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Sample records for synthase inhibition results

  1. Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

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    Louw Abraham I

    2010-04-01

    Full Text Available Abstract Background Plasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn were examined on a morphological, transcriptomic, proteomic and metabolic level. Results Morphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in

  2. Inhibition or knock out of Inducible nitric oxide synthase result in resistance to bleomycin-induced lung injury

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    Crimi Nunzio

    2005-06-01

    Full Text Available Abstract Background In the present study, by comparing the responses in wild-type mice (WT and mice lacking (KO the inducible (or type 2 nitric oxide synthase (iNOS, we investigated the role played by iNOS in the development of on the lung injury caused by bleomycin administration. When compared to bleomycin-treated iNOSWT mice, iNOSKO mice, which had received bleomycin, exhibited a reduced degree of the (i lost of body weight, (ii mortality rate, (iii infiltration of the lung with polymorphonuclear neutrophils (MPO activity, (iv edema formation, (v histological evidence of lung injury, (vi lung collagen deposition and (vii lung Transforming Growth Factor beta1 (TGF-β1 expression. Methods Mice subjected to intratracheal administration of bleomycin developed a significant lung injury. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from bleomycin-treated iNOSWT mice. Results The intensity and degree of nitrotyrosine staining was markedly reduced in tissue section from bleomycin-iNOSKO mice. Treatment of iNOSWT mice with of GW274150, a novel, potent and selective inhibitor of iNOS activity (5 mg/kg i.p. also significantly attenuated all of the above indicators of lung damage and inflammation. Conclusion Taken together, our results clearly demonstrate that iNOS plays an important role in the lung injury induced by bleomycin in the mice.

  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. Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 ...

    African Journals Online (AJOL)

    Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 and Lipid Peroxidation by Methanol Extract of Pericarpium Zanthoxyli. ... Production of iNOS induced by LPS was significantly (p < 0.05) inhibited by the extract, suggesting that the extract inhibits nitric oxide (NO) production by suppressing iNOS expression.

  5. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S. (Univ. of Texas Medical School, Houston (United States))

    1991-03-15

    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-({sup 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.

  7. 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

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

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    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.

  9. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    International Nuclear Information System (INIS)

    Nishida, Yoshihiro; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-01-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion

  10. Binding and inhibition of human spermidine synthase by decarboxylated S-adenosylhomocysteine

    Energy Technology Data Exchange (ETDEWEB)

    Še; #269; kut; #279; , Jolita; McCloskey, Diane E.; Thomas, H. Jeanette; Secrist III, John A.; Pegg, Anthony E.; Ealick, Steven E. (Cornell); (Southern Research); (UPENN-MED)

    2011-11-17

    Aminopropyltransferases are essential enzymes that form polyamines in eukaryotic and most prokaryotic cells. Spermidine synthase (SpdS) is one of the most well-studied enzymes in this biosynthetic pathway. The enzyme uses decarboxylated S-adenosylmethionine and a short-chain polyamine (putrescine) to make a medium-chain polyamine (spermidine) and 5'-deoxy-5'-methylthioadenosine as a byproduct. Here, we report a new spermidine synthase inhibitor, decarboxylated S-adenosylhomocysteine (dcSAH). The inhibitor was synthesized, and dose-dependent inhibition of human, Thermatoga maritima, and Plasmodium falciparum spermidine synthases, as well as functionally homologous human spermine synthase, was determined. The human SpdS/dcSAH complex structure was determined by X-ray crystallography at 2.0 {angstrom} resolution and showed consistent active site positioning and coordination with previously known structures. Isothermal calorimetry binding assays confirmed inhibitor binding to human SpdS with K{sub d} of 1.1 {+-} 0.3 {mu}M in the absence of putrescine and 3.2 {+-} 0.1 {mu}M in the presence of putrescine. These results indicate a potential for further inhibitor development based on the dcSAH scaffold.

  11. Inhibition of NO-synthase and degranulation of rat omental mast cells in vitro

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    A. M. Northover

    1996-01-01

    Full Text Available Mast cell amines, platelet-activating factor (PAF, thromboxanes and leukotrienes have been shown to be released during nitric oxide-synthase inhibition in the rat intestine. Mast cells in rat isolated omentum (OMCs or isolated from the rat peritoneal cavity (PMCs have been used here to investigate the relationship(s between these agents. N-nitro-L-arginine methyl ester (L-NAME, 100 μM caused some degranulation of OMCs, but no enhancement of histamine release from PMCs. PAF (5 μM and U46619 (1 μM degranulated OMCs and enhanced histamine release from PMCs. Pre-treatment of the omentum with BN52021 (10 μM inhibited degranulation of OMCs in response to L-NAME, PAF or U46619. Pretreatment with 1-benzylimidazole (5 or 50 μM inhibited the effect of L-NAME but not that of PAF. Indomethacin (1 μM or sodium nitroprusside (10 μM also inhibited the effects of L-NAME, but nordihydroguaiaretic acid (30 μM did not. In PMCs BN52021 inhibited PAF-induced, but not U46619-induced, release of histamine. These results suggest that inhibition of nitric oxidesynthase in the omentum by L-NAME allows thromboxanes to release PAF, which in turn degranulates and releases histamine from OMCs.

  12. Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 ...

    African Journals Online (AJOL)

    HP

    Purpose: To explore the antioxidant properties of the methanol extract of Pericarpium Zanthoxyli and its effect on inducible nitric oxide synthase (iNOS), cycleooxygenase-2 (COX-2) and lipopolysaccharides (LPS)-induced cell damage in macrophage cells. Methods: Anti-oxidant activities were tested by measuring free ...

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

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    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.

  14. Silencing of xylose isomerase and cellulose synthase by siRNA inhibits encystation in Acanthamoeba castellanii.

    Science.gov (United States)

    Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Khan, Naveed Ahmed

    2013-03-01

    A key challenge in the successful treatment of Acanthamoeba infections is its ability to transform into a dormant cyst form that is resistant to physiological conditions and pharmacological therapies, resulting in recurrent infections. The carbohydrate linkage analysis of cyst walls of Acanthamoeba castellanii showed variously linked sugar residues, including xylofuranose/xylopyranose, glucopyranose, mannopyranose, and galactopyranose. Here, it is shown that exogenous xylose significantly reduced A. castellanii differentiation in encystation assays (P castellanii. Inhibition of both enzymes using siRNA against xylose isomerase and cellulose synthase but not scrambled siRNA attenuated A. castellanii metamorphosis, as demonstrated by the arrest of encystation of A. castellanii. Neither inhibitor nor siRNA probes had any effect on the viability and extracellular proteolytic activities of A. castellanii.

  15. A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease

    NARCIS (Netherlands)

    McEachern, Kerry Anne; Fung, John; Komarnitsky, Svetlana; Siegel, Craig S.; Chuang, Wei-Lien; Hutto, Elizabeth; Shayman, James A.; Grabowski, Gregory A.; Aerts, Johannes M. F. G.; Cheng, Seng H.; Copeland, Diane P.; Marshall, John

    2007-01-01

    An approach to treating Gaucher disease is substrate inhibition therapy which seeks to abate the aberrant lysosomal accumulation of glucosylceramide. We have identified a novel inhibitor of glucosylceramide synthase (Genz-112638) and assessed its activity in a murine model of Gaucher disease

  16. A Systems Chemical Biology Study of Malate Synthase and Isocitrate Lyase Inhibition in Mycobacterium tuberculosis During Active and NRP Growth

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    May, Elebeoba E.; Leitão, Andrei; Tropsha, Alexander; Oprea, Tudor I.

    2013-01-01

    The ability of Mycobacterium tuberculosis (Mtb) to survive in low oxygen environments enables the bacterium to persist in a latent state within host tissues. In vitro studies of Mtb growth have identified changes in isocitrate lyase (ICL) and malate synthase (MS) that enable bacterial persistent under low oxygen and other environmentally limiting conditions. Systems chemical biology (SCB) enables us to evaluate the effects of small molecule inhibitors not only on the reaction catalyzed by malate synthase and isocitrate lyase, but the effect on the complete tricarboxylic acid cycle (TCA) by taking into account complex network relationships within that system. To study the kinetic consequences of inhibition on persistent bacilli, we implement a systems-chemical biology (SCB) platform and perform a chemistry-centric analysis of key metabolic pathways believed to impact Mtb latency. We explore consequences of disrupting the function of malate synthase (MS) and isocitrate lyase (ICL) during aerobic and hypoxic non-replicating persistence (NRP) growth by using the SCB method to identify small molecules that inhibit the function of MS and ICL, and simulating the metabolic consequence of the disruption. Results indicate variations in target and non-target reaction steps, clear differences in the normal and low oxygen models, as well as dosage dependent response. Simulation results from singular and combined enzyme inhibition strategies suggest ICL may be the more effective target for chemotherapeutic treatment against Mtb growing in a microenvironment where oxygen is slowly depleted, which may favor persistence. PMID:24121675

  17. Glycogen Synthase Kinase 3β Inhibition as a Therapeutic Approach in the Treatment of Endometrial Cancer

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    Liang Ma

    2013-08-01

    Full Text Available Alternative strategies beyond current chemotherapy and radiation therapy regimens are needed in the treatment of advanced stage and recurrent endometrial cancers. There is considerable promise for biologic agents targeting the extracellular signal-regulated kinase (ERK pathway for treatment of these cancers. Many downstream substrates of the ERK signaling pathway, such as glycogen synthase kinase 3β (GSK3β, and their roles in endometrial carcinogenesis have not yet been investigated. In this study, we tested the importance of GSK3β inhibition in endometrial cancer cell lines and in vivo models. Inhibition of GSK3β by either lithium chloride (LiCl or specific GSK3β inhibitor VIII showed cytostatic and cytotoxic effects on multiple endometrial cancer cell lines, with little effect on the immortalized normal endometrial cell line. Flow cytometry and immunofluorescence revealed a G2/M cell cycle arrest in both type I (AN3CA, KLE, and RL952 and type II (ARK1 endometrial cancer cell lines. In addition, LiCl pre-treatment sensitized AN3CA cells to the chemotherapy agent paclitaxel. Administration of LiCl to AN3CA tumor-bearing mice resulted in partial or complete regression of some tumors. Thus, GSK3β activity is associated with endometrial cancer tumorigenesis and its pharmacologic inhibition reduces cell proliferation and tumor growth.

