protein kinase target for psychotropics used in these disorders. We previously reported that serotonin, a major neurotransmitter involved in mood disorders, regulates GSK3 by acutely increasing its N-terminal serine phosphorylation. The present study was undertaken to further determine if atypical antipsychotics, which have therapeutic effects in both mood disorders and schizophrenia, can regulate phospho-Ser-GSK3 and inhibit its activity. The results showed that acute treatment of mice with risperidone rapidly increased the level of brain phospho-Ser-GSK3 in the cortex, hippocampus, striatum, and cerebellum in a dose-dependent manner. Regulation of phospho-Ser-GSK3 was a shared effect among several atypical antipsychotics, including olanzapine, clozapine, quetiapine, and ziprasidone. In addition, combination treatment of mice with risperidone and a monoamine reuptake inhibitor antidepressant imipramine or fluoxetine elicited larger increases in brain phospho-Ser-GSK3 than each agent alone. Taken together, these results provide new information suggesting that atypical antipsychotics, in addition to mood stabilizers and antidepressants, can inhibit the activity of GSK3. These findings may support the pharmacological mechanisms of atypical antipsychotics in the treatment of mood disorders.

  5. Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

    Directory of Open Access Journals (Sweden)

    Kawamukai Makoto

    2004-11-01

    Full Text Available Abstract Background Isopentenyl diphosphate (IPP, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

  6. Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family.

    Science.gov (United States)

    Wilson, Wayne A; Pradhan, Prajakta; Madhan, Nayasha; Gist, Galen C; Brittingham, Andrew

    2017-07-01

    Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  7. Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases.

    Science.gov (United States)

    Lohman, Jeremy R; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2015-10-13

    Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

  8. Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

    Science.gov (United States)

    Lohman, Jeremy R.; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2015-01-01

    Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs. PMID:26420866

  9. Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases

    Energy Technology Data Exchange (ETDEWEB)

    Lohman, Jeremy; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne E.; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, B G

    2015-10-13

    Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

  10. Resveratrol suppresses growth of cancer stem-like cells by inhibiting fatty acid synthase.

    Science.gov (United States)

    Pandey, Puspa R; Okuda, Hiroshi; Watabe, Misako; Pai, Sudha K; Liu, Wen; Kobayashi, Aya; Xing, Fei; Fukuda, Koji; Hirota, Shigeru; Sugai, Tamotsu; Wakabayashi, Go; Koeda, Keisuke; Kashiwaba, Masahiro; Suzuki, Kazuyuki; Chiba, Toshimi; Endo, Masaki; Fujioka, Tomoaki; Tanji, Susumu; Mo, Yin-Yuan; Cao, Deliang; Wilber, Andrew C; Watabe, Kounosuke

    2011-11-01

    Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. Resveratrol has been shown to have strong hypolipidemic effect on normal adipocytes and as hyper-lipogenesis is a hallmark of cancer cell physiology, the effect of resveratrol on lipid synthesis in cancer stem-like cells (CD24(-)/CD44(+)/ESA(+)) that were isolated from both ER+ and ER- breast cancer cell lines was examined. The authors found that resveratrol significantly reduced the cell viability and mammosphere formation followed by inducing apoptosis in cancer stem-like cells. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the fatty acid synthase (FAS) gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by TOFA and by Fumonisin B1, suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of cancer stem-like cells in an animal model of xenograft without showing apparental toxicity. Taken together, the results of this study indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol.

  11. Thymidilate synthase and p53 primary tumour expression as predictive factors for advanced colorectal cancer patients.

    Science.gov (United States)

    Paradiso, A; Simone, G; Petroni, S; Leone, B; Vallejo, C; Lacava, J; Romero, A; Machiavelli, M; De Lena, M; Allegra, C J; Johnston, P G

    2000-02-01

    The purpose of this work was to analyse the ability of p53 and thymidilate synthase (TS) primary tumour expression to retrospectively predict clinical response to chemotherapy and long-term prognosis in patients with advanced colorectal cancers homogeneously treated by methotrexate (MTX)-modulated-5-fluorouracil (5-FU-FA). A total of 108 advanced colorectal cancer patients entered the present retrospective study. Immunohistochemical p53 (pAb 1801 mAb) and TS (TS106 mAb) expression on formalin-fixed paraffin-embedded primary tumour specimens was related to probability of clinical response to chemotherapy, time to progression and overall survival. p53 was expressed in 53/108 (49%) tumours, while 54/108 (50%) showed TS immunostaining. No relationship was demonstrated between p53 positivity and clinical response to chemotherapy (objective response (OR): 20% vs 23%, in p53+ and p53- cases respectively) or overall survival. Percent of OR was significantly higher in TS-negative with respect to TS-positive tumours (30% vs 15% respectively; P < 0.04); simultaneous analysis of TS and p53 indicated 7% OR for p53-positive/TS-positive tumours vs 46% for p53-positive/TS-negative tumours (P < 0.03). Logistic regression analysis confirmed a significant association between TS tumour status and clinical response to chemotherapy (hazard ratio (HR): 2.91; 95% confidence interval (CI) 8.34-1.01; two-sided P < 0.05). A multivariate analysis of overall survival showed that only a small number of metastatic sites was statistically relevant (HR 1.89; 95% CI 2.85-1.26; two-sided P < 0.03). Our study suggests that immunohistochemical expression of p53 and TS could assist the clinician in predicting response of colorectal cancer patients to modulated MTX-5-FU therapy.

  12. Stereochemical course of enzyme-catalyzed aminopropyl transfer: spermidine synthase

    International Nuclear Information System (INIS)

    Kullberg, D.W.; Orr, G.R.; Coward, J.K.

    1986-01-01

    The R and S enantionmers of S-adenosyl-3-[ 2 H]3-(methylthio)-1-propylamine (decarboxylated S-adenosylmethionine), previously synthesized in this laboratory, were incubated with [1,4- 2 H 4 ]-putrescine in the presence of spermidine synthase from E. coli. The resulting chiral [ 2 H 5 ]spermidines were isolated and converted to their N 1 ,N 7 -dibocspermidine-N 4 -(1S,4R)-camphanamides. The derivatives were analyzed by 500 MHz 1 H-NMR and the configuration of the chiral center assigned by correlation with the spectra of synthetic chiral [ 2 H 3 ]dibocspermidine camphanamide standards. The enzyme-catalyzed aminopropyl transfer was shown to occur with net retention of configuration, indicative of a double-displacement mechanism. This result concurs with that of a previous steady-state kinetics study of spermidine synthase isolated from E. coli, but contradicts the single-displacement mechanism suggested by a stereochemical analysis of chiral spermidines biosynthesized in E. coli treated with chirally deuterated methionines. It also indicates that this aminopropyltransferase is mechanistically distinct from the methyltransferases, which have been shown to act via a single-displacement mechanism (net inversion at -CH 3 ) in all cases studied to date

  13. In Vitro Biochemical Characterization of All Barley Endosperm Starch Synthases

    Directory of Open Access Journals (Sweden)

    Jose Antonio Cuesta-Seijo

    2016-01-01

    Full Text Available Starch is the main storage polysaccharide in cereals and the major source of calories in the human diet. It is synthesized by a panel of enzymes including five classes of starch synthases (SSs. While the overall starch synthase (SS reaction is known, the functional differences between the five SS classes are poorly understood. Much of our knowledge comes from analyzing mutant plants with altered SS activities, but the resulting data are often difficult to interpret as a result of pleitropic effects, competition between enzymes, overlaps in enzyme activity and disruption of multi-enzyme complexes. Here we provide a detailed biochemical study of the activity of all five classes of SSs in barley endosperm. Each enzyme was produced recombinantly in E. coli and the properties and modes of action in vitro were studied in isolation from other SSs and other substrate modifying activities. Our results define the mode of action of each SS class in unprecedented detail; we analyze their substrate selection, temperature dependence and stability, substrate affinity and temporal abundance during barley development. Our results are at variance with some generally accepted ideas about starch biosynthesis and might lead to the reinterpretation of results obtained in planta. In particular, they indicate that granule bound SS is capable of processive action even in the absence of a starch matrix, that SSI has no elongation limit, and that SSIV, believed to be critical for the initiation of starch granules, has maltoligosaccharides and not polysaccharides as its preferred substrates.

  14. Glycogen synthase kinase 3: more than a namesake.

    Science.gov (United States)

    Rayasam, Geetha Vani; Tulasi, Vamshi Krishna; Sodhi, Reena; Davis, Joseph Alex; Ray, Abhijit

    2009-03-01

    Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, tau protein and beta catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target.

  15. Polyketide synthases from poison hemlock (Conium maculatum L.).

    Science.gov (United States)

    Hotti, Hannu; Seppänen-Laakso, Tuulikki; Arvas, Mikko; Teeri, Teemu H; Rischer, Heiko

    2015-11-01

    Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by the condensation of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). We isolated PKS genes or their fragments from poison hemlock (Conium maculatum L.) by using random amplification of cDNA ends (RACE) and transcriptome analysis, and characterized three full-length enzymes by feeding different starter-CoAs in vitro. On the basis of our in vitro experiments, two of the three characterized PKS genes in poison hemlock encode chalcone synthases (CPKS1 and CPKS2), and one encodes a novel type of PKS (CPKS5). We show that CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. Our results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs. © 2015 FEBS.

  16. Amaryllidaceae Alkaloids as Potential Glycogen Synthase Kinase-3β Inhibitors

    Directory of Open Access Journals (Sweden)

    Daniela Hulcová

    2018-03-01

    Full Text Available Glycogen synthase kinase-3β (GSK-3β is a multifunctional serine/threonine protein kinase that was originally identified as an enzyme involved in the control of glycogen metabolism. It plays a key role in diverse physiological processes including metabolism, the cell cycle, and gene expression by regulating a wide variety of well-known substances like glycogen synthase, tau-protein, and β-catenin. Recent studies have identified GSK-3β as a potential therapeutic target in Alzheimer´s disease, bipolar disorder, stroke, more than 15 types of cancer, and diabetes. GSK-3β is one of the most attractive targets for medicinal chemists in the discovery, design, and synthesis of new selective potent inhibitors. In the current study, twenty-eight Amaryllidaceae alkaloids of various structural types were studied for their potency to inhibit GSK-3β. Promising results have been demonstrated by alkaloids of the homolycorine-{9-O-demethylhomolycorine (IC50 = 30.00 ± 0.71 µM, masonine (IC50 = 27.81 ± 0.01 μM}, and lycorine-types {caranine (IC50 = 30.75 ± 0.04 μM}.

  17. The crystal structure of human GDP-L-fucose synthase.

    Science.gov (United States)

    Zhou, Huan; Sun, Lihua; Li, Jian; Xu, Chunyan; Yu, Feng; Liu, Yahui; Ji, Chaoneng; He, Jianhua

    2013-09-01

    Human GDP-l-fucose synthase, also known as FX protein, synthesizes GDP-l-fucose from its substrate GDP-4-keto-6-deoxy-d-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4. In this paper, the first crystal structure of human FX protein was determined at 2.37 Å resolution. The asymmetric unit of the crystal structure contains four molecules which form two homodimers. Each molecule consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain. Compared with the Escherichia coli GDP-l-fucose synthase, the overall structures of these two enzymes have four major differences. There are four loops in the structure of human FX protein corresponding to two α-helices and two β-sheets in that of the E. coli enzyme. Besides, there are seven different amino acid residues binding with NAPDH comparing human FX protein with that from E. coli. The structure of human FX reveals the key catalytic residues and could be useful for the design of drugs for the treatment of inflammation, auto-immune diseases, and possibly certain types of cancer.

  18. Prostaglandin E(2) synthase inhibition as a therapeutic target.

    Science.gov (United States)

    Iyer, Jitesh P; Srivastava, Punit K; Dev, Rishabh; Dastidar, Sunanda G; Ray, Abhijit

    2009-07-01

    Most NSAIDs function by inhibiting biosynthesis of PGE(2) by inhibition of COX-1 and/or COX-2. Since COX-1 has a protective function in the gastro-intestinal tract (GIT), non-selective inhibition of both cycloxy genases leads to moderate to severe gastro-intestinal intolerance. Attempts to identify selective inhibitors of COX-2, led to the identification of celecoxib and rofecoxib. However, long-term use of these drugs has serious adverse effects of sudden myocardial infarction and thrombosis. Drug-mediated imbalance in the levels of prostaglandin I(2) (PGI(2)) and thromboxane A(2) (TXA(2)) with a bias towards TXA(2) may be the primary reason for these events. This resulted in the drugs being withdrawn from the market, leaving a need for an effective and safe anti-inflammatory drug. Recently, the focus of research has shifted to enzymes downstream of COX in the prosta glandin biosynthetic pathway such as prostaglandin E(2) synthases. Microsomal prostaglandin E(2) synthase-1 (mPGES-1) specifically isomerizes PGH(2) to PGE(2), under inflammatory conditions. In this review, we examine the biology of mPGES-1 and its role in disease. Progress in designing molecules that can selectively inhibit mPGES-1 is reviewed. mPGES-1 has the potential to be a target for anti-inflammatory therapy, devoid of adverse GIT and cardiac effects and warrants further investigation.

  19. Chromosomal localization of the human and mouse hyaluronan synthase genes

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, A.P.; McDonald, J.A. [Mayo Clinic Scottsdale, AZ (United States); Seldin, M.F. [Univ. of California Davis, CA (United States)] [and others

    1997-05-01

    We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17. HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome. 33 refs., 2 figs.

  20. The effect of a selective neuronal nitric oxide synthase inhibitor 3-bromo 7-nitroindazole on spatial learning and memory in rats.

    Science.gov (United States)

    Gocmez, Semil Selcen; Yazir, Yusufhan; Sahin, Deniz; Karadenizli, Sabriye; Utkan, Tijen

    2015-04-01

    Since the discovery of nitric oxide (NO) as a neuronal messenger, its way to modulate learning and memory functions is subject of intense research. NO is an intercellular messenger in the central nervous system and is formed on demand through the conversion of L-arginine to L-citrulline via the enzyme nitric oxide synthase (NOS). Neuronal form of nitric oxide synthase may play an important role in a wide range of physiological and pathological conditions. Therefore the aim of this study was to investigate the effects of chronic 3-bromo 7-nitroindazole (3-Br 7-NI), specific neuronal nitric oxide synthase (nNOS) inhibitor, administration on spatial learning and memory performance in rats using the Morris water maze (MWM) paradigm. Male rats received either 3-Br 7-NI (20mg/kg/day) or saline via intraperitoneal injection for 5days. Daily administration of the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-Br 7-NI impaired the acquisition of the MWM task. 3-Br 7-NI also impaired the probe trial. The MWM training was associated with a significant increase in the brain-derived neurotrophic factor (BDNF) mRNA expression in the hippocampus. BDNF mRNA expression in the hippocampus did not change after 3-Br 7-NI treatment. L-arginine significantly reversed behavioural parameters, and the effect of 3-Br 7-NI was found to be NO-dependent. There were no differences in locomotor activity and blood pressure in 3-Br 7-NI treated rats. Our results may suggest that nNOS plays a key role in spatial memory formation in rats. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Virus-induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence-related genes resulting in reduced withanolides and biotic stress tolerance.

    Science.gov (United States)

    Singh, Anup Kumar; Dwivedi, Varun; Rai, Avanish; Pal, Shaifali; Reddy, Sajjalavarahalli Gangireddy Eswara; Rao, Dodaghatta Krishnarao Venkata; Shasany, Ajit Kumar; Nagegowda, Dinesh A

    2015-12-01

    Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (β-amyrin synthase) and β-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides

  2. Selectivity of the surface binding site (SBS) on barley starch synthase I

    DEFF Research Database (Denmark)

    Wilkens, Casper; Cuesta-Seijo, Jose A.; Palcic, Monica

    2014-01-01

    Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was ...

  3. Inhibition of the ATP Synthase Eliminates the Intrinsic Resistance of Staphylococcus aureus towards Polymyxins

    DEFF Research Database (Denmark)

    Vestergaard, Martin; Nøhr-Meldgaard, Katrine; Bojer, Martin Saxtorph

    2017-01-01

    , linezolid, daptomycin, and oxacillin were unchanged. ATP synthase activity is known to be inhibited by oligomycin A, and the presence of this compound increased polymyxin B-mediated killing of S. aureus Our results demonstrate that the ATP synthase contributes to intrinsic resistance of S. aureus towards...

  4. Creation of a high-amylose durum wheat through mutagenesis of starch synthase II (SSIIa)

    Science.gov (United States)

    In cereal seeds mutations in one or more starch synthases lead to decreased amylopectin and increased amylose content. Here, the impact of starch synthase IIa (SSIIa or SGP-1) mutations upon durum starch was investigated. A screen of durum accessions identified two lines lacking SGP-A1, the A geno...

  5. Cytidine triphosphate synthase activity and mRNA expression in normal human blood cells

    NARCIS (Netherlands)

    Verschuur, A. C.; van Gennip, A. H.; Muller, E. J.; Voûte, P. A.; Vreken, P.; van Kuilenburg, A. B.

    1999-01-01

    Cytidine triphosphate (CTP) synthase is one of the key enzymes in pyrimidine nucleotide anabolic pathways. The activity of this enzyme is elevated in various malignancies including acute lymphocytic leukemia (ALL). In this study we investigated the activity of CTP synthase in various human blood

  6. Cloning and sequence analysis of putative type II fatty acid synthase ...

    Indian Academy of Sciences (India)

    Prakash

    Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. ... acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP .... Helix II plays a dominant role in the interaction ... main distinguishing features of plant ACPs in plastids and ..... synthase component; J. Biol.

  7. Reduced methylation of the thromboxane synthase gene is correlated with its increased vascular expression in preeclampsia.

    Science.gov (United States)

    Mousa, Ahmad A; Strauss, Jerome F; Walsh, Scott W

    2012-06-01

    Preeclampsia is characterized by increased thromboxane and decreased prostacyclin levels, which predate symptoms, and can explain some of the clinical manifestations of preeclampsia, including hypertension and thrombosis. In this study, we examined DNA methylation of the promoter region of the thromboxane synthase gene (TBXAS1) and the expression of thromboxane synthase in systemic blood vessels of normal pregnant and preeclamptic women. Thromboxane synthase is responsible for the synthesis of thromboxane A(2), a potent vasoconstrictor and activator of platelets. We also examined the effect of experimentally induced DNA hypomethylation on the expression of thromboxane synthase in a neutrophil-like cell line (HL-60 cells) and in cultured vascular smooth muscle and endothelial cells. We found that DNA methylation of the TBXAS1 promoter was decreased and thromboxane synthase expression was increased in omental arteries of preeclamptic women as compared with normal pregnant women. Increased thromboxane synthase expression was observed in vascular smooth muscles cells, endothelial cells, and infiltrating neutrophils. Experimentally induced DNA hypomethylation only increased expression of thromboxane synthase in the neutrophil-like cell line, whereas tumor necrosis factor-α, a neutrophil product, increased its expression in cultured vascular smooth muscle cells. Our study suggests that epigenetic mechanisms and release of tumor necrosis factor-α by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in maternal systemic blood vessels, contributing to the hypertension and coagulation abnormalities associated with preeclampsia.

  8. Pain modulation by nitric oxide in the spinal cord.

    Directory of Open Access Journals (Sweden)

    Marco Aurelio M Freire

    2009-09-01

    Full Text Available Nitric oxide (NO is a versatile messenger molecule first associated with endothelial relaxing effects. In the central nervous system (CNS, NO synthesis is primarily triggered by activation of N-methyl-D-aspartate (NMDA receptors and has a Janus face, with both beneficial and harmful properties, depending on concentration and the identity of its synthetic enzyme isoform. There are three isoforms of the NO synthesizing enzyme nitric oxide synthase (NOS: neuronal (nNOS, endothelial (eNOS, and inducible nitric oxide synthase (iNOS, each one involved with specific events in the brain. In CNS, nNOS is involved with modulation of synaptic transmission through long-term potentiation in several regions, including nociceptive circuits in the spinal cord. Here, we review the role played by NO on central pain sensitization.

  9. Functional specificity of cardiolipin synthase revealed by the identification of a cardiolipin synthase CrCLS1 in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Chun-Hsien eHung

    2016-01-01

    Full Text Available Phosphatidylglycerol (PG and cardiolipin (CL are two essential classes of phospholipid in plants and algae. Phosphatidylglycerophosphate synthase (PGPS and cardiolipin synthase (CLS involved in the biosynthesis of PG and CL belong to CDP-alcohol phosphotransferase and share overall amino acid sequence homology. However, it remains elusive whether PGPS and CLS are functionally distinct in vivo. Here, we report identification of a gene encoding CLS in Chlamydomonas reinhardtii, CrCLS1, and its functional compatibility. Whereas CrCLS1 did not complement the growth phenotype of a PGPS mutant of Synechocystis sp. PCC 6803, it rescued the temperature-sensitive growth phenotype, growth profile with different carbon sources, phospholipid composition and enzyme activity of ∆crd1, a CLS mutant of Saccharomyces cerevisiae. These results suggest that CrCLS1 encodes a functional CLS of C. reinhardtii as the first identified algal CLS, whose enzyme function is distinct from that of PGPSs from C. reinhardtii. Comparison of CDP-alcohol phosphotransferase motif between PGPS and CLS among different species revealed a possible additional motif that might define the substrate specificity of these closely related enzymes.

  10. Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis.

    Science.gov (United States)

    Sitthithaworn, W; Kojima, N; Viroonchatapan, E; Suh, D Y; Iwanami, N; Hayashi, T; Noji, M; Saito, K; Niwa, Y; Sankawa, U

    2001-02-01

    cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method. Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence. When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP). The structural factors determining the product length in plant GGPPSs were investigated by constructing S. dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase. The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length. Further, when a chimeric gene comprised of the putative transit peptide of the S. dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S. dulcis GGPPS is a chloroplast protein.

  11. Riboflavin accumulation and characterization of cDNAs encoding lumazine synthase and riboflavin synthase in bitter melon (Momordica charantia).

    Science.gov (United States)

    Tuan, Pham Anh; Kim, Jae Kwang; Lee, Sanghyun; Chae, Soo Cheon; Park, Sang Un

    2012-12-05

    Riboflavin (vitamin B2) is the universal precursor of the coenzymes flavin mononucleotide and flavin adenine dinucleotide--cofactors that are essential for the activity of a wide variety of metabolic enzymes in animals, plants, and microbes. Using the RACE PCR approach, cDNAs encoding lumazine synthase (McLS) and riboflavin synthase (McRS), which catalyze the last two steps in the riboflavin biosynthetic pathway, were cloned from bitter melon (Momordica charantia), a popular vegetable crop in Asia. Amino acid sequence alignments indicated that McLS and McRS share high sequence identity with other orthologous genes and carry an N-terminal extension, which is reported to be a plastid-targeting sequence. Organ expression analysis using quantitative real-time RT PCR showed that McLS and McRS were constitutively expressed in M. charantia, with the strongest expression levels observed during the last stage of fruit ripening (stage 6). This correlated with the highest level of riboflavin content, which was detected during ripening stage 6 by HPLC analysis. McLS and McRS were highly expressed in the young leaves and flowers, whereas roots exhibited the highest accumulation of riboflavin. The cloning and characterization of McLS and McRS from M. charantia may aid the metabolic engineering of vitamin B2 in crops.

  12. Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production

    Energy Technology Data Exchange (ETDEWEB)

    Yuzawa, Satoshi [Univ. of California, Berkeley, CA (United States); Deng, Kai [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States); Wang, George [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Baidoo, Edward E. K. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Northen, Trent R. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Adams, Paul D. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Katz, Leonard [Univ. of California, Berkeley, CA (United States); Synthetic Biology Research Center, Emeryville, CA (United States); Keasling, Jay D. [Univ. of California, Berkeley, CA (United States); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Synthetic Biology Research Center, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2016-08-22

    Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed in this paper the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. Finally, these results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production.

  13. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    OpenAIRE

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and...

  14. Cloning and heterologous expression of a novel subgroup of class IV polyhydroxyalkanoate synthase genes from the genus Bacillus.

    Science.gov (United States)

    Mizuno, Kouhei; Kihara, Takahiro; Tsuge, Takeharu; Lundgren, Benjamin R; Sarwar, Zaara; Pinto, Atahualpa; Nomura, Christopher T

    2017-01-01

    Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.

  15. Altering the expression of two chitin synthase genes differentially affects the growth and morphology of Aspergillus oryzae

    DEFF Research Database (Denmark)

    Müller, Christian; Hjort, C.M.; Hansen, K.

    2002-01-01

    In Aspergillus oryzae, one full-length chitin synthase (chsB) and fragments of two other chitin synthases (csmA and chsC) were identified. The deduced amino acid sequence of chsB was similar (87% identity) to chsB from Aspergillus nidulans, which encodes a class III chitin synthase. The sequence...

  16. Corn silk induces nitric oxide synthase in murine macrophages.

    Science.gov (United States)

    Kim, Kyung A; Choi, Sang Kyu; Choi, Hye Seon

    2004-12-31

    Corn silk has been purified as an anticoagulant previously and the active component is a polysaccharide with a molecular mass of 135 kDa. It activates murine macrophages to induce nitric oxide synthase (NOS) and generate substantial amounts of NO in time and dose-dependent manners. It was detectable first at 15 h after stimulation by corn silk, peaked at 24 h, and undetectable by 48 h. Induction of NOS is inhibited by pyrolidine dithiocarbamate (PDTC) and genistein, an inhibitor of nuclear factor kappa B (NF-kappaB) and tyrosine kinase, respectively, indicating that iNOS stimulated by corn silk is associated with tyrosine kinase and NF-kappaB signaling pathways. IkappaB-alpha degradation was detectible at 10 min, and the level was restored at 120 min after treatment of corn silk. Corn silk induced nuclear translocation of NF-kappaB by phosphorylation and degradation of IkappaB-alpha.

  17. CTP synthase forms the cytoophidium in human hepatocellular carcinoma.

    Science.gov (United States)

    Chang, Chia-Chun; Jeng, Yung-Ming; Peng, Min; Keppeke, Gerson Dierley; Sung, Li-Ying; Liu, Ji-Long

    2017-12-15

    CTP synthase (CTPS) can aggregate into an intracellular macrostructure, the cytoophidium, in various organisms including human cells. Previous studies have shown that assembly of human CTPS cytoophidia may be correlated with the cellular metabolic status, and is able to promote the activity of CTPS. A correlation between the cytoophidium and cancer metabolism has been proposed but not yet been revealed. In the current study we provide clear evidence of the presence of CTPS cytoophidia in various human cancers and some non-cancerous tissues. Moreover, among 203 tissue samples of hepatocellular carcinoma, 56 (28%) samples exhibited many cytoophidia, whereas no cytoophidia were detected in adjacent non-cancerous hepatocytes for all samples. Our findings suggest that the CTPS cytoophidium may participate in the adaptive metabolism of human hepatocellular carcinoma. Copyright © 2017. Published by Elsevier Inc.

  18. Reduced ceramide synthase 2 activity causes progressive myoclonic epilepsy

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Olsen, Anne S B; Neess, Ditte

    2014-01-01

    between genes involved in SL metabolism and epilepsy. METHODS: We used quantitative real-time PCR, Western blotting, and enzymatic assays to determine the mRNA, protein, and activity levels of ceramide synthase 2 (CERS2) in fiibroblasts isolated from parental control subjects and from a patient diagnosed...... with progressive myoclonic epilepsy (PME). Mass spectrometry and fluorescence microscopy were used to examine the effects of reduced CERS2 activity on cellular lipid composition and plasma membrane functions. RESULTS: We identify a novel 27 kb heterozygous deletion including the CERS2 gene in a proband diagnosed...... with PME. Compared to parental controls, levels of CERS2 mRNA, protein, and activity were reduced by ˜50% in fibroblasts isolated from this proband, resulting in significantly reduced levels of ceramides and sphingomyelins containing the very long-chain fatty acids C24:0 and C26:0. The change in SL...

  19. Sphingomyelin Synthase 1 Is Essential for Male Fertility in Mice.

    Directory of Open Access Journals (Sweden)

    Anke Wittmann

    Full Text Available Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1-/- mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility.

  20. Cellulose synthases: new insights from crystallography and modeling.

    Science.gov (United States)

    Slabaugh, Erin; Davis, Jonathan K; Haigler, Candace H; Yingling, Yaroslava G; Zimmer, Jochen

    2014-02-01

    Detailed information about the structure and biochemical mechanisms of cellulose synthase (CelS) proteins remained elusive until a complex containing the catalytic subunit (BcsA) of CelS from Rhodobacter sphaeroides was crystalized. Additionally, a 3D structure of most of the cytosolic domain of a plant CelS (GhCESA1 from cotton, Gossypium hirsutum) was produced by computational modeling. This predicted structure contributes to our understanding of how plant CelS proteins may be similar and different as compared with BcsA. In this review, we highlight how these structures impact our understanding of the synthesis of cellulose and other extracellular polysaccharides. We show how the structures can be used to generate hypotheses for experiments testing mechanisms of glucan synthesis and translocation in plant CelS. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Two Cycloartenol Synthases for Phytosterol Biosynthesis in Polygala tenuifolia Willd.