  18. Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages

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    Smith Milton

    2006-11-01

    Full Text Available Abstract Background 2-Chloroethyl ethyl sulphide (CEES is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD. Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. Results We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA or dichlorofluorescin diacetate (DCFH-DA. Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity Conclusion CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-κB signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS

  19. Propolis attenuates oxidative injury in brain and lung of nitric oxide synthase inhibited rats

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    Zeliha Selamoglu-Talas

    2015-10-01

    Full Text Available Background: The blocking of nitric oxide synthase (NOS activity may reason vasoconstriction with formation of reactive oxygen species. Propolis has biological and pharmacological properties, such as antioxidant. The aim of this study was to examine the antioxidant effects of propolis which natural product on biochemical parameters in brain and lung tissues of acute nitric oxide synthase inhibited rats by Nω-nitro-L-arginine methyl ester (L-NAME.Methods: Rats have been received L-NAME (40 mg/kg, intraperitoneally, NOS inhibitor for 15 days to produce hypertension and propolis (200mg/kg, by gavage the lastest 5 of 15 days.Results: There  were  the  increase  (P<0.001  in  the  malondialdehyde  levels  in  the  L-NAME treatment groups when compared to control rats, but the decrease (P<0.001 in the catalase activities in both brain and lung tissues. There were statistically changes (P<0.001 in these parameters of L-NAME+propolis treated rats as compared with L-NAME-treated group.Conclusion: The application of L-NAME to the Wistar rats resulted in well developed oxidative stress. Also, propolis may influence endothelial NO production. Identification of such compounds and characterisation of their cellular actions may increase our knowledge of the regulation of endothelial NO production and could provide valuable clues for the prevention or treatment of hypertensive diseases and oxidative stress.

  20. Chlorine gas exposure causes systemic endothelial dysfunction by inhibiting endothelial nitric oxide synthase-dependent signaling.

    Science.gov (United States)

    Honavar, Jaideep; Samal, Andrey A; Bradley, Kelley M; Brandon, Angela; Balanay, Joann; Squadrito, Giuseppe L; MohanKumar, Krishnan; Maheshwari, Akhil; Postlethwait, Edward M; Matalon, Sadis; Patel, Rakesh P

    2011-08-01

    Chlorine gas (Cl(2)) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl(2) exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl(2) promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl(2) for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl(2) dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl(2) (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl(2)-exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl(2) exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl(2)-exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl(2) exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.

  1. Lycopene inhibits LPS-induced proinflammatory mediator inducible nitric oxide synthase in mouse macrophage cells.

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    Rafi, Mohamed M; Yadav, Prem Narayan; Reyes, Marynell

    2007-01-01

    Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 microM) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene.

  2. Human farnesyl pyrophosphate synthase inhibition by nitrogen bisphosphonates: a 3D-QSAR study

    Science.gov (United States)

    Fernández, David; Ortega-Castro, Joaquín; Frau, Juan

    2013-08-01

    We report the results of a comparative molecular field analysis and comparative molecular similarity index analysis of the human farnesyl pyrophosphate synthase (FPPS) inhibition by nitrogen bisphosphonates (NBPs) taking into account their time-dependent inhibition efficacies. The 3D-QSAR models obtained provide steric, electrostatic and hydrophobic contour maps consistent with the interactions into the active site of human FPPS observed in available crystallographic structures. Furthermore, the 3D-QSAR models obtained provide accurately IC50 values of the NBPs of the training set. The predictive ability of these 3D-QSAR models was found to rely on the choice of biologically active conformations of the target molecules and on a careful examination of the protonation status of the NBPs in the training set. The best models obtained can be useful to predict biological values of a high number of NBPs that have been used for the treatment of different diseases as potential inhibitors of the activity of the FPPS enzyme.

  3. Inhibition of human lanosterol synthase by the constituents of Colocasia esculenta (taro).

    Science.gov (United States)

    Sakano, Yuichi; Mutsuga, Motoh; Tanaka, Rie; Suganuma, Hiroyuki; Inakuma, Takahiro; Toyoda, Masatake; Goda, Yukihiro; Shibuya, Masaaki; Ebizuka, Yutaka

    2005-02-01

    Ethanol extracts of lyophilized vegetables were tested for inhibition of human lanosterol synthase (hOSC) in order to find the compounds to suppress cholesterol biosynthesis. Of 130 samples tested, twelve samples showed significant inhibition. Among them, Colocasia esculenta (taro) showed the highest inhibition (55% inhibition at 300 microg/ml). Examination of activity variation among eight taro cultivars indicated that "Aichi-wase" and "Yatsugashira" had the most potent activity for hOSC inhibition. In order to identify the active constituent of taro, ethanol extracts of "Aichi-wase" were partitioned with hexane and aqueous methanol, and fractionated by silica gel column chromatography. Inhibitory activity was concentrated in two major active fractions. Further purification of these fractions by preparative HPLC gave three monogalactosyldiacylglycerols and five digalactosyldiacylglycerols as active compounds that showed 28 to 67% inhibitory activities at the concentration 300 microg/ml.

  4. Normalization of Supine Blood Pressure After Nitric Oxide Synthase Inhibition in Persons With Tetraplegia

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    Wecht, Jill M; Weir, Joseph P; Krothe, AnnMarie H; Spungen, Ann M; Bauman, William A

    2007-01-01

    Background/Objective: Orthostatic hypotension is a well-defined clinical consequence of spinal cord injury (SCI), particularly in those with tetraplegia. The etiology of orthostatic hypotension is thought to be loss of sympathetic vasomotor control, although other factors may play a role. There is evidence of up-regulation of nitric oxide synthase (NOS) activity after hind-limb suspension in rats, a condition of antigravity that may have similar vascular effects as shown in persons with tetraplegia caused by paralysis. The study objective was to determine the effect of a NOS inhibitor (nitro-L-arginine methyl ester [L-NAME]) on supine mean arterial pressure in persons with chronic tetraplegia compared with non-SCI controls. Methods: Fourteen individuals participated (7 with tetraplegia and 7 controls). Subjects visited the laboratory twice for placebo on day 1 and L-NAME (1 mg/kg) on day 2; both were infused intravenously over 60 minutes. Blood pressure was monitored for 3 hours after infusion at the brachial artery using a standard manual cuff. Results: Mean arterial pressure (MAP) was lower at baseline (P tetraplegia group compared with the control group. L-NAME increased MAP in both groups; however, the relative increase was greater in the tetraplegia group compared with the control group, such that group differences for MAP were eliminated. Supine MAP was normalized with L-NAME, and there was an increased sensitivity to NOS inhibition in the group with tetraplegia. Conclusions: These findings indicate that blood pressure dysregulation in persons with tetraplegia may reflect increased vascular NO and suggest a novel treatment of hypotension using NOS inhibition in this population. PMID:17385265

  5. Alpha-mangostin inhibits intracellular fatty acid synthase and induces apoptosis in breast cancer cells.

    Science.gov (United States)

    Li, Ping; Tian, Weixi; Ma, Xiaofeng

    2014-06-03

    Fatty acid synthase (FAS) has been proven over-expressed in human breast cancer cells and consequently, has been recognized as a target for breast cancer treatment. Alpha-mangostin, a natural xanthone found in mangosteen pericarp, has a variety of biological activities, including anti-cancer effect. In our previous study, alpha-mangostin had been found both fast-binding and slow-binding inhibitions to FAS in vitro. This study was designed to investigate the activity of alpha-mangostin on intracellular FAS activity in FAS over-expressed human breast cancer cells, and to testify whether the anti-cancer activity of alpha-mangostin may be related to its inhibitory effect on FAS. We evaluated the cytotoxicity of alpha-mangostin in human breast cancer MCF-7 and MDA-MB-231 cells. Intracellular FAS activity was measured by a spectrophotometer at 340 nm of NADPH absorption. Cell Counting Kit assay was used to test the cell viability. Immunoblot analysis was performed to detect FAS expression level, intracellular fatty acid accumulation and cell signaling (FAK, ERK1/2 and AKT). Apoptotic effects were detected by flow cytometry and immunoblot analysis of PARP, Bax and Bcl-2. Small interfering RNA was used to down-regulate FAS expression and/or activity. Alpha-mangostin could effectively suppress FAS expression and inhibit intracellular FAS activity, and result in decrease of intracellular fatty acid accumulation. It could also reduce cell viability, induce apoptosis in human breast cancer cells, increase in the levels of the PARP cleavage product, and attenuate the balance between anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family. Moreover, alpha-mangostin inhibited the phosphorylation of FAK. However, the active forms of AKT, and ERK1/2 proteins were not involved in the changes of FAS expression induced by alpha-mangostin. Alpha-mangostin induced breast cancer cell apoptosis by inhibiting FAS, which provide a basis for the development of xanthone as an agent for

  6. Crystallographic and enzymatic insights into the mechanisms of Mg-ADP inhibition in the A1complex of the A1AOATP synthase.

    Science.gov (United States)

    Singh, Dhirendra; Grüber, Gerhard

    2018-01-01

    F-ATP synthases are described to have mechanisms which regulate the unnecessary depletion of ATP pool during an energy limited state of the cell. Mg-ADP inhibition is one of the regulatory features where Mg-ADP gets entrapped in the catalytic site, preventing the binding of ATP and further inhibiting ATP hydrolysis. Knowledge about the existence and regulation of the related archaeal-type A 1 A O ATP synthases (A 3 B 3 CDE 2 FG 2 ac) is limited. We demonstrate MgADP inhibition of the enzymatically active A 3 B 3 D- and A 3 B 3 DF complexes of Methanosarcina mazei Gö1 A-ATP synthase and reveal the importance of the amino acids P235 and S238 inside the P-loop (GPFGSGKTV) of the catalytic A subunit. Substituting these two residues by the respective P-loop residues alanine and cysteine (GAFGCGKTV) of the related eukaryotic V-ATPase increases significantly the ATPase activity of the enzyme variant and abolishes MgADP inhibition. The atomic structure of the P235A, S238C double mutant of subunit A of the Pyrococcus horikoshii OT3 A-ATP synthase provides details of how these critical residues affect nucleotide-binding and ATP hydrolysis in this molecular engine. The qualitative data are confirmed by quantitative results derived from fluorescence correlation spectroscopy experiments. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Hemodynamic effects of nitric oxide synthase inhibition before and after cardiac arrest in infant piglets.