    Science.gov (United States)

    Jin, Mei Lan; Lee, Woo Moon; Kim, Ok Tae

    2017-11-15

    Oxidosqualene cyclases (OSCs) are enzymes that play a key role in control of the biosynthesis of phytosterols and triterpene saponins. In order to uncover OSC genes from Polygala tenuifolia seedlings induced by methyl jasmonate (MeJA), RNA-sequencing analysis was performed using the Illumina sequencing platform. A total of 148,488,632 high-quality reads from two samples (control and the MeJA treated) were generated. We screened genes related to phytosterol and triterpene saponin biosynthesis and analyzed the transcriptional changes of differentially expressed unigene (DEUG) values calculated by fragments per kilobase million (FPKM). In our datasets, two full-length cDNAs of putative OSC genes, PtCAS1 , and PtCAS2 , were found, in addition to the PtBS (β-amyrin synthase) gene reported in our previous studies and the two cycloartenol synthase genes of P. tenuifolia . All genes were isolated and characterized in yeast cells. The functional expression of the two PtCAS genes in yeast cells showed that the genes all produce a cycloartenol as the sole product. When qRT-PCR analysis from different tissues was performed, the expressions of PtCAS1 and PtCAS2 were highest in flowers and roots, respectively. After MeJA treatment, the transcripts of PtCAS1 and PtCAS2 genes increased by 1.5- and 2-fold, respectively. Given these results, we discuss the potential roles of the two PtCAS genes in relation to triterpenoid biosynthesis.

  2. Two Cycloartenol Synthases for Phytosterol Biosynthesis in Polygala tenuifolia Willd

    Directory of Open Access Journals (Sweden)

    Mei Lan Jin

    2017-11-01

    Full Text Available Oxidosqualene cyclases (OSCs are enzymes that play a key role in control of the biosynthesis of phytosterols and triterpene saponins. In order to uncover OSC genes from Polygala tenuifolia seedlings induced by methyl jasmonate (MeJA, RNA-sequencing analysis was performed using the Illumina sequencing platform. A total of 148,488,632 high-quality reads from two samples (control and the MeJA treated were generated. We screened genes related to phytosterol and triterpene saponin biosynthesis and analyzed the transcriptional changes of differentially expressed unigene (DEUG values calculated by fragments per kilobase million (FPKM. In our datasets, two full-length cDNAs of putative OSC genes, PtCAS1, and PtCAS2, were found, in addition to the PtBS (β-amyrin synthase gene reported in our previous studies and the two cycloartenol synthase genes of P. tenuifolia. All genes were isolated and characterized in yeast cells. The functional expression of the two PtCAS genes in yeast cells showed that the genes all produce a cycloartenol as the sole product. When qRT-PCR analysis from different tissues was performed, the expressions of PtCAS1 and PtCAS2 were highest in flowers and roots, respectively. After MeJA treatment, the transcripts of PtCAS1 and PtCAS2 genes increased by 1.5- and 2-fold, respectively. Given these results, we discuss the potential roles of the two PtCAS genes in relation to triterpenoid biosynthesis.

  3. The evolution of function in strictosidine synthase-like proteins.

    Science.gov (United States)

    Hicks, Michael A; Barber, Alan E; Giddings, Lesley-Ann; Caldwell, Jenna; O'Connor, Sarah E; Babbitt, Patricia C

    2011-11-01

    The exponential growth of sequence data provides abundant information for the discovery of new enzyme reactions. Correctly annotating the functions of highly diverse proteins can be difficult, however, hindering use of this information. Global analysis of large superfamilies of related proteins is a powerful strategy for understanding the evolution of reactions by identifying catalytic commonalities and differences in reaction and substrate specificity, even when only a few members have been biochemically or structurally characterized. A comparison of >2500 sequences sharing the six-bladed β-propeller fold establishes sequence, structural, and functional links among the three subgroups of the functionally diverse N6P superfamily: the arylesterase-like and senescence marker protein-30/gluconolactonase/luciferin-regenerating enzyme-like (SGL) subgroups, representing enzymes that catalyze lactonase and related hydrolytic reactions, and the so-called strictosidine synthase-like (SSL) subgroup. Metal-coordinating residues were identified as broadly conserved in the active sites of all three subgroups except for a few proteins from the SSL subgroup, which have been experimentally determined to catalyze the quite different strictosidine synthase (SS) reaction, a metal-independent condensation reaction. Despite these differences, comparison of conserved catalytic features of the arylesterase-like and SGL enzymes with the SSs identified similar structural and mechanistic attributes between the hydrolytic reactions catalyzed by the former and the condensation reaction catalyzed by SS. The results also suggest that despite their annotations, the great majority of these >500 SSL sequences do not catalyze the SS reaction; rather, they likely catalyze hydrolytic reactions typical of the other two subgroups instead. This prediction was confirmed experimentally for one of these proteins. Copyright © 2011 Wiley-Liss, Inc.

  4. Intestinal nitric oxide synthase activity changes during experimental colon obstruction.

    Science.gov (United States)

    Palásthy, Zsolt; Kaszaki, József; Lázár, György; Nagy, Sándor; Boros, Mihály

    2006-08-01

    The experiments in this study were designed to follow the time course of nitric oxide (NO) synthesis in the large bowel during acute mechanical ileus. Occlusion of the mid-transverse colon was maintained for 420 min in anesthetized dogs. Strain-gauge transducers were used to analyze motility changes on the hepatic and lienal flexures, respectively. Constitutive NO synthase (cNOS) and inducible NOS (iNOS) activities were determined in tissue biopsies, and plasma nitrite/nitrate (NOx) level was measured in the portal blood. Following completion of the baseline studies, the animals were treated with either 7-nitroindazole (7-NI, selective neuronal NOS inhibitor), or N-nitro-L-arginine (NNA, non-selective NOS inhibitor). In the sham-operated group the cNOS activities differed significantly in the oral and aboral tissue samples (oral: 102.9; versus aboral: 62.1 fmol/mg protein/min). The obstruction elicited a significant increase in portal NOx and elevated tissue inducible NO synthase (iNOS) activity. NNA treatment decreased the motility index in both intestinal segments for 60 min, but 120 min later the motility index was significantly elevated (2.5-fold increase in the oral part, and 1.8-fold enhancement in the aboral segment, respectively). Treatment with 7-NI decreased the cNOS activity in the oral and aboral parts by approximately 40% and 70%, respectively, and suppressed the motility increase in the aboral colon segment. The motility of the colon was either significantly increased or decreased, depending on the type and selectivity of the NOS inhibitor compounds applied. NO of neuronal origin is a transmitter that stimulates peristaltic activity; but an increased iNOS/nNOS ratio significantly moderates the obstruction-induced motility increase.

  5. Mutational, Phylogeny and Evolution Analyses of Salvia Copalyl Diphosphate Synthase

    International Nuclear Information System (INIS)

    Hao, D. C.; Thimmappa, R. B.; Xiao, P. G.

    2016-01-01

    The cyclization of geranylgeranyl diphosphate (GGPP) is catalyzed by copalyl diphosphate synthase (CPS), a class II diterpene synthase (diTPS), to form copalyl diphosphate (CPP), which is an essential substrate of a variety of diterpenes in secondary metabolism of angiosperm including Salvia medicinal plants. The protein environment of the N-terminal class II active site stabilizes the carbocation intermediates and maintains the catalytic activity of angiosperm class II diTPS. The virtual modeling and mutagenesis of the class II diTPS of Salvia miltiorrhiza (SmCPS) were accomplished to illuminate the catalytic activity of SmCPS. Terminal truncations and point mutations established the role of the Beta-Gamma domain and Alpha domain, i.e., they facilitate the flexible conformational change of the class II active site after substrate binding. E203 and K238 in the N-ter Gamma domain of SmCPS1 are functional in the substrate binding and conformational transition and might be essential in catalysis. Similar to other CPSs, the ensuing protonation of the GGPP substrate and coordination of the diphosphate group are governed by highly conserved residues in the DxDD motif of SmCPS, e.g., D372 of CPS1. Moreover, F256 and Y505 stabilize the carbocation and control the enzymatic activity during CPP formation. The amino acids of the predicted active sites, despite under purifying selection, vary greatly, corresponding to the functional flexibility of angiosperm CPSs. Molecular phylogeny and evolution analyses suggest early and ongoing evolution of labdane-related diterpenoid metabolism in angiosperm. (author)

  6. Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

    Science.gov (United States)

    Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

    1985-01-29

    Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

  7. MEMORY MODULATION

    Science.gov (United States)

    Roozendaal, Benno; McGaugh, James L.

    2011-01-01

    Our memories are not all created equally strong: Some experiences are well remembered while others are remembered poorly, if at all. Research on memory modulation investigates the neurobiological processes and systems that contribute to such differences in the strength of our memories. Extensive evidence from both animal and human research indicates that emotionally significant experiences activate hormonal and brain systems that regulate the consolidation of newly acquired memories. These effects are integrated through noradrenergic activation of the basolateral amygdala which regulates memory consolidation via interactions with many other brain regions involved in consolidating memories of recent experiences. Modulatory systems not only influence neurobiological processes underlying the consolidation of new information, but also affect other mnemonic processes, including memory extinction, memory recall and working memory. In contrast to their enhancing effects on consolidation, adrenal stress hormones impair memory retrieval and working memory. Such effects, as with memory consolidation, require noradrenergic activation of the basolateral amygdala and interactions with other brain regions. PMID:22122145

  8. Structure of the dimeric form of CTP synthase from Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank

    2011-01-01

    CTP synthase catalyzes the last committed step in de novo pyrimidine-nucleotide biosynthesis. Active CTP synthase is a tetrameric enzyme composed of a dimer of dimers. The tetramer is favoured in the presence of the substrate nucleotides ATP and UTP; when saturated with nucleotide, the tetramer...... completely dominates the oligomeric state of the enzyme. Furthermore, phosphorylation has been shown to regulate the oligomeric states of the enzymes from yeast and human. The crystal structure of a dimeric form of CTP synthase from Sulfolobus solfataricus has been determined at 2.5 Å resolution...

  9. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  10. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

    Science.gov (United States)

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  11. A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

    Energy Technology Data Exchange (ETDEWEB)

    Datta, Supratim; Koutmos, Markos; Pattridge, Katherine A.; Ludwig, Martha L.; Matthews, Rowena G. (Michigan)

    2008-07-08

    B{sub 12}-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme.

  12. Low-Dose Ribavirin Treatments Attenuate Neuroinflammatory Activation of BV-2 Cells by Interfering with Inducible Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Iva Bozic

    2015-01-01

    Full Text Available Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 μM modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 μM to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 μM ribavirin promoted LPS induced apoptosis. We determined that 1 μM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 μM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation.

  13. Low-Dose Ribavirin Treatments Attenuate Neuroinflammatory Activation of BV-2 Cells by Interfering with Inducible Nitric Oxide Synthase

    Science.gov (United States)

    Bozic, Iva; Savic, Danijela; Jovanovic, Marija; Bjelobaba, Ivana; Laketa, Danijela; Nedeljkovic, Nadezda; Stojiljkovic, Mirjana; Pekovic, Sanja; Lavrnja, Irena

    2015-01-01

    Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 μM) modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 μM) to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS) stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 μM ribavirin promoted LPS induced apoptosis. We determined that 1 μM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 μM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation. PMID:26413464

  14. Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning.

    Science.gov (United States)

    Amour, Julien; Brzezinska, Anna K; Weihrauch, Dorothee; Billstrom, Amie R; Zielonka, Jacek; Krolikowski, John G; Bienengraeber, Martin W; Warltier, David C; Pratt, Philip F; Kersten, Judy R

    2009-02-01

    Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or N-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or N-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. N-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection. The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.

  15. Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses.

    Science.gov (United States)

    Pépin, Geneviève; Nejad, Charlotte; Ferrand, Jonathan; Thomas, Belinda J; Stunden, H James; Sanij, Elaine; Foo, Chwan-Hong; Stewart, Cameron R; Cain, Jason E; Bardin, Philip G; Williams, Bryan R G; Gantier, Michael P

    2017-10-03

    Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans. Copyright © 2017 Pépin et al.

  16. Possible roles of long-chain sphingomyelines and sphingomyelin synthase 2 in mouse macrophage inflammatory response

    International Nuclear Information System (INIS)

    Sakamoto, Hideaki; Yoshida, Tetsuya; Sanaki, Takao; Shigaki, Shuhei; Morita, Hirotoshi; Oyama, Miki; Mitsui, Masaru; Tanaka, Yoshikazu; Nakano, Toru; Mitsutake, Susumu; Igarashi, Yasuyuki; Takemoto, Hiroshi

    2017-01-01

    To evaluate the precise role of sphingomyelin synthase 2 (SMS2) in sphingomyelin (SM) metabolism and their anti-inflammatory properties, we analyzed species of major SM and ceramide (Cer) (18:1, 18:0 sphingoid backbone, C14 - C26 N-acyl part) in SMS2 knockout and wild-type mouse plasma and liver using HPLC-MS. SMS2 deficiency significantly decreased very long chain SM (SM (d18:1/22:0) and SM (d18:1/24:0 or d18:0/24:1)) and increased very long chain Cer (Cer (d18:1/24:0 or d18:0/24:1) and Cer (d18:1/24:1)), but not long chain SM (SM (d18:1/16:0), SM (d18:1/18:0 or d18:0/18:1) and SM (d18:1/18:1)) in plasma. To examine the effects of SM on inflammation, we studied the role of very long chain SM in macrophage activation. Addition of SM (d18:1/24:0) strongly upregulated several macrophage activation markers, SM (d18:1/6:0) and Cer (d18:1/24:0) however, did not. It was suggested that very long chain SM but not long chain SM were decreased in SMS2-deficient mice liver and plasma. And the exogenously added very long chain SM (d18:1/24:0) could activate macrophages directly, suggesting a novel role of plasma very long chain SM in modulating macrophage activation and resulting inflammation. - Highlights: • Very long-chain SM species were decreased in SMS2 knockout mouse plasma and liver. • Very long-chain ceramide species were increased in SMS2 knockout mouse plasma. • SMS2 deficiency diminished the inflammatory response of macrophages. • Very long-chain SM enhanced ICAM1 and iNOS expression in peritoneal macrophages.

  17. Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme.

    Science.gov (United States)

    Babbitt, Shalon E; San Francisco, Brian; Bretsnyder, Eric C; Kranz, Robert G

    2014-08-19

    C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS-heme-cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical "correction" supports the proposed role of heme binding for the corresponding residues.

  18. Fatty acid synthase regulates the chemosensitivity of breast cancer cells to cisplatin-induced apoptosis.

    Science.gov (United States)

    Al-Bahlani, Shadia; Al-Lawati, Hanaa; Al-Adawi, Moza; Al-Abri, Nadia; Al-Dhahli, Buthaina; Al-Adawi, Kawther

    2017-06-01

    Fatty acid synthase (FASN) is a key enzyme in fat biosynthesis that is over-expressed in advanced breast cancer stages. Cisplatin (CDDP) is a platinum-based drug used in the treatment of certain types of this disease. Although it was shown that FASN inhibition induced apoptosis by enhancing the cytotoxicity of certain drugs in breast cancer, its role in regulating the chemosensitivity of different types of breast cancer cells to CDDP-induced apoptosis is not established yet. Therefore, two different breast cancer cell lines; triple negative breast cancer (TNBC; MDA-MB-231) and triple positive breast cancer (TPBC; BT-474) cells were used to examine such role. We show that TNBC cells had naturally less fat content than TPBC cells. Subsequently, the fat content increased in both cells when treated with Palmitate rather than Oleate, whereas both fatty acids produced apoptotic ultra-structural effects and attenuated FASN expression. However, Oleate increased FASN expression in TPBC cells. CDDP decreased FASN expression and increased apoptosis in TNBC cells. These effects were further enhanced by combining CDDP with fatty acids. We also illustrate that the inhibition of FASN by either siRNA or exogenous inhibitor decreased CDDP-induced apoptosis in TPBC cells suggesting its role as an apoptotic factor, while an opposite finding was observed in TNBC cells when siRNA and fatty acids were used, suggesting its role as a survival factor. To our knowledge, we are the first to demonstrate a dual role of FASN in CDDP-induced apoptosis in breast cancer cells and how it can modulate their chemosensitivity.

  19. Molecular size estimation of plasma membrane β-glucan synthase from red beet root

    International Nuclear Information System (INIS)

    Sloan, M.E.; Eiberger, L.L.; Wasserman, B.P.

    1986-01-01

    Cellulose and cell wall β-D-glucans in higher plants are thought to be synthesized by the plasma membrane enzyme, β-glucan synthase. This enzyme has never been purified to homogeneity, hence its subunit composition is unknown. Partial purification of red beet root glucan synthase by glycerol density gradient centrifugation followed by SDS-PAGE yielded a highly enriched subunit of 68 kDa. Radiation inactivation of plasma membranes gave a molecular size the 450 kDa for the holoenzyme complex. This suggests that glucan synthase consists of 6 to 7 subunits and confirms electron microscope studies showing that glucan synthases exist as multi-subunit complexes embedded within the membrane

  20. SCREENING OF 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE ACTIVITY DEFICIENCY AMONG HYPERP HENYLALANINEMIC PATIENTS

    Directory of Open Access Journals (Sweden)

    DURDI QUJEQ

    1999-10-01

    Full Text Available A deficiency of the phenylalanine hydroxylase activity or its cofactor tetrahydrobiopterin may"nlead to hyperphenylalamnemia and as a result, loss of IQ, poor school performance, and"nbehavior problems occurs. Deficiency in 6-pyruvoyl-tetrahydropterin synthase activity is the"nmajor cause of tetrahydrobiopterin deficient phenylketonuria. In this study, blood specimens"nfrom 165 healthy volunteers and 127 children with phenylketonuria were used to determine"nthe 6-pyruvoyl-tetrahydropterin synthase activity. It was found that the activity of 6-"npyruvoyl- tetrahydropterin synthase was decreased in comparison with control [23.46 +/-"n2.94, (mean +/- SD, mmol/ ml/h, n=I27 vs. 127.63 +/- 4.52, n=165, p<0.05]. Results of"nthis study indicate that examination of 6-pyruvoyl-tetrahydropterin synthase activity is helpful"nand may lead to the diagnosis cause of hyperphenylalaninemia.

  1. Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

    KAUST Repository

    Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

    2010-01-01

    Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations

  2. Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Sandrini, Michael; Piskur, Jure

    2001-01-01

    no pyrimidine catabolic pathway, it enabled growth on N-carbamyl- beta -alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta -alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial......beta -Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamy-beta -alanine as the sole nitrogen source and exhibits diminished beta -alanine synthase...... N- carbamyl amidohydrolases. All three beta -alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N...

  3. Citric acid production and citrate synthase genes in distinct strains of ...

    African Journals Online (AJOL)

    SAM

    2014-05-28

    May 28, 2014 ... synthase in lactic acid production by A. niger and with the ... A number of microorganisms, including both bacteria and fungi, possess the capacity ..... citric acid production by solid-state fermentation from cassava bagasse and ...

  4. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    OpenAIRE

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-01-01

    An oligonucleotide representing a regulatory element of human thymidylate synthase mRNA has been crystallized as a dimer. The structure of the asymmetric dimer has been determined at 1.97 Å resolution.

  5. Modulators of arginine metabolism support cancer immunosurveillance

    Directory of Open Access Journals (Sweden)

    Freschi Massimo

    2009-01-01

    Full Text Available Abstract Background Tumor-associated accrual of myeloid derived suppressor cells (MDSC in the blood, lymphoid organs and tumor tissues may lead to perturbation of the arginine metabolism and impairment of the endogenous antitumor immunity. The objective of this study was to evaluate whether accumulation of MDSC occurred in Th2 prone BALB/c and Th1 biased C57BL/6 mice bearing the C26GM colon carcinoma and RMA T lymphoma, respectively, and to investigate whether N(G nitro-L-arginine methyl ester (L-NAME and sildenafil, both modulators of the arginine metabolism, restored antitumor immunity. Results We report here that MDSC accumulate in the spleen and blood of mice irrespective of the mouse and tumor model used. Treatment of tumor-bearing mice with either the phosphodiesterase-5 inhibitor sildenafil or the nitric-oxide synthase (NOS inhibitor L-NAME significantly restrained tumor growth and expanded the tumor-specific immune response. Conclusion Our data emphasize the role of MDSC in modulating the endogenous tumor-specific immune response and underline the anti-neoplastic therapeutic potential of arginine metabolism modulators.

  6. Seasonal influence on gene expression of monoterpene synthases in Salvia officinalis (Lamiaceae).

    Science.gov (United States)

    Grausgruber-Gröger, Sabine; Schmiderer, Corinna; Steinborn, Ralf; Novak, Johannes

    2012-03-01

    Garden sage (Salvia officinalis L., Lamiaceae) is one of the most important medicinal and aromatic plants and possesses antioxidant, antimicrobial, spasmolytic, astringent, antihidrotic and specific sensorial properties. The essential oil of the plant, formed mainly in very young leaves, is in part responsible for these activities. It is mainly composed of the monoterpenes 1,8-cineole, α- and β-thujone and camphor synthesized by the 1,8-cineole synthase, the (+)-sabinene synthase and the (+)-bornyl diphosphate synthase, respectively, and is produced and stored in epidermal glands. In this study, the seasonal influence on the formation of the main monoterpenes in young, still expanding leaves of field-grown sage plants was studied in two cultivars at the level of mRNA expression, analyzed by qRT-PCR, and at the level of end-products, analyzed by gas chromatography. All monoterpene synthases and monoterpenes were significantly influenced by cultivar and season. 1,8-Cineole synthase and its end product 1,8-cineole remained constant until August and then decreased slightly. The thujones increased steadily during the vegetative period. The transcript level of their corresponding terpene synthase, however, showed its maximum in the middle of the vegetative period and declined afterwards. Camphor remained constant until August and then declined, exactly correlated with the mRNA level of the corresponding terpene synthase. In summary, terpene synthase mRNA expression and respective end product levels were concordant in the case of 1,8-cineole (r=0.51 and 0.67 for the two cultivars, respectively; p<0.05) and camphor (r=0.75 and 0.82; p<0.05) indicating basically transcriptional control, but discordant for α-/β-thujone (r=-0.05 and 0.42; p=0.87 and 0.13, respectively). Copyright © 2011 Elsevier GmbH. All rights reserved.

  7. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment

    International Nuclear Information System (INIS)

    Tanaka, Hiroaki; Tsurumura, Toshiharu; Aritake, Kosuke; Furubayashi, Naoki; Takahashi, Sachiko; Yamanaka, Mari; Hirota, Erika; Sano, Satoshi; Sato, Masaru; Kobayashi, Tomoyuki; Tanaka, Tetsuo; Inaka, Koji; Urade, Yoshihiro

    2011-01-01

    Crystals of hematopoietic prostaglandin D synthase grown in microgravity show improved quality. Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein

  8. Use of octaketide synthases to produce kermesic acid and flavokermesic acid

    DEFF Research Database (Denmark)

    2017-01-01

    A method for producing an octaketide derived aromatic compound of interest (e.g. carminic acid), wherein the method comprises (I): heterologous expression of a recombinantly introduced Type III polyketide synthase (PKS) gene encoding an octaketide synthase (OKS) to obtain non-reduced octaketide...... in vivo within the recombinant host cell and (II): converting in vivo the non-reduced octaketide of step (I) into a C14-C34 aromatic compound of interest (e.g. carminic acid)....

  9. Use of heterologous expressed polyketide synthase and small molecule foldases to make aromatic and cyclic compounds

    DEFF Research Database (Denmark)

    2016-01-01

    A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell.......A method for producing individual or libraries of tri- to pentadecaketide-derived aromatic compounds of interest by heterologous expression of polyketide synthase and aromatase/cyclase in a recombinant host cell....

  10. Use of octaketide synthases to produce kermesic acid and flavokermesic acid

    DEFF Research Database (Denmark)

    2016-01-01

    A method for producing an octaketide derived aromatic compound of interest (e.g. carminic acid), wherein the method comprises (I): heterologous expression of a recombinantly introduced Type III polyketide synthase (PKS) gene encoding an octaketide synthase (OKS) to obtain non-reduced octaketide...... in vivo within the recombinant host cell and (II): converting in vivo the non-reduced octaketide of step (I) into a C14-C34 aromatic compound of interest (e.g. carminic acid)....

  11. The subcellular localization of yeast glycogen synthase is dependent upon glycogen content

    OpenAIRE

    Wilson, Wayne A.; Boyer, Michael P.; Davis, Keri D.; Burke, Michael; Roach, Peter J.

    2010-01-01

    The budding yeast, Saccharomyces cerevisiae, accumulates the storage polysaccharide glycogen in response to nutrient limitation. Glycogen synthase, the major form of which is encoded by the GSY2 gene, catalyzes the key regulated step in glycogen storage. Here, we utilize Gsy2p fusions to green fluorescent protein (GFP) to determine where glycogen synthase is located within cells. We demonstrate that the localization pattern of Gsy2-GFP depends upon the glycogen content of the cell. When glyco...

  12. The polyketide components of waxes and the Cer-cqu gene cluster encoding a novel polyketide synthase, the β-diketone synthase, DKS

    DEFF Research Database (Denmark)

    von Wettstein, Penny

    2017-01-01

    The primary function of the outermost, lipophilic layer of plant aerial surfaces, called the cuticle, is preventing non-stomatal water loss. Its exterior surface is often decorated with wax crystals, imparting a blue-grey color. Identification of the barley Cer-c, -q and -u genes forming the 101 kb...... Cer-cqu gene cluster encoding a novel polyketide synthase-the β-diketone synthase (DKS), a lipase/carboxyl transferase, and a P450 hydroxylase, respectively, establishes a new, major pathway for the synthesis of plant waxes. The major product is a β-diketone (14,16-hentriacontane) aliphatic that forms...

  13. Fast Convolution Module (Fast Convolution Module)

    National Research Council Canada - National Science Library

    Bierens, L

    1997-01-01

    This report describes the design and realisation of a real-time range azimuth compression module, the so-called 'Fast Convolution Module', based on the fast convolution algorithm developed at TNO-FEL...

  14. Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot1[OPEN

    Science.gov (United States)

    Yahyaa, Mosaab; Matsuba, Yuki; Brandt, Wolfgang; Doron-Faigenboim, Adi; Bar, Einat; McClain, Alan; Davidovich-Rikanati, Rachel; Lewinsohn, Efraim; Pichersky, Eran; Ibdah, Mwafaq

    2015-01-01

    Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms. PMID:26157114

  15. Protein modelling of triterpene synthase genes from mangrove plants using Phyre2 and Swiss-model

    Science.gov (United States)

    Basyuni, M.; Wati, R.; Sulistiyono, N.; Hayati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

    2018-03-01

    Molecular cloning of five oxidosqualene cyclases (OSC) genes from Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa had previously been cloned, characterized, and encoded mono and -multi triterpene synthases. The present study analyzed protein modelling of triterpene synthase genes from mangrove using Phyre2 and Swiss-model. The diversity was noted within protein modelling of triterpene synthases using Phyre2 from sequence identity (38-43%) and residue (696-703). RsM2 was distinguishable from others for template structure; it used lanosterol synthase as a template (PDB ID: w6j.1.A). By contrast, other genes used human lanosterol synthase (1w6k.1.A). The predicted bind sites were correlated with the product of triterpene synthase, the product of BgbAS was β-amyrin, while RsM1 contained a significant amount of β-amyrin. Similarly BgLUS and KcMS, both main products was lupeol, on the other hand, RsM2 with the outcome of taraxerol. Homology modelling revealed that 696 residues of BgbAS, BgLUS, RsM1, and RsM2 (91-92% of the amino acid sequence) had been modelled with 100% confidence by the single highest scoring template using Phyre2. This coverage was higher than Swiss-model (85-90%). The present study suggested that molecular cloning of triterpene genes provides useful tools for studying the protein modelling related regulation of isoprenoids biosynthesis in mangrove forests.

  16. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    International Nuclear Information System (INIS)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S.

    1991-01-01

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[ 35 S]methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate

  17. A high-throughput colorimetric screening assay for terpene synthase activity based on substrate consumption.

    Directory of Open Access Journals (Sweden)

    Maiko Furubayashi

    Full Text Available Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

  18. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  19. Effects and mechanism of acid rain on plant chloroplast ATP synthase.

    Science.gov (United States)

    Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

    2016-09-01

    Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

  20. Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene.

    Science.gov (United States)

    Beekwilder, Jules; van Houwelingen, Adèle; Cankar, Katarina; van Dijk, Aalt D J; de Jong, René M; Stoopen, Geert; Bouwmeester, Harro; Achkar, Jihane; Sonke, Theo; Bosch, Dirk

    2014-02-01

    Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  1. Platelet-derived growth factor (PDGF) stimulates glycogen synthase activity in 3T3 cells

    International Nuclear Information System (INIS)

    Chan, C.P.; Bowen-Pope, D.F.; Ross, R.; Krebs, E.G.

    1986-01-01

    Hormonal regulation of glycogen synthase, an enzyme that can be phosphorylated on multiple sites, is often associated with changes in its phosphorylation state. Enzyme activation is conventionally monitored by determining the synthase activity ratio [(activity in the absence of glucose 6-P)/(activity in the presence of glucose 6-P)]. Insulin causes an activation of glycogen synthase with a concomitant decrease in its phosphate content. In a previous report, the authors showed that epidermal growth factor (EGF) increases the glycogen synthase activity ratio in Swiss 3T3 cells. The time and dose-dependency of this response was similar to that of insulin. Their recent results indicate that PDGF also stimulates glycogen synthase activity. Enzyme activation was maximal after 30 min. of incubation with PDGF; the time course observed was very similar to that with insulin and EGF. At 1 ng/ml (0.03nM), PDGF caused a maximal stimulation of 4-fold in synthase activity ratio. Half-maximal stimulation was observed at 0.2 ng/ml (6 pM). The time course of changes in enzyme activity ratio closely followed that of 125 I-PDGF binding. The authors data suggest that PDGF, as well as EFG and insulin, may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms

  2. Substrate specificity of Arabidopsis 3-ketoacyl-CoA synthases

    International Nuclear Information System (INIS)

    Blacklock, Brenda J.; Jaworski, Jan G.