    Science.gov (United States)

    Schleien, C L; Kuluz, J W; Gelman, B

    1998-04-01

    Using infant piglets, we studied the effects of nonspecific inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg) on vascular pressures, regional blood flow, and cerebral metabolism before 8 min of cardiac arrest, during 6 min of cardiopulmonary resuscitation (CPR), and at 10 and 60 min of reperfusion. We tested the hypotheses that nonspecific NO synthase inhibition 1) will attenuate early postreperfusion hyperemia while still allowing for successful resuscitation after cardiac arrest, 2) will allow for normalization of blood flow to the kidneys and intestines after cardiac arrest, and 3) will maintain cerebral metabolism in the face of altered cerebral blood flow after reperfusion. Before cardiac arrest, L-NAME increased vascular pressures and cardiac output and decreased blood flow to brain (by 18%), heart (by 36%), kidney (by 46%), and intestine (by 52%) compared with placebo. During CPR, myocardial flow was maintained in all groups to successfully resuscitate 24 of 28 animals [P value not significant (NS)]. Significantly, L-NAME attenuated postresuscitation hyperemia in cerebellum, diencephalon, anterior cerebral, and anterior-middle watershed cortical brain regions and to the heart. Likewise, cerebral metabolic rates of glucose (CMRGluc) and of lactate production (CMRLac) were not elevated at 10 min of reperfusion. These cerebral blood flow and metabolic effects were reversed by L-arginine. Flows returned to baseline levels by 60 min of reperfusion. Kidney and intestinal flow, however, remained depressed throughout reperfusion in all three groups. Thus nonspecific inhibition of NO synthase did not adversely affect the rate of resuscitation from cardiac arrest while attenuating cerebral and myocardial hyperemia. Even though CMRGluc and CMRLac early after resuscitation were decreased, they were maintained at baseline levels. This may be clinically advantageous in protecting the brain and heart from the damaging effects of

  8. Alpha-mangostin inhibits intracellular fatty acid synthase and induces apoptosis in breast cancer cells

    OpenAIRE

    Li, Ping; Tian, Weixi; Ma, Xiaofeng

    2014-01-01

    Background Fatty acid synthase (FAS) has been proven over-expressed in human breast cancer cells and consequently, has been recognized as a target for breast cancer treatment. Alpha-mangostin, a natural xanthone found in mangosteen pericarp, has a variety of biological activities, including anti-cancer effect. In our previous study, alpha-mangostin had been found both fast-binding and slow-binding inhibitions to FAS in vitro. This study was designed to investigate the activity of alpha-mangos...

  9. Insulin induces an increase in cytosolic glucose levels in 3T3-L1 cells with inhibited glycogen synthase activation.

    Science.gov (United States)

    Chowdhury, Helena H; Kreft, Marko; Jensen, Jørgen; Zorec, Robert

    2014-10-02

    Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

  10. Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans.

    Science.gov (United States)

    Pacheco-Moisés, Fermín; Minauro-Sanmiguel, Fernando; Bravo, Concepción; García, José J

    2002-08-01

    The F1F0 complex of Paracoccus denitrificans (PdF1F0) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF1F0-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur J. Biochem. 267, 993-1000) but its effect on the PdF1F0-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and 32Pi ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal delta pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K0.5 for Mg2+-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC50 of Mg2+-ADP for inhibition of the PdF1F0-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF1F0 complex. These effects are not due to membrane or PdF1F0 uncoupling. Kinetic and structural modifications that could account for these results are discussed.

  11. Effect of acute nitric oxide synthase inhibition in the modulation of heart rate in rats

    Directory of Open Access Journals (Sweden)

    A.L. Fellet

    2003-05-01

    Full Text Available Acute nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME on chronotropic and pressor responses was studied in anesthetized intact rats and rats submitted to partial and complete autonomic blockade. Blood pressure and heart rate were monitored intra-arterially. Intravenous L-NAME injection (7.5 mg/kg elicited the same hypertensive response in intact rats and in rats with partial (ganglionic and parasympathetic blockade and complete autonomic blockade (38 ± 3, 55 ± 6, 54 ± 5, 45 ± 5 mmHg, respectively; N = 9, P = NS. L-NAME-induced bradycardia at the time when blood pressure reached the peak plateau was similar in intact rats and in rats with partial autonomic blockade (43 ± 8, 38 ± 5, 46 ± 6 bpm, respectively; N = 9, P = NS. Rats with combined autonomic blockade showed a tachycardic response to L-NAME (10 ± 3 bpm, P<0.05 vs intact animals, N = 9. Increasing doses of L-NAME (5.0, 7.5 and 10 mg/kg, N = 9 caused a similar increase in blood pressure (45 ± 5, 38 ± 3, 44 ± 9 mmHg, respectively; P = NS and heart rate (31 ± 4, 34 ± 3, 35 ± 4 bpm, respectively; P = NS. Addition of L-NAME (500 µM to isolated atria from rats killed by cervical dislocation and rats previously subjected to complete autonomic blockade did not affect spontaneous beating or contractile strength (N = 9. In vivo results showed that L-NAME promoted a tachycardic response in rats with complete autonomic blockade, whereas the in vitro experiments showed no effect on intrinsic heart rate, suggesting that humoral mechanisms may be involved in the L-NAME-induced cardiac response.

  12. Chamomile: an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity.

    Science.gov (United States)

    Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K; Gupta, Sanjay

    2010-12-01

    Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we investigated the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and explored its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β, IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ, the upstream kinase regulating NF-κB/Rel activity, and degradation of inhibitory factor-κB. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent.

  13. Chamomile, an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity

    Science.gov (United States)

    Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K; Gupta, Sanjay

    2010-01-01

    Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we aimed to investigate the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and to explore its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β , IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ , the upstream kinase regulating NF-κ B/Rel activity, and degradation of inhibitory factor-κ B. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent. PMID:21042790

  14. Biochemical and Structural Basis for Inhibition of Enterococcus faecalis Hydroxymethylglutaryl-CoA Synthase, mvaS, by Hymeglusin

    Energy Technology Data Exchange (ETDEWEB)

    Skaff, D. Andrew; Ramyar, Kasra X.; McWhorter, William J.; Barta, Michael L.; Geisbrecht, Brian V.; Miziorko, Henry M. (UMKC)

    2012-07-25

    Hymeglusin (1233A, F244, L-659-699) is established as a specific {beta}-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 {angstrom}) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.

  15. Lithium Impairs Kidney Development and Inhibits Glycogen Synthase Kinase-3β in Collecting Duct Principal Cells

    DEFF Research Database (Denmark)

    Kjærsgaard, Gitte; Madsen, Kirsten; Marcussen, Niels

    on serine9 (pGSK-3β)and subsequent epithelial to mesenchymal dedifferentiation (EMT). GSK-3β immunoreactive protein was associated with collecting ducts in developing and adult human and rat kidney. Total GSK-3β protein abundance was stable in medulla while it decreased in cortex in the postnatal period......GSK-3β abundance in collecting duct. The data are compatible with the notion that increased GSK-3β activity in the postnatal kidney medulla is necessary for kidney development.......The postnatal rat kidney is highly susceptible to Lithium (Li+), which leads to significant tissue injury. We hypothesized that Li+ impairs development of the kidney through entry into epithelial cells of the distal nephron, inhibition of Glycogen Synthase Kinase-3β (GSK-3β) through phosphorylation...

  16. Target-site resistance to acetolactate synthase (ALS)-inhibiting herbicides in Amaranthus palmeri from Argentina.

    Science.gov (United States)

    Larran, Alvaro S; Palmieri, Valeria E; Perotti, Valeria E; Lieber, Lucas; Tuesca, Daniel; Permingeat, Hugo R

    2017-12-01

    Herbicide-resistant weeds are a serious problem worldwide. Recently, two populations of Amaranthus palmeri with suspected cross-resistance to acetolactate synthase (ALS)-inhibiting herbicides (R1 and R2) were found by farmers in two locations in Argentina (Vicuña Mackenna and Totoras, respectively). We conducted studies to confirm and elucidate the mechanism of resistance. We performed in vivo dose-response assays, and confirmed that both populations had strong resistance to chlorimuron-ethyl, diclosulam and imazethapyr when compared with a susceptible population (S). In vitro ALS activity inhibition tests only indicated considerable resistance to imazethapyr and chlorimuron-ethyl, indicating that other non-target mechanisms could be involved in diclosulam resistance. Subsequently, molecular analysis of als nucleotide sequences revealed three single base-pair mutations producing substitutions in amino acids previously associated with resistance to ALS inhibitors, A122, W574, and S653. This is the first report of als resistance alleles in A. palmeri in Argentina. The data support the involvement of a target-site mechanism of resistance to ALS-inhibiting herbicides. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  17. Extracts of Magnoliae flos inhibit inducible nitric oxide synthase via ERK in human respiratory epithelial cells.

    Science.gov (United States)

    Baek, Jin Ah; Lee, Yang Deok; Lee, Chan Bog; Go, Hyeon Kyu; Kim, Jin Pyo; Seo, Jeong Ju; Rhee, Yang Keun; Kim, A Mi; Na, Dong Jib

    2009-03-01

    Nitric oxide (NO) is a marker of pulmonary inflammation. In asthma, the levels of exhaled NO are elevated and the source of this increased NO is inducible nitric oxide synthase (iNOS) within airway epithelial cells. Epimagnolin and fargesin are compounds isolated from the ethanol extract of Magnoliae flos, the seed of the Magnolia plant and are used to treat nasal congestion, headache and sinusitis in Asian countries. This study investigated whether epimagnolin and fargesin inhibit extracellular signal-regulated kinase (ERK) activation and decrease iNOS expression and NO production in stimulated human respiratory epithelial cells. An immortal Type II alveolar cell line of human origin (A549) was stimulated by cytomix (CM), composed of IL-1beta, TNF-alpha and IFN-gamma, with or without concurrent exposure to M. flos extract (epimagnolin or fargesin). CM-induced levels of NO production, iNOS expression and ERK activation were evaluated. A549 cells stimulated with CM showed increases in iNOS mRNA and protein expression, and NO synthesis. However, treatment with epimagnolin or fargesin decreased levels of iNOS mRNA and protein expression, and NO synthesis. CM stimulated a rapid increase in the activity of ERK, whereas epimagnolin and fargesin inhibited ERK phosphorylation. Epimagnolin and fargesin inhibit iNOS expression and decrease production of NO via ERK pathway in cytokine-stimulated human respiratory epithelial cells.