    2006-01-01

    The very long chain fatty acids (VLCFA) incorporated into plant lipids are derived from the iterative addition of C2 units provided by malonyl-CoA to an acyl-CoA by the 3-ketoacyl-CoA synthase (KCS) component of a fatty acid elongase (FAE) complex. Mining of the Arabidopsis genome sequence database revealed 20 genes with homology to seed-specific FAE1 KCS. Eight of the 20 putative KCSs were cloned, expressed in yeast, and isolated as (His) 6 fusion proteins. Five of the eight (At1g71160, At1g19440, At1g07720, At5g04530, and At4g34250) had little or no activity with C16 to C20 substrates while three demonstrated activity with C16, C18, and C20 saturated acyl-CoA substrates. At1g01120 KCS (KCS1) and At2g26640 KCS had broad substrate specificities when assayed with saturated and mono-unsaturated C16 to C24 acyl-CoAs while At4g34510 KCS was specific for saturated fatty acyl-CoA substrates

  3. Kinetics and equilibria of cyanide binding to prostaglandin H synthase.

    Science.gov (United States)

    MacDonald, I D; Dunford, H B

    1989-09-01

    Cyanide binding to prostaglandin H (PGH) synthase results in a spectral shift in the Soret region. This shift was exploited to determine equilibrium and kinetic parameters of the cyanide binding process. At pH 8.0, ionic strength 0.22 M, 4 degrees C, the cyanide dissociation constant, determined from equilibrium experiments, is (65 +/- 10) microM. The binding rate constant is (2.8 +/- 0.2) x 10(3) M-1 s-1, and the dissociation rate constant is zero within experimental error. Through a kinetic study of the binding process as a function of pH, from pH 3.96 to 8.00, it was possible to determine the pKa of a heme-linked acid group on the enzyme of 4.15 +/- 0.10 with citrate buffer. An apparent pKa of 4.75 +/- 0.03 was determined with acetate buffer; this different value is attributed to complexation of the enzyme with one of the components of the acetate buffer.

  4. Circulation of Pneumocystis dihydropteroate synthase mutants in France.

    Science.gov (United States)

    Le Gal, Solène; Damiani, Céline; Perrot, Maëla; Rouillé, Amélie; Virmaux, Michèle; Quinio, Dorothée; Moalic, Elodie; Saliou, Philippe; Berthou, Christian; Le Meur, Yann; Totet, Anne; Nevez, Gilles

    2012-10-01

    Data on the prevalence of Pneumocystis jirovecii (P. jirovecii) dihydropteroate synthase (DHPS) mutants in France are still limited. In this study, mutant prevalence in the Brest region (western France) was determined. Archival pulmonary specimens from 85 patients infected with P. jirovecii and admitted to our institution (University Hospital, Brest) from October 2007 to February 2010 were retrospectively typed at the DHPS locus using a polymerase chain reaction-restriction fragment length polymorphism assay. Type identification was successful in 66 of 85 patients. Sixty-four patients were infected with a wild type, whereas mutants were found in 2 patients (2/66, 3%). Medical chart analysis revealed that these 2 patients usually lived in Paris. Another patient usually lived on the French Riviera, whereas 63 patients were from the city of Brest. Thus, the corrected prevalence of mutants in patients who effectively lived in our geographic area was 0% (0/63). Taking into account that i) Paris is characterized by a high prevalence of mutants from 18.5% to 40%, ii) infection diagnoses were performed in the 2 Parisians during their vacation Paris to Brest through infected vacationers. The study shows that the usual city of patient residence, rather than the city of infection diagnosis, is a predictor of mutants and that P. jirovecii infections involving mutants do not represent a public health issue in western France. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Inhibition of fatty acid synthase prevents preadipocyte differentiation

    International Nuclear Information System (INIS)

    Schmid, Bernhard; Rippmann, Joerg F.; Tadayyon, Moh; Hamilton, Bradford S.

    2005-01-01

    Inhibition of fatty acid synthase (FAS) reduces food intake in rodents. As adipose tissue expresses FAS, we sought to investigate the effect of reduced FAS activity on adipocyte differentiation. FAS activity was suppressed either pharmacologically or by siRNA during differentiation of 3T3-L1 cells. Cerulenin (10 μM), triclosan (50 μM), and C75 (50 μM) reduced dramatically visible lipid droplet accumulation, while incorporation of [1- 14 C]acetate into lipids was reduced by 75%, 70%, and 90%, respectively. Additionally, the substances reduced FAS, CEBPα, and PPARγ mRNA by up to 85% compared to that of control differentiated cells. Transient transfection with FAS siRNA suppressed FAS mRNA and FAS activity, and this was accompanied by reduction of CEBPα and PPARγ mRNA levels, and complete prevention of lipid accumulation. CD36, a late marker of differentiation, was also reduced. Together, these results suggest that FAS generated signals may be essential to support preadipocyte differentiation

  6. Hyperbaric oxygen upregulates cochlear constitutive nitric oxide synthase

    Directory of Open Access Journals (Sweden)

    Kao Ming-Ching

    2011-02-01

    Full Text Available Abstract Background Hyperbaric oxygen therapy (HBOT is a known adjuvant for treating ischemia-related inner ear diseases. Controversies still exist in the role of HBOT in cochlear diseases. Few studies to date have investigated the cellular changes that occur in inner ears after HBOT. Nitric oxide, which is synthesized by nitric oxide synthase (NOS, is an important signaling molecule in cochlear physiology and pathology. Here we investigated the effects of hyperbaric oxygen on eardrum morphology, cochlear function and expression of NOS isoforms in cochlear substructures after repetitive HBOT in guinea pigs. Results Minor changes in the eardrum were observed after repetitive HBOT, which did not result in a significant hearing threshold shift by tone burst auditory brainstem responses. A differential effect of HBOT on the expression of NOS isoforms was identified. Upregulation of constitutive NOS (nNOS and eNOS was found in the substructures of the cochlea after HBOT, but inducible NOS was not found in normal or HBOT animals, as shown by immunohistochemistry. There was no obvious DNA fragmentation present in this HBOT animal model. Conclusions The present evidence indicates that the customary HBOT protocol may increase constitutive NOS expression but such upregulation did not cause cell death in the treated cochlea. The cochlear morphology and auditory function are consequently not changed through the protocol.

  7. Comparative study of Chalcone synthase promoters across plant families

    Directory of Open Access Journals (Sweden)

    Francisco Buitrago

    2009-07-01

    Full Text Available En la era post-genómica, el entendimiento de la regulación génica se ha convertido en un reto y una prioridad de investigación. En este trabajo realizamos un estudio comparativo de las secuencias reguladoras del gen de la chalcón sintetasa de varias familias botánicas. Veintidós secuencias de promotores de Chalcone Synthase fueron comparados teniendo en cuenta tres elementos Cis reguladores: Caja-G, Caja-H y Caja-TATA, que podrían estar actuando como una sola unidad cooperativa. Nuestra comparación muestra que estos elementos puede que se conserven en algunas especies e inclusive que se conserven a nivel de familia. Sin embargo, en algunas especies no todos los elementos Cis fueron encontrados, mostrando que no todas las especies se regulan bajo los mismos parámetros. Adicionalmente, una comparación entre promotores de una misma especie con una familia de multigenes Chs, mostró que los genes duplicados son variables en la composición del elemento Cis, sugiriendo que estos genes pueden estarse expresando de maneras diferentes.

  8. Library of Norcoclaurine Synthases and Their Immobilization for Biocatalytic Transformations.

    Science.gov (United States)

    Lechner, Horst; Soriano, Pablo; Poschner, Roman; Hailes, Helen C; Ward, John M; Kroutil, Wolfgang

    2018-03-01

    Norcoclaurine synthases (NCS), catalyzing a Pictet-Spengler reaction in plants as one of the first enzymes in the biosynthetic benzylisoquinoline pathway, are investigated for biocatalytic transformations. The library of NCS available is extended by two novel NCSs from Argemone mexicana (AmNCS1, AmNCS2) and one new NCS from Corydalis saxicola (CsNCS); furthermore, it is shown that the NCS from Papaver bracteatum (PbNCS) is a highly productive catalyst leading to the isoquinoline product with up to >99% e.e. Under certain conditions lyophilized whole Escherichia coli cells containing the various overexpressed NCS turned out to be suitable catalysts. The reaction using dopamine as substrate bears several challenges such as the spontaneous non-stereoselective background reaction and side reactions. The PbNCS enzyme is successfully immobilized on various carriers whereby EziG3 proved to be the best suited for biotransformations. Dopamine showed limited stability in solution resulting in the coating of the catalyst over time, which could be solved by the addition of ascorbic acid (e.g., 1 mg ml -1 ) as antioxidant. © 2017 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH &Co. KGaA.

  9. Identification and Functional Characterization of Sesquiterpene Synthases from Xanthium strumarium.

    Science.gov (United States)

    Li, Yuanjun; Chen, Fangfang; Li, Zhenqiu; Li, Changfu; Zhang, Yansheng

    2016-03-01

    Xanthium strumarium synthesizes various pharmacologically active sesquiterpenes. The molecular characterization of sesquiterpene biosynthesis in X. strumarium has not been reported so far. In this study, the cDNAs coding for three sesquiterpene synthases (designated as XsTPS1, XsTPS2 and XsTPS3) were isolated using the X. strumarium transcriptome that we recently constructed. XsTPS1, XsTPS2 and XsTPS3 were revealed to have primary activities forming germacrene D, guaia-4,6-diene and germacrene A, respectively, by either ectopic expression in yeast cells or purified recombinant protein-based in vitro assays. Quantitative real-time PCRs and metabolite analysis for the different plant parts showed that the transcript abundance of XsTPS1-XsTPS3 is consistent with the accumulation pattern of their enzymatic products, supporting their biochemical functions in vivo. In particular, we discovered that none of the XsTPS2 product, guaia-4,6-diene, can be detected in one of the X. strumarium cultivars used in this study (it was named the Hubei-cultivar), in which a natural deletion of two A bases in the XsTPS2 cDNA disrupts its activity, which further confirmed the proposed biochemical role of XsTPS2 in X. strumarium in vivo. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  10. Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

    Directory of Open Access Journals (Sweden)

    Vinciane Régnier

    Full Text Available The cystathionine β-synthase (CBS gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA metabolism, a pathway important for several brain physiological processes.Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1 expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

  11. Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

    International Nuclear Information System (INIS)

    Shams-Eldin, Hosam; Santos de Macedo, Cristiana; Niehus, Sebastian; Dorn, Caroline; Kimmel, Juergen; Azzouz, Nahid; Schwarz, Ralph T.

    2008-01-01

    Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker's yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups

  12. Inducible expression of trehalose synthase in Bacillus licheniformis.

    Science.gov (United States)

    Li, Youran; Gu, Zhenghua; Zhang, Liang; Ding, Zhongyang; Shi, Guiyang

    2017-02-01

    Trehalose synthase (TreS) could transform maltose into trehalose via isomerization. It is a crucial enzyme in the process of trehalose enzymatical transformation. In this study, plasmid-based inducible expression systems were constructed to produce Thermomonospora curvata TreS in B. licheniformis. Xylose operons from B. subtilis, B. licheniformis and B. megaterium were introduced to regulate the expression of the gene encoding TreS. It was functionally expressed, and the BlsTs construct yielded the highest enzyme activity (12.1 U/mL). Furthermore, the effect of different cultural conditions on the inducible expression of BlsTs was investigated, and the optimal condition was as follows: 4% maltodextrin, 0.4% soybean powder, 1% xylose added after 10 h of growth and an induction time of 12 h at 37 °C. As a result, the maximal yield reached 24.7 U/mL. This study contributes to the industrial application of B. licheniformis, a GRAS workhorse for enzyme production. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Phylogenomic and functional domain analysis of polyketide synthases in Fusarium

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Daren W.; Butchko, Robert A.; Baker, Scott E.; Proctor, Robert H.

    2012-02-01

    Fusarium species are ubiquitous in nature, cause a range of plant diseases, and produce a variety of chemicals often referred to as secondary metabolites. Although some fungal secondary metabolites affect plant growth or protect plants from other fungi and bacteria, their presence in grain based food and feed is more often associated with a variety of diseases in plants and in animals. Many of these structurally diverse metabolites are derived from a family of related enzymes called polyketide synthases (PKSs). A search of genomic sequence of Fusarium verticillioides, F. graminearum, F. oxysporum and Nectria haematococca (anamorph F. solani) identified a total of 58 PKS genes. To gain insight into how this gene family evolved and to guide future studies, we conducted a phylogenomic and functional domain analysis. The resulting genealogy suggested that Fusarium PKSs represent 34 different groups responsible for synthesis of different core metabolites. The analyses indicate that variation in the Fusarium PKS gene family is due to gene duplication and loss events as well as enzyme gain-of-function due to the acquisition of new domains or of loss-of-function due to nucleotide mutations. Transcriptional analysis indicate that the 16 F. verticillioides PKS genes are expressed under a range of conditions, further evidence that they are functional genes that confer the ability to produce secondary metabolites.

  14. Conservation and Role of Electrostatics in Thymidylate Synthase.

    Science.gov (United States)

    Garg, Divita; Skouloubris, Stephane; Briffotaux, Julien; Myllykallio, Hannu; Wade, Rebecca C

    2015-11-27

    Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome.

  15. Expression, Purification, and Characterisation of Dehydroquinate Synthase from Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Leonardo Negron

    2011-01-01

    Full Text Available Dehydroquinate synthase (DHQS catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30∘C followed by a heat treatment at 70∘C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry and is active over broad pH and temperature ranges. The kinetic parameters are KM (3-deoxy-D-arabino-heptulosonate 7-phosphate 3.7 μM and kcat 3.0 sec-1 at 60∘C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness Cd2+, Co2+, Zn2+, and Mn2+. High activity of a DHQS in the presence of Cd2+ has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd2+ for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay.

  16. SUCROSE SYNTHASE: ELUCIDATION OF COMPLEX POST-TRANSLATIONAL REGULATORY MECHANISMS

    Energy Technology Data Exchange (ETDEWEB)

    Steven C. Huber

    2009-05-12

    Studies have focused on the enzyme sucrose synthase, which plays an important role in the metabolism of sucrose in seeds and tubers. There are three isoforms of SUS in maize, referred to as SUS1, SUS-SH1, and SUS2. SUS is generally considered to be tetrameric protein but recent evidence suggests that SUS can also occur as a dimeric protein. The formation of tetrameric SUS is regulated by sucrose concentration in vitro and this could also be an important factor in the cellular localization of the protein. We found that high sucrose concentrations, which promote tetramer formation, also inhibit the binding of SUS1 to actin filaments in vitro. Previously, high sucrose concentrations were shown to promote SUS association with the plasma membrane. The specific regions of the SUS molecule involved in oligomerization are not known, but we identified a region of the SUS1 moelcule by bioinformatic analysis that was predicted to form a coiled coil. We demonstrated that this sequence could, in fact, self-associate as predicted for a coiled coil, but truncation analysis with the full-length recombinant protein suggested that it was not responsible for formation of dimers or tetramers. However, the coiled coil may function in binding of other proteins to SUS1. Overall, sugar availability may differentially influence the binding of SUS to cellular structures, and these effects may be mediated by changes in the oligomeric nature of the enzyme.

  17. Glycogen synthase kinase-3 regulation of urinary concentrating ability.

    Science.gov (United States)

    Rao, Reena

    2012-09-01

    Glycogen synthase kinase-3 (GSK3) is an enzyme that is gaining prominence as a critical signaling molecule in the epithelial cells of renal tubules. This review will focus on recent findings exploring the role of GSK3 in renal collecting ducts, especially its role in urine concentration involving vasopressin signaling. Recent studies using inhibition or tissue-specific gene deletion of GSK3 revealed the mechanism by which GSK3 regulates aquaporin 2 water channels via adenylate cyclase or the prostaglandin-E2 pathway. In other studies, postnatal treatment with lithium, an inhibitor of GSK3, increased cell proliferation and led to microcyst formation in rat kidneys. These studies suggest that loss of GSK3 activity could interfere with renal water transport at two levels. In the short term, it could disrupt vasopressin signaling in collecting duct cells and in the long term it could alter the structure of the collecting ducts, making them less responsive to the hydro-osmotic effects of vasopressin. Ongoing studies reveal the crucial role played by GSK3 in the regulation of vasopressin action in the renal collecting ducts and suggest a possible use of GSK3 inhibitors in disease conditions associated with disrupted vasopressin signaling.

  18. Glycogen synthase kinase-3 regulates inflammatory tolerance in astrocytes

    Science.gov (United States)

    Beurel, Eléonore; Jope, Richard S.

    2010-01-01

    Inflammatory tolerance is the down-regulation of inflammation upon repeated stimuli, which is well-established to occur in peripheral immune cells. However, less is known about inflammatory tolerance in the brain although it may provide an important protective mechanism from detrimental consequences of prolonged inflammation, which appears to occur in many psychiatric and neurodegenerative conditions. Array analysis of 308 inflammatory molecules produced by mouse primary astrocytes after two sequential stimulations with lipopolysaccharide (LPS) distinguished three classes, tolerant, sensitized and unaltered groups. For many of these inflammatory molecules, inhibition of glycogen synthase kinase-3 (GSK3) increased tolerance and reduced sensitization. Focusing on LPS-tolerance in interleukin-6 (IL-6) production, we found that microglia exhibited a strong tolerance response that matched that of macrophages, whereas astrocytes exhibited only partial tolerance. The astrocyte semi-tolerance was found to be regulated by GSK3. GSK3 inhibitors or knocking down GSK3 levels promoted LPS-tolerance and astrocytes expressing constitutively active GSK3 did not develop LPS-tolerance. These findings identify the critical role of GSK3 in counteracting IL-6 inflammatory tolerance in cells of the CNS, supporting the therapeutic potential of GSK3 inhibitors to reduce neuroinflammation by promoting tolerance. PMID:20553816

  19. Brain phenotype of transgenic mice overexpressing cystathionine β-synthase.

    Science.gov (United States)

    Régnier, Vinciane; Billard, Jean-Marie; Gupta, Sapna; Potier, Brigitte; Woerner, Stéphanie; Paly, Evelyne; Ledru, Aurélie; David, Sabrina; Luilier, Sabrina; Bizot, Jean-Charles; Vacano, Guido; Kraus, Jan P; Patterson, David; Kruger, Warren D; Delabar, Jean M; London, Jaqueline

    2012-01-01

    The cystathionine β-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes. Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line. We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

  20. Mechanics of Cellulose Synthase Complexes in Living Plant Cells

    Science.gov (United States)

    Zehfroosh, Nina; Liu, Derui; Ramos, Kieran P.; Yang, Xiaoli; Goldner, Lori S.; Baskin, Tobias I.

    The polymer cellulose is one of the major components of the world's biomass with unique and fascinating characteristics such as its high tensile strength, renewability, biodegradability, and biocompatibility. Because of these distinctive aspects, cellulose has been the subject of enormous scientific and industrial interest, yet there are still fundamental open questions about cellulose biosynthesis. Cellulose is synthesized by a complex of transmembrane proteins called ``Cellulose Synthase A'' (CESA) in the plasma membrane. Studying the dynamics and kinematics of the CESA complex will help reveal the mechanism of cellulose synthesis and permit the development and validation of models of CESA motility. To understand what drives these complexes through the cell membrane, we used total internal reflection fluorescence microscopy (TIRFM) and variable angle epi-fluorescence microscopy to track individual, fluorescently-labeled CESA complexes as they move in the hypocotyl and root of living plants. A mean square displacement analysis will be applied to distinguish ballistic, diffusional, and other forms of motion. We report on the results of these tracking experiments. This work was funded by NSF/PHY-1205989.

  1. CTP limitation increases expression of CTP synthase in Lactococcus lactis

    DEFF Research Database (Denmark)

    Jørgensen, C.M.; Hammer, Karin; Martinussen, Jan

    2003-01-01

    CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which beta-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold...... decrease in the CTP pool induced by cytidine limitation was found to immediately increase expression of the L. lactis pyrG gene. The final level of expression of pyrG is 37-fold higher than the uninduced level. CTP limitation has pronounced effects on central cellular metabolism, and both RNA and protein...... for regulation of the pyrG gene. It is possible to fold the pyrG leader in an alternative structure that would prevent the formation of the terminator. We suggest a model for pyrG regulation in L. lactis, and probably in other gram-positive bacteria as well, in which pyrG expression is directly dependent...

  2. Module theory, extending modules and generalizations

    CERN Document Server

    Tercan, Adnan

    2016-01-01

    The main focus of this monograph is to offer a comprehensive presentation of known and new results on various generalizations of CS-modules and CS-rings. Extending (or CS) modules are generalizations of injective (and also semisimple or uniform) modules. While the theory of CS-modules is well documented in monographs and textbooks, results on generalized forms of the CS property as well as dual notions are far less present in the literature. With their work the authors provide a solid background to module theory, accessible to anyone familiar with basic abstract algebra. The focus of the book is on direct sums of CS-modules and classes of modules related to CS-modules, such as relative (injective) ejective modules, (quasi) continuous modules, and lifting modules. In particular, matrix CS-rings are studied and clear proofs of fundamental decomposition results on CS-modules over commutative domains are given, thus complementing existing monographs in this area. Open problems round out the work and establish the...

  3. Structural basis for substrate activation and regulation by cystathionine beta-synthase (CBS) domains in cystathionine [beta]-synthase

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Kabil, Omer; Smith, Janet L.; Banerjee, Ruma (Michigan-Med)

    2011-08-17

    The catalytic potential for H{sub 2}S biogenesis and homocysteine clearance converge at the active site of cystathionine {beta}-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes {beta}-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H{sub 2}S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 {angstrom}) and an aminoacrylate intermediate (1.55 {angstrom}) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory 'energy-sensing' CBS domains, named after this protein, suggests a mechanism for allosteric activation by S-adenosylmethionine.

  4. Ceramide synthases expression and role of ceramide synthase-2 in the lung: insight from human lung cells and mouse models.

    Directory of Open Access Journals (Sweden)

    Irina Petrache

    Full Text Available Increases in ceramide levels have been implicated in the pathogenesis of both acute or chronic lung injury models. However, the role of individual ceramide species, or of the enzymes that are responsible for their synthesis, in lung health and disease has not been clarified. We now show that C24- and C16-ceramides are the most abundant lung ceramide species, paralleled by high expression of their synthetic enzymes, ceramide synthase 2 (CerS2 and CerS5, respectively. Furthermore, the ceramide species synthesis in the lung is homeostatically regulated, since mice lacking very long acyl chain C24-ceramides due to genetic deficiency of CerS2 displayed a ten-fold increase in C16-ceramides and C16-dihydroceramides along with elevation of acid sphingomyelinase and CerS5 activities. Despite relatively preserved total lung ceramide levels, inhibition of de novo sphingolipid synthesis at the level of CerS2 was associated with significant airflow obstruction, airway inflammation, and increased lung volumes. Our results suggest that ceramide species homeostasis is crucial for lung health and that CerS2 dysfunction may predispose to inflammatory airway and airspace diseases.

  5. Identification of a Fungal 1,8-Cineole Synthase from Hypoxylon sp. with Specificity Determinants in Common with the Plant Synthases*

    Science.gov (United States)

    Shaw, Jeffrey J.; Berbasova, Tetyana; Sasaki, Tomoaki; Jefferson-George, Kyra; Spakowicz, Daniel J.; Dunican, Brian F.; Portero, Carolina E.; Narváez-Trujillo, Alexandra; Strobel, Scott A.

    2015-01-01

    Terpenes are an important and diverse class of secondary metabolites widely produced by fungi. Volatile compound screening of a fungal endophyte collection revealed a number of isolates in the family Xylariaceae, producing a series of terpene molecules, including 1,8-cineole. This compound is a commercially important component of eucalyptus oil used in pharmaceutical applications and has been explored as a potential biofuel additive. The genes that produce terpene molecules, such as 1,8-cineole, have been little explored in fungi, providing an opportunity to explore the biosynthetic origin of these compounds. Through genome sequencing of cineole-producing isolate E7406B, we were able to identify 11 new terpene synthase genes. Expressing a subset of these genes in Escherichia coli allowed identification of the hyp3 gene, responsible for 1,8-cineole biosynthesis, the first monoterpene synthase discovered in fungi. In a striking example of convergent evolution, mutational analysis of this terpene synthase revealed an active site asparagine critical for water capture and specificity during cineole synthesis, the same mechanism used in an unrelated plant homologue. These studies have provided insight into the evolutionary relationship of fungal terpene synthases to those in plants and bacteria and further established fungi as a relatively untapped source of this important and diverse class of compounds. PMID:25648891

  6. Cellulose synthase complex organization and cellulose microfibril structure.

    Science.gov (United States)

    Turner, Simon; Kumar, Manoj

    2018-02-13

    Cellulose consists of linear chains of β-1,4-linked glucose units, which are synthesized by the cellulose synthase complex (CSC). In plants, these chains associate in an ordered manner to form the cellulose microfibrils. Both the CSC and the local environment in which the individual chains coalesce to form the cellulose microfibril determine the structure and the unique physical properties of the microfibril. There are several recent reviews that cover many aspects of cellulose biosynthesis, which include trafficking of the complex to the plasma membrane and the relationship between the movement of the CSC and the underlying cortical microtubules (Bringmann et al. 2012 Trends Plant Sci. 17 , 666-674 (doi:10.1016/j.tplants.2012.06.003); Kumar & Turner 2015 Phytochemistry 112 , 91-99 (doi:10.1016/j.phytochem.2014.07.009); Schneider et al. 2016 Curr. Opin. Plant Biol. 34 , 9-16 (doi:10.1016/j.pbi.2016.07.007)). In this review, we will focus on recent advances in cellulose biosynthesis in plants, with an emphasis on our current understanding of the structure of individual catalytic subunits together with the local membrane environment where cellulose synthesis occurs. We will attempt to relate this information to our current knowledge of the structure of the cellulose microfibril and propose a model in which variations in the structure of the CSC have important implications for the structure of the cellulose microfibril produced.This article is part of a discussion meeting issue 'New horizons for cellulose nanotechnology'. © 2017 The Author(s).

  7. On the function of chitin synthase extracellular domains in biomineralization.

    Science.gov (United States)

    Weiss, Ingrid M; Lüke, Florian; Eichner, Norbert; Guth, Christina; Clausen-Schaumann, Hauke

    2013-08-01

    Molluscs with various shell architectures evolved around 542-525 million years ago, as part of a larger phenomenon related to the diversification of metazoan phyla. Molluscs deposit minerals in a chitin matrix. The mollusc chitin is synthesized by transmembrane enzymes that contain several unique extracellular domains. Here we investigate the assembly mechanism of the chitin synthase Ar-CS1 via its extracellular domain ArCS1_E22. The corresponding transmembrane protein ArCS1_E22TM accumulates in membrane fractions of the expression host Dictyostelium discoideum. Soluble recombinant ArCS1_E22 proteins can be purified as monomers only at basic pH. According to confocal fluorescence microscopy experiments, immunolabeled ArCS1_E22 proteins adsorb preferably to aragonitic nacre platelets at pH 7.75. At pH 8.2 or pH 9.0 the fluorescence signal is less intense, indicating that protein-mineral interaction is reduced with increasing pH. Furthermore, ArCS1_E22 forms regular nanostructures on cationic substrates as revealed by atomic force microscopy (AFM) experiments on modified mica cleavage planes. These experiments suggest that the extracellular domain ArCS1_E22 is involved in regulating the multiple enzyme activities of Ar-CS1 such as chitin synthesis and myosin movements by interaction with mineral surfaces and eventually by protein assembly. The protein complexes could locally probe the status of mineralization according to pH unless ions and pCO2 are balanced with suitable buffer substances. Taking into account that the intact enzyme could act as a force sensor, the results presented here provide further evidence that shell formation is coordinated physiologically with precise adjustment of cellular activities to the structure, topography and stiffness at the mineralizing interface. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Nitric oxide synthase-3 promotes embryonic development of atrioventricular valves.

    Directory of Open Access Journals (Sweden)

    Yin Liu

    Full Text Available Nitric oxide synthase-3 (NOS3 has recently been shown to promote endothelial-to-mesenchymal transition (EndMT in the developing atrioventricular (AV canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT and NOS3(-/- mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3(-/- compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3(-/- mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1(+ cells in the AV cushion were decreased in NOS3(-/- compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFβ, bone morphogenetic protein (BMP2 and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3(-/- compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency.

  9. Discriminating the reaction types of plant type III polyketide synthases.

    Science.gov (United States)

    Shimizu, Yugo; Ogata, Hiroyuki; Goto, Susumu

    2017-07-01

    Functional prediction of paralogs is challenging in bioinformatics because of rapid functional diversification after gene duplication events combined with parallel acquisitions of similar functions by different paralogs. Plant type III polyketide synthases (PKSs), producing various secondary metabolites, represent a paralogous family that has undergone gene duplication and functional alteration. Currently, there is no computational method available for the functional prediction of type III PKSs. We developed a plant type III PKS reaction predictor, pPAP, based on the recently proposed classification of type III PKSs. pPAP combines two kinds of similarity measures: one calculated by profile hidden Markov models (pHMMs) built from functionally and structurally important partial sequence regions, and the other based on mutual information between residue positions. pPAP targets PKSs acting on ring-type starter substrates, and classifies their functions into four reaction types. The pHMM approach discriminated two reaction types with high accuracy (97.5%, 39/40), but its accuracy decreased when discriminating three reaction types (87.8%, 43/49). When combined with a correlation-based approach, all 49 PKSs were correctly discriminated, and pPAP was still highly accurate (91.4%, 64/70) even after adding other reaction types. These results suggest pPAP, which is based on linear discriminant analyses of similarity measures, is effective for plant type III PKS function prediction. pPAP is freely available at ftp://ftp.genome.jp/pub/tools/ppap/. goto@kuicr.kyoto-u.ac.jp. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  10. Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis.

    Science.gov (United States)

    Noar, Roslyn D; Daub, Margaret E

    2016-01-01

    Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity) for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity) to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that they may encode

  11. Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development.

    Science.gov (United States)

    Schmölzer, Katharina; Gutmann, Alexander; Diricks, Margo; Desmet, Tom; Nidetzky, Bernd

    2016-01-01

    Sucrose synthase (SuSy, EC 2.4.1.13) is a glycosyltransferase (GT) long known from plants and more recently discovered in bacteria. The enzyme catalyzes the reversible transfer of a glucosyl moiety between fructose and a nucleoside diphosphate (NDP) (sucrose+NDP↔NDP-glucose+fructose). The equilibrium for sucrose conversion is pH dependent, and pH values between 5.5 and 7.5 promote NDP-glucose formation. The conversion of a bulk chemical to high-priced NDP-glucose in a one-step reaction provides the key aspect for industrial interest. NDP-sugars are important as such and as key intermediates for glycosylation reactions by highly selective Leloir GTs. SuSy has gained renewed interest as industrially attractive biocatalyst, due to substantial scientific progresses achieved in the last few years. These include biochemical characterization of bacterial SuSys, overproduction of recombinant SuSys, structural information useful for design of tailor-made catalysts, and development of one-pot SuSy-GT cascade reactions for production of several relevant glycosides. These advances could pave the way for the application of Leloir GTs to be used in cost-effective processes. This review provides a framework for application requirements, focusing on catalytic properties, heterologous enzyme production and reaction engineering. The potential of SuSy biocatalysis will be presented based on various biotechnological applications: NDP-sugar synthesis; sucrose analog synthesis; glycoside synthesis by SuSy-GT cascade reactions. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Insights into the reactivation of cobalamin-dependent methionine synthase

    Energy Technology Data Exchange (ETDEWEB)

    Koutmos, Markos; Datta, Supratim; Pattridge, Katherine A.; Smith, Janet L.; Matthews, Rowena G.; (Michigan)

    2009-12-10

    Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every {approx}2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetH{sup CT}) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetH{sup CT} ({sub s-s}MetH{sup CT}) that offer further insight into the reactivation of MetH. The structure of {sub s-s}MetH{sup CT} with cob(II)alamin and S-adenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of {sub s-s}MetH{sub CT} with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.

  13. Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis.

    Directory of Open Access Journals (Sweden)

    Roslyn D Noar

    Full Text Available Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that

  14. Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation.

    Science.gov (United States)

    Liu, Jiayan; Seibold, Steve A; Rieke, Caroline J; Song, Inseok; Cukier, Robert I; Smith, William L

    2007-06-22

    The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

  15. Hyaluronan synthase mediates dye translocation across liposomal membranes

    Directory of Open Access Journals (Sweden)

    Medina Andria P

    2012-01-01

    Full Text Available Abstract Background Hyaluronan (HA is made at the plasma membrane and secreted into the extracellular medium or matrix by phospolipid-dependent hyaluronan synthase (HAS, which is active as a monomer. Since the mechanism by which HA is translocated across membranes is still unresolved, we assessed the presence of an intraprotein pore within HAS by adding purified Streptococcus equisimilis HAS (SeHAS to liposomes preloaded with the fluorophore Cascade Blue (CB. Results CB translocation (efflux was not observed with mock-purified material from empty vector control E. coli membranes, but was induced by SeHAS, purified from membranes, in a time- and dose-dependent manner. CB efflux was eliminated or greatly reduced when purified SeHAS was first treated under conditions that inhibit enzyme activity: heating, oxidization or cysteine modification with N-ethylmaleimide. Reduced CB efflux also occurred with SeHAS K48E or K48F mutants, in which alteration of K48 within membrane domain 2 causes decreased activity and HA product size. The above results used liposomes containing bovine cardiolipin (BCL. An earlier study testing many synthetic lipids found that the best activating lipid for SeHAS is tetraoleoyl cardiolipin (TO-CL and that, in contrast, tetramyristoyl cardiolipin (TM-CL is an inactivating lipid (Weigel et al, J. Biol. Chem. 281, 36542, 2006. Consistent with the effects of these CL species on SeHAS activity, CB efflux was more than 2-fold greater in liposomes made with TO-CL compared to TM-CL. Conclusions The results indicate the presence of an intraprotein pore in HAS and support a model in which HA is translocated to the exterior by HAS itself.

  16. p63 promotes cell survival through fatty acid synthase.

    Directory of Open Access Journals (Sweden)

    Venkata Sabbisetti

    2009-06-01

    Full Text Available There is increasing evidence that p63, and specifically DeltaNp63, plays a central role in both development and tumorigenesis by promoting epithelial cell survival. However, few studies have addressed the molecular mechanisms through which such important function is exerted. Fatty acid synthase (FASN, a key enzyme that synthesizes long-chain fatty acids and is involved in both embryogenesis and cancer, has been recently proposed as a direct target of p53 family members, including p63 and p73. Here we show that knockdown of either total or DeltaN-specific p63 isoforms in squamous cell carcinoma (SCC9 or immortalized prostate epithelial (iPrEC cells caused a decrease in cell viability by inducing apoptosis without affecting the cell cycle. p63 silencing significantly reduced both the expression and the activity of FASN. Importantly, stable overexpression of either FASN or myristoylated AKT (myr-AKT was able to partially rescue cells from cell death induced by p63 silencing. FASN induced AKT phosphorylation and a significant reduction in cell viability was observed when FASN-overexpressing SCC9 cells were treated with an AKT inhibitor after p63 knockdown, indicating that AKT plays a major role in FASN-mediated survival. Activated AKT did not cause any alteration in the FASN protein levels but induced its activity, suggesting that the rescue from apoptosis documented in the p63-silenced cells expressing myr-AKT cells may be partially mediated by FASN. Finally, we demonstrated that p63 and FASN expression are positively associated in clinical squamous cell carcinoma samples as well as in the developing prostate. Taken together, our findings demonstrate that FASN is a functionally relevant target of p63 and is required for mediating its pro-survival effects.

  17. Regulation of glycogen synthase kinase-3 during bipolar mania treatment.

    Science.gov (United States)

    Li, Xiaohong; Liu, Min; Cai, Zhuoji; Wang, Gang; Li, Xiaohua

    2010-11-01

    Bipolar disorder is a debilitating psychiatric illness presenting with recurrent mania and depression. The pathophysiology of bipolar disorder is poorly understood, and molecular targets in the treatment of bipolar disorder remain to be identified. Preclinical studies have suggested that glycogen synthase kinase-3 (GSK3) is a potential therapeutic target in bipolar disorder, but evidence of abnormal GSK3 in human bipolar disorder and its response to treatment is still lacking. This study was conducted in acutely ill type I bipolar disorder subjects who were hospitalized for a manic episode. The protein level and the inhibitory serine phosphorylation of GSK3 in peripheral blood mononuclear cells of bipolar manic and healthy control subjects were compared, and the response of GSK3 to antimanic treatment was evaluated. The levels of GSK3α and GSK3β in this group of bipolar manic subjects were higher than healthy controls. Symptom improvement during an eight-week antimanic treatment with lithium, valproate, and atypical antipsychotics was accompanied by a significant increase in the inhibitory serine phosphorylation of GSK3, but not the total level of GSK3, whereas concomitant electroconvulsive therapy treatment during a manic episode appeared to dampen the response of GSK3 to pharmacological treatment. Results of this study suggest that GSK3 can be modified during the treatment of bipolar mania. This finding in human bipolar disorder is in agreement with preclinical data suggesting that inhibition of GSK3 by increasing serine phosphorylation is a response of GSK3 to psychotropics used in bipolar disorder, supporting the notion that GSK3 is a promising molecular target in the pharmacological treatment of bipolar disorder. © 2010 John Wiley and Sons A/S.

  18. Molecular cloning and characterization of glycogen synthase in Eriocheir sinensis.

    Science.gov (United States)

    Li, Ran; Zhu, Li-Na; Ren, Li-Qi; Weng, Jie-Yang; Sun, Jin-Sheng

    2017-12-01

    Glycogen plays an important role in glucose and energy homeostasis at cellular and organismal levels. In glycogen synthesis, glycogen synthase (GS) is a rate-limiting enzyme catalysing the addition of α-1,4-linked glucose units from (UDP) 3 -glucose to a nascent glycogen chain using glycogenin (GN) as a primer. While studies on mammalian liver GS (GYS2) are numerous, enzymes from crustaceans, which also use glycogen and glucose as their main energy source, have received less attention. In the present study, we amplified full-length GS cDNA from Eriocheir sinensis. Tissue expression profiling revealed the highest expression of GS in the hepatopancreas. During moulting, GS expression and activity declined, and glycogen levels in the hepatopancreas were reduced. Recombinant GS was expressed in Escherichia coli Rosetta (DE3), and induction at 37°C or 16°C yielded EsGS in insoluble inclusion bodies (EsGS-I) or in soluble form (EsGS-S), respectively. Enzyme activity was measured in a cell-free system containing glucose-6-phosphate (G6P), and both forms possessed glycosyltransferase activity, but refolded EsGS-I was more active. Enzyme activity of both GS and EsGS-I in the hepatopancreas was optimum at 25°C, which is coincident with the optimum growth temperature of Chinese mitten crab, and higher (37°C) or lower (16°C) temperatures resulted in lower enzyme activity. Taken together, the results suggest that GS may be important for maintaining normal physiological functions such as growth and reproduction. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    Science.gov (United States)

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Old Yellow Enzyme from Trypanosoma cruzi Exhibits In Vivo Prostaglandin F2α Synthase Activity and Has a Key Role in Parasite Infection and Drug Susceptibility

    Directory of Open Access Journals (Sweden)

    Florencia Díaz-Viraqué

    2018-03-01

    Full Text Available The discovery that trypanosomatids, unicellular organisms of the order Kinetoplastida, are capable of synthesizing prostaglandins raised questions about the role of these molecules during parasitic infections. Multiple studies indicate that prostaglandins could be related to the infection processes and pathogenesis in trypanosomatids. This work aimed to unveil the role of the prostaglandin F2α synthase TcOYE in the establishment of Trypanosoma cruzi infection, the causative agent of Chagas disease. This chronic disease affects several million people in Latin America causing high morbidity and mortality. Here, we propose a prokaryotic evolutionary origin for TcOYE, and then we used in vitro and in vivo experiments to show that T. cruzi prostaglandin F2α synthase plays an important role in modulating the infection process. TcOYE overexpressing parasites were less able to complete the infective cycle in cell culture infections and increased cardiac tissue parasitic load in infected mice. Additionally, parasites overexpressing the enzyme increased PGF2α synthesis from arachidonic acid. Finally, an increase in benznidazole and nifurtimox susceptibility in TcOYE overexpressing parasites showed its participation in activating the currently anti-chagasic drugs, which added to its observed ability to confer resistance to hydrogen peroxide, highlights the relevance of this enzyme in multiple events including host–parasite interaction.

  1. Reduced multiplication modules

    Indian Academy of Sciences (India)

    if M is a von Neumann regular module (VNM); i.e., every principal submodule of M is a summand submodule. Also if M is an injective R-module, then M is a VNM. Keywords. Multiplication module; reduced module; minimal prime submodule;. Zariski topology; extremally disconnected. 1. Introduction. In this paper all rings are ...

  2. Transcutaneous electrical nerve stimulation attenuates CFA-induced hyperalgesia and inhibits spinal ERK1/2-COX-2 pathway activation in rats

    OpenAIRE

    Fang, Jun-Fan; Liang, Yi; Du, Jun-Ying; Fang, Jian-Qiao

    2013-01-01

    Background Transcutaneous electrical nerve stimulation (TENS) is a non-pharmacologic treatment for pain relief. In previous animal studies, TENS effectively alleviated Complete Freund?s Adjuvant (CFA)- or carrageenan-induced inflammatory pain. Although TENS is known to produce analgesia via opioid activation in the brain and at the spinal level, few reports have investigated the signal transduction pathways mediated by TENS. Prior studies have verified the importance of the activation of extr...

  3. Topical dura mater application of CFA induces enhanced expression of c-fos and glutamate in rat trigeminal nucleus caudalis: attenuated by KYNA derivate (SZR72).

    Science.gov (United States)

    Lukács, M; Warfvinge, K; Tajti, J; Fülöp, F; Toldi, J; Vécsei, L; Edvinsson, L

    2017-12-01

    Migraine is a debilitating neurological disorder where trigeminovascular activation plays a key role. We have previously reported that local application of Complete Freund's Adjuvant (CFA) onto the dura mater caused activation in rat trigeminal ganglion (TG) which was abolished by a systemic administration of kynurenic acid (KYNA) derivate (SZR72). Here, we hypothesize that this activation may extend to the trigeminal complex in the brainstem and is attenuated by treatment with SZR72. Activation in the trigeminal nucleus caudalis (TNC) and the trigeminal tract (Sp5) was achieved by application of CFA onto the dural parietal surface. SZR72 was given intraperitoneally (i.p.), one dose prior CFA deposition and repeatedly daily for 7 days. Immunohistochemical studies were performed for mapping glutamate, c-fos, PACAP, substance P, IL-6, IL-1β and TNFα in the TNC/Sp5 and other regions of the brainstem and at the C 1 -C 2 regions of the spinal cord. We found that CFA increased c-fos and glutamate immunoreactivity in TNC and C 1 -C 2 neurons. This effect was mitigated by SZR72. PACAP positive fibers were detected in the fasciculus cuneatus and gracilis. Substance P, TNFα, IL-6 and IL-1β immunopositivity were detected in fibers of Sp5 and neither of these molecules showed any change in immunoreactivity following CFA administration. This is the first study demonstrating that dural application of CFA increases the expression of c-fos and glutamate in TNC neurons. Treatment with the KYNA analogue prevented this expression.

  4. Environmental cold exposure increases blood flow and affects pain sensitivity in the knee joints of CFA-induced arthritic mice in a TRPA1-dependent manner.

    Science.gov (United States)

    Fernandes, Elizabeth S; Russell, Fiona A; Alawi, Khadija M; Sand, Claire; Liang, Lihuan; Salamon, Robin; Bodkin, Jennifer V; Aubdool, Aisah A; Arno, Matthew; Gentry, Clive; Smillie, Sarah-Jane; Bevan, Stuart; Keeble, Julie E; Malcangio, Marzia; Brain, Susan D

    2016-01-11

    The effect of cold temperature on arthritis symptoms is unclear. The aim of this study was to investigate how environmental cold affects pain and blood flow in mono-arthritic mice, and examine a role for transient receptor potential ankyrin 1 (TRPA1), a ligand-gated cation channel that can act as a cold sensor. Mono-arthritis was induced by unilateral intra-articular injection of complete Freund's adjuvant (CFA) in CD1 mice, and in mice either lacking TRPA1 (TRPA1 KO) or respective wildtypes (WT). Two weeks later, nociception and joint blood flow were measured following exposure to 10 °C (1 h) or room temperature (RT). Primary mechanical hyperalgesia in the knee was measured by pressure application apparatus; secondary mechanical hyperalgesia by automated von Frey system; thermal hyperalgesia by Hargreaves technique, and weight bearing by the incapacitance test. Joint blood flow was recorded by full-field laser perfusion imager (FLPI) and using clearance of (99m)Technetium. Blood flow was assessed after pretreatment with antagonists of either TRPA1 (HC-030031), substance P neurokinin 1 (NK1) receptors (SR140333) or calcitonin gene-related peptide (CGRP) (CGRP8-37). TRPA1, TAC-1 and CGRP mRNA levels were examined in dorsal root ganglia, synovial membrane and patellar cartilage samples. Cold exposure caused bilateral primary mechanical hyperalgesia 2 weeks after CFA injection, in a TRPA1-dependent manner. In animals maintained at RT, clearance techniques and FLPI showed that CFA-treated joints exhibited lower blood flow than saline-treated joints. In cold-exposed animals, this reduction in blood flow disappears, and increased blood flow in the CFA-treated joint is observed using FLPI. Cold-induced increased blood flow in CFA-treated joints was blocked by HC-030031 and not observed in TRPA1 KOs. Cold exposure increased TRPA1 mRNA levels in patellar cartilage, whilst reducing it in synovial membranes from CFA-treated joints. We provide evidence that environmental cold exposure enhances pain and increases blood flow in a mono-arthritis model. These changes are dependent on TRPA1. Thus, TRPA1 may act locally within the joint to influence blood flow via sensory nerves, in addition to its established nociceptive actions.

  5. GluN2B/CaMKII mediates CFA-induced hyperalgesia via HDAC4-modified spinal COX2 transcription.

    Science.gov (United States)

    Lai, Cheng-Yuan; Hsieh, Ming-Chun; Ho, Yu-Cheng; Chen, Gin-Den; Chou, Dylan; Ruan, Ting; Lee, An-Sheng; Wang, Hsueh-Hsiao; Chau, Yat-Pang; Peng, Hsien-Yu; Lai, Cheng-Hung

    2018-06-01

    Histone deacetylase 4 (HDAC4), which actively shuttles between the nucleus and cytoplasm, is an attractive candidate for a repressor mechanism in epigenetic modification. However, the potential role of HDAC4-dependent epigenetics in the neural plasticity underlying the development of inflammatory pain has not been well established. By injecting complete Freund's adjuvant (CFA) into the hind-paw of Sprague-Dawley rats (200-250 g), we found animals displayed behavioral hyperalgesia was accompanied with HDAC4 phosphorylation and cytoplasmic redistribution in the dorsal horn neurons. Cytoplasmic HDAC4 retention led to its uncoupling with the COX2 promoter, hence prompting spinal COX2 transcription and expression in the dorsal horn. Moreover, the GluN2B-bearing N-methyl-d-aspartate receptor (GluN2B-NMDAR)/calmodulin-dependent protein kinase II (CaMKII) acted as an upstream cascade to facilitate HDAC4 phosphorylation/redistribution-associated spinal COX2 expression after inflammatory insults. The results of this pilot study demonstrated that the development and/or maintenance of inflammatory pain involved the spinal HDAC4-dependent epigenetic mechanisms. Our findings open up a new avenue for the development of a novel medical strategy for the relief of inflammatory pain. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. High-fat diet exacerbates pain-like behaviors and periarticular bone loss in mice with CFA-induced knee arthritis.

    Science.gov (United States)

    Loredo-Pérez, Aleyda A; Montalvo-Blanco, Carlos E; Hernández-González, Luis I; Anaya-Reyes, Maricruz; Fernández Del Valle-Laisequilla, Cecilia; Reyes-García, Juan G; Acosta-González, Rosa I; Martínez-Martínez, Arisai; Villarreal-Salcido, Jaira C; Vargas-Muñoz, Virginia M; Muñoz-Islas, Enriqueta; Ramírez-Rosas, Martha B; Jiménez-Andrade, Juan M

    2016-05-01

    Our aim was to quantify nociceptive spontaneous behaviors, knee edema, proinflammatory cytokines, bone density, and microarchitecture in high-fat diet (HFD)-fed mice with unilateral knee arthritis. ICR male mice were fed either standard diet (SD) or HFD starting at 3 weeks old. At 17 weeks, HFD and SD mice received intra-articular injections either with Complete Freund's Adjuvant (CFA) or saline into the right knee joint every 7 days for 4 weeks. Spontaneous pain-like behaviors and knee edema were assessed for 26 days. At day 26 post-first CFA injection, serum levels of IL-1β, IL-6, and RANKL were measured by ELISA, and microcomputed tomography analysis of knee joints was performed. HFD-fed mice injected with CFA showed greater spontaneous pain-like behaviors of the affected extremity as well as a decrease in the weight-bearing index compared to SD-fed mice injected with CFA. Knee edema was not significantly different between diets. HFD significantly exacerbated arthritis-induced bone loss at the distal femoral metaphysis but had no effect on femoral diaphyseal cortical bone. HFD did not modify serum levels of proinflammatory cytokines. HFD exacerbates pain-like behaviors and significantly increases the magnitude of periarticular trabecular bone loss in a murine model of unilateral arthritis. © 2016 The Obesity Society.

  7. Electroacupuncture Attenuates CFA-induced Inflammatory Pain by suppressing Nav1.8 through S100B, TRPV1, Opioid, and Adenosine Pathways in Mice.

    Science.gov (United States)

    Liao, Hsien-Yin; Hsieh, Ching-Liang; Huang, Chun-Ping; Lin, Yi-Wen

    2017-02-13

    Pain is associated with several conditions, such as inflammation, that result from altered peripheral nerve properties. Electroacupuncture (EA) is a common Chinese clinical medical technology used for pain management. Using an inflammatory pain mouse model, we investigated the effects of EA on the regulation of neurons, microglia, and related molecules. Complete Freund's adjuvant (CFA) injections produced a significant mechanical and thermal hyperalgesia that was reversed by EA or a transient receptor potential V1 (TRPV1) gene deletion. The expression of the astrocytic marker glial fibrillary acidic protein (GFAP), the microglial marker Iba-1, S100B, receptor for advanced glycation end-products (RAGE), TRPV1, and other related molecules was dramatically increased in the dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) of CFA-treated mice. This effect was reversed by EA and TRPV1 gene deletion. In addition, endomorphin (EM) and N 6 -cyclopentyladenosine (CPA) administration reliably reduced mechanical and thermal hyperalgesia, thereby suggesting the involvement of opioid and adenosine receptors. Furthermore, blocking of opioid and adenosine A1 receptors reversed the analgesic effects of EA. Our study illustrates the substantial therapeutic effects of EA against inflammatory pain and provides a novel and detailed mechanism underlying EA-mediated analgesia via neuronal and non-neuronal pathways.

  8. Anti-hyperalgesic activity of the aqueous and methanol extracts of the leaves of Pittosporum mannii Hook on CFA-induced persistent inflammatory pain.

    Science.gov (United States)

    Wandji, Bibiane Aimée; Bomba, Francis Desire Tatsinkou; Nkeng-Efouet, Pepin Alango; Piegang, Basile Nganmegne; Kamanyi, Albert; Nguelefack, Télesphore Benoît

    2018-02-01

    Previous study showed that aqueous (AEPM) and methanol (MEPM) extracts from the leaves of Pittosporum mannii have analgesic effects in acute pain models. The present study evaluates the acute and chronic anti-hypernociceptive and anti-inflammatory effects of AEPM and MEPM in a model of persistent inflammatory pain. The third day after induction of inflammatory pain by subplantar injection of 100 µL of CFA in Wistar rats, AEPM and MEPM were administered orally (75, 150 and 300 mg/kg/day) and their anti-hyperalgesic and anti-inflammatory effects were follow in acute (1-24 h) and chronic (for 14 days) treatments. At the end of the chronic treatment, oxidative stress and liver parameters were assessed. Effects of plant extracts were also evaluated on nociception induced by Phorbol 12-Myristate 13-Acetate (PMA) and 8-bromo 3',5'-cAMP (8-Br-cAMP) in mice. AEPM and MEPM significantly reversed the mechanical hyperalgesia caused by CFA in acute and chronic treatment. Moreover, AEPM and MEPM also significantly reduced the nociception caused by PMA (60%) and 8-Br-cAMP (87%). Nevertheless, AEPM and MEPM failed to inhibit the paw edema caused by CFA. Plant extracts significantly reduced the nitric oxide content in the spinal cord and the plasmatic concentration of alanine aminotransferase. MEPM also significantly increased the glutathione content in the spinal cord. AEPM and MEPM given orally are effective in inhibiting mechanical hyperalgesia in persistent inflammatory pain caused by CFA. Their mechanisms of action seem to involve an interaction with PKC, PKA and nitric oxide pathways. These extracts might be devoid of hepatotoxic effects.

  9. Early, middle, or late administration of zoledronate alleviates spontaneous nociceptive behavior and restores functional outcomes in a mouse model of CFA-induced arthritis.

    Science.gov (United States)

    Morado-Urbina, Carlos Eduardo; Alvarado-Vázquez, Perla Abigail; Montiel-Ruiz, Rosa Mariana; Acosta-González, Rosa Issel; Castañeda-Corral, Gabriela; Jiménez-Andrade, Juan Miguel

    2014-11-01

    This study was performed to evaluate whether early, middle, or late treatment of zoledronate, an approved bisphosphonate that blocks bone resorption, can reduce nociceptive behaviors in a mouse arthritis model. Arthritis was produced by repeated intra-articular knee injections of complete Freund's adjuvant (CFA). A dose-response curve with zoledronate (3, 30, 100, and 300 μg/kg, i.p., day 4 to day 25, twice weekly for 3 weeks) was performed, and the most effective dose of zoledronate (100 μg/kg, i.p.) was initially administered at different times of disease progression: day 4 (early), day 15 (middle), or day 21 (late) and continued until day 25 after the first CFA injection. Flinching of the injected extremity (spontaneous nociceptive behavior), vertical rearings and horizontal activity (functional outcomes), and knee edema were assessed. Zoledronate improved both functional outcomes and reduced flinching behavior. At day 25, the effect of zoledronate on flinching behavior and vertical rearings was greater in magnitude when it was given early or middle rather than late in the treatment regimen. Chronic zoledronate did not reduce knee edema in CFA-injected mice nor functional outcomes in naïve mice by itself. These results suggest that zoledronate may have a positive effect on arthritis-induced nociception and functional disabilities. © 2014 Wiley Periodicals, Inc.

  10. Probing the Effects and Mechanisms of Electroacupuncture at Ipsilateral or Contralateral ST36-ST37 Acupoints on CFA-induced Inflammatory Pain.

    Science.gov (United States)

    Lu, Kung-Wen; Hsu, Chao-Kuei; Hsieh, Ching-Liang; Yang, Jun; Lin, Yi-Wen

    2016-02-24

    Transient receptor potential vanilloid 1 (TRPV1) and associated signaling pathways have been reported to be increased in inflammatory pain signaling. There are accumulating evidences surrounding the therapeutic effect of electroacupuncture (EA). EA can reliably attenuate the increase of TRPV1 in mouse inflammatory pain models with unclear signaling mechanisms. Moreover, the difference in the clinical therapeutic effects between using the contralateral and ipsilateral acupoints has been rarely studied. We found that inflammatory pain, which was induced by injecting the complete Freund's adjuvant (CFA), (2.14 ± 0.1, p CFA injection; this expression can be further attenuated significantly in EA treatment. TRPV1 and associated signaling pathways can be prevented in TRPV1 knockout mice, suggesting that TRPV1 knockout mice are resistant to inflammatory pain. Through this study, we have increased the understanding of the mechanism that both ipsilateral and contralateral EA might alter TRPV1 and associated signaling pathways to reduce inflammatory pain.

  11. Probing the Effects and Mechanisms of Electroacupuncture at Ipsilateral or Contralateral ST36–ST37 Acupoints on CFA-induced Inflammatory Pain

    Science.gov (United States)

    Lu, Kung-Wen; Hsu, Chao-Kuei; Hsieh, Ching-Liang; Yang, Jun; Lin, Yi-Wen

    2016-01-01

    Transient receptor potential vanilloid 1 (TRPV1) and associated signaling pathways have been reported to be increased in inflammatory pain signaling. There are accumulating evidences surrounding the therapeutic effect of electroacupuncture (EA). EA can reliably attenuate the increase of TRPV1 in mouse inflammatory pain models with unclear signaling mechanisms. Moreover, the difference in the clinical therapeutic effects between using the contralateral and ipsilateral acupoints has been rarely studied. We found that inflammatory pain, which was induced by injecting the complete Freund’s adjuvant (CFA), (2.14 ± 0.1, p < 0.05, n = 8) can be alleviated after EA treatment at either ipsilateral (3.91 ± 0.21, p < 0.05, n = 8) or contralateral acupoints (3.79 ± 0.25, p < 0.05, n = 8). EA may also reduce nociceptive Nav sodium currents in dorsal root ganglion (DRG) neurons. The expression of TRPV1 and associated signaling pathways notably increased after the CFA injection; this expression can be further attenuated significantly in EA treatment. TRPV1 and associated signaling pathways can be prevented in TRPV1 knockout mice, suggesting that TRPV1 knockout mice are resistant to inflammatory pain. Through this study, we have increased the understanding of the mechanism that both ipsilateral and contralateral EA might alter TRPV1 and associated signaling pathways to reduce inflammatory pain. PMID:26906464

  12. Role of Polymorphisms of Inducible Nitric Oxide Synthase and Endothelial Nitric Oxide Synthase in Idiopathic Environmental Intolerances

    Directory of Open Access Journals (Sweden)

    Chiara De Luca

    2015-01-01

    Full Text Available Oxidative stress and inflammation play a pathogenetic role in idiopathic environmental intolerances (IEI, namely, multiple chemical sensitivity (MCS, fibromyalgia (FM, and chronic fatigue syndrome (CFS. Given the reported association of nitric oxide synthase (NOS gene polymorphisms with inflammatory disorders, we aimed to investigate the distribution of NOS2A −2.5 kb (CCTTTn as well as Ser608Leu and NOS3 −786T>C variants and their correlation with nitrite/nitrate levels, in a study cohort including 170 MCS, 108 suspected MCS (SMCS, 89 FM/CFS, and 196 healthy subjects. Patients and controls had similar distributions of NOS2A Ser608Leu and NOS3 −786T>C polymorphisms. Interestingly, the NOS3 −786TT genotype was associated with increased nitrite/nitrate levels only in IEI patients. We also found that the NOS2A −2.5 kb (CCTTT11 allele represents a genetic determinant for FM/CFS, and the (CCTTT16 allele discriminates MCS from SMCS patients. Instead, the (CCTTT8 allele reduces by three-, six-, and tenfold, respectively, the risk for MCS, SMCS, and FM/CFS. Moreover, a short number of (CCTTT repeats is associated with higher concentrations of nitrites/nitrates. Here, we first demonstrate that NOS3 −786T>C variant affects nitrite/nitrate levels in IEI patients and that screening for NOS2A −2.5 kb (CCTTTn polymorphism may be useful for differential diagnosis of various IEI.