  18. Osteopontin protects against hyperoxia-induced lung injury by inhibiting nitric oxide synthases.

    Science.gov (United States)

    Zhang, Xiang-Feng; Liu, Shuang; Zhou, Yu-Jie; Zhu, Guang-Fa; Foda, Hussein D

    2010-04-05

    Exposure of adult mice to more than 95% O(2) produces a lethal injury by 72 hours. Nitric oxide synthase (NOS) is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury (ALI). Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of nitric oxide production. However, the relationship between nitric oxide and endogenous OPN in lung tissue during hyperoxia-induced ALI has not yet been elucidated, thus we examined the role that OPN plays in the hyperoxia-induced lung injury and its relationships with NOS. One hundred and forty-four osteopontin knock-out (KO) mice and their matched wild type background control (WT) were exposed in sealed cages > 95% oxygen or room air for 24- 72 hours, and the severity of lung injury was assessed; expression of OPN, endothelial nitric oxide synthase (eNOS) and iNOS mRNA in lung tissues at 24, 48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction (RT-PCR); immunohistochemistry (IHC) was performed for the detection of iNOS, eNOS, and OPN protein in lung tissues. OPN KO mice developed more severe acute lung injury at 72 hours of hyperoxia. The wet/dry weight ratio increased to 6.85 +/- 0.66 in the KO mice at 72 hours of hyperoxia as compared to 5.31 +/- 0.92 in the WT group (P < 0.05). iNOS mRNA (48 hours: 1.04 +/- 0.08 vs. 0.63 +/- 0.09, P < 0.01; 72 hours: 0.89 +/- 0.08 vs. 0.72 +/- 0.09, P < 0.05) and eNOS mRNA (48 hours: 0.62 +/- 0.08 vs. 0.43 +/- 0.09, P < 0.05; 72 hours: 0.67 +/- 0.08 vs. 0.45 +/- 0.09, P < 0.05) expression was more significantly increased in OPN KO mice than their matched WT mice when exposed to hyperoxia. IHC study showed higher expression of iNOS (20.54 +/- 3.18 vs. 12.52 +/- 2.46, P < 0.05) and eNOS (19.83 +/- 5.64 vs. 9.45 +/- 3.82, P < 0.05) in lung tissues of OPN KO mice at 72 hours of hyperoxia. OPN can protect against

  19. Effects of long-term inhibition of neuronal nitric oxide synthase on blood pressure and renin release

    DEFF Research Database (Denmark)

    Ollerstam, A.; Skøtt, O.; Ek, J.

    2001-01-01

    Nitric oxide (NO) produced by neuronal NO-synthase (nNOS) in macula densa cells may be involved in the control of renin release. 7-Nitro indazole (7-NI) inhibits nNOS, and we investigated the effect of short- (4 days) and long-term (4 weeks) 7-NI treatment on blood pressure (BP), plasma renin...

  20. Inhibition of nitric oxide synthase expression in activated microglia and peroxynitrite scavenging activity by Opuntia ficus indica var. saboten.

    Science.gov (United States)

    Lee, Ming Hong; Kim, Jae Yeon; Yoon, Jeong Hoon; Lim, Hyo Jin; Kim, Tae Hee; Jin, Changbae; Kwak, Wie-Jong; Han, Chang-Kyun; Ryu, Jae-Ha

    2006-09-01

    Activated microglia by neuronal injury or inflammatory stimulation overproduce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) such as superoxide anion, resulting in neurodegenerative diseases. The toxic peroxynitrite (ONOO-), the reaction product of NO and superoxide anion further contributes to oxidative neurotoxicity. A butanol fraction obtained from 50% ethanol extracts of Opuntia ficus indica var. saboten (Cactaceae) stem (SK OFB901) and its hydrolysis product (SK OFB901H) inhibited the production of NO in LPS-activated microglia in a dose dependent manner (IC50 15.9, 4.2 microg/mL, respectively). They also suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells at higher than 30 microg/mL as observed by western blot analysis and RT-PCR experiment. They also inhibited the degradation of I-kappaB-alpha in activated microglia. Moreover, they showed strong activity of peroxynitrite scavenging in a cell free bioassay system. These results imply that Opuntia ficus indica may have neuroprotective activity through the inhibition of NO production by activated microglial cells and peroxynitrite scavenging activity. Copyright (c) 2006 John Wiley & Sons, Ltd.

  1. Mitochondrial dysfunction is responsible for fatty acid synthase inhibition-induced apoptosis in breast cancer cells by PdpaMn.

    Science.gov (United States)

    Wang, Qiang; Du, Xia; Zhou, Bingjie; Li, Jing; Lu, Wenlong; Chen, Qiuyun; Gao, Jing

    2017-12-01

    Targeting cellular metabolism is becoming a hallmark to overcome drug resistance in breast cancer treatment. Activation of fatty acid synthase (FASN) has been shown to promote breast cancer cell growth. However, there is no concrete report underlying the mechanism associated with mitochondrial dysfunction in relation to fatty acid synthase inhibition-induced apoptosis in breast cancer cells. The current study is aimed at exploring the effect of the novel manganese (Mn) complex, labeled as PdpaMn, on lipid metabolism and mitochondrial function in breast cancer cells. Herein, we observed that PdpaMn displayed strong cytotoxicity on breast cancer cell lines and selectively targeted the tumor without affecting the normal organs or cells in vivo. We also observed that PdpaMn could bind to TE domain of FASN and decrease the activity and the level of expression of FASN, which is an indication that FASN could serve as a target of PdpaMn. In addition, we demonstrated that PdpaMn increased intrinsic apoptosis in breast cancer cells relayed by a suppressed the level of expression of FASN, followed by the release of mitochondrial cytochrome c and the activation of caspases-9. Instigated by the above observations, we hypothesized that PdpaMn-induced apoptosis events are dependent on mitochondrial dysfunction. Indeed, we found that mitochondrial membrane potential (MMP) collapse, mitochondrial oxygen consumption reduction and adenosine triphosphate (ATP) release were deeply repressed. Furthermore, our results showed that PdpaMn significantly increased the reactive oxygen species (ROS) production, and the protection conferred by the free radical scavenger N-acetyl-cysteine (NAC) indicates that PdpaMn-induced apoptosis through an oxidative stress-associated mechanism. More so, the above results have demonstrated that mitochondrial dysfunction participated in FASN inhibition-induce apoptosis in breast cancer cells by PdpaMn. Therefore, PdpaMn may be considered as a good candidate

  2. Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

    International Nuclear Information System (INIS)

    Manceur, Aziza P.; Tseng, Michael; Holowacz, Tamara; Witterick, Ian; Weksberg, Rosanna; McCurdy, Richard D.; Warsh, Jerry J.; Audet, Julie

    2011-01-01

    The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

  3. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  4. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    Energy Technology Data Exchange (ETDEWEB)

    Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  5. Role of nitric oxide synthase inhibition in the acute hypertensive response to intracerebroventricular cadmium

    Science.gov (United States)

    Demontis, Maria Piera; Varoni, Maria Vittoria; Volpe, Anna Rita; Emanueli, Costanza; Madeddu, Paolo

    1998-01-01

    In the rat, intracerebroventricular (i.c.v.) injection of cadmium, a pollutant with long biological half-life, causes a sustained increase in blood pressure at doses that are ineffective by peripheral route. Since cadmium inhibits calcium-calmodulin constitutive nitric oxide (NO) synthase in cytosolic preparations of rat brain, this mechanism may be responsible for the acute pressor action of this heavy metal.To test this possibility, we evaluated the effect of i.c.v. injection of 88 nmol cadmium in normotensive unanaesthetized Wistar rats, which were i.c.v. pre-treated with: (1) saline (control), (2) L-arginine (L-Arg), to increase the availability of substrate for NO biosynthesis, (3) D-arginine (D-Arg), (4) 3-[4-morpholinyl]-sydnonimine-hydrochloride (SIN-1), an NO donor, or (5) CaCl2, a cofactor of brain calcium-calmodulin-dependent cNOSI. In additional experiments, the levels of L-citrulline (the stable equimolar product derived from enzymatic cleavage of L-Arg by NO synthase) were determined in the brain of vehicle- or cadmium-treated rats.The pressor response to cadmium reached its nadir at 5 min (43±4 mmHg) and lasted over 20 min in controls. L-Citrulline/protein content was reduced from 35 up to 50% in the cerebral cortex, pons, hippocampus, striatus, hypothalamus (P<0.01) of cadmium-treated rats compared with controls. Central injection of NG nitro-L-arginine-methylester (L-NAME) also reduced the levels of L-citrulline in the brain.Both the magnitude and duration of the response were attenuated by 1.21 and 2.42 μmol SIN-1 (32±3 and 15±4 mmHg, P<0.05), or 1 μmol CaCl2 (6±4 mmHg, P<0.05). Selectivity of action exerted by SIN-1 was confirmed by the use of another NO donor, S-nitroso-N-acetyl-penicillamine (SNAP). Both L-Arg and D-Arg caused a mild but significant attenuation in the main phase of the pressor response evoked by cadmium. However, only L-Arg reduced the magnitude of the delayed, pressor response. Despite their similarity in

  6. Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Mahalingan, Krishna K.; Baskaran†, Sulochanadevi; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D. (Indiana-Med)

    2017-01-10

    Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.

  7. Pharmacological inhibition of glycogen synthase kinase 3 regulates T cell development in vitro.

    Directory of Open Access Journals (Sweden)

    Jan-Hendrik Schroeder

    Full Text Available The development of functional T cells requires receptor-mediated transition through multiple checkpoints in the thymus. Double negative 3 (DN3 thymocytes are selected for the presence of a rearranged TCR beta chain in a process termed β-selection which requires signalling via the pre-TCR, Notch1 and CXCL12. Signal integration by these receptors converges on core pathways including the Phosphatidylinositol-3-kinase (PI3K pathway. Glycogen Synthase Kinase 3 (GSK3 is generally thought to be negatively regulated by the PI3K pathway but its role in β-selection has not been characterised. Here we show that developmental progression of DN3 thymocytes is promoted following inhibition of GSK3 by the synthetic compound CHIR99021. CHIR99021 allows differentiation in the absence of pre-TCR-, Notch1- or CXCL12-mediated signalling. It antagonizes IL-7-mediated inhibition of DP thymocyte differentiation and increases IL-7-promoted cell recovery. These data indicate a potentially important role for inactivation of GSK3 during β-selection. They might help to establish an in vitro stromal cell-free culture system of thymocyte development and offer a new platform for screening regulators of proliferation, differentiation and apoptosis.

  8. New insights into human farnesyl pyrophosphate synthase inhibition by second-generation bisphosphonate drugs

    Science.gov (United States)

    Fernández, D.; Ramis, R.; Ortega-Castro, J.; Casasnovas, R.; Vilanova, B.; Frau, J.

    2017-07-01

    Pamidronate, alendronate, APHBP and neridronate are a group of drugs, known as second-generation bisphosphonates (2G-BPs), commonly used in the treatment of bone-resorption disorders, and recently their use has been related to some collateral side effects. The therapeutic activity of 2G-BPs is related to the inhibition of the human Farnesyl Pyrophosphate Synthase (hFPPS). Available inhibitory activity values show that 2G-BPs act time-dependently, showing big differences in their initial inhibitory activities but similar final IC50 values. However, there is a lack of information explaining this similar final inhibitory potency. Although different residues have been identified in the stabilization of the R2 side chain of 2G-BPs into the active site, similar free binding energies were obtained that highlighted a similar stability of the ternary complexes, which in turns justified the similar IC50 values reported. Free binding energy calculations also demonstrated that the union of 2G-BPs to the active site were 38 to 54 kcal mol-1 energetically more favourable than the union of the natural substrate, which is the basis of the inhibition potency of the hFPPS activity.

  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. Orthostatic responses to nitric oxide synthase inhibition in persons with tetraplegia.

    Science.gov (United States)

    Wecht, Jill M; Radulovic, Miroslav; Lafountaine, Michael F; Rosado-Rivera, Dwindally; Zhang, Run-Lin; Bauman, William A

    2009-08-01

    To determine the effects of 1.0 mg/kg nitro-L-arginine methyl ester (L-NAME) on orthostatic mean arterial pressure (MAP), serum aldosterone, and plasma renin concentrations in persons with chronic tetraplegia compared with nonspinal cord-injured controls. Prospective placebo-controlled intervention study. James J. Peters Veterans Affairs Medical Center. Patients (n=5) with tetraplegia and controls (n=7) participated. The groups were matched for age, height, and weight; the average duration of injury in the tetraplegia group was 22+/-14 years. Subjects with tetraplegia visited the laboratory twice, receiving placebo on day 1 and L-NAME (1.0 mg/kg) on day 2. The agents were infused via an intravenous catheter over 60 minutes with the patient in the supine position. Data were collected during the infusion and then during head-up tilt to 45 degrees for 30 minutes. Control subjects visited the laboratory once for placebo infusion and the head-up tilt maneuver. Orthostatic MAP. Orthostatic MAP was reduced after placebo infusion in subjects with tetraplegia compared with controls (69+/-11 vs 89+/-9 mmHg, respectively; Ptetraplegia group with controls after L-NAME infusion. Orthostatic aldosterone levels were increased after placebo compared with L-NAME infusion in persons with tetraplegia; plasma renin levels did not differ among the groups. These data suggest that nitric oxide synthase inhibition may have clinical potential for treatment of orthostatic hypotension in persons with chronic tetraplegia.

  11. Inhibition of glycogen synthase kinase-3β by falcarindiol isolated from Japanese Parsley (Oenanthe javanica).

    Science.gov (United States)

    Yoshida, Jun; Seino, Hiroko; Ito, Yoshiaki; Nakano, Toshimitsu; Satoh, Takumi; Ogane, Yoshiko; Suwa, Saori; Koshino, Hiroyuki; Kimura, Ken-Ichi

    2013-08-07

    A new biological activity of falcarindiol isolated from Japanese parsley (Oenanthe javanica) using the mutant yeast YNS17 strain (zds1Δ erg3Δ pdr1Δ pdr3Δ) was discovered as an inhibitor of glycogen synthase kinase-3β (GSK-3β). Falcarindiol inhibited GSK-3β in an ATP noncompetitive manner with a Ki value of 86.9 μM using a human enzyme and luminescent kinase assay platform. Falcarindiol also both suppressed gene expression of glucose-6-phosphatase (G6Pase) in rat hepatoma H4IIE cells and protected mouse neuroblastoma HT22 cells from glutamate-induced oxidative cell death at 10 μM. During an oral glucose tolerance test (OGTT), the blood glucose level was significantly decreased in the rats treated with oral administration of O. javanica extract containing falcarindiol (15 mg/kg). These findings indicate that Japanese parsley could be a useful food ingredient against type-2 diabetes and Alzheimer's disease.

  12. Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

    Energy Technology Data Exchange (ETDEWEB)

    Min, Li; Jin, Zhongmin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang (GUW); (Maryland); (GWU); (Georgia)

    2010-01-07

    N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in L-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by L-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with L-arginine bound and in the active R-state complexed with CoA and L-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of L-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by {approx}10 {angstrom} and decreases its height by {approx}20{angstrom}. AAK dimers move 5{angstrom} outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by {approx}4{sup o}. The NAT domains rotate {approx}109{sup o} relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the L-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.

  13. Packed red blood cells are an abundant and proximate potential source of nitric oxide synthase inhibition.

    Directory of Open Access Journals (Sweden)

    Charles F Zwemer

    Full Text Available We determined, for packed red blood cells (PRBC and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS inhibitors asymmetric dimethylarginine (ADMA and monomethylarginine (LNMMA.ADMA and LNMMA are near equipotent NOS inhibitors forming blood's total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined.We measured total (free and protein incorporated ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete in vitro acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis.In vitro strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM and LNMMA (58.9 ± 28.9 μM that persisted over 42-d at 6° or -80°C. In vitro 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma.The compelling physiological ramifications are that regardless of storage age, 1 PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2 PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate risk for iatrogenic NOS inhibition upon

  14. Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Dong Ju Son

    2014-08-01

    Full Text Available PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum and long pepper (Piper longum, was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2, COX-1, COX-2, and thromboxane A2 (TXA2 synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PGE2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.

  15. Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice

    Directory of Open Access Journals (Sweden)

    Shailendra P. Singh

    2015-08-01

    Full Text Available Glycogen synthase kinase-3β (GSK3β is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3β expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-β1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-β1 treatment increased GSK3β expression and GSK3 inhibition abolished TGF-β1-induced SMAD3 activation and α-smooth muscle actin (α-SMA expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3β stimulated α-SMA expression even in the absence of TGF-β1 treatment. These results suggest that TGF-β regulates GSK3β, which in turn is important for TGF-β–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-β signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury.

  16. Arginase Inhibition Restores Peroxynitrite-Induced Endothelial Dysfunction via L-Arginine-Dependent Endothelial Nitric Oxide Synthase Phosphorylation

    Science.gov (United States)

    Nguyen, Minh Cong; Park, Jong Taek; Jeon, Yeong Gwan; Jeon, Byeong Hwa; Hoe, Kwang Lae; Kim, Young Myeong

    2016-01-01

    Purpose Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism. Materials and Methods Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay. Results SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. NG-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition. Conclusion These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions. PMID:27593859

  17. Pharmacologic inhibition of sphingomyelin synthase (SMS) activity reduces apolipoprotein-B secretion from hepatocytes and attenuates endotoxin-mediated macrophage inflammation.

    Science.gov (United States)

    Lou, Bin; Dong, Jibin; Li, Yali; Ding, Tingbo; Bi, Tingting; Li, Yue; Deng, Xiaodong; Ye, Deyong; Jiang, Xian-Cheng

    2014-01-01

    Sphingomyelin synthase (SMS) plays an important role in plasma atherogenic lipoprotein metabolism, inflammation, and the development of atherosclerosis. To understand whether the impaired apoB secretion and inflammation response is a direct result from lack of SMS activity, in this study, we prepared a series of compounds that inhibit SMS activity. Further, we characterized Dy105, the most potent inhibitor. We found that Dy105 treatment significantly reduces SM levels in SM-rich microdomain on cell membranes. Moreover, we found that SMS inhibition reduces apoB secretion in a human hepatoma cell line and reduces the activation of NFκB and p38, a MAP kinase, in bone marrow derived macrophages. These studies provided further evidence that SMS activity regulates atherogenic lipoprotein metabolism and inflammatory responses. Pharmacologic inhibition of SMS may be a new therapy for atherosclerosis by reducing apoB secretion, and reducing inflammation.

  18. Sunscreen ingredients inhibit inducible nitric oxide synthase (iNOS): a possible biochemical explanation for the sunscreen melanoma controversy.

    Science.gov (United States)

    Chiang, Thomas M; Sayre, Robert M; Dowdy, John C; Wilkin, Nathaniel K; Rosenberg, E William

    2005-02-01

    Sunscreen products are rated upon their ability to inhibit visible redness of the skin 24 h after measured doses of ultraviolet (UV) exposure (Sun Protection Factor, SPF). Although sunscreens prevent UV-induced redness, their ability to protect against melanoma or the development of moles is less clear. UV-induced redness occurs in part by the action of nitric oxide (NO), synthesized in the skin. NO is also an important immunoregulatory molecule in the induction of the cell-mediated tumour immune response. In this study, various sunscreen ingredients were tested for their ability to inhibit the production of NO. Four of the five sunscreens tested directly inhibited the conversion of arginine to citrulline by inducible nitric oxide synthase (iNOS) in vitro. These findings suggest that sunscreens may prevent redness partly by UV absorption and partly by inhibition of the skin's inflammatory response. As such, sunscreens might promote instead of protect against melanoma.

  19. Neuronal nitric oxide synthase supports Renin release during sodium restriction through inhibition of phosphodiesterase 3

    DEFF Research Database (Denmark)

    Sällström, Johan; Jensen, Boye L; Skøtt, Ole

    2010-01-01

    by measurements of inulin- and para-amino hippuric acid (PAH) clearances, respectively. RESULTS: The basal PRC was reduced in nNOS⁻(/)⁻ compared to the wild types. Administration of milrinone caused a more pronounced PRC increase in nNOS⁻(/)⁻, resulting in normalized renin levels, whereas PDE5 inhibition did...

  20. Tyrosinase inhibition due to interaction of homocyst(e)ine with copper: the mechanism for reversible hypopigmentation in homocystinuria due to cystathionine beta-synthase deficiency.