  13. Modulational effects in accelerators

    International Nuclear Information System (INIS)

    Satogata, T.

    1997-01-01

    We discuss effects of field modulations in accelerators, specifically those that can be used for operational beam diagnostics and beam halo control. In transverse beam dynamics, combined effects of nonlinear resonances and tune modulations influence diffusion rates with applied tune modulation has been demonstrated. In the longitudinal domain, applied RF phase and voltage modulations provide mechanisms for parasitic halo transport, useful in slow crystal extraction. Experimental experiences with transverse tune and RF modulations are also discussed

  14. Isolation and Characterization of Three New Monoterpene Synthases from Artemisia annua

    Science.gov (United States)

    Ruan, Ju-Xin; Li, Jian-Xu; Fang, Xin; Wang, Ling-Jian; Hu, Wen-Li; Chen, Xiao-Ya; Yang, Chang-Qing

    2016-01-01

    Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5, and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg2+ and Mn2+ were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn2+ for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography–mass spectrometry detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate, salicylic acid, and gibberellin, suggesting a role of these monoterpene synthases in plant–environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant. PMID:27242840

  15. Isolation, characterization, and mechanistic studies of (-)-alpha-gurjunene synthase from Solidago canadensis.

    Science.gov (United States)

    Schmidt, C O; Bouwmeester, H J; Bülow, N; König, W A

    1999-04-15

    The leaves of the composite Solidago canadensis (goldenrod) were shown to contain (-)-alpha-gurjunene synthase activity. This sesquiterpene is likely to be the precursor for cyclocolorenone, a sesquiterpene ketone present in high amounts in S. canadensis leaves. (-)-alpha-Gurjunene synthase was purified to apparent homogeneity (741-fold) by anion-exchange chromatography (on several matrices), dye ligand chromatography, hydroxylapatite chromatography, and gel filtration. Chromatography on a gel filtration matrix indicated a native molecular mass of 48 kDa, and SDS-PAGE showed the enzyme to be composed of one subunit with a denatured mass of 60 kDa. Its maximum activity was observed at pH 7.8 in the presence of 10 mM Mg2+ and the KM value for the substrate farnesyl diphosphate was 5.5 microM. Over a range of purification steps (-)-alpha-gurjunene and (+)-gamma-gurjunene synthase activities copurified. In addition, the product ratio of the enzyme activity under several different assay conditions was always 91% (-)-alpha-gurjunene and 9% (+)-gamma-gurjunene. This suggests that the formation of these two structurally related products is catalyzed by one enzyme. For further confirmation, we carried out a number of mechanistic studies with (-)-alpha-gurjunene synthase, in which an enzyme preparation was incubated with deuterated substrate analogues. Based on mass spectrometry analysis of the products formed, a cyclization mechanism was postulated which makes it plausible that the synthase catalyzes the formation of both sesquiterpenes. Copyright 1999 Academic Press.

  16. Monoterpene synthase from Dracocephalum kotschyi and SPME-GC-MS analysis of its aroma profile

    Directory of Open Access Journals (Sweden)

    S. Saeidnia

    2014-04-01

    Full Text Available Dracocephalum kotschyi (Lamiaceae, as one of the remarkable aromatic plants, widely grows and also is cultivated in various temperate regions of Iran. There are diverse reports about the composition of the oil of this plant representing limonene derivatives as its major compounds. There is no report on cloning of mono- or sesquiterpene synthases from this plant. In the present study, the aroma profile of D. kotschyi has been extracted and analyzed via Headspace Solid-Phase Microextraction technique coupled with Gas Chromatography- Mass Spectroscopy. In order to determine the sequence of the active terpene synthase in this plant, first mRNA was prepared and cloning was performed by 3’ and 5’-RACEs-PCR method, then cDNA was sequenced and finally aligned with other recognized terpene synthases. The results showed that the plant leaves mainly comprised geranial (37.2%, limonene-10-al (28.5%, limonene (20.1% and 1,1-dimethoxy decane (14.5%. Sequencing the cDNA cloned from this plant revealed the presence of a monoterpene synthase absolutely similar to limonene synthase, responsible in formation of limonene, terpinolene, camphene and some other cyclic monoterpenes in its young leaves.

  17. Pivotal role of glycogen synthase kinase-3: A therapeutic target for Alzheimer's disease.

    Science.gov (United States)

    Maqbool, Mudasir; Mobashir, Mohammad; Hoda, Nasimul

    2016-01-01

    Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism of cause and paucity of complete cure. Out of all the neurodegenerative diseases, Alzheimer's disease is the most devastating and loosening of thinking and judging ability disease that occurs in the old age people. Many hypotheses came forth in order to explain its causes. In this review, we have enlightened Glycogen Synthase Kinase-3 which has been considered as a concrete cause for Alzheimer's disease. Plaques and Tangles (abnormal structures) are the basic suspects in damaging and killing of nerve cells wherein Glycogen Synthase Kinase-3 has a key role in the formation of these fatal accumulations. Various Glycogen Synthase Kinase-3 inhibitors have been reported to reduce the amount of amyloid-beta as well as the tau hyperphosphorylation in both neuronal and nonneuronal cells. Additionally, Glycogen Synthase Kinase-3 inhibitors have been reported to enhance the adult hippocampal neurogenesis in vivo as well as in vitro. Keeping the chemotype of the reported Glycogen Synthase Kinase-3 inhibitors in consideration, they may be grouped into natural inhibitors, inorganic metal ions, organo-synthetic, and peptide like inhibitors. On the basis of their mode of binding to the constituent enzyme, they may also be grouped as ATP, nonATP, and allosteric binding sites competitive inhibitors. ATP competitive inhibitors were known earlier inhibitors but they lack efficient selectivity. This led to find the new ways for the enzyme inhibition. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  18. Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia).

    Science.gov (United States)

    Landmann, Christian; Fink, Barbara; Festner, Maria; Dregus, Márta; Engel, Karl-Heinz; Schwab, Wilfried

    2007-09-15

    The essential oil of lavender (Lavandula angustifolia) is mainly composed of mono- and sesquiterpenes. Using a homology-based PCR strategy, two monoterpene synthases (LaLIMS and LaLINS) and one sesquiterpene synthase (LaBERS) were cloned from lavender leaves and flowers. LaLIMS catalyzed the formation of (R)-(+)-limonene, terpinolene, (1R,5S)-(+)-camphene, (1R,5R)-(+)-alpha-pinene, beta-myrcene and traces of alpha-phellandrene. The proportions of these products changed significantly when Mn(2+) was supplied as the cofactor instead of Mg(2+). The second enzyme LaLINS produced exclusively (R)-(-)-linalool, the main component of lavender essential oil. LaBERS transformed farnesyl diphosphate and represents the first reported trans-alpha-bergamotene synthase. It accepted geranyl diphosphate with higher affinity than farnesyl diphosphate and also produced monoterpenes, albeit at low rates. LaBERS is probably derived from a parental monoterpene synthase by the loss of the plastidial signal peptide and by broadening its substrate acceptance spectrum. The identification and description of the first terpene synthases from L. angustifolia forms the basis for the biotechnological modification of essential oil composition in lavender.

  19. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    International Nuclear Information System (INIS)

    Nemazanyy, Ivan; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T.

    2006-01-01

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy β and originally identified CoA synthase, CoASy α. The transcript specific for CoASy β was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy β. In contrast to CoASy α, which shows ubiquitous expression, CoASy β is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation

  20. Bifunctional activity of deoxyhypusine synthase/hydroxylase from Trichomonas vaginalis.

    Science.gov (United States)

    Quintas-Granados, Laura Itzel; Carvajal Gamez, Bertha Isabel; Villalpando, Jose Luis; Ortega-Lopez, Jaime; Arroyo, Rossana; Azuara-Liceaga, Elisa; Álvarez-Sánchez, María Elizbeth

    2016-04-01

    The Trichomonas vaginalis genome analysis suggested the presence of a putative deoxyhypusine synthase (TvDHS) that catalyzes the posttranslational modification of eIF-5A. Herein, we expressed and purified the recombinant TvDHS (rTvDHS) protein (43 kDa) and the recombinant TveIF-5A (rTveIF-5A) precursor protein (46 kDa). A 41 kDa band of the native TvDHS was recognized by western blot analysis in T. vaginalis total protein extract by a mouse polyclonal anti-rTvDHS antibody. The enzymatic activity of rTvDHS was determined by in vitro rTveIF-5A precursor modification. The modification reaction was performed by using ((3)H)-spermidine, and the biochemical analysis showed that rTvDHS exhibited Km value of 0.6 μM. The rTvDHS activity was inhibited by the spermidine analog, N″-guanyl-1,7-diamino-heptane (GC7). Native gel electrophoresis analysis showed two bands corresponding to an rTvDHS-rTveIF-5A complex and an intermediate form of rTveIF-5A. The two forms were subsequently separated by ion exchange chromatography to identify the hypusine residue by MS/MS analysis. Moreover, mutations in TvDHS showed that the putative HE motif present in this enzyme is involved in the hydroxylation of TveIF-5A. We observed that only hypusine-containing TveIF-5A was bound to an RNA hairpin ERE structure from the cox-2 gene, which contains the AAAUGUCACAC consensus sequence. Interestingly, 2DE-WB assays, using parasites that were grown in DAB-culture conditions and transferred to exogenous putrescine, showed the new isoform of TveIF-5A. In summary, our results indicate that T. vaginalis contains an active TvDHS capable of modifying the precursor TveIF-5A protein, which subsequently exhibits RNA binding activity. Copyright © 2015. Published by Elsevier B.V.

  1. Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system.

    Science.gov (United States)

    Slama, Nawel; Leiba, Jade; Eynard, Nathalie; Daffé, Mamadou; Kremer, Laurent; Quémard, Annaïk; Molle, Virginie

    2011-09-02

    The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. Fatty acid synthase as a factor required for exercise-induced cognitive enhancement and dentate gyrus cellular proliferation.

    Directory of Open Access Journals (Sweden)

    Nataliya E Chorna

    Full Text Available Voluntary running is a robust inducer of adult hippocampal neurogenesis. Given that fatty acid synthase (FASN, the key enzyme for de novo fatty acid biosynthesis, is critically involved in proliferation of embryonic and adult neural stem cells, we hypothesized that FASN could mediate both exercise-induced cell proliferation in the subgranular zone (SGZ of the dentate gyrus (DG and enhancement of spatial learning and memory. In 20 week-old male mice, voluntary running-induced hippocampal-specific upregulation of FASN was accompanied also by hippocampal-specific accumulation of palmitate and stearate saturated fatty acids. In experiments addressing the functional role of FASN in our experimental model, chronic intracerebroventricular (i.c.v. microinfusions of C75, an irreversible FASN inhibitor, and significantly impaired exercise-mediated improvements in spatial learning and memory in the Barnes maze. Unlike the vehicle-injected mice, the C75 group adopted a non-spatial serial escape strategy and displayed delayed escape latencies during acquisition and memory tests. Furthermore, pharmacologic blockade of FASN function with C75 resulted in a significant reduction, compared to vehicle treated controls, of the number of proliferative cells in the DG of running mice as measured by immunoreactive to Ki-67 in the SGZ. Taken together, our data suggest that FASN plays an important role in exercise-mediated cognitive enhancement, which might be associated to its role in modulating exercise-induced stimulation of neurogenesis.

  3. Defective bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase.

    Science.gov (United States)

    Armour, K E; Armour, K J; Gallagher, M E; Gödecke, A; Helfrich, M H; Reid, D M; Ralston, S H

    2001-02-01

    Nitric oxide (NO) is a pleiotropic signaling molecule that is produced by bone cells constitutively and in response to diverse stimuli such as proinflammatory cytokines, mechanical strain, and sex hormones. Endothelial nitric oxide synthase (eNOS) is the predominant NOS isoform expressed in bone, but its physiological role in regulating bone metabolism remains unclear. Here we studied various aspects of bone metabolism in female mice with targeted disruption of the eNOS gene. Mice with eNOS deficiency (eNOS KO) had reduced bone mineral density, and cortical thinning when compared with WT controls and histomorphometric analysis of bone revealed profound abnormalities of bone formation, with reduced osteoblast numbers, surfaces and mineral apposition rate. Studies in vitro showed that osteoblasts derived from eNOS KO mice had reduced rates of growth when compared with WT and were less well differentiated as reflected by lower levels of alkaline phosphatase activity. Mice with eNOS deficiency lost bone normally following ovariectomy but exhibited a significantly blunted anabolic response to high dose exogenous estrogen. We conclude that the eNOS pathway plays an essential role in regulating bone mass and bone turnover by modulating osteoblast function.

  4. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    Energy Technology Data Exchange (ETDEWEB)

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A. [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States); Nusrat, Asma, E-mail: anusrat@emory.edu [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States)

    2010-07-02

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  5. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    International Nuclear Information System (INIS)

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A.; Nusrat, Asma

    2010-01-01

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  6. 2C-Methyl- D- erythritol 2,4-cyclodiphosphate synthase from Stevia rebaudiana Bertoni is a functional gene.

    Science.gov (United States)

    Kumar, Hitesh; Singh, Kashmir; Kumar, Sanjay

    2012-12-01

    Stevia [Stevia rebaudiana (Bertoni)] is a perennial herb which accumulates sweet diterpenoid steviol glycosides (SGs) in its leaf tissue. SGs are synthesized by 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Of the various enzymes of the MEP pathway, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS) (encoded by MDS) catalyzes the cyclization of 4-(cytidine 5' diphospho)-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate. Complementation of the MDS knockout mutant strain of Escherichia coli, EB370 with putative MDS of stevia (SrMDS) rescued the lethal mutant, suggesting SrMDS to be a functional gene. Experiments conducted in plant growth chamber and in the field suggested SrMDS to be a light regulated gene. Indole 3-acetic acid (IAA; 50, 100 μM) down-regulated the expression of SrMDS at 4 h of the treatment, whereas, abscisic acid did not modulate its expression. A high expression of SrMDS was observed during the light hours of the day as compared to the dark hours. The present work established functionality of SrMDS and showed the role of light and IAA in regulating expression of SrMDS.

  7. Policing starter unit selection of the enterocin type II polyketide synthase by the type II thioesterase EncL.

    Science.gov (United States)

    Kalaitzis, John A; Cheng, Qian; Meluzzi, Dario; Xiang, Longkuan; Izumikawa, Miho; Dorrestein, Pieter C; Moore, Bradley S

    2011-11-15

    Enterocin is an atypical type II polyketide synthase (PKS) product from the marine actinomycete 'Streptomyces maritimus'. The enterocin biosynthesis gene cluster (enc) codes for proteins involved in the assembly and attachment of the rare benzoate primer that initiates polyketide assembly with the addition of seven malonate molecules and culminates in a Favorskii-like rearrangement of the linear poly-β-ketone to give its distinctive non-aromatic, caged core structure. Fundamental to enterocin biosynthesis, which utilizes a single acyl carrier protein (ACP), EncC, for both priming with benzoate and elongating with malonate, involves maintaining the correct balance of acyl-EncC substrates for efficient polyketide assembly. Here, we report the characterization of EncL as a type II thioesterase that functions to edit starter unit (mis)priming of EncC. We performed a series of in vivo mutational studies, heterologous expression experiments, in vitro reconstitution studies, and Fourier-transform mass spectrometry-monitored competitive enzyme assays that together support the proposed selective hydrolase activity of EncL toward misprimed acetyl-ACP over benzoyl-ACP to facilitate benzoyl priming of the enterocin PKS complex. While this system resembles the R1128 PKS that also utilizes an editing thioesterase (ZhuC) to purge acetate molecules from its initiation module ACP in favor of alkylacyl groups, the enterocin system is distinct in its usage of a single ACP for both priming and elongating reactions with different substrates. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Clp Protease and OR Directly Control the Proteostasis of Phytoene Synthase, the Crucial Enzyme for Carotenoid Biosynthesis in Arabidopsis.

    Science.gov (United States)

    Welsch, Ralf; Zhou, Xiangjun; Yuan, Hui; Álvarez, Daniel; Sun, Tianhu; Schlossarek, Dennis; Yang, Yong; Shen, Guoxin; Zhang, Hong; Rodriguez-Concepcion, Manuel; Thannhauser, Theodore W; Li, Li

    2018-01-08

    Phytoene synthase (PSY) is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits (i.e., ClpS1, ClpC1, and ClpD). High levels of PSY and several other carotenogenic enzyme proteins overaccumulate in the clpc1, clpp4, and clpr1-2 mutants. The overaccumulated PSY was found to be partially enzymatically active. Impairment of Clp activity in clpc1 results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE (OR) protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpc1, counterbalancing Clp-mediated proteolysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the quality control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  9. Cyclopropane fatty acid synthase mutants of probiotic human-derived Lactobacillus reuteri are defective in TNF inhibition.

    Science.gov (United States)

    Jones, Sara E; Whitehead, Kristi; Saulnier, Delphine; Thomas, Carissa M; Versalovic, James; Britton, Robert A

    2011-01-01

    Although commensal microbes have been shown to modulate host immune responses, many of the bacterial factors that mediate immune regulation remain unidentified. Select strains of human-derived Lactobacillus reuteri synthesize immunomodulins that potently inhibit production of the inflammatory cytokine TNF. In this study, genetic and genomic approaches were used to identify and investigate L. reuteri genes required or human TNF immunomodulatory activity. Analysis of membrane fatty acids from multiple L. reuteri strains cultured in MRS medium showed that only TNF inhibitory strains produced the cyclopropane fatty acid (CFA) lactobacillic acid. The enzyme cyclopropane fatty acid synthase is required for synthesis of CFAs such as lactobacillic acid, therefore the cfa gene was inactivated and supernatants from the cfa mutant strain were assayed for TNF inhibitory activity. We found that supernatants from the wild-type strain, but not the cfa mutant, suppressed TNF production by activated THP-1 human monocytoid cells Although this suggested a direct role for lactobacillic acid in immunomodulation, purified lactobacillic acid did not suppress TNF at physiologically relevant concentrations. We further analyzed TNF inhibitory and TNF non-inhibitory strains under different growth conditions and found that lactobacillic acid production did not correlate with TNF inhibition. These results indicate that cfa indirectly contributed to L. reuter immunomodulatory activity and suggest that other mechanisms, such as decreased membrane fluidity or altered expression of immunomodulins, result in the loss of TNF inhibitory activity. By increasing our understanding of immunomodulation by probiotic species, beneficial microbes can be rationally selected to alleviate intestinal inflammation.

  10. Oxidative stress inactivates cobalamin-independent methionine synthase (MetE in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Elise R Hondorp

    2004-11-01

    Full Text Available In nature, Escherichia coli are exposed to harsh and non-ideal growth environments-nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE. We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert

  11. NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with μ-Opioid Agonist Activity.

    Science.gov (United States)

    Renton, Paul; Green, Brenda; Maddaford, Shawn; Rakhit, Suman; Andrews, John S

    2012-03-08

    A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the μ-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the μ-opioid GPCR was predicated on the modulatory role of nitric oxide on μ-opioid receptor function. Structure-activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 μM), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent μ-opioid binding affinity, K i = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 μM). This work represents a novel approach in the development of new analgesics for the treatment of pain.

  12. Increased and Altered Fragrance of Tobacco Plants after Metabolic Engineering Using Three Monoterpene Synthases from Lemon

    Science.gov (United States)

    Lücker, Joost; Schwab, Wilfried; van Hautum, Bianca; Blaas, Jan; van der Plas, Linus H. W.; Bouwmeester, Harro J.; Verhoeven, Harrie A.

    2004-01-01

    Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting β-pinene, limonene, and γ-terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes. PMID:14718674

  13. Modified cellulose synthase gene from Arabidopsis thaliana confers herbicide resistance to plants

    Science.gov (United States)

    Somerville, Chris R [Portola Valley, CA; Scheible, Wolf [Golm, DE

    2007-07-10

    Cellulose synthase ("CS"), a key enzyme in the biosynthesis of cellulose in plants is inhibited by herbicides comprising thiazolidinones such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl)phenyl-4-thiazolidinone (TZ), isoxaben and 2,6-dichlorobenzonitrile (DCB). Two mutant genes encoding isoxaben and TZ-resistant cellulose synthase have been isolated from isoxaben and TZ-resistant Arabidopsis thaliana mutants. When compared with the gene coding for isoxaben or TZ-sensitive cellulose synthase, one of the resistant CS genes contains a point mutation, wherein glycine residue 998 is replaced by an aspartic acid. The other resistant mutation is due to a threonine to isoleucine change at amino acid residue 942. The mutant CS gene can be used to impart herbicide resistance to a plant; thereby permitting the utilization of the herbicide as a single application at a concentration which ensures the complete or substantially complete killing of weeds, while leaving the transgenic crop plant essentially undamaged.

  14. Crystallization and preliminary crystallographic analysis of an octaketide-producing plant type III polyketide synthase

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Hiroyuki [Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511 (Japan); Kondo, Shin; Kato, Ryohei [Innovation Center Yokohama, Mitsubishi Chemical Corporation, 1000 Kamoshida, Aoba, Yokohama, Kanagawa 227-8502 (Japan); Wanibuchi, Kiyofumi; Noguchi, Hiroshi [School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526 (Japan); Sugio, Shigetoshi, E-mail: sugio.shigetoshi@mw.m-kagaku.co.jp [Innovation Center Yokohama, Mitsubishi Chemical Corporation, 1000 Kamoshida, Aoba, Yokohama, Kanagawa 227-8502 (Japan); Abe, Ikuro, E-mail: sugio.shigetoshi@mw.m-kagaku.co.jp [School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526 (Japan); PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kohno, Toshiyuki, E-mail: sugio.shigetoshi@mw.m-kagaku.co.jp [Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511 (Japan)

    2007-11-01

    Octaketide synthase from A. arborescens has been overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to 2.6 Å. Octaketide synthase (OKS) from Aloe arborescens is a plant-specific type III polyketide synthase that produces SEK4 and SEK4b from eight molecules of malonyl-CoA. Recombinant OKS expressed in Escherichia coli was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group I422, with unit-cell parameters a = b = 110.2, c = 281.4 Å, α = β = γ = 90.0°. Diffraction data were collected to 2.6 Å resolution using synchrotron radiation at BL24XU of SPring-8.

  15. Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii

    International Nuclear Information System (INIS)

    Huynh, Frederick; Tan, Tien-Chye; Swaminathan, Kunchithapadam; Patel, Bharat K. C.

    2004-01-01

    The first crystallographic study of a sucrose phosphate synthase from H. orenii, an organism that is both thermophilic and halophilic, is reported. The protein crystal diffracts X-rays to 3.01 Å. This is the first report of the crystallization of a sucrose phosphate synthase (SPS; EC 2.4.1.14). It also constitutes the first study of a sucrose phosphate synthase from a non-photosynthetic thermohalophilic anaerobic bacterium, Halothermothrix orenii. The purified recombinant spsA protein has been crystallized in the monoclinic space group C2, with unit-cell parameters a = 154.2, b = 47.9, c = 72.3 Å, β = 103.16°, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 3.01 Å. Heavy-metal and halide-soaking trials are currently in progress to solve the structure

  16. Broad Substrate Specificity of the Loading Didomain of the Lipomycin Polyketide Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Yuzawa, S; Eng, CH; Katz, L; Keasling, JD

    2013-06-04

    LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze the polyketide chain initiation reaction using isobutyryl-CoA as a substrate, followed by an elongation reaction with methylmalonyl-CoA to start the biosynthesis of antibiotic alpha-lipomycin in Streptomyces aureofaciens Tu117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in Escherichia coli, and its substrate specificity was investigated in vitro. Surprisingly, several different acyl-CoAs, including isobutyryl-CoA, were accepted as the starter substrates, while no product was observed with acetyl-CoA. These results demonstrate the broad substrate specificity of LipPks1 and may be applied to producing new antibiotics.

  17. Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

    Science.gov (United States)

    Cheng, Jiujun; Charles, Trevor C

    2016-09-01

    Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

  18. Occurrence of theobromine synthase genes in purine alkaloid-free species of Camellia plants.

    Science.gov (United States)

    Ishida, Mariko; Kitao, Naoko; Mizuno, Kouichi; Tanikawa, Natsu; Kato, Misako

    2009-02-01

    Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are purine alkaloids that are present in high concentrations in plants of some species of Camellia. However, most members of the genus Camellia contain no purine alkaloids. Tracer experiments using [8-(14)C]adenine and [8-(14)C]theobromine showed that the purine alkaloid pathway is not fully functional in leaves of purine alkaloid-free species. In five species of purine alkaloid-free Camellia plants, sufficient evidence was obtained to show the occurrence of genes that are homologous to caffeine synthase. Recombinant enzymes derived from purine alkaloid-free species showed only theobromine synthase activity. Unlike the caffeine synthase gene, these genes were expressed more strongly in mature tissue than in young tissue.

  19. Human METTL12 is a mitochondrial methyltransferase that modifies citrate synthase.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2017-06-01

    The protein methylome in mammalian mitochondria has been little studied until recently. Here, we describe that lysine-368 of human citrate synthase is methylated and that the modifying enzyme, localized in the mitochondrial matrix, is methyltransferase-like protein 12 (METTL12), a member of the family of 7β-strand methyltransferases. Lysine-368 is near the active site of citrate synthase, but removal of methylation has no effect on its activity. In mitochondria, it is possible that some or all of the enzymes of the citric acid cycle, including citrate synthase, are organized in metabolons to facilitate the channelling of substrates between participating enzymes. Thus, possible roles for the methylation of Lys-368 are in controlling substrate channelling itself, or in influencing protein-protein interactions in the metabolon. © 2017 The Authors FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  20. Interaction between neuronal nitric oxide synthase signaling and temperature influences sarcoplasmic reticulum calcium leak: role of nitroso-redox balance.

    Science.gov (United States)

    Dulce, Raul A; Mayo, Vera; Rangel, Erika B; Balkan, Wayne; Hare, Joshua M

    2015-01-02

    Although nitric oxide (NO) signaling modulates cardiac function and excitation-contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects. To test the hypothesis that temperature profoundly affects nitroso-redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca(2+)) leak. We measured SR Ca(2+) leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase-deficient mice-1 [NOS1(-/-)]), and hyper S-nitrosoglutathione reductase-deficient (GSNOR(-/-)) mice. In WT cardiomyocytes, SR Ca(2+) leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1(-/-) cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR(-/-) cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1(-/-) cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1(-/-) cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1(-/-) cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca(2+) content in NOS1(-/-) cells but had no effect in WT or GSNOR(-/-). Ca(2+) leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR

  1. Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms With Acute Rejection in Liver Transplant Recipients.

    Science.gov (United States)

    Azarpira, Negar; Namazi, Soha; Malahi, Sayan; Kazemi, Kourosh

    2016-06-01

    Polymorphisms of the endothelial nitric oxide synthase gene have been associated with altered endothelial nitric oxide synthase activity. The purpose of this study was to investigate the relation between endothelial nitric oxide synthase -786T/C and 894G/T polymorphism and their haplotypes on the occurrence of acute rejection episodes in liver transplant recipients. We conducted a case control study in which 100 liver transplant recipients and 100 healthy controls were recruited from Shiraz Transplant Center. The patients used triple therapy including tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression maintenance. DNA was extracted from peripheral blood and endothelial nitric oxide synthase polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism. Patients included 60 men and 40 women (mean age, 32.35 ± 10.2 y). There was a significant association of endothelial nitric oxide synthase 894G/T and acute rejection episode. The GT* gen-otype and acute rejection episodes had a significant association (odds ratio, 2.42; 95% confidence interval, 0.97-6.15; P = .03). The GG and GT* genotype and T* allele frequency were significantly different between patients and control subjects (P = .001). Haplotype TT* was higher in recipients than control subjects (odds ratio, 2.17; 95% confidence interval, 1.12-4.25; P = .01). Haplotype TG was higher in the control group (odds ratio, 0.62; 95% confidence interval, 0.40-0.96; P = .02). Our results suggest a relation between different endothelial nitric oxide synthase geno-types and risk of acute rejection episodes. However, further study is necessary to determine genetic susceptibility for transplant patients.