    Science.gov (United States)

    Reish, O; Townsend, D; Berry, S A; Tsai, M Y; King, R A

    1995-01-01

    Deficiency of cystathionine beta-synthase (CBS) is a genetic disorder of transsulfuration resulting in elevated plasma homocyst(e)ine and methionine and decreased cysteine. Affected patients have multisystem involvement, which may include light skin and hair. Reversible hypopigmentation in treated homocystinuric patients has been infrequently reported, and the mechanism is undefined. Two CBS-deficient homocystinuric patients manifested darkening of their hypopigmented hair following treatment that decreased plasma homocyst(e)ine. We hypothesized that homocyst(e)ine inhibits tyrosinase, the major pigment enzyme. The activity of tyrosinase extracted from pigmented human melanoma cells (MNT-1) that were grown in the presence of homocysteine was reduced in comparison to that extracted from cells grown without homocysteine. Copper sulfate restored homocyst(e)ine-inhibited tyrosinase activity when added to the culture cell media at a proportion of 1.25 mol of copper sulfate per 1 mol of DL-homocysteine. Holo-tyrosinase activity was inhibited by adding DL-homocysteine to the assay reaction mixture, and the addition of copper sulfate to the reaction mixture prevented this inhibition. Other tested compounds, L-cystine and betaine did not affect tyrosinase activity. Our data suggest that reversible hypopigmentation in homocystinuria is the result of tyrosinase inhibition by homocyst(e)ine and that the probable mechanism of this inhibition is the interaction of homocyst(e)ine with copper at the active site of tyrosinase. Images Figure 1 PMID:7611281

  1. Short Hairpin RNA Suppression of Thymidylate Synthase Produces DNA Mismatches and Results in Excellent Radiosensitization

    International Nuclear Information System (INIS)

    Flanagan, Sheryl A.; Cooper, Kristin S.; Mannava, Sudha; Nikiforov, Mikhail A.; Shewach, Donna S.

    2012-01-01

    Purpose: To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. Methods and Materials: shRNA suppression of TS was compared with 5-fluoro-2′-deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. Results: TS shRNA produced profound (≥90%) and prolonged (≥8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. Conclusions: TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA

  2. shRNA suppression of thymidylate synthase produces DNA mismatches and results in excellent radiosensitization

    Science.gov (United States)

    Flanagan, Sheryl A.; Cooper, Kristin S.; Mannava, Sudha; Nikiforov, Mikhail A.; Shewach, Donna S.

    2012-01-01

    Purpose To determine the effect of shRNA-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. Methods and Materials shRNA suppression of TS was compared to FdUrd inactivation of TS ± ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured via clonogenic assay. Cell cycle effects were measured by flow cytometry. Effects of FdUrd or shRNA suppression of TS on dNTP imbalances and consequent nucleotide mis-incorporations into DNA were analyzed by HPLC and as pSP189 plasmid mutations, respectively. Results TS shRNA produced profound (≥ 90%) and prolonged (≥ 8 days) suppression of TS in HCT116 and HT29 cells while FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs compared to FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, though its effects were not as long-lasting as FdUrd. Both treatments resulted in phosphorylation of chk1. TS shRNA alone was less cytotoxic than FdUrd, but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio (RER): TS shRNA, 1.5 – 1.7; FdUrd, 1.4 – 1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. Conclusions TS shRNA produced less cytotoxicity than FdUrd, but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches following TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP and consequent DNA mismatches underlying radiosensitization. Importantly, shRNA suppression of TS avoids FP-mediated TS elevation and its negative prognostic role. These studies support further exploration of TS suppression as a novel

  3. Short Hairpin RNA Suppression of Thymidylate Synthase Produces DNA Mismatches and Results in Excellent Radiosensitization

    Energy Technology Data Exchange (ETDEWEB)

    Flanagan, Sheryl A., E-mail: sflan@umich.edu [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Cooper, Kristin S. [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Mannava, Sudha; Nikiforov, Mikhail A. [Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York (United States); Shewach, Donna S. [Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2012-12-01

    Purpose: To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. Methods and Materials: shRNA suppression of TS was compared with 5-fluoro-2 Prime -deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. Results: TS shRNA produced profound ({>=}90%) and prolonged ({>=}8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. Conclusions: TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA

  4. Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

    Energy Technology Data Exchange (ETDEWEB)

    Aslan, Mutay, E-mail: mutayaslan@akdeniz.edu.tr [Department of Medical Biochemistry, Akdeniz University Faculty of Medicine, Antalya (Turkey); Basaranlar, Goksun [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Unal, Mustafa [Department of Ophthalmology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Ciftcioglu, Akif [Department of Pathology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Derin, Narin [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Mutus, Bulent [Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario (Canada)

    2014-11-01

    Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress. - Highlights: • Inhibition of N-SMase decreases NOS2 levels in ocular hypertension. • Inhibition of N-SMase decreases protein nitration in ocular hypertension. • Inhibition of N-SMase decreases caspase activation in ocular hypertension. • Inhibition of N-SMase decreases apoptosis in ocular hypertension.

  5. Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

    International Nuclear Information System (INIS)

    Miziorko, H.M.; Behnke, C.E.

    1985-01-01

    3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1- 14 C]-3-Chloropropionyl-CoA binds covalently to the enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of the enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [ 14 C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue

  6. Chamomile, an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity

    OpenAIRE

    Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K; Gupta, Sanjay

    2010-01-01

    Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we aimed to investigate the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and to explore its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β , IL-6 and TNFα-induced NO levels in RAW 264.7 macropha...

  7. Binding and Inhibition of Spermidine Synthase from Plasmodium falciparum and Implications for In Vitro Inhibitor Testing.

    Directory of Open Access Journals (Sweden)

    Janina Sprenger

    Full Text Available The aminopropyltransferase spermidine synthase (SpdS is a promising drug target in cancer and in protozoan diseases including malaria. Plasmodium falciparum SpdS (PfSpdS transfers the aminopropyl group of decarboxylated S-adenosylmethionine (dcAdoMet to putrescine or to spermidine to form spermidine or spermine, respectively. In an effort to understand why efficient inhibitors of PfSpdS have been elusive, the present study uses enzyme activity assays and isothermal titration calorimetry with verified or predicted inhibitors of PfSpdS to analyze the relationship between binding affinity as assessed by KD and inhibitory activity as assessed by IC50. The results show that some predicted inhibitors bind to the enzyme with high affinity but are poor inhibitors. Binding studies with PfSpdS substrates and products strongly support an ordered sequential mechanism in which the aminopropyl donor (dcAdoMet site must be occupied before the aminopropyl acceptor (putrescine site can be occupied. Analysis of the results also shows that the ordered sequential mechanism adequately accounts for the complex relationship between IC50 and KD and may explain the limited success of previous efforts at structure-based inhibitor design for PfSpdS. Based on PfSpdS active-site occupancy, we suggest a classification of ligands that can help to predict the KD-IC50 relations in future design of new inhibitors. The present findings may be relevant for other drug targets that follow an ordered sequential mechanism.

  8. Chronic nitric oxide synthase inhibition exacerbates renal dysfunction in cirrhotic rats

    DEFF Research Database (Denmark)

    Graebe, Martin; Brond, Lone; Christensen, Sten

    2004-01-01

    The present study investigated sodium balance and renal tubular function in cirrhotic rats with chronic blockade of the nitric oxide (NO) system. Rats were treated with the nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) starting on the day of common bile duct ligation...

  9. A new amino acid substitution (Ala-205-Phe) in acetolactate synthase (ALS) confers broad spectrum resistance to ALS-inhibiting herbicides.

    Science.gov (United States)

    Brosnan, James T; Vargas, Jose J; Breeden, Gregory K; Grier, Logan; Aponte, Raphael A; Tresch, Stefan; Laforest, Martin

    2016-01-01

    This is a first report of an Ala-205-Phe substitution in acetolactate synthase conferring resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl-triazolinones, and pyrimidinyl (thio) benzoate herbicides. Resistance to acetolactate synthase (ALS) and photosystem II inhibiting herbicides was confirmed in a population of allotetraploid annual bluegrass (Poa annua L.; POAAN-R3) selected from golf course turf in Tennessee. Genetic sequencing revealed that seven of eight POAAN-R3 plants had a point mutation in the psbA gene resulting in a known Ser-264-Gly substitution on the D1 protein. Whole plant testing confirmed that this substitution conferred resistance to simazine in POAAN-R3. Two homeologous forms of the ALS gene (ALSa and ALSb) were detected and expressed in all POAAN-R3 plants sequenced. The seven plants possessing the Ser-264-Gly mutation conferring resistance to simazine also had a homozygous Ala-205-Phe substitution on ALSb, caused by two nucleic acid substitutions in one codon. In vitro ALS activity assays with recombinant protein and whole plant testing confirmed that this Ala-205-Phe substitution conferred resistance to imidazolinone, sulfonylurea, triazolopyrimidines, sulfonylamino-carbonyl- triazolinones, and pyrimidinyl (thio) benzoate herbicides. This is the first report of Ala-205-Phe mutation conferring wide spectrum resistance to ALS inhibiting herbicides.

  10. Inhibition of Nitric Oxide Synthase 1 Induces Salt-Sensitive Hypertension in Nitric Oxide Synthase 1α Knockout and Wild-Type Mice.

    Science.gov (United States)

    Wang, Ximing; Chandrashekar, Kiran; Wang, Lei; Lai, En Yin; Wei, Jin; Zhang, Gensheng; Wang, Shaohui; Zhang, Jie; Juncos, Luis A; Liu, Ruisheng

    2016-04-01

    We recently showed that α, β, and γ splice variants of neuronal nitric oxide synthase (NOS1) expressed in the macula densa and NOS1β accounts for most of the NO generation. We have also demonstrated that the mice with deletion of NOS1 specifically from the macula densa developed salt-sensitive hypertension. However, the global NOS1 knockout (NOS1KO) strain is neither hypertensive nor salt sensitive. This global NOS1KO strain is actually an NOS1αKO model. Consequently, we hypothesized that inhibition of NOS1β in NOS1αKO mice induces salt-sensitive hypertension. NOS1αKO and C57BL/6 wild-type (WT) mice were implanted with telemetry transmitters and divided into 7-nitroindazole (10 mg/kg/d)-treated and nontreated groups. All of the mice were fed a normal salt (0.4% NaCl) diet for 5 days, followed by a high-salt diet (4% NaCl). NO generation by the macula densa was inhibited by >90% in WT and NOS1αKO mice treated with 7-nitroindazole. Glomerular filtration rate in conscious mice was increased by ≈ 40% after a high-salt diet in both NOS1αKO and WT mice. In response to acute volume expansion, glomerular filtration rate, diuretic and natriuretic response were significantly blunted in the WT and knockout mice treated with 7-nitroindazole. Mean arterial pressure had no significant changes in mice fed a high-salt diet, but increased ≈ 15 mm Hg similarly in NOS1αKO and WT mice treated with 7-nitroindazole. We conclude that NOS1β, but not NOS1α, plays an important role in control of sodium excretion and hemodynamics in response to either an acute or a chronic salt loading. © 2016 American Heart Association, Inc.