  2. Molecular cloning of a seed specific multifunctional RFO synthase/ galactosylhydrolase in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Roman eGangl

    2015-09-01

    Full Text Available Stachyose is among the raffinose family oligosaccharides one of the major water-soluble carbohydrates next to sucrose in seeds of a number of plant species. Especially in leguminous seeds, e.g. chickpea, stachyose is reported as the major component. In contrast to their ambiguous potential as essential source of carbon for germination, raffinose family oligosaccharides are indigestible for humans and can contribute to diverse abdominal disorders.In the genome of Arabidopsis thaliana, six putative raffinose synthase genes are reported, whereas little is known about these putative raffinose synthases and their biochemical characteristics or their contribution to the raffinose family oligosaccharide physiology in A. thaliana.In this paper, we report on the molecular cloning, functional expression in Escherichia coli and purification of recombinant AtRS4 from A. thaliana and the biochemical characterisation of the putative stachyose synthase (AtSTS, At4g01970 as a raffinose and high affinity stachyose synthase (Km for raffinose 259.2 ± 21.15 µM as well as stachyose and galactinol specific galactosylhydrolase. A T-DNA insertional mutant in the AtRS4 gene was isolated. Only sqPCR from WT siliques showed a specific transcriptional AtRS4 PCR product. Metabolite measurements in seeds of ΔAtRS4 mutant plants revealed a total loss of stachyose in ΔAtRS4 mutant seeds. We conclude that AtRS4 is the only stachyose synthase in the genome of A. thaliana that AtRS4 represents a key regulation mechanism in the raffinose family oligosaccharide physiology of A. thaliana due to its multifunctional enzyme activity and that AtRS4 is possibly the second seed specific raffinose synthase beside AtRS5, which is responsible for Raf-accumulation under abiotic stress.

  3. Homocysteine threshold value based on cystathionine beta synthase and paraoxonase 1 activities in mice.

    Science.gov (United States)

    Hamelet, J; Aït-Yahya-Graison, E; Matulewicz, E; Noll, C; Badel-Chagnon, A; Camproux, A-C; Demuth, K; Paul, J-L; Delabar, J M; Janel, N

    2007-12-01

    Hyperhomocysteinaemia is a metabolic disorder associated with the development of premature atherosclerosis. Among the determinants which predispose to premature thromboembolic and atherothrombotic events, serum activity of paraoxonase 1, mainly synthesized in the liver, has been shown to be a predictor of cardiovascular disease and to be negatively correlated with serum homocysteine levels in human. Even though treatments of hyperhomocysteinaemic patients ongoing cardiovascular complications are commonly used, it still remains unclear above which homocysteine level a preventive therapy should be started. In order to establish a threshold of plasma homocysteine concentration we have analyzed the hepatic cystathionine beta synthase and paraoxonase 1 activities in a moderate to intermediate murine model of hyperhomocysteinaemia. Using wild type and heterozygous cystathionine beta synthase deficient mice fed a methionine enriched diet or a control diet, we first studied the link between cystathionine beta synthase and paraoxonase 1 activities and plasma homocysteine concentration. Among the animals used in this study, we observed a negative correlation between plasma homocysteine level and cystathionine beta synthase activity (rho=-0.52, P=0.0008) or paraoxonase 1 activity (rho=-0.49, P=0.002). Starting from these results, a homocysteine cut-off value of 15 microm has been found for both cystathionine beta synthase (P=0.0003) and paraoxonase 1 (P=0.0007) activities. Our results suggest that both cystathionine beta synthase and paraoxonase 1 activities are significantly decreased in mice with a plasma homocysteine value greater than 15 microm. In an attempt to set up preventive treatment for cardiovascular disease our results indicate that treatments should be started from 15 microm of plasma homocysteine.

  4. Isolation and characterization of three new monoterpene synthases from Artemisia annua

    OpenAIRE

    Ju-Xin eRuan; Jian-Xu eLi; Xin eFang; Ling-Jian eWang; Wen-Li eHu; Xiao-Ya eChen; Changqing eYang

    2016-01-01

    Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5 and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with...

  5. Mechanistic studies of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

    International Nuclear Information System (INIS)

    Dotson, G.D.; Woodard, R.W.

    1994-01-01

    The enzyme 3-deOXY-D-manno-octulosonic acid 8-phosphate synthase (KDO 8-P synthase) catalyses the condensation of arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give the unique eight-carbon acidic sugar 3-deoxy-D-nianno-octulosonic acid 8-phosphate (KDO 8-P) found only in gram-negative bacteria and required for lipid A maturation and cellular growth. The E. coli gene kdsA that encodes KDO 8-P synthase has been amplified by standard PCR methodologies. The synthetic gene, subcloned into the expression vector pT7-7 was used to infect E. coli BL 21 (DE 3). Purification of crude supernatant from this transformant on Q Sepharose yields >200 mg of near-homogeneous KDO 8-P synthase per liter of cell culture. To explore the mechanism of KDO 8-P synthase, we prepared (E)- and (Z)-(3 2 H)PEP, (2- 13 C)PEP, and (2- 13 C, 18 O)PEP chemically from the appropriately labeled 3-bromopyruvates by reaction with trimethylphosphite under Perkow reaction conditions. Our 1 H-NMR analysis of the stereochemistry at C3 of the KDO 8-Ps, obtained by separate incubation of (E)- and (Z)-(3- 2 H)PEP with A 5-P in the presence of KDO 8-P synthase, demonstrated that the reaction is stereospecific with respect to both the C3 of PEP and the C1 carbonyl of A 5-P. (Z)-(3- 2 H)PEP gave predominantly (3S)-(3 2 H)KDO 8-P and (E)-(3- 2 H)PEP gave predominantly (3R)-(3 2 H)KDO-8P, which indicates condensation of the si face of PEP upon the re face of A 5-P-an orientation analogous to that seen with the similar aldehyde Iyase DAH 7-P synthase. The fate of the enolic oxygen of (2- 13 C, 18 O)PEP, during the course of the KDO 8-P synthase-catalyzed reaction as monitored by both 13 C- and 31 P-NMR spectroscopy demonstrated that the inorganic phosphate (Pi) and not the KDO 8-P contained the 18 O

  6. Isolation and identification of a thermophilic strain producing trehalose synthase from geothermal water in China.

    Science.gov (United States)

    Zhu, Yueming; Zhang, Jun; Wei, Dongsheng; Wang, Yufan; Chen, Xiaoyun; Xing, Laijun; Li, Mingchun

    2008-08-01

    A slightly thermophilic strain, CBS-01, producing trehalose synthase (TreS), was isolated from geothermal water in this study. According to the phenotypic characteristics and phylogenetic analysis of the 16s rRNA gene sequence, it was identified as Meiothermus ruber. The trehalose synthase gene of Meiothermus ruber CBS-01 was cloned by polymerase chain reaction and sequenced. The TreS gene consisted of 2,895 nucleotides, which specified a 964-amino-acid protein. This novel TreS catalyzed reversible interconversion of maltose and trehalose.

  7. Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

    Directory of Open Access Journals (Sweden)

    Ro Dae-Kyun

    2009-07-01

    Full Text Available Abstract Background Sesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development. Results Glandular trichomes of sunflower (Helianthus annuus L. were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of in vitro and in vivo characterization of sesquiterpene synthase gene products in Escherichia coli and Saccharomyces cerevisiae, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low in vitro activity, the third enzyme was expressed in vivo in yeast as a thioredoxin-fusion protein for functional characterization. In in vivo assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon δ-cadinene as one of the two main products with α-muuorlene, β-caryophyllene, α-humulene and α-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes

  8. Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare

    DEFF Research Database (Denmark)

    Zerbe, Philipp; Chiang, Angela; Dullat, Harpreet

    2014-01-01

    Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five...... different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3...

  9. Mechanistic studies of 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Dotson, G.D.; Woodard, R.W. [Univ. of Michigan, Ann Arbor, MI (United States)

    1994-12-01

    The enzyme 3-deOXY-D-manno-octulosonic acid 8-phosphate synthase (KDO 8-P synthase) catalyses the condensation of arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give the unique eight-carbon acidic sugar 3-deoxy-D-nianno-octulosonic acid 8-phosphate (KDO 8-P) found only in gram-negative bacteria and required for lipid A maturation and cellular growth. The E. coli gene kdsA that encodes KDO 8-P synthase has been amplified by standard PCR methodologies. The synthetic gene, subcloned into the expression vector pT7-7 was used to infect E. coli BL 21 (DE 3). Purification of crude supernatant from this transformant on Q Sepharose yields >200 mg of near-homogeneous KDO 8-P synthase per liter of cell culture. To explore the mechanism of KDO 8-P synthase, we prepared (E)- and (Z)-(3{sup 2}H)PEP, (2-{sup 13}C)PEP, and (2-{sup 13}C,{sup 18}O)PEP chemically from the appropriately labeled 3-bromopyruvates by reaction with trimethylphosphite under Perkow reaction conditions. Our {sup 1}H-NMR analysis of the stereochemistry at C3 of the KDO 8-Ps, obtained by separate incubation of (E)- and (Z)-(3-{sup 2}H)PEP with A 5-P in the presence of KDO 8-P synthase, demonstrated that the reaction is stereospecific with respect to both the C3 of PEP and the C1 carbonyl of A 5-P. (Z)-(3-{sup 2}H)PEP gave predominantly (3S)-(3{sup 2}H)KDO 8-P and (E)-(3-{sup 2}H)PEP gave predominantly (3R)-(3{sup 2}H)KDO-8P, which indicates condensation of the si face of PEP upon the re face of A 5-P-an orientation analogous to that seen with the similar aldehyde Iyase DAH 7-P synthase. The fate of the enolic oxygen of (2-{sup 13}C, {sup 18}O)PEP, during the course of the KDO 8-P synthase-catalyzed reaction as monitored by both {sup 13}C- and {sup 31}P-NMR spectroscopy demonstrated that the inorganic phosphate (Pi) and not the KDO 8-P contained the {sup 18}O.

  10. Development of intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase for discriminating Curcuma species.

    Science.gov (United States)

    Kita, Tomoko; Komatsu, Katsuko; Zhu, Shu; Iida, Osamu; Sugimura, Koji; Kawahara, Nobuo; Taguchi, Hiromu; Masamura, Noriya; Cai, Shao-Qing

    2016-03-01

    Various Curcuma rhizomes have been used as medicines or spices in Asia since ancient times. It is very difficult to distinguish them morphologically, especially when they are boiled and dried, which causes misidentification leading to a loss of efficacy. We developed a method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. This method could apply to identification of not only fresh plants but also samples of crude drugs or edible spices. By applying this method to Curcuma specimens and samples, and constructing a dendrogram based on these markers, seven Curcuma species were clearly distinguishable. Moreover, Curcuma longa specimens were geographically distinguishable. On the other hand, Curcuma kwangsiensis (gl type) specimens also showed intraspecies polymorphism, which may have occurred as a result of hybridization with other Curcuma species. The molecular method we developed is a potential tool for global classification of the genus Curcuma. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. The Polyketide Components of Waxes and the Cer-cqu Gene Cluster Encoding a Novel Polyketide Synthase, the β-Diketone Synthase, DKS.

    Science.gov (United States)

    von Wettstein-Knowles, Penny

    2017-07-10

    The primary function of the outermost, lipophilic layer of plant aerial surfaces, called the cuticle, is preventing non-stomatal water loss. Its exterior surface is often decorated with wax crystals, imparting a blue-grey color. Identification of the barley Cer-c , -q and -u genes forming the 101 kb Cer-cqu gene cluster encoding a novel polyketide synthase-the β-diketone synthase (DKS), a lipase/carboxyl transferase, and a P450 hydroxylase, respectively, establishes a new, major pathway for the synthesis of plant waxes. The major product is a β-diketone (14,16-hentriacontane) aliphatic that forms long, thin crystalline tubes. A pathway branch leads to the formation of esterified alkan-2-ols.

  12. Predicting the functions and specificity of triterpenoid synthases: a mechanism-based multi-intermediate docking approach.

    Directory of Open Access Journals (Sweden)

    Bo-Xue Tian

    2014-10-01

    Full Text Available Terpenoid synthases construct the carbon skeletons of tens of thousands of natural products. To predict functions and specificity of triterpenoid synthases, a mechanism-based, multi-intermediate docking approach is proposed. In addition to enzyme function prediction, other potential applications of the current approach, such as enzyme mechanistic studies and enzyme redesign by mutagenesis, are discussed.

  13. Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

    Science.gov (United States)

    Souza-Moreira, Tatiana M.; Alves, Thaís B.; Pinheiro, Karina A.; Felippe, Lidiane G.; de Lima, Gustavo M. A.; Watanabe, Tatiana F.; Barbosa, Cristina C.; Santos, Vânia A. F. F. M.; Lopes, Norberto P.; Valentini, Sandro R.; Guido, Rafael V. C.; Furlan, Maysa; Zanelli, Cleslei F.

    2016-11-01

    Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

  14. Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

    DEFF Research Database (Denmark)

    Kadziola, Anders; Jepsen, Clemens H; Johansson, Eva

    2005-01-01

    The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and...

  15. Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Gaucher-Wieczorek, Florence; Guérineau, Vincent; Touboul, David; Thétiot-Laurent, Sophie; Pelissier, Franck; Badet-Denisot, Marie-Ange; Badet, Bernard; Durand, Philippe

    2014-08-01

    Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. The C-terminal peptide of Aquifex aeolicus riboflavin synthase directs encapsulation of native and foreign guests by a cage-forming lumazine synthase.

    Science.gov (United States)

    Azuma, Yusuke; Zschoche, Reinhard; Hilvert, Donald

    2017-06-23

    Encapsulation of specific enzymes in self-assembling protein cages is a hallmark of bacterial compartments that function as counterparts to eukaryotic organelles. The cage-forming enzyme lumazine synthase (LS) from Bacillus subtilis (BsLS), for example, encapsulates riboflavin synthase (BsRS), enabling channeling of lumazine from the site of its generation to the site of its conversion to vitamin B 2 Elucidating the molecular mechanisms underlying the assembly of these supramolecular complexes could help inform new approaches for metabolic engineering, nanotechnology, and drug delivery. To that end, we investigated a thermostable LS from Aquifex aeolicus (AaLS) and found that it also forms cage complexes with the cognate riboflavin synthase (AaRS) when both proteins are co-produced in the cytosol of Escherichia coli A 12-amino acid-long peptide at the C terminus of AaRS serves as a specific localization sequence responsible for targeting the guest to the protein compartment. Sequence comparisons suggested that analogous peptide segments likely direct RS complexation by LS cages in other bacterial species. Covalent fusion of this peptide tag to heterologous guest molecules led to their internalization into AaLS assemblies both in vivo and in vitro , providing a firm foundation for creating tailored biomimetic nanocompartments for medical and biotechnological applications. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Electroabsorption optical modulator

    Energy Technology Data Exchange (ETDEWEB)

    Skogen, Erik J.

    2017-11-21

    An electroabsorption modulator incorporates waveguiding regions along the length of the modulator that include quantum wells where at least two of the regions have quantum wells with different bandgaps. In one embodiment of the invention, the regions are arranged such that the quantum wells have bandgaps with decreasing bandgap energy along the length of the modulator from the modulator's input to its output. The bandgap energy of the quantum wells may be decreased in discrete steps or continuously. Advantageously, such an arrangement better distributes the optical absorption as well as the carrier density along the length of the modulator. Further advantageously, the modulator may handle increased optical power as compared with prior art modulators of similar dimensions, which allows for improved link gain when the optical modulator is used in an analog optical communication link.

  18. CDC 7600 module slice

    CERN Multimedia

    Each module contained 8 circuit cards for a total of about 300-500 uncovered transistors packaged with face plates so the Freon plates wouldn't touch the circuits. (cooling plates that surrounded each module).

  19. Exploration Augmentation Module Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The Exploration Augmentation Module (EAM) project goal is to design and deliver a flight module that is to be deployed to Earth-Lunar Distant Retrograde Orbit (DRO)....

  20. Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

    Directory of Open Access Journals (Sweden)

    Mirian Perez Maluf

    2009-01-01

    Full Text Available In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

  1. CDC 6600 Cordwood Module

    CERN Multimedia

    1964-01-01

    The CDC 6600 cordwood module containing 64 silicon transistors. The module was mounted between two plates that were cooled conductive by a refrigeration unit via the front panel. The construction of this module uses the cord method, so called because the resistors seem to be stacked like cord between the two circuit boards in order to obtain a high density. The 6600 model contained nearly 6,000 such modules.

  2. Modulating lignin in plants

    Science.gov (United States)

    Apuya, Nestor; Bobzin, Steven Craig; Okamuro, Jack; Zhang, Ke

    2013-01-29

    Materials and methods for modulating (e.g., increasing or decreasing) lignin content in plants are disclosed. For example, nucleic acids encoding lignin-modulating polypeptides are disclosed as well as methods for using such nucleic acids to generate transgenic plants having a modulated lignin content.

  3. An investigation into modulators

    International Nuclear Information System (INIS)

    Heine, E.

    1988-01-01

    In the framework of the MEA-update it is important to establish which modulators are necessary. In this report it is lookedif the existing modulators can be maintained or new modulators have to be made. Besides technical aspects also material expenses and necessary manpower play a role. 12 figs.; 6 tabs

  4. Weakly Coretractable Modules

    Science.gov (United States)

    Hadi, Inaam M. A.; Al-aeashi, Shukur N.

    2018-05-01

    If R is a ring with identity and M is a unitary right R-module. Here we introduce the class of weakly coretractable module. Some basic properties are investigated and some relationships between these modules and other related one are introduced.

  5. Amplitude modulation detection with concurrent frequency modulation.

    Science.gov (United States)

    Nagaraj, Naveen K

    2016-09-01

    Human speech consists of concomitant temporal modulations in amplitude and frequency that are crucial for speech perception. In this study, amplitude modulation (AM) detection thresholds were measured for 550 and 5000 Hz carriers with and without concurrent frequency modulation (FM), at AM rates crucial for speech perception. Results indicate that adding 40 Hz FM interferes with AM detection, more so for 5000 Hz carrier and for frequency deviations exceeding the critical bandwidth of the carrier frequency. These findings suggest that future cochlear implant processors, encoding speech fine-structures may consider limiting the FM to narrow bandwidth and to low frequencies.

  6. Arctigenin reduces blood pressure by modulation of nitric oxide synthase and NADPH oxidase expression in spontaneously hypertensive rats.

    Science.gov (United States)

    Liu, Ying; Wang, Guoyuan; Yang, Mingguang; Chen, Haining; zhao, Yan; Yang, Shucai; Sun, Changhao

    2015-12-25

    Arctigenin is a bioactive constituent from dried seeds of Arctium lappa L., which was traditionally used as medicine. Arctigenin exhibits various bioactivities, but its effects on blood pressure regulation are still not widely studied. In this study, we investigated antihypertensive effects of arctigenin by long-term treatment in spontaneously hypertensive rats (SHRs). Arctigenin (50 mg/kg) or vehicle was administered to SHRs or Wistar rats as negative control by oral gavage once a day for total 8 weeks. Nifedipine (3 mg/kg) was used as a positive drug control. After treatment, hemodynamic and physical parameters, vascular reactivity in aorta, the concentration of plasma arctigenin and serum thromboxane B2, NO release and vascular p-eNOS, p-Akt, caveolin-1 protein expression, and vascular superoxide anion generation and p47phox protein expression were detected and analyzed. The results showed that arctigenin significantly reduced systolic blood pressure and ameliorated endothelial dysfunction of SHRs. Arctigenin reduced the levels of thromboxane B2 in plasma and superoxide anion in thoracic aorta of SHRs. Furthermore, arctigenin increased the NO production by enhancing the phosphorylation of Akt and eNOS (Ser 1177), and inhibiting the expression of NADPH oxidase in thoracic aorta of SHRs. Our data suggested that antihypertensive mechanisms of arctigenin were associated with enhanced eNOS phosphorylation and decreased NADPH oxidase-mediated superoxide anion generation. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon

    NARCIS (Netherlands)

    Lücker, J.; Schwab, W.; Hautum, van B.; Blaas, J.; Plas, van der L.H.W.; Bouwmeester, H.J.; Verhoeven, H.A.

    2004-01-01

    Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one

  8. The influence of monoterpene synthase transformation on the odour of tabacco.

    NARCIS (Netherlands)

    Tamer, el M.K.; Smeets, M.A.M.; Holthuysen, N.T.E.; Lucker, J.; Tang, A.; Roozen, J.P.; Bouwmeester, H.J.; Voragen, A.G.J.

    2003-01-01

    Monoterpenes are an important class of terpenoids that are commonly present in plant essential oils. These can be extracted from plants and are used in the flavouring and perfumery industry. Monoterpene synthases are the key enzymes in monoterpene biosynthesis, as they catalyse the cyclisation of

  9. Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

    International Nuclear Information System (INIS)

    Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao; Yu, Xiyan; Wang, Xiufeng

    2010-01-01

    To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

  10. Lack of endothelial nitric oxide synthase aggravates murine accelerated anti-glomerular basement membrane glomerulonephritis

    NARCIS (Netherlands)

    Heeringa, P; van Goor, H; Itoh-Lindstrom, Y; Maeda, N; Falk, RJ; Assmann, KJM; Kallenberg, CGM; Jennette, JC

    Nitric oxide (NO) radicals generated by endothelial nitric oxide synthase (eNOS) are involved in the regulation of vascular tone. In addition, NO radicals derived from eNOS inhibit platelet aggregation and leukocyte adhesion to the endothelium and, thus, may have anti-inflammatory effects. To study

  11. Nitric oxide synthase expression and apoptotic cell death in brains of AIDS and AIDS dementia patients

    NARCIS (Netherlands)

    Vincent, V. A.; de Groot, C. J.; Lucassen, P. J.; Portegies, P.; Troost, D.; Tilders, F. J.; van Dam, A. M.

    1999-01-01

    To determine the occurrence and cellular localization of inducible nitric oxide synthase (iNOS), NOS activity and its association with cell death in brains of AIDS and AIDS dementia complex (ADC) patients. Post-mortem cerebral cortex tissue of eight AIDS patients, eight ADC patients and eight

  12. Pre- and posttranslational upregulation of muscle-specific glycogen synthase in athletes

    DEFF Research Database (Denmark)

    Vestergaard, H; Andersen, P H; Lund, S

    1994-01-01

    Expression of muscle-specific glycogen synthase (GS) and phosphofructokinase (PFK) was analyzed in seven athletes and eight control subjects who were characterized using the euglycemic, hyperinsulinemic (2 mU.kg-1.min-1) clamp technique in combination with indirect calorimetry and biopsy sampling...

  13. Crystallization of Δ{sup 1}-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

    Energy Technology Data Exchange (ETDEWEB)

    Shoyama, Yoshinari; Takeuchi, Ayako; Taura, Futoshi [Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Tamada, Taro; Adachi, Motoyasu; Kuroki, Ryota [Neutron Science Research Center, Japan Atomic Energy Research Institute, 2-4 Shirakata-Shirane, Tokai, Ibaraki 319-1195 (Japan); Shoyama, Yukihiro; Morimoto, Satoshi [Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

    2005-08-01

    Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase from C. sativa was crystallized. The crystal diffracted to 2.7 Å resolution with sufficient quality for further structure determination. Δ{sup 1}-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å{sup 3} Da{sup −1} assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

  14. Molecular characterization of two alkylresorcylic acid synthases from Sordariomycetes fungi

    DEFF Research Database (Denmark)

    Ramakrishnan, Dhivya; Tiwari, Manish Kumar; Manoharan, Gomathi

    2018-01-01

    Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were...

  15. Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency

    NARCIS (Netherlands)

    Morris, A.A.; Kozich, V.; Santra, S.; Andria, G.; Ben-Omran, T.I.; Chakrapani, A.B.; Crushell, E.; Henderson, M.J.; Hochuli, M.; Huemer, M.; Janssen, M.C.H.; Maillot, F.; Mayne, P.D.; McNulty, J.; Morrison, T.M.; Ogier, H.; O'Sullivan, S.; Pavlikova, M.; Almeida, I.T. de; Terry, A.; Yap, S.; Blom, H.J.; Chapman, K.A.

    2017-01-01

    Cystathionine beta-synthase (CBS) deficiency is a rare inherited disorder in the methionine catabolic pathway, in which the impaired synthesis of cystathionine leads to accumulation of homocysteine. Patients can present to many different specialists and diagnosis is often delayed. Severely affected

  16. Sucrose Synthase Is Associated with the Cell Wall of Tobacco Pollen Tubes

    NARCIS (Netherlands)

    Persia, D.; Cai, G.; Casino, C.; Willemse, M.T.M.; Cresti, M.

    2008-01-01

    Sucrose synthase (Sus; EC 2.4.1.13) is a key enzyme of sucrose metabolism in plant cells, providing carbon for respiration and for the synthesis of cell wall polymers and starch. Since Sus is important for plant cell growth, insights into its structure, localization, and features are useful for

  17. Erratum Aldosterone synthase C-344T, angiotensin II type 1 receptor ...

    Indian Academy of Sciences (India)

    Aldosterone synthase C-344T, angiotensin II type 1 receptor A1166C and 11-β hydroxysteroid dehydrogenase G534A gene polymorphisms and essential hypertension in the population of Odisha, India. Manisha Patnaik, Pallabi Pati, Surendra N. Swain, Manoj K. Mohapatra, Bhagirathi Dwibedi, Shantanu K. Kar.

  18. Isolation of an ATP synthase cDNA from Sinonovacula constricta ...

    African Journals Online (AJOL)

    Yomi

    2012-01-24

    Jan 24, 2012 ... protein involved in temperature challenge in S. constricta. Key words: Sinonovacula constricta, ATP synthase, ... MATERIALS AND METHODS. Experimental animals. Sinonovacula constricta (7 to 8 g ... Dissociation curve analysis of amplification products was performed at the end of each PCR reaction to ...

  19. Crystallization of Δ1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa

    International Nuclear Information System (INIS)

    Shoyama, Yoshinari; Takeuchi, Ayako; Taura, Futoshi; Tamada, Taro; Adachi, Motoyasu; Kuroki, Ryota; Shoyama, Yukihiro; Morimoto, Satoshi

    2005-01-01

    Δ 1 -Tetrahydrocannabinolic acid (THCA) synthase from C. sativa was crystallized. The crystal diffracted to 2.7 Å resolution with sufficient quality for further structure determination. Δ 1 -Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 Å resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 Å. The calculated Matthews coefficient was approximately 4.1 or 2.0 Å 3 Da −1 assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively

  20. Structure of Salmonella typhimurium OMP Synthase in a Complete Substrate Complex

    DEFF Research Database (Denmark)

    Grubmeyer, Charles; Hansen, Michael Riis; Fedorov, Alexander A.

    2012-01-01

    Dimeric Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10), a key enzyme in de novo pyrimidine nucleotide synthesis, has been cocrystallized in a complete substrate E·MgPRPP·orotate complex and the structure determined to 2.2 Å resolution. This structure resem...

  1. Cloning and characterization of ATP synthase CF1 α gene from ...

    African Journals Online (AJOL)

    ATP synthase CF1 α subunit protein is a key enzyme for energy metabolism in plant kingdom, and plays an important role in multiple cell processes. In this study, the complete atpA gene (accession no. JN247444) was cloned from sweet potato (Ipomoea batatas L. Lam) by reverse transcriptasepolymerase chain reaction ...

  2. Organellar and cytosolic localization of four phosphoribosyl diphosphate synthase isozymes in spinach

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1999-01-01

    Four cDNAs encoding phosphoribosyl diphosphate (PRPP) synthase were isolated from a spinach (Spinacia oleracea) cDNA library by complementation of an Escherichia coli Δprs mutation. The four gene products produced PRPP in vitro from ATP and ribose-5-phosphate. Two of the enzymes (isozymes 1 and 2...

  3. In situ localization of chalcone synthase mRNA in pea root nodule development.

    NARCIS (Netherlands)

    Yang, W.C.; Canter Cremers, H.C.J.; Hogendijk, P.; Katinakis, P.; Wijffelman, C.A.; Franssen, H.J.; Kammen, van A.; Bisseling, T.

    1992-01-01

    In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it

  4. Metabolite profiling of Arabidopsis thaliana (L.) plants transformed with an antisense chalcone synthase gene

    DEFF Research Database (Denmark)

    Le Gall, G.; Metzdorff, Stine Broeng; Pedersen, Jan W.

    2005-01-01

    A metabolite profiling study has been carried out on Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija and a series of transgenic lines of the ecotype transformed with a CHS (chalcone synthase) antisense construct. Compound identifications by LC/MS and H-1 NMR are discussed. The glucosinolate...

  5. Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)

    NARCIS (Netherlands)

    Lunkenbein, S.; Coiner, H.; Vos, de C.H.; Schaart, J.G.; Boone, M.J.; Krens, F.A.; Schwab, W.; Salentijn, E.M.J.

    2006-01-01

    An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens.

  6. of endothelial nitric oxide synthase gene and serum level of vascular ...

    African Journals Online (AJOL)

    uwerhiavwe

    Davignon and Ganz, 2004). NO is synthe- sized via a reaction that includes the conversion of L- arginine to L-citruline catalyzed by endothelial nitric oxide synthase (eNOS), which is one of the three isoforms of the enzyme (Mayer and Hemmens, 1997) ...

  7. Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Kelley, M.J.; Carman, G.M.

    1987-01-01

    The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase was purified 2300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism

  8. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...

  9. Factors influencing gene silencing of granule-bound starch synthase in potato

    NARCIS (Netherlands)

    Heilersig, H.J.B.