  11. Local inhibition of hippocampal nitric oxide synthase does not impair place learning in the Morris water escape task in rats.

    Science.gov (United States)

    Blokland, A; de Vente, J; Prickaerts, J; Honig, W; Markerink-van Ittersum, M; Steinbusch, H

    1999-01-01

    Recent studies have provided evidence that nitric oxide (NO) has a role in certain forms of memory formation. Spatial learning is one of the cognitive abilities that has been found to be impaired after systemic administration of an NO-synthase inhibitor. As the hippocampus has a pivotal role in spatial orientation, the present study examined the role of hippocampal NO in spatial learning and reversal learning in a Morris task in adult rats. It was found that N omega-nitro-L-arginine infusions into the dorsal hippocampus affected the manner in which the rats were searching the submerged platform during training, but did not affect the efficiency to find the spatial location of the escape platform. Hippocampal NO-synthase inhibition did not affect the learning of a new platform position in the same water tank (i.e. reversal learning). Moreover, no treatment effects were observed in the probe trials (i.e. after acquisition and after reversal learning), indicating that the rats treated with N omega-nitro-L-arginine had learned the spatial location of the platform. These findings were obtained under conditions where the NO synthesis in the dorsal hippocampus was completely inhibited. On the basis of the present data it was concluded that hippocampal NO is not critically involved in place learning in rats.

  12. Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera).

    Science.gov (United States)

    Richter, A; Jacobsen, H-J; de Kathen, A; de Lorenzo, G; Briviba, K; Hain, R; Ramsay, G; Kiesecker, H

    2006-11-01

    The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated.

  13. Attenuation by creatine of myocardial metabolic stress in Brattleboro rats caused by chronic inhibition of nitric oxide synthase.

    Science.gov (United States)

    Constantin-Teodosiu, D; Greenhaff, P L; Gardiner, S M; Randall, M D; March, J E; Bennett, T

    1995-12-01

    1. The present experiment was undertaken to investigate: (a) the effect of nitric oxide synthase (NOS) inhibition, mediated by oral supplementation of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on measures of myocardial energy metabolism and function: (b) the effect of oral creatine supplementation on these variables, in the absence and presence of L-NAME. 2. In one series of experiments, 4 weeks oral administration of L-NAME (0.05 mg ml-1 day-1 in the drinking water) to Brattleboro rats caused significant reductions in myocardial ATP, creatine, and total creatine concentrations and an accumulation of tissue lactate when compared with control animals. Administration of creatine (0.63 mg ml-1 day-1 in the drinking water) for 4 weeks elevated myocardial creatine and total creatine concentrations and reduced lactate accumulation, but did not significantly affect ATP or phosphocreatine (PCr). Concurrent treatment with creatine and L-NAME prevented the reduction in creatine and total creatine concentrations, and significantly attenuated the accumulation of lactate and the reduction in ATP seen with L-NAME alone. 3. In a second series of experiments, 4 weeks treatment with L-NAME and creatine plus L-NAME increased mean arterial blood pressure in conscious Brattleboro rats. Hearts isolated from these animals showed decreased coronary flow and left ventricular developed pressure (LVDP), and total mechanical performance. Treatment with creatine alone had no measurable effect on either mean arterial blood pressure or coronary flow in isolated hearts. However, there was an increase in LVDP, but not in total mechanical performance, because there was a bradycardia. 4. These results indicate that creatine supplementation can attenuate the metabolic stress associated with L-NAME administration and that this effect occurs as a consequence of the action of creatine on myocardial energy metabolism.

  14. Fatty acid synthase inhibition by amentoflavone suppresses HER2/neu (erbB2) oncogene in SKBR3 human breast cancer cells.

    Science.gov (United States)

    Lee, Jin Sun; Sul, Ji Young; Park, Jun Beom; Lee, Myung Sun; Cha, Eun Young; Song, In Sang; Kim, Je Ryong; Chang, Eil Sung

    2013-05-01

    Fatty acid synthase (FASN) is a potential therapeutic target for treatment of cancer and obesity, and is highly elevated in 30% of HER2-overexpressing breast cancers. Considerable interest has developed in searching for novel FASN inhibitors as therapeutic agents in treatment of HER2-overexpressing breast cancers. Amentoflavone was found to be effective in suppressing FASN expression in HER2-positive SKBR3 cells. Pharmacological inhibition of FASN by amentoflavone specifically down-regulated HER2 protein and mRNA, and caused an up-regulation of PEA3, a transcriptional repressor of HER2. In addition, pharmacological blockade of FASN by amentoflavone preferentially decreased cell viability and induced cell death in SKBR3 cells. Palmitate reduced the cytotoxic effect of amentoflavone, as the percentage of viable cells was increased after the addition of exogenous palmitate. Amentoflavone-induced FASN inhibition inhibited the translocation of SREBP-1 in SKBR3 cells. Amentoflavone inhibited phosphorylation of AKT, mTOR, and JNK. The use of pharmacological inhibitors revealed that the modulation of AKT, mTOR, and JNK phosphorylation required synergistic amentoflavone-induced FASN inhibition and HER2 activation in SKBR3 cells. These results suggest that amentoflavone modulated FASN expression by regulation of HER2-pathways, and induced cell death to enhance chemopreventive or chemotherapeutic activity in HER2-positive breast cancers. Copyright © 2012 John Wiley & Sons, Ltd.

  15. Modulation of cerebral RAGE expression following nitric oxide synthase inhibition in rats subjected to focal cerebral ischemia.

    Science.gov (United States)

    Greco, Rosaria; Demartini, Chiara; Zanaboni, Anna Maria; Blandini, Fabio; Amantea, Diana; Tassorelli, Cristina

    2017-04-05

    The receptor for advanced glycation endproducts (RAGE) is a key mediator of neuroinflammation following cerebral ischemia. Nitric oxide (NO) plays a dualistic role in cerebral ischemia, depending on whether it originates from neuronal, inducible or endothelial synthase. Although a dynamic interplay between RAGE and NO pathways exists, its relevance in ischemic stroke has not been investigated. The aim of this study is to evaluate the effect of the NO synthase (NOS) inhibition on RAGE expression in rats subjected to transient middle cerebral artery occlusion (tMCAo). Full-length (fl-RAGE) gene expression was elevated in the striatum and, to a lesser extent, in the cortex of rats undergone tMCAo. The exacerbation of cortical damage caused by systemic administration of L-N-(1-iminoethyl)ornithine (L-NIO), a relatively selective inhibitor of endothelial NOS (eNOS), was associated with elevated mRNA levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and fl-RAGE in both the cortex and the striatum. Conversely, NG-nitro-l-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, decreased cortical damage, did not affect cerebral cytokine mRNA levels, while it increased fl-RAGE mRNA expression only in the striatum. Fl-RAGE striatal protein levels varied accordingly with observed mRNA changes in the striatum, while in the cortex, RAGE protein levels were reduced by tMCAo and further decreased following L-NIO treatment. Modulation of RAGE expression by different inhibitors of NOS may have opposite effects on transient cortical ischemia: the non selective inhibition of NOS activity is protective, while the selective inhibition of eNOS is harmful, probably via the activation of inflammatory pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Acetolactate Synthase-Inhibiting Gametocide Amidosulfuron Causes Chloroplast Destruction, Tissue Autophagy, and Elevation of Ethylene Release in Rapeseed

    Directory of Open Access Journals (Sweden)

    Xi-Qiong Liu

    2017-09-01

    Full Text Available Background: Acetolactate synthase (ALS-inhibiting herbicides amidosulfuron (Hoestar is an efficient gametocide that can induce male sterility in rapeseed (Brassica napus L.. We conducted an integrated study of cytological, transcriptomic, and physiological analysis to decipher the gametocidal effect of amidosulfuron.Results: In the first several days after exposure to amidosulfuron at a gametocidal dose of ca. 1 μg per plant, the plants showed the earliest symptoms including short retard of raceme elongation, slight chlorosis on leaf, and decrease of photosynthesis rate. Chloroplasts in leaf and anther epidermis, and tapetal plastids were deformed. Both tapetal cell and uni-nucleate microspore showed autophagic vacuoles and degenerated quickly. The amidosulfuron treatment caused reduction of photosynthetic rate and the contents of leaf chlorophyll, soluble sugar and pyruvate, as well as content alteration of several free amino acids in the treated plants. A comparison of transcriptomic profiling data of the young flower buds of the treated plants with the control identified 142 up-regulated and 201 down-regulated differential expression transcripts with functional annotations. Down-regulation of several interesting genes encoding PAIR1, SDS, PPD2, HFM1, CSTF77, A6, ALA6, UGE1, FLA20, A9, bHLH91, and putative cell wall protein LOC106368794, and up-regulation of autophagy-related protein ATG8A indicated functional abnormalities about cell cycle, cell wall formation, chloroplast structure, and tissue autophagy. Ethylene-responsive transcription factor RAP2-11-like was up-regulated in the flower buds and ethylene release rate was also elevated. The transcriptional regulation in the amidosulfuron-treated plants was in line with the cytological and physiological changes.Conclusions: The results suggested that metabolic decrease related to photosynthesis and energy supply are associated with male sterility induced by amidosulfuron. The results provide

  17. Pu-erh Tea Reduces Nitric Oxide Levels in Rats by Inhibiting Inducible Nitric Oxide Synthase Expression through Toll-Like Receptor 4

    Science.gov (United States)

    Xu, Yang; Wang, Guan; Li, Chunjie; Zhang, Min; Zhao, Hang; Sheng, Jun; Shi, Wei

    2012-01-01

    Pu-erh tea undergoes a unique fermentation process and contains theabrownins, polysaccharides and caffeine; although it is unclear about which component is associated with the down regulation of nitric oxide levels or how this process is mediated. To address this question we examined the effects of pu-erh tea on nitric oxide synthase (NOS) genes. Cohorts of rats were separately given four-week treatments of water as control, pu-erh tea, or the tea components: theabrownins, caffeine or polysaccharides. Five experimental groups were injected with lipopolysaccharides (LPS) to induce nitric oxide (NO) production, while the corresponding five control groups were injected with saline as a negative control. The serum and liver NO concentrations were examined and the NOS expression of both mRNA and protein was measured in liver. The results showed that the rats which were fed pu-erh tea or polysaccharides had lower levels of NO which corresponded with the down-regulation of inducible nitric oxide synthase (iNOS) expression. We further demonstrate that this effect is mediated through reduction of Toll-like receptor 4 (TLR4) signaling. Thus we find that the polysaccharide components in pu-erh tea reduce NO levels in an animal model by inhibiting the iNOS expression via signaling through TLR4. PMID:22837686

  18. Ferulic acid and its water-soluble derivatives inhibit nitric oxide production and inducible nitric oxide synthase expression in rat primary astrocytes.

    Science.gov (United States)

    Kikugawa, Masaki; Ida, Tomoaki; Ihara, Hideshi; Sakamoto, Tatsuji

    2017-08-01

    We recently reported that two water-soluble derivatives of ferulic acid (1-feruloyl glycerol, 1-feruloyl diglycerol) previously developed by our group exhibited protective effects against amyloid-β-induced neurodegeneration in vitro and in vivo. In the current study, we aimed to further understand this process by examining the derivatives' ability to suppress abnormal activation of astrocytes, the key event of neurodegeneration. We investigated the effects of ferulic acid (FA) derivatives on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat primary astrocytes. The results showed that these compounds inhibited NO production and iNOS expression in a concentration-dependent manner and that the mechanism underlying these effects was the suppression of the nuclear factor-κB pathway. This evidence suggests that FA and its derivatives may be effective neuroprotective agents and could be useful in the treatment of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

  19. β-Escin sodium inhibits inducible nitric oxide synthase expression via downregulation of the JAK/STAT pathway in A549 cells.

    Science.gov (United States)

    Ji, Deng Bo; Xu, Bo; Liu, Jing Tao; Ran, Fu Xiang; Cui, Jing Rong

    2011-12-01

    β-escin, a triterpene saponin, is one of the major active compounds extracted from horse chestnut (Aesculus hippocastanum) seed. Previous work has found that β-escin sodium has antiinflammatory and antitumor effects. In the present study, we investigated its effect on cell proliferation and inducible nitric-oxide synthase (iNOS) expression in human lung carcinoma A549 cells. β-escin sodium (5-40 µg/mL) inhibited cytokine mixture (CM)-induced nitric oxide (NO) production in A549 cells by reducing the expression of iNOS. β-escin sodium suppressed phosphorylation and nuclear translocation of STAT1 (Tyr701) and STAT3 (Tyr705) induced by CM but did not affect the activation of c-Jun and NF-κB. β-escin sodium inhibited the activation of protein tyrosine kinase JAK2. Pervanadate treatment reversed the β-escin sodium-induced downregulation of STAT3 and STAT1. β-escin sodium treatment enhanced an activating phosphorylation of the phosphatase SHP2. Small interfering RNA-mediated knockdown of SHP2 inhibited β-escin sodium-induced phospho-STAT dephosphorylation. Moreover β-escin sodium reduced the activation of p38 MAPK. Finally, β-escin sodium inhibited the proliferation of A549 cells, did not change the cell membrane's permeability, nuclear morphology and size and the mitochondria's transmembrane potential of A549 cells. Taken together, these results demonstrate that β-escin sodium could downregulate iNOS expression through inhibiting JAK/STAT signaling and p38 MAPK activation in A549 cells. β-escin sodium has a marked antiproliferative effect on A549 cells at least in part by inhibiting the JAK/STAT signaling pathway, but not by a cytotoxic effect. β-escin sodium would be useful as a chemopreventive agent or a therapeutic against inflammatory-associated tumor. © 2011 Wiley Periodicals, Inc. Copyright © 2011 Wiley Periodicals, Inc.

  20. Nitric Oxide Synthase Gene Transfer Overcomes the Inhibition of Wound Healing by Sulfur Mustard in a Human Keratinocyte In Vitro Model

    Science.gov (United States)

    Ishida, Hiroshi; Ray, Radharaman; Amnuaysirikul, Jack; Ishida, Keiko; Ray, Prabhati

    2012-01-01

    Sulfur mustard (SM) is a chemical warfare agent that causes extensive skin injury. Previously we reported that SM exposure resulted in suppression of inducible nitric oxide synthase (iNOS) expression to inhibit the healing of scratch wounds in a cultured normal human epidermal keratinocyte (NHEK) model. Based on this finding, the present study was to use adenovirus-mediated gene transfer of iNOS to restore the nitric oxide (NO) supply depleted by exposure to SM and to evaluate the effect of NO on wound healing inhibited by SM in NHEKs. The effect of the iNOS gene transfer on iNOS protein expression and NO generation were monitored by Western blot and flow cytometry, respectively. Wound healing with or without the iNOS gene transfer after SM exposure was assessed by light and confocal microscopy. The iNOS gene transfer via adenovirus resulted in overexpression of the iNOS and an increase in NO production regardless of SM exposure in the NHEK model. The gene transfer was also effective in overcoming the inhibition of wound healing due to SM exposure leading to the promotion of wound closure. The findings in this study suggest that the iNOS gene transfer is a promising therapeutic strategy for SM-induced skin injury. PMID:23762631

  1. Arginase inhibition reduces interleukin-1β-stimulated vascular smooth muscle cell proliferation by increasing nitric oxide synthase-dependent nitric oxide production

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Jeongyeon; Ryoo, Sungwoo, E-mail: ryoosw08@kangwon.ac.kr

    2013-06-07

    Highlights: •Arginase inhibition suppressed proliferation of IL-1β-stimulated VSMCs in dose-dependent manner. •NO production from IL-1β-induced iNOS expression was augmented by arginase inhibition, reducing VSMC proliferation. •Incubation with cGMP analogues abolished IL-1β-dependent proliferation of VSMCs. -- Abstract: We investigated whether arginase inhibition suppressed interleukin (IL)-1β-stimulated proliferation in vascular smooth muscle cells (VSMCs) and the possible mechanisms involved. IL-1β stimulation increased VSMC proliferation, while the arginase inhibitor BEC and transfection of the antisense (AS) oligonucleotide against arginase I decreased VSMC proliferation and was associated with increased protein content of the cell cycle regulator p21Waf1/Cip1. IL-1β incubation induced inducible nitric oxide synthase (iNOS) mRNA expression and protein levels in a dose-dependent manner, but did not affect arginase I and II expression. Consistent with this data, IL-1β stimulation resulted in increase in NO production that was significantly augmented by arginase inhibition. The specific iNOS inhibitor 1400W abolished IL-1β-mediated NO production and further accentuated IL-1β-stimulated cell proliferation. Incubation with NO donors GSNO and DETA/NO in the presence of IL-1β abolished VSMCs proliferation and increased p21Waf1/Cip1 protein content. Furthermore, incubation with the cGMP analogue 8-Br-cGMP prevented IL-1β-induced VSMCs proliferation. In conclusion, arginase inhibition augmented iNOS-dependent NO production that resulted in suppression of IL-1β-induced VSMCs proliferation in a cGMP-dependent manner.

  2. Arginase inhibition reduces interleukin-1β-stimulated vascular smooth muscle cell proliferation by increasing nitric oxide synthase-dependent nitric oxide production

    International Nuclear Information System (INIS)

    Yoon, Jeongyeon; Ryoo, Sungwoo

    2013-01-01

    Highlights: •Arginase inhibition suppressed proliferation of IL-1β-stimulated VSMCs in dose-dependent manner. •NO production from IL-1β-induced iNOS expression was augmented by arginase inhibition, reducing VSMC proliferation. •Incubation with cGMP analogues abolished IL-1β-dependent proliferation of VSMCs. -- Abstract: We investigated whether arginase inhibition suppressed interleukin (IL)-1β-stimulated proliferation in vascular smooth muscle cells (VSMCs) and the possible mechanisms involved. IL-1β stimulation increased VSMC proliferation, while the arginase inhibitor BEC and transfection of the antisense (AS) oligonucleotide against arginase I decreased VSMC proliferation and was associated with increased protein content of the cell cycle regulator p21Waf1/Cip1. IL-1β incubation induced inducible nitric oxide synthase (iNOS) mRNA expression and protein levels in a dose-dependent manner, but did not affect arginase I and II expression. Consistent with this data, IL-1β stimulation resulted in increase in NO production that was significantly augmented by arginase inhibition. The specific iNOS inhibitor 1400W abolished IL-1β-mediated NO production and further accentuated IL-1β-stimulated cell proliferation. Incubation with NO donors GSNO and DETA/NO in the presence of IL-1β abolished VSMCs proliferation and increased p21Waf1/Cip1 protein content. Furthermore, incubation with the cGMP analogue 8-Br-cGMP prevented IL-1β-induced VSMCs proliferation. In conclusion, arginase inhibition augmented iNOS-dependent NO production that resulted in suppression of IL-1β-induced VSMCs proliferation in a cGMP-dependent manner

  3. Effects of Nitric Oxide Synthase Inhibition on Fiber-Type Composition, Mitochondrial Biogenesis, and SIRT1 Expression in Rat Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    Masataka Suwa, Hiroshi Nakano, Zsolt Radak, Shuzo Kumagai

    2015-09-01

    Full Text Available It was hypothesized that nitric oxide synthases (NOS regulated SIRT1 expression and lead to a corresponding changes of contractile and metabolic properties in skeletal muscle. The purpose of the present study was to investigate the influence of long-term inhibition of nitric oxide synthases (NOS on the fiber-type composition, metabolic regulators such as and silent information regulator of transcription 1 (SIRT1 and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, and components of mitochondrial biogenesis in the soleus and plantaris muscles of rats. Rats were assigned to two groups: control and NOS inhibitor (Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, ingested for 8 weeks in drinking water-treated groups. The percentage of Type I fibers in the L-NAME group was significantly lower than that in the control group, and the percentage of Type IIA fibers was concomitantly higher in soleus muscle. In plantaris muscle, muscle fiber composition was not altered by L-NAME treatment. L-NAME treatment decreased the cytochrome C protein expression and activity of mitochondrial oxidative enzymes in the plantaris muscle but not in soleus muscle. NOS inhibition reduced the SIRT1 protein expression level in both the soleus and plantaris muscles, whereas it did not affect the PGC-1α protein expression. L-NAME treatment also reduced the glucose transporter 4 protein expression in both muscles. These results suggest that NOS plays a role in maintaining SIRT1 protein expression, muscle fiber composition and components of mitochondrial biogenesis in skeletal muscle.

  4. Glycogen synthase kinase-3 inhibition disrupts nuclear factor-kappaB activity in pancreatic cancer, but fails to sensitize to gemcitabine chemotherapy

    Directory