    2005-01-01

    In the past, antisense RNA technology was used to modify the composition of potato tuber starch. Potato starch comprises amylose and amylopectin, polymers of glucose. Amylose production in potato is completely dependent on the presence of granule-bound starch synthase I (GBSSI). Inhibition of GBSSI

  10. The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex.

    Science.gov (United States)

    Gay, Darren C; Wagner, Drew T; Meinke, Jessica L; Zogzas, Charles E; Gay, Glen R; Keatinge-Clay, Adrian T

    2016-03-01

    Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

    Science.gov (United States)

    Lewin, Anna; Hederstedt, Lars

    2016-02-01

    Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Nitric Oxide Synthases Reveal a Role for Calmodulin in Controlling Electron Transfer

    Science.gov (United States)

    Abu-Soud, Husam M.; Stuehr, Dennis J.

    1993-11-01

    Nitric oxide (NO) is synthesized within the immune, vascular, and nervous systems, where it acts as a wide-ranging mediator of mammalian physiology. The NO synthases (EC 1.14.13.39) isolated from neurons or endothelium are calmodulin dependent. Calmodulin binds reversibly to neuronal NO synthase in response to elevated Ca2+, triggering its NO production by an unknown mechanism. Here we show that calmodulin binding allows NADPH-derived electrons to pass onto the heme group of neuronal NO synthase. Calmodulin-triggered electron transfer to heme was independent of substrate binding, caused rapid enzymatic oxidation of NADPH in the presence of O_2, and was required for NO synthesis. An NO synthase isolated from cytokine-induced macrophages that contains tightly bound calmodulin catalyzed spontaneous electron transfer to its heme, consistent with bound calmodulin also enabling electron transfer within this isoform. Together, these results provide a basis for how calmodulin may regulate NO synthesis. The ability of calmodulin to trigger electron transfer within an enzyme is unexpected and represents an additional function for calcium-binding proteins in biology.

  13. Microsatellite instability and the association with plasma homocysteine and thymidylate synthase in colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Lindebjerg, Jan; Crüger, Dorthe G.

    2008-01-01

    , carcinoembryonic antigen, vitamin B12, and folate. Microsatellite instability of tumors was associated with higher levels of plasma homocysteine (p = 0.008) and higher protein expression of thymidylate synthase (p ... factors. CEA was not associated with neither homocysteine nor microsatellite instability. The data suggests that there is a more pronounced methyl unit deficiency in microsatellite instable tumors....

  14. Methionine synthase A2756G and reduced folate carrier1 A80G ...

    African Journals Online (AJOL)

    Aim of the study: To analyze the effect of methionine synthase (MTR) A2756G, and reduced folate carrier (RFC1) A80G gene polymorphisms on the maternal risk for DS. Patients: This study was conducted in the Medical Genetics Center, Ain-Shams University hospitals, on a total of 170 mothers of children, diagnosed with ...

  15. Methionine synthase A2756G and reduced folate carrier1 A80G ...

    African Journals Online (AJOL)

    Background: Polymorphisms of genes encoding enzymes involved in folate metabolism have long been hypothesized to be maternal risk factors for Down syndrome, however, results are conflicting and inconclusive. Aim of the study: To analyze the effect of methionine synthase (MTR) A2756G, and reduced folate carrier ...

  16. Aldosterone-Synthase Gene Polymorphism is Associated with Blood Pressure Levels and Left Ventricle Mass Index

    Czech Academy of Sciences Publication Activity Database

    Horký, K.; Jáchymová, M.; Heller, S.; Linhart, A.; Hlubocká, Z.; Umnerová, V.; Peleška, Jan; Pavlíková, Markéta; Jindra, A.

    2004-01-01

    Roč. 204, 1 suppl. (2004), s. 35 ISSN 0014-2565. [World Congress of Internal Medicine /27./. 26.09.2004-01.10.2004, Granada] R&D Projects: GA MŠk LN00B107 Keywords : aldosterone synthase (CYP11B) * genetic polymorphism * arterial hypertension * left ventricular hypertrophy Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

  17. Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China); Yu, Xiyan, E-mail: yuxiyan@sdau.edu.cn [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China); Wang, Xiufeng, E-mail: xfwang@sdau.edu.cn [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China)

    2010-03-12

    To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

  18. Expanding the product portfolio of fungal type I fatty acid synthases

    DEFF Research Database (Denmark)

    Zhu, Zhiwei; Zhou, Yongjin J.; Krivoruchko, Anastasia

    2017-01-01

    Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes into ...

  19. In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

    Science.gov (United States)

    Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

    2014-04-01

    A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

  20. Divisible ℤ-modules

    Directory of Open Access Journals (Sweden)

    Futa Yuichi

    2016-03-01

    Full Text Available In this article, we formalize the definition of divisible ℤ-module and its properties in the Mizar system [3]. We formally prove that any non-trivial divisible ℤ-modules are not finitely-generated.We introduce a divisible ℤ-module, equivalent to a vector space of a torsion-free ℤ-module with a coefficient ring ℚ. ℤ-modules are important for lattice problems, LLL (Lenstra, Lenstra and Lovász base reduction algorithm [15], cryptographic systems with lattices [16] and coding theory [8].

  1. A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module.

    Science.gov (United States)

    Matthews, Peter R; Schindler, Michael; Howles, Paul; Arioli, Tony; Williamson, Richard E

    2010-10-01

    Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5' and 3' rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the protein's first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed.

  2. A CESA from Griffithsia monilis (Rhodophyta, Florideophyceae) has a family 48 carbohydrate-binding module

    Science.gov (United States)

    Matthews, Peter R.; Schindler, Michael; Howles, Paul; Arioli, Tony; Williamson, Richard E.

    2010-01-01

    Cellulose synthases form rosette terminal complexes in the plasma membranes of Streptophyta and various linear terminal complexes in other taxa. The sequence of a putative CESA from Griffithsia monilis (Rhodophyta, Floridiophyceae) was deduced using a cloning strategy involving degenerate primers, a cDNA library screen, and 5′ and 3′ rapid amplification of cDNA ends (RACE). RACE identified two alternative transcriptional starts and four alternative polyadenylation sites. The first translation start codon provided an open reading frame of 2610 bp encoding 870 amino acids and was PCR amplified without introns from genomic DNA. Southern hybridization indicated one strongly hybridizing gene with possible weakly related genes or pseudogenes. Amino acid sequence analysis identified a family 48 carbohydrate-binding module (CBM) upstream of the protein's first predicted transmembrane domain. There are broad similarities in predicted 3D structures of the family 48 modules from CESA, from several glycogen- and starch-binding enzymes, and from protein kinases, but there are substitutions at some residues thought to be involved in ligand binding. The module in G. monilis CESA will be on the cytoplasmic face of the plasma membrane so that it could potentially bind either low molecular weight ligands or starch which is cytosolic rather than inside membrane-bound plastids in red algae. Possible reasons why red algal CESAs have evolved family 48 modules perhaps as part of a system to regulate cellulose synthase activity in relation to cellular carbohydrate status are briefly discussed. PMID:20702566

  3. SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides

    International Nuclear Information System (INIS)

    Hartwig, S.; Frister, T.; Alemdar, S.; Li, Z.; Scheper, T.; Beutel, S.

    2015-01-01

    An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L −1 were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni 2+ -IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg 2+ containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC–MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis–Menten model, kinetic parameters of K M  = 1.111 μM (±0.113), v max  = 0.3245 μM min −1 (±0.0035), k cat  = 2.95 min −1 , as well as a catalytic efficiency k cat /K M  = 4.43 × 10 4  M −1 s −1 were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. - Highlights: • Uncharacterized (+)-zizaene synthase from C. zizanoides was cloned and expressed. • Fusion to SUMO and cold-shock induction

  4. SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides

    Energy Technology Data Exchange (ETDEWEB)

    Hartwig, S.; Frister, T.; Alemdar, S.; Li, Z.; Scheper, T.; Beutel, S., E-mail: beutel@iftc.uni-hannover.de

    2015-03-20

    An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to ∼325 mg/L{sup −1} were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni{sup 2+}-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg{sup 2+} containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC–MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis–Menten model, kinetic parameters of K{sub M} = 1.111 μM (±0.113), v{sub max} = 0.3245 μM min{sup −1} (±0.0035), k{sub cat} = 2.95 min{sup −1}, as well as a catalytic efficiency k{sub cat}/K{sub M} = 4.43 × 10{sup 4} M{sup −1} s{sup −1} were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. - Highlights: • Uncharacterized (+)-zizaene synthase from C. zizanoides was cloned

  5. The effect of anandamide on uterine nitric oxide synthase activity depends on the presence of the blastocyst.

    Directory of Open Access Journals (Sweden)

    Micaela S Sordelli

    2011-04-01

    Full Text Available Nitric oxide production, catalyzed by nitric oxide synthase (NOS, should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot(-1 h(-1 compared to days 4 (0.34±0.03 and 5 (0.35±0.02 of pregnancy and to day 6 implantation sites (0.33±0.01. This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA, an endocannabinoid, binds to cannabinoid receptors type 1 (CB1 and type 2 (CB2, and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04 and URB-597 (1.08±0.09 vs 0.83±0.06 inhibited NOS activity in the absence of a blastocyst (pseudopregnancy through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05. While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02, a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01. Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These

  6. Isolation and characterization of three new monoterpene synthases from Artemisia annua

    Directory of Open Access Journals (Sweden)

    Ju-Xin eRuan

    2016-05-01

    Full Text Available Artemisia annua, an annual herb used in traditional Chinese medicine, produces a wealth of monoterpenes and sesquiterpenes, including the well-known sesquiterpene lactone artemisinin, an active ingredient in the treatment for malaria. Here we report three new monoterpene synthases of A. annua. From a glandular trichome cDNA library, monoterpene synthases of AaTPS2, AaTPS5 and AaTPS6, were isolated and characterized. The recombinant proteins of AaTPS5 and AaTPS6 produced multiple products with camphene and 1,8-cineole as major products, respectively, and AaTPS2 produced a single product, β-myrcene. Although both Mg2+ and Mn2+ were able to support their catalytic activities, altered product spectrum was observed in the presence of Mn2+ for AaTPS2 and AaTPS5. Analysis of extracts of aerial tissues and root of A. annua with gas chromatography-mass spectrometry (GC-MS detected more than 20 monoterpenes, of which the three enzymes constituted more than 1/3 of the total. Mechanical wounding induced the expression of all three monoterpene synthase genes, and transcript levels of AaTPS5 and AaTPS6 were also elevated after treatments with phytohormones of methyl jasmonate (MeJA, salicylic acid (SA and gibberellin (GA, suggesting a role of these monoterpene synthases in plant-environment interactions. The three new monoterpene synthases reported here further our understanding of molecular basis of monoterpene biosynthesis and regulation in plant.

  7. Molecular and biochemical characterization of caffeine synthase and purine alkaloid concentration in guarana fruit.

    Science.gov (United States)

    Schimpl, Flávia Camila; Kiyota, Eduardo; Mayer, Juliana Lischka Sampaio; Gonçalves, José Francisco de Carvalho; da Silva, José Ferreira; Mazzafera, Paulo

    2014-09-01

    Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism.

    Science.gov (United States)

    Xie, Zhengzhi; Ma, Xiaoqiang; Gang, David R

    2009-01-01

    Turmeric is an excellent example of a plant that produces large numbers of metabolites from diverse metabolic pathways or networks. It is hypothesized that these metabolic pathways or networks contain biosynthetic modules, which lead to the formation of metabolite modules-groups of metabolites whose production is co-regulated and biosynthetically linked. To test whether such co-regulated metabolite modules do exist in this plant, metabolic profiling analysis was performed on turmeric rhizome samples that were collected from 16 different growth and development treatments, which had significant impacts on the levels of 249 volatile and non-volatile metabolites that were detected. Importantly, one of the many co-regulated metabolite modules that were indeed readily detected in this analysis contained the three major curcuminoids, whereas many other structurally related diarylheptanoids belonged to separate metabolite modules, as did groups of terpenoids. The existence of these co-regulated metabolite modules supported the hypothesis that the 3-methoxyl groups on the aromatic rings of the curcuminoids are formed before the formation of the heptanoid backbone during the biosynthesis of curcumin and also suggested the involvement of multiple polyketide synthases with different substrate selectivities in the formation of the array of diarylheptanoids detected in turmeric. Similar conclusions about terpenoid biosynthesis could also be made. Thus, discovery and analysis of metabolite modules can be a powerful predictive tool in efforts to understand metabolism in plants.

  9. Impact of drought stress on specialised metabolism: Biosynthesis and the expression of monoterpene synthases in sage (Salvia officinalis).

    Science.gov (United States)

    Radwan, Alzahraa; Kleinwächter, Maik; Selmar, Dirk

    2017-09-01

    In previous experiments, we demonstrated that the amount of monoterpenes in sage is increased massively by drought stress. Our current study is aimed to elucidate whether this increase is due, at least in part, to elevated activity of the monoterpene synthases responsible for the biosynthesis of essential oils in sage. Accordingly, the transcription rates of the monoterpene synthases were analyzed. Salvia officinalis plants were cultivated under moderate drought stress. The concentrations of monoterpenes as well as the expression of the monoterpene synthases were analyzed. The amount of monoterpenes massively increased in response to drought stress; it doubled after just two days of drought stress. The observed changes in monoterpene content mostly match with the patterns of monoterpene synthase expressions. The expression of bornyl diphosphate synthase was strongly up-regulated; its maximum level was reached after two days. Sabinene synthase increased gradually and reached a maximum after two weeks. In contrast, the transcript level of cineole synthase continuously declined. This study revealed that the stress related increase of biosynthesis is not only due to a "passive" shift caused by the stress related over-reduced status, but also is due - at least in part-to an "active" up-regulation of the enzymes involved. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Identification and characterization of two bisabolene synthases from linear glandular trichomes of sunflower (Helianthus annuus L., Asteraceae).

    Science.gov (United States)

    Aschenbrenner, Anna-Katharina; Kwon, Moonhyuk; Conrad, Jürgen; Ro, Dae-Kyun; Spring, Otmar

    2016-04-01

    Sunflower is known to produce a variety of bisabolene-type sesquiterpenes and accumulates these substances in trichomes of leaves, stems and flowering parts. A bioinformatics approach was used to identify the enzyme responsible for the initial step in the biosynthesis of these compounds from its precursor farnesyl pyrophosphate. Based on sequence similarity with a known bisabolene synthases from Arabidopsis thaliana AtTPS12, candidate genes of Helianthus were searched in EST-database and used to design specific primers. PCR experiments identified two candidates in the RNA pool of linear glandular trichomes of sunflower. Their sequences contained the typical motifs of sesquiterpene synthases and their expression in yeast functionally characterized them as bisabolene synthases. Spectroscopic analysis identified the stereochemistry of the product of both enzymes as (Z)-γ-bisabolene. The origin of the two sunflower bisabolene synthase genes from the transcripts of linear trichomes indicates that they may be involved in the synthesis of sesquiterpenes produced in these trichomes. Comparison of the amino acid sequences of the sunflower bisabolene synthases showed high similarity with sesquiterpene synthases from other Asteracean species and indicated putative evolutionary origin from a β-farnesene synthase. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Uncoupling of Vascular Nitric Oxide Synthase Caused by Intermittent Hypoxia

    Directory of Open Access Journals (Sweden)

    Mohammad Badran

    2016-01-01

    Full Text Available Objective. Obstructive sleep apnea (OSA, characterized by chronic intermittent hypoxia (CIH, is often present in diabetic (DB patients. Both conditions are associated with endothelial dysfunction and cardiovascular disease. We hypothesized that diabetic endothelial dysfunction is further compromised by CIH. Methods. Adult male diabetic (BKS.Cg-Dock7m +/+ Leprdb/J (db/db mice (10 weeks old and their heterozygote littermates were subjected to CIH or intermittent air (IA for 8 weeks. Mice were separated into 4 groups: IA (intermittent air nondiabetic, IH (intermittent hypoxia nondiabetic, IADB (intermittent air diabetic, and IHDB (intermittent hypoxia diabetic groups. Endothelium-dependent and endothelium-independent relaxation and modulation by basal nitric oxide (NO were analyzed using wire myograph. Plasma 8-isoprostane, interleukin-6 (IL-6, and asymmetric dimethylarginine (ADMA were measured using ELISA. Uncoupling of eNOS was measured using dihydroethidium (DHE staining. Results. Endothelium-dependent vasodilation and basal NO production were significantly impaired in the IH and IADB group compared to IA group but was more pronounced in IHDB group. Levels of 8-isoprostane, IL-6, ADMA, and eNOS uncoupling were ≈2-fold higher in IH and IADB groups and were further increased in the IHDB group. Conclusion. Endothelial dysfunction is more pronounced in diabetic mice subjected to CIH compared to diabetic or CIH mice alone. Oxidative stress, ADMA, and eNOS uncoupling were exacerbated by CIH in diabetic mice.

  12. Structure of the ent-Copalyl Diphosphate Synthase PtmT2 from Streptomyces platensis CB00739, a Bacterial Type II Diterpene Synthase.

    Science.gov (United States)

    Rudolf, Jeffrey D; Dong, Liao-Bin; Cao, Hongnan; Hatzos-Skintges, Catherine; Osipiuk, Jerzy; Endres, Michael; Chang, Chin-Yuan; Ma, Ming; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2016-08-31

    Terpenoids are the largest and most structurally diverse family of natural products found in nature, yet their presence in bacteria is underappreciated. The carbon skeletons of terpenoids are generated through carbocation-dependent cyclization cascades catalyzed by terpene synthases (TSs). Type I and type II TSs initiate cyclization via diphosphate ionization and protonation, respectively, and protein structures of both types are known. Most plant diterpene synthases (DTSs) possess three α-helical domains (αβγ), which are thought to have arisen from the fusion of discrete, ancestral bacterial type I TSs (α) and type II TSs (βγ). Type II DTSs of bacterial origin, of which there are no structurally characterized members, are a missing piece in the structural evolution of TSs. Here, we report the first crystal structure of a type II DTS from bacteria. PtmT2 from Streptomyces platensis CB00739 was verified as an ent-copalyl diphosphate synthase involved in the biosynthesis of platensimycin and platencin. The crystal structure of PtmT2 was solved at a resolution of 1.80 Å, and docking studies suggest the catalytically active conformation of geranylgeranyl diphosphate (GGPP). Site-directed mutagenesis confirmed residues involved in binding the diphosphate moiety of GGPP and identified DxxxxE as a potential Mg(2+)-binding motif for type II DTSs of bacterial origin. Finally, both the shape and physicochemical properties of the active sites are responsible for determining specific catalytic outcomes of TSs. The structure of PtmT2 fundamentally advances the knowledge of bacterial TSs, their mechanisms, and their role in the evolution of TSs.

  13. Classes of modules

    CERN Document Server

    Dauns, John

    2006-01-01

    Because traditional ring theory places restrictive hypotheses on all submodules of a module, its results apply only to small classes of already well understood examples. Often, modules with infinite Goldie dimension have finite-type dimension, making them amenable to use with type dimension, but not Goldie dimension. By working with natural classes and type submodules (TS), Classes of Modules develops the foundations and tools for the next generation of ring and module theory. It shows how to achieve positive results by placing restrictive hypotheses on a small subset of the complement submodules, Furthermore, it explains the existence of various direct sum decompositions merely as special cases of type direct sum decompositions. Carefully developing the foundations of the subject, the authors begin by providing background on the terminology and introducing the different module classes. The modules classes consist of torsion, torsion-free, s[M], natural, and prenatural. They expand the discussion by exploring...

  14. Amplification and diversity analysis of keto synthase domains of putative polyketide synthase genes in Aspergillus ochraceus and Aspergillus carbonarius producers of ochratoxin A

    International Nuclear Information System (INIS)

    Atoui, A.; Phong Dao, H.; Mathieu, F.; Lebrihi, A.

    2006-01-01

    The diversity of polyketide synthase (PKS) genes in Aspergillus ochraceus NRRL 3174 and Aspergil- lus carbonarius 2Mu134 has been investigated using different primer pairs previously developed for the ketosynthase (KS) domain of fungal PKSs. Nine different KS domain sequences in A. ochraceus NRRL 3174 as well as five different KS domain sequences in A. carbonarius 2Mu134 have been identified. The identified KS fragments were distributed in five different clusters on the phylogenetic tree, indicating that they most probably represent PKSs responsible for different functions. (author)

  15. Near infrared radiation protects against oxygen-glucose deprivation-induced neurotoxicity by down-regulating neuronal nitric oxide synthase (nNOS) activity in vitro.

    Science.gov (United States)

    Yu, Zhanyang; Li, Zhaoyu; Liu, Ning; Jizhang, Yunneng; McCarthy, Thomas J; Tedford, Clark E; Lo, Eng H; Wang, Xiaoying

    2015-06-01

    Near infrared radiation (NIR) has been shown to be neuroprotective against neurological diseases including stroke and brain trauma, but the underlying mechanisms remain poorly understood. In the current study we aimed to investigate the hypothesis that NIR may protect neurons by attenuating oxygen-glucose deprivation (OGD)-induced nitric oxide (NO) production and modulating cell survival/death signaling. Primary mouse cortical neurons were subjected to 4 h OGD and NIR was applied at 2 h reoxygenation. OGD significantly increased NO level in primary neurons compared to normal control, which was significantly ameliorated by NIR at 5 and 30 min post-NIR. Neither OGD nor NIR significantly changed neuronal nitric oxide synthase (nNOS) mRNA or total protein levels compared to control groups. However, OGD significantly increased nNOS activity compared to normal control, and this effect was significantly diminished by NIR. Moreover, NIR significantly ameliorated the neuronal death induced by S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a NO donor. Finally, NIR significantly rescued OGD-induced suppression of p-Akt and Bcl-2 expression, and attenuated OGD-induced upregulation of Bax, BAD and caspase-3 activation. These results suggest NIR may protect against OGD at least partially through reducing NO production by down-regulating nNOS activity, and modulating cell survival/death signaling.

  16. FASTBUS Snoop Diagnostic Module

    International Nuclear Information System (INIS)

    Walz, H.V.; Downing, R.

    1980-11-01

    Development of the FASTBUS Snoop Module, undertaken as part of the prototype program for the new interlaboratory data bus standard, is described. The Snoop Module resides on a FASTBUS crate segment and provides diagnostic monitoring and testing capability. Communication with a remote host computer is handled independent of FASTBUS through a serial link. The module consists of a high-speed ECL front-end to monitor and single-step FASTBUS cycles, a master-slave interface, and a control microprocessor with serial communication ports. Design details and performance specifications of the prototype module are reported. 9 figures, 1 table

  17. Bracket for photovoltaic modules

    Science.gov (United States)

    Ciasulli, John; Jones, Jason

    2014-06-24

    Brackets for photovoltaic ("PV") modules are described. In one embodiment, a saddle bracket has a mounting surface to support one or more PV modules over a tube, a gusset coupled to the mounting surface, and a mounting feature coupled to the gusset to couple to the tube. The gusset can have a first leg and a second leg extending at an angle relative to the mounting surface. Saddle brackets can be coupled to a torque tube at predetermined locations. PV modules can be coupled to the saddle brackets. The mounting feature can be coupled to the first gusset and configured to stand the one or more PV modules off the tube.

  18. Crossed modules of racks

    OpenAIRE

    Crans, Alissa S.; Wagemann, Friedrich

    2014-01-01

    We generalize the notion of a crossed module of groups to that of a crossed module of racks. We investigate the relation to categorified racks, namely strict 2-racks, and trunk-like objects in the category of racks, generalizing the relation between crossed modules of groups and strict 2-groups. Then we explore topological applications. We show that by applying the rack-space functor, a crossed module of racks gives rise to a covering. Our main result shows how the fundamental racks associate...

  19. Model theory and modules

    CERN Document Server

    Prest, M

    1988-01-01

    In recent years the interplay between model theory and other branches of mathematics has led to many deep and intriguing results. In this, the first book on the topic, the theme is the interplay between model theory and the theory of modules. The book is intended to be a self-contained introduction to the subject and introduces the requisite model theory and module theory as it is needed. Dr Prest develops the basic ideas concerning what can be said about modules using the information which may be expressed in a first-order language. Later chapters discuss stability-theoretic aspects of module

  20. Delphi Accounts Receivable Module -

    Data.gov (United States)

    Department of Transportation — Delphi accounts receivable module contains the following data elements, but are not limited to customer information, cash receipts, line of accounting details, bill...

  1. Molecular characterization and functional analysis of chalcone synthase from Syringa oblata Lindl. in the flavonoid biosynthetic pathway.

    Science.gov (United States)

    Wang, Yu; Dou, Ying; Wang, Rui; Guan, Xuelian; Hu, Zenghui; Zheng, Jian

    2017-11-30

    The flower color of Syringa oblata Lindl., which is often modulated by the flavonoid content, varies and is an important ornamental feature. Chalcone synthase (CHS) catalyzes the first key step in the flavonoid biosynthetic pathway. However, little is known about the role of S. oblata CHS (SoCHS) in flavonoid biosynthesis in this species. Here, we isolate and analyze the cDNA (SoCHS1) that encodes CHS in S. oblata. We also sought to analyzed the molecular characteristics and function of flavonoid metabolism by SoCHS1. We successfully isolated the CHS-encoding genomic DNA (gDNA) in S. oblata (SoCHS1), and the gene structural analysis indicated it had no intron. The opening reading frame (ORF) sequence of SoCHS1 was 1170bp long and encoded a 389-amino acid polypeptide. Multiple sequence alignment revealed that both the conserved CHS active site residues and CHS signature sequence were in the deduced amino acid sequence of SoCHS1. Crystallographic analysis revealed that the protein structure of SoCHS1 is highly similar to that of FnCHS1 in Freesia hybrida. The quantitative real-time polymerase chain reaction (PCR) performed to detect the SoCHS1 transcript expression levels in flowers, and other tissues revealed the expression was significantly correlated with anthocyanin accumulation during flower development. The ectopic expression results of Nicotiana tabacum showed that SoCHS1 overexpression in transgenic tobacco changed the flower color from pale pink to pink. In conclusion, these results suggest that SoCHS1 plays an essential role in flavonoid biosynthesis in S. oblata, and could be used to modify flavonoid components in other plant species. Copyright © 2017. Published by Elsevier B.V.

  2. Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition

    International Nuclear Information System (INIS)

    Wang, Wenwen; Yang, Yanqin; Xiong, Zhewen; Kong, Jiamin; Fu, Xinlu; Shen, Feihai; Huang, Zhiying

    2016-01-01

    Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3β in TP-induced cardiotoxicity. Inhibition of GSK-3β activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3β overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3β overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3β interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3β inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3β could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP. - Highlights: • GSK-3β inhibition ameliorates TP-induced cardiotoxicity in vitro and in vivo. • GSK-3β controls Cyp-F activation, and regulates mPTP and apoptosis in H9c2 cells. • The protective effect is attributed to GSK-3β activity rather than to protein level. • GSK-3β may be a promising target against TP-induced cardiotoxicity.

  3. Functional effect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat flavour formation

    Science.gov (United States)

    Battilana, Juri; Emanuelli, Francesco; Gambino, Giorgio; Gribaudo, Ivana; Gasperi, Flavia; Boss, Paul K.; Grando, Maria Stella

    2011-01-01

    Grape berries of Muscat cultivars (Vitis vinifera L.) contain high levels of monoterpenols and exhibit a distinct aroma related to this composition of volatiles. A structural gene of the plastidial methyl-erythritol-phosphate (MEP) pathway, 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS), was recently suggested as a candidate gene for this trait, having been co-localized with a major quantitative trait locus for linalool, nerol, and geraniol concentrations in berries. In addition, a structured association study discovered a putative causal single nucleotide polymorphism (SNP) responsible for the substitution of a lysine with an asparagine at position 284 of the VvDXS protein, and this SNP was significantly associated with Muscat-flavoured varieties. The significance of this nucleotide difference was investigated by comparing the monoterpene profiles with the expression of VvDXS alleles throughout berry development in Moscato Bianco, a cultivar heterozygous for the SNP mutation. Although correlation was detected between the VvDXS transcript profile and the accumulation of free monoterpenol odorants, the modulation of VvDXS expression during berry development appears to be independent of nucleotide variation in the coding sequence. In order to assess how the non-synonymous mutation may enhance Muscat flavour, an in vitro characterization of enzyme isoforms was performed followed by in vivo overexpression of each VvDXS allele in tobacco. The results showed that the amino acid non-neutral substitution influences the enzyme kinetics by increasing the catalytic efficiency and also dramatically affects monoterpene levels in transgenic lines. These findings confirm a functional effect of the VvDXS gene polymorphism and may pave the way for metabolic engineering of terpenoid contents in grapevine. PMID:21868399

  4. Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wenwen; Yang, Yanqin; Xiong, Zhewen; Kong, Jiamin [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Fu, Xinlu [Laboratory Animals Center, Sun Yat-sen University, Guangzhou 510006 (China); Shen, Feihai, E-mail: shenfh3@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Huang, Zhiying, E-mail: hzhiying@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China)

    2016-12-15

    Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3β in TP-induced cardiotoxicity. Inhibition of GSK-3β activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3β overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3β overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3β interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3β inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3β could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP. - Highlights: • GSK-3β inhibition ameliorates TP-induced cardiotoxicity in vitro and in vivo. • GSK-3β controls Cyp-F activation, and regulates mPTP and apoptosis in H9c2 cells. • The protective effect is attributed to GSK-3β activity rather than to protein level. • GSK-3β may be a promising target against TP-induced cardiotoxicity.

  5. Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

    Science.gov (United States)

    Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

    2016-10-01

    Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

  6. Neuropeptides and nitric oxide synthase in the gill and the air-breathing organs of fishes.

    Science.gov (United States)

    Zaccone, Giacomo; Mauceri, Angela; Fasulo, Salvatore

    2006-05-01

    Anatomical and histochemical studies have demonstrated that the bulk of autonomic neurotransmission in fish gill is attributed to cholinergic and adrenergic mechanisms (Nilsson. 1984. In: Hoar WS, Randall DJ, editors. Fish physiology, Vol. XA. Orlando: Academic Press. p 185-227; Donald. 1998. In: Evans DH, editor. The physiology of fishes, 2nd edition. Boca Raton: CRC Press. p 407-439). In many tissues, blockade of adrenergic and cholinergic transmission results in residual responses to nerve stimulation, which are termed NonAdrenergic, NonCholinergic (NANC). The discovery of nitric oxide (NO) has provided a basis for explaining many examples of NANC transmissions with accumulated physiological and pharmacological data indicating its function as a primary NANC transmitter. Little is known about the NANC neurotransmission, and studies on neuropeptides and NOS (Nitric Oxide Synthase) are very fragmentary in the gill and the air-breathing organs of fishes. Knowledge of the distribution of nerves and effects of perfusing agonists may help to understand the mechanisms of perfusion regulation in the gill (Olson. 2002. J Exp Zool 293:214-231). Air breathing as a mechanism for acquiring oxygen has evolved independently in several groups of fishes, necessitating modifications of the organs responsible for the exchange of gases. Aquatic hypoxia in freshwaters has been probably the more important selective force in the evolution of air breathing in vertebrates. Fishes respire with gills that are complex structures with many different effectors and potential control systems. Autonomic innervation of the gill has received considerable attention. An excellent review on branchial innervation includes Sundin and Nilsson's (2002. J Exp Zool 293:232-248) with an emphasis on the anatomy and basic functioning of afferent and efferent fibers of the branchial nerves. The chapters by Evans (2002. J Exp Zool 293:336-347) and Olson (2002) provide new challenges about a variety of

  7. (+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

    Science.gov (United States)

    Prosser, Ian; Phillips, Andy L; Gittings, Simon; Lewis, Mervyn J; Hooper, Antony M; Pickett, John A; Beale, Michael H

    2002-08-01

    Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

  8. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of SAICAR synthase from Streptococcus suis serotype 2

    International Nuclear Information System (INIS)

    Cheng, Xia; Lu, Guangwen; Qi, Jianxun; Cheng, Hao; Gao, Feng; Wang, Jundong; Yan, Jinghua

    2010-01-01

    Crystals of SAICAR synthase from S. suis serotype 2 were obtained in the presence of 40 mM aspartic acid substrate; they belonged to space group P2 and diffracted to 2.8 Å resolution. Phosphoribosylaminoimidazole-succinocarboxamide synthase (SAICAR synthase) plays an essential role in the de novo biosynthesis of purine nucleotides. In this study, the SAICAR synthase from Streptococcus suis was cloned and overexpressed in Escherichia coli. The subsequent product was purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.8 Å resolution and belonged to space group P2, with unit-cell parameters a = 70.2, b = 52.2, c = 153.9 Å, β = 102.8°

  9. Dihydrofolate reductase and dihydropteroate synthase genotypes associated with in vitro resistance of Plasmodium falciparum to pyrimethamine, trimethoprim, sulfadoxine, and sulfamethoxazole

    DEFF Research Database (Denmark)

    Khalil, Insaf; Rønn, Anita M; Alifrangis, Michael

    2003-01-01

    A total of 70 Plasmodium falciparum isolates were tested in vitro against pyrimethamine (PYR), trimethoprim (TRM), sulfadoxine (SDX), and sulfamethoxazole (SMX), and their dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genotypes were determined. dhfr genotypes correlated...

  10. The FoF1-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Zíková, Alena; Schnaufer, A.; Dalley, A. R.; Panigrahi, A. K.; Stuart, K. D.

    2009-01-01

    Roč. 5, č. 5 (2009), s. 1-15 E-ISSN 1932-6203 Keywords : mitochondrion * respiratory chain * / ATP synthase * TAP-tag * RNAi Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.351, year: 2009

  11. Expression of prostaglandin synthases (pgds and pges) during zebrafish gonadal differentiation

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Nielsen, John E; Nielsen, Betina Frydenlund

    2010-01-01

    The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E2 synthase (pges) and especially the lipocalin-type prostaglandin D2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found....... In this study, a sexually dimorphic expression of pgds was found in gonads of adult zebrafish with expression in testis but not in ovaries. To determine whether the sex-specific expression pattern of pgds was present in gonads of juvenile zebrafish and therefore could be an early marker of sex in zebrafish, we...... microdissected gonads from four randomly selected individual zebrafish for every second day in the period 2-20 days post hatch (dph) and 0-1 dph. The temporal expression of pgds and pges was investigated in the microdissected gonads, however, no differential expression that could indicate sex-specific difference...

  12. Expression of prostaglandin synthases (pgds and pges) duringzebrafishgonadal differentiation

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Nielsen, John E.; Nielsen, Betina F.

    2010-01-01

    The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E-2 synthase (pges) and especially the lipocalin-type prostaglandin D-2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found...... In this study, a sexually dimorphic expression of pgds was found in gonads of adult zebrafish with expression in testis but not in ovaries. To determine whether the sex-specific expression pattern of pgds was present in gonads of juvenile zebrafish and therefore could be an early marker of sex in zebrafish, we...... microdissected gonads from four randomly selected individual zebrafish for every second day in the period 2-20 days post hatch (dph) and 0-1 dph The temporal expression of pgds and pges was investigated in the microdissected gonads, however, no differential expression that could indicate sex-specific difference...

  13. Expression, crystallization and preliminary crystallographic study of octaprenyl pyrophosphate synthase from Helicobacter pylori

    International Nuclear Information System (INIS)

    Zhang, Jinyong; Zhang, Xiaoli; Mao, Xuhu; Zou, Quanming; Li, Defeng

    2011-01-01

    Octaprenyl pyrophosphate synthase from H. pylori has been expressed, purified and crystallized, and a diffraction data set was collected to 2.00 Å resolution. Octaprenyl pyrophosphate synthase (OPPs) is involved in the synthesis of the side chains of ubiquinone and menaquinone and catalyzes consecutive condensation reactions of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate polyprenyl pyrophosphate and pyrophosphate. In order to investigate the roles played by OPPs in the metabolism of ubiquinone and menaquinone and the enzymatic mechanisms of these enzymes, analysis of the structure–function relationship of OPPs from Helicobacter pylori was initiated. The gene for OPPs was cloned, the protein was expressed, purified and crystallized and a diffraction data set was collected to 2.00 Å resolution. The crystals belonged to space group P4 1 2 1 2 or P4 3 2 1 2, with unit-cell parameters a = b = 109.33, c = 103.41 Å

  14. Microsatellite instability in colorectal cancer and association with thymidylate synthase and dihydropyrimidine dehydrogenase expression

    DEFF Research Database (Denmark)

    Jensen, Søren A; Vainer, Ben; Kruhøffer, Mogens

    2009-01-01

    unclarified. The association of MSI and MMR status with outcome and with thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) expression in colorectal cancer were evaluated. METHODS: MSI in five reference loci, MMR enzymes (hMSH2, hMSH6, hMLH1 and hPMS2), thymidylate synthase (TS....... Absence of repair protein expression was assessed in 52 (17.0%) tumors, which had primarily lost hMLH1 in 39 (12.7%), hMSH2 in 5 (1.6%), and hMSH6 in 8 (2.6%) tumors. In multivariate analysis MSI (instable) compared to MSS (stable) tumors were significantly associated with lower risk of recurrence (hazard...

  15. Enzymatic synthesis of S-phenyl-L-cysteine from keratin hydrolysis industries wastewater with tryptophan synthase.

    Science.gov (United States)

    Xu, Lisheng; Wang, Zhiyuan; Mao, Pingting; Liu, Junzhong; Zhang, Hongjuan; Liu, Qian; Jiao, Qing-Cai

    2013-04-01

    An economical method for production of S-phenyl-L-cysteine from keratin acid hydrolysis wastewater (KHW) containing L-serine was developed by recombinant tryptophan synthase. This study provides us with an alternative KHW utilization strategy to synthesize S-phenyl-L-cysteine. Tryptophan synthase could efficiently convert L-serine contained in KHW to S-phenyl-L-cysteine at pH 9.0, 40°C and Trion X-100 of 0.02%. In a scale up study, L-serine conversion rate reach 97.1% with a final S-phenyl-L-cysteine concentration of 38.6 g l(-1). Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

    Science.gov (United States)

    Sauge-Merle, Sandrine; Cuiné, Stéphan; Carrier, Patrick; Lecomte-Pradines, Catherine; Luu, Doan-Trung; Peltier, Gilles

    2003-01-01

    Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes. PMID:12514032

  17. IDENTIFICATION AND CHARACTERIZATION OF THE SUCROSE SYNTHASE 2 GENE (Sus2 IN DURUM WHEAT

    Directory of Open Access Journals (Sweden)

    Mariateresa eVolpicella

    2016-03-01

    Full Text Available Sucrose transport is the central system for the allocation of carbon resources in vascular plants. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, represents a key enzyme in the control of the flow of carbon into starch biosynthesis. In the present study the genomic identification and characterization of the Sus2-2A and Sus2-2B genes coding for sucrose synthase in durum wheat (cultivars Ciccio and Svevo is reported. The genes were analyzed for their expression in different tissues and at different seed maturation stages, in four tetraploid wheat genotypes (Svevo, Ciccio, Primadur and 5-BIL42. The activity of the encoded proteins was evaluated by specific activity assays on endosperm extracts and their structure established by modelling approaches. The combined results of SUS2 expression and activity levels were then considered in the light of their possible involvement in starch yield.

  18. Strategies in megasynthase engineering – fatty acid synthases (FAS as model proteins

    Directory of Open Access Journals (Sweden)

    Manuel Fischer

    2017-06-01

    Full Text Available Megasynthases are large multienzyme proteins that produce a plethora of important natural compounds by catalyzing the successive condensation and modification of precursor units. Within the class of megasynthases, polyketide synthases (PKS are responsible for the production of a large spectrum of bioactive polyketides (PK, which have frequently found their way into therapeutic applications. Rational engineering approaches have been performed during the last 25 years that seek to employ the “assembly-line synthetic concept” of megasynthases in order to deliver new bioactive compounds. Here, we highlight PKS engineering strategies in the light of the newly emerging structural information on megasynthases, and argue that fatty acid synthases (FAS are and will be valuable objects for further developing this field.

  19. The Dictyostelium discoideum cellulose synthase: Structure/function analysis and identification of interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Richard L. Blanton

    2004-02-19

    OAK-B135 The major accomplishments of this project were: (1) the initial characterization of dcsA, the gene for the putative catalytic subunit of cellulose synthase in the cellular slime mold Dictyostelium discoideum; (2) the detection of a developmentally regulated event (unidentified, but perhaps a protein modification or association with a protein partner) that is required for cellulose synthase activity (i.e., the dcsA product is necessary, but not sufficient for cellulose synthesis); (3) the continued exploration of the developmental context of cellulose synthesis and DcsA; (4) the isolation of a GFP-DcsA-expressing strain (work in progress); and (5) the identification of Dictyostelium homologues for plant genes whose products play roles in cellulose biosynthesis. Although our progress was slow and many of our results negative, we did develop a number of promising avenues of investigation that can serve as the foundation for future projects.

  20. Energy Equivalence of Information in the Mitochondrion and the Thermodynamic Efficiency of ATP Synthase.

    Science.gov (United States)

    Matta, Chérif F; Massa, Lou

    2015-09-01

    Half a century ago, Johnson and Knudsen resolved the puzzle of the apparent low efficiency of the kidney (∼ 0.5%) compared to most other bodily organs (∼ 40%) by taking into account the entropic cost of ion sorting, the principal function of this organ. Similarly, it is shown that the efficiency of energy transduction of the chemiosmotic proton-motive force by ATP synthase is closer to 90% instead of the oft-quoted textbook value of only 60% when information theoretic considerations are applied to the mitochondrion. This high efficiency is consistent with the mechanical energy transduction of ATP synthase known to be close to the 100% thermodynamic limit. It would have been wasteful for evolution to maximize the mechanical energy transduction to 100% while wasting 40% of the chemiosmotic free energy in the conversion of the proton-motive force into mechanical work before being captured as chemical energy in adenosine 5'-triphosphate.

  1. Involvement of Glycogen Synthase Kinase-3 in the Mechanisms of Conditioned Food Aversion Memory Reconsolidation.

    Science.gov (United States)

    Nikitin, V P; Solntseva, S V; Kozyrev, S A

    2017-02-01

    Experiments were performed on the snails trained in conditioned food aversion for 3 days. Injection of TDZD-8 (glycogen synthase kinase-3 inhibitor, 2 mg/kg) in combination with reminder (presentation of a conditioned food stimulus) led to memory impairment developing 3 days after inhibitor/reminder exposure and followed by spontaneous recovery in 10 days. Injections of TDZD-8 in a dose of 4 or 20 mg/kg before reminder were shown to cause amnesia that persisted for more than 10 days. Memory recovery during repeated training was observed at the earlier period than after initial training. The impairment of memory reconsolidation by TDZD-8 after training of snails for 1 day was less pronounced than under standard training conditions (3 days). The effect of a glycogen synthase kinase-3 inhibitor during memory reconsolidation is probably followed by impairment of memory retrieval and/or partial loss, which can be compensated spontaneously or after repeated training.

  2. PCR cloning of Polyhydroxybutyrate Synthase Gene (phbC) from Aeromonashydrophila

    International Nuclear Information System (INIS)

    Enan, M. R.; Bashandy, S.A.

    2006-01-01

    Plastic wastes are considered to be severe environmental contaminantscausing waste disposal problems. Widespread use of biodegradable plastics isone of the solutions, but it is limited by high production cost. A polymerasechain reaction (PCR) protocol was developed for the specific for the specificdetection and isolation of full-length gene coding for polyhydroxybutyrate(PBH). (PCR) strategy using (PHB) primers resulted in the amplification of(DNA) fragments with the expected size from all isolated bacteria (PBH)synthase gene was cloned directly from Aeromonas hydrophila genome for thefirst time. The clonec fragment was named (phbCAh) gene exhibits similarly to(PHB) synthase genes of Alcaligenes latus and Pseudomonas oleovorans (97%),Alcaligenes sp. (81%) and Comamonas acidovorans (84%). (author)

  3. The effect of high pressure on the intracellular trehalose synthase activity of Thermus aquaticus.

    Science.gov (United States)

    Dong, Yongsheng; Ma, Lei; Duan, Yuanliang

    2016-01-01

    To understand the effect of high pressure on the intracellular trehalose synthase activity, Thermus aquaticus (T. aquaticus) in the logarithmic growth phase was treated with high-pressure air, and its intracellular trehalose synthase (TSase) activity was determined. Our results indicated that pressure is a factor strongly affecting the cell growth. High pressure significantly attenuated the growth rate of T. aquaticus and shortened the duration of stationary phase. However, after 2 h of culture under 1.0 MPa pressure, the activity of intracellular TSase in T. aquaticus reached its maximum value, indicating that pressure can significantly increase the activity of intracellular TSase in T. aquaticus. Thus the present study provides an important guide for the enzymatic production of trehalose.

  4. Functional loss of two ceramide synthases elicits autophagy-dependent lifespan extension in C. elegans

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Kruse, Rikke; Harvald, Eva Bang

    2013-01-01

    Ceramide and its metabolites constitute a diverse group of lipids, which play important roles as structural entities of biological membranes as well as regulators of cellular growth, differentiation, and development. The C. elegans genome comprises three ceramide synthase genes; hyl-1, hyl-2...... that hyl-1;lagr-1 animals display reduced feeding, increased resistance to heat, and reduced reproduction. Collectively, our data suggest that specific sphingolipids produced by different ceramide synthases have opposing roles in determination of C. elegans lifespan. We propose that loss of HYL-1 and LAGR......, and lagr-1. HYL-1 function is required for synthesis of ceramides and sphingolipids containing very long acyl-chains (≥C24), while HYL-2 is required for synthesis of ceramides and sphingolipids containing shorter acyl-chains (≤C22). Here we show that functional loss of HYL-2 decreases lifespan, while loss...

  5. Stochastic thermodynamics of a chemical nanomachine: The channeling enzyme tryptophan synthase.

    Science.gov (United States)

    Loutchko, Dimitri; Eisbach, Maximilian; Mikhailov, Alexander S

    2017-01-14

    The enzyme tryptophan synthase is characterized by a complex pattern of allosteric interactions that regulate the catalytic activity of its two subunits and opening or closing of their ligand gates. As a single macromolecule, it implements 13 different reaction steps, with an intermediate product directly channeled from one subunit to another. Based on experimental data, a stochastic model for the operation of tryptophan synthase has been earlier constructed [D. Loutchko, D. Gonze, and A. S. Mikhailov, J. Phys. Chem. B 120, 2179 (2016)]. Here, this model is used to consider stochastic thermodynamics of such a chemical nanomachine. The Gibbs energy landscape of the internal molecular states is determined, the production of entropy and its flow within the enzyme are analyzed, and the information exchange between the subunits resulting from allosteric cross-regulations and channeling is discussed.

  6. Interleukin-2-induced survival of natural killer (NK) cells involving phosphatidylinositol-3 kinase-dependent reduction of ceramide through acid sphingomyelinase, sphingomyelin synthase, and glucosylceramide synthase.

    Science.gov (United States)

    Taguchi, Yoshimitsu; Kondo, Tadakazu; Watanabe, Mitsumasa; Miyaji, Michihiko; Umehara, Hisanori; Kozutsumi, Yasunori; Okazaki, Toshiro

    2004-11-15

    Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.

  7. Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

    Science.gov (United States)

    Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

    2012-07-27

    Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

  8. Strengthening Triterpene Saponins Biosynthesis by Over-Expression of Farnesyl Pyrophosphate Synthase Gene and RNA Interference of Cycloartenol Synthase Gene in Panax notoginseng Cells

    Directory of Open Access Journals (Sweden)

    Yan Yang

    2017-04-01

    Full Text Available To conform to the multiple regulations of triterpene biosynthesis, the gene encoding farnesyl pyrophosphate synthase (FPS was transformed into Panax notoginseng (P. notoginseng cells in which RNA interference (RNAi of the cycloartenol synthase (CAS gene had been accomplished. Transgenic cell lines showed both higher expression levels of FPS and lower expression levels of CAS compared to the wild-type (WT cells. In the triterpene and phytosterol analysis, transgenic cell lines provided a higher accumulation of total triterpene saponins, and a lower amount of phytosterols in comparison with the WT cells. Compared with the cells in which RNAi of the CAS gene was achieved, the cells with simultaneously over-expressed FPS and silenced CAS showed higher triterpene contents. These results demonstrate that over-expression of FPS can break the rate-limiting reaction catalyzed by FPS in the triterpene saponins biosynthetic pathway; and inhibition of CAS expression can decrease the synthesis metabolic flux of the phytosterol branch. Thus, more precursors flow in the direction of triterpene synthesis, and ultimately promote the accumulation of P. notoginseng saponins. Meanwhile, silencing and over-expressing key enzyme genes simultaneously is more effective than just manipulating one gene in the regulation of saponin biosynthesis.

  9. Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening

    Science.gov (United States)

    Zhang, Xiu-Mei; Wang, Wei; Du, Li-Qing; Xie, Jiang-Hui; Yao, Yan-Li; Sun, Guang-Ming

    2012-01-01

    Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion. PMID:22949808

  10. Disruption of Bcchs4, Bcchs6 or Bcchs7 chitin synthase genes in Botrytis cinerea and the essential role of class VI chitin synthase (Bcchs6).

    Science.gov (United States)

    Morcx, Serena; Kunz, Caroline; Choquer, Mathias; Assie, Sébastien; Blondet, Eddy; Simond-Côte, Elisabeth; Gajek, Karina; Chapeland-Leclerc, Florence; Expert, Dominique; Soulie, Marie-Christine

    2013-03-01

    Chitin synthases play critical roles in hyphal development and fungal pathogenicity. Previous studies on Botrytis cinerea, a model organism for necrotrophic pathogens, have shown that disruption of Bcchs1 and more particularly Bcchs3a genes have a drastic impact on virulence (Soulié et al., 2003, 2006). In this work, we investigate the role of other CHS including BcCHS4, BcCHS6 and BcCHS7 during the life cycle of B. cinerea. Single deletions of corresponding genes were carried out. Phenotypic analysis indicates that: (i) BcCHS4 enzyme is not essential for development and pathogenicity of the fungus; (ii) BcCHS7 is required for pathogenicity in a host dependant manner. For Bcchs6 gene disruption, we obtained only heterokaryotic strains. Indeed, sexual or asexual purification assays were unsuccessful. We concluded that class VI chitin synthase could be essential for B. cinerea and therefore BcCHS6 represents a valuable antifungal target. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Cell-Specific Expression of Homospermidine Synthase, the Entry Enzyme of the Pyrrolizidine Alkaloid Pathway in Senecio vernalis, in Comparison with Its Ancestor, Deoxyhypusine Synthase1

    Science.gov (United States)

    Moll, Stefanie; Anke, Sven; Kahmann, Uwe; Hänsch, Robert; Hartmann, Thomas; Ober, Dietrich

    2002-01-01

    Pyrrolizidine alkaloids (PAs) are constitutive plant defense compounds with a sporadic taxonomic occurrence. The first committed step in PA biosynthesis is catalyzed by homospermidine synthase (HSS). Recent evidence confirmed that HSS evolved by gene duplication from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of the eukaryotic translation initiation factor 5A. To better understand the evolutionary relationship between these two enzymes, which are involved in completely different biological processes, we studied their tissue-specific expression. RNA-blot analysis, reverse transcriptase-PCR, and immunolocalization techniques demonstrated that DHS is constitutively expressed in shoots and roots of Senecio vernalis (Asteraceae), whereas HSS expression is root specific and restricted to distinct groups of endodermis and neighboring cortex cells located opposite to the phloem. All efforts to detect DHS by immunolocalization failed, but studies with promoter-β-glucuronidase fusions confirmed a general expression pattern, at least in young seedlings of tobacco (Nicotiana tabacum). The expression pattern for HSS differs completely from its ancestor DHS due to the adaptation of HSS to the specific requirements of PA biosynthesis. PMID:12226485

  12. Growth Modulation in Achondroplasia.

    Science.gov (United States)

    McClure, Philip K; Kilinc, Eray; Birch, John G

    2017-09-01

    Achondroplasia is the most common skeletal dysplasia with a rate of nearly 1/10,000. The development of lower extremity deformity is well documented, and various modes of correction have been reported. There are no reports on the use of growth modulation to correct angular deformity in achondroplasia. Medical Records from 1985 to 2015 were reviewed for the diagnosis of achondroplasia and growth modulation procedures. Patients who had been treated for angular deformity of the legs by growth modulation were identified. A detailed analysis of their medical record and preoperative and final lower extremity radiographs was completed. Four patients underwent growth modulation procedures, all to correct existing varus deformity of the legs. Three of the 4 patients underwent bilateral distal femoral and proximal tibial growth modulation. The remaining patient underwent tibial correction only. Two of the 4 patients had a combined proximal fibular epiphysiodesis. All limbs had some improvement of alignment; however, 1 patient went on to bilateral osteotomies. Only 1 limb corrected to a neutral axis with growth modulation alone at last follow-up, initial implantation was done before 5 years of age. Growth modulation is an effective means for deformity correction in the setting of achondroplasia. However implantation may need to be done earlier than would be typical for patients without achondroplasia. Osteotomy may still be required after growth modulation for incomplete correction.

  13. Defect detection module

    International Nuclear Information System (INIS)

    Ernwein, R.; Westermann, G.

    1986-01-01

    The ''defect detector'' module is aimed at exceptional event or state recording. Foreseen for voltage presence monitoring on high supply voltage module of drift chambers, its characteristics can also show up the vanishing of supply voltage and take in account transitory fast signals [fr

  14. The Strip Module

    DEFF Research Database (Denmark)

    Pedersen, Tommy

    1996-01-01

    When the behaviour of a ship in waves is to be predicted it is convenient to have a tool which includes different approaches to the problem.The aim of this project is to develop such a tool named the strip theory module. The strip theory module will consist of submodules dependent on the I...

  15. The FPAX fastbus module

    International Nuclear Information System (INIS)

    Barlag, S.; Bouquet, B.; Lavigne, B.; Rypko, J.

    1989-07-01

    The FPAX is a Fastbus module with 4 independent, 2 slave and 2 master, ports on two segments. It operates as a normal master on either segment or as a Block-Mover on both. The processor board is based on a 68020 microprocessor. A local/network switch allows operation as a host or as a normal module on the Fastbus network

  16. [Interspecific polymorphism of the glucosyltransferase domain of the sucrose synthase gene in the genus Malus and related species of Rosaceae].

    Science.gov (United States)

    Boris, K V; Kochieva, E Z; Kudryavtsev, A M

    2014-12-01

    The sequences that encode the main functional glucosyltransferase domain of sucrose synthase genes have been identified for the first time in 14 species of the genus Malus and related species of the family Rosaceae, and their polymorphism was investigated. Single nucleotide substitutions leading to amino acid substitutions in the protein sequence, including the conservative transmembrane motif sequence common to all sucrose synthase genes of higher plants, were detected in the studied sequences.

  17. Leukotriene C4 synthase and ischemic cardiovascular disease and obstructive pulmonary disease in 13,000 individuals

    DEFF Research Database (Denmark)

    Freiberg, Jacob J; Dahl, Morten; Tybjaerg-Hansen, Anne

    2009-01-01

    Ischemic cardiovascular disease and obstructive pulmonary disease involve inflammation. Leukotrienes may be important pro-inflammatory mediators. We tested the hypothesis that the (-1072)G > A and (-444)A > C promoter polymorphisms of leukotriene C4 synthase confer risk of transient ischemic atta...... with risk of asthma or COPD. Leukotriene C4 synthase promoter genotypes influence risk of TIA and ischemic stroke, but not risk of IHD/coronary atherosclerosis, asthma, or COPD....

  18. Synthesis of N-(Methoxycarbonylthienylmethylthioureas and Evaluation of Their Interaction with Inducible and Neuronal Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Michael D. Threadgill

    2010-04-01

    Full Text Available Two isomeric N-(methoxycarbonylthienylmethylthioureas were synthesised by a sequence of radical bromination of methylthiophenecarboxylic esters, substitution with trifluoroacetamide anion, deprotection, formation of the corresponding isothiocyanates and addition of ammonia. The interaction of these new thiophene-based thioureas with inducible and neuronal nitric oxide synthase was evaluauted. These novel thienylmethylthioureas stimulated the activity of inducible Nitric Oxide Synthase (iNOS.

  19. Effects of hypercapnia and NO synthase inhibition in sustained hypoxic pulmonary vasoconstriction

    Science.gov (United States)

    2012-01-01

    Background Acute respiratory disorders may lead to sustained alveolar hypoxia with hypercapnia resulting in impaired pulmonary gas exchange. Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange during local acute (0-30 min), as well as sustained (> 30 min) hypoxia by matching blood perfusion to alveolar ventilation. Hypercapnia with acidosis improves pulmonary gas exchange in repetitive conditions of acute hypoxia by potentiating HPV and preventing pulmonary endothelial dysfunction. This study investigated, if the beneficial effects of hypercapnia with acidosis are preserved during sustained hypoxia as it occurs, e.g in permissive hypercapnic ventilation in intensive care units. Furthermore, the effects of NO synthase inhibitors under such conditions were examined. Method We employed isolated perfused and ventilated rabbit lungs to determine the influence of hypercapnia with or without acidosis (pH corrected with sodium bicarbonate), and inhibitors of endothelial as well as inducible NO synthase on acute or sustained HPV (180 min) and endothelial permeability. Results In hypercapnic acidosis, HPV was intensified in sustained hypoxia, in contrast to hypercapnia without acidosis when HPV was amplified during both phases. L-NG-Nitroarginine (L-NNA), a non-selective NO synthase inhibitor, enhanced acute as well as sustained HPV under all conditions, however, the amplification of sustained HPV induced by hypercapnia with or without acidosis compared to normocapnia disappeared. In contrast 1400 W, a selective inhibitor of inducible NO synthase (iNOS), decreased HPV in normocapnia and hypercapnia without acidosis at late time points of sustained HPV and selectively reversed the amplification of sustained HPV during hypercapnia without acidosis. Hypoxic hypercapnia without acidosis increased capillary filtration coefficient (Kfc). This increase disappeared after administration of 1400 W. Conclusion Hypercapnia with and without acidosis increased HPV during

  20. Role of the Colletotrichum acutatum sesquiterpene synthase CaTPS in the biosynthesis of sesquiterpenoids

    DEFF Research Database (Denmark)

    Amby, Daniel Buchvaldt; Manczak, Tom; Petersen, Mikael Agerlin

    2016-01-01

    biosynthesis is performed by sesquiterpene synthases (TPS). Only a few TPSs have been functionally characterized from filamentous fungi and none from the genus Colletotrichum. Despite being an important fungal pathogen to agriculture, it is poorly understood at the molecular and chemical levels. The terpenoid...... characterization of TPS in Colletotrichum spp. and terpenoid profiles of Coll. acutatum, which could facilitate studies on the role of terpenoids in the ecology of Coll. acutatum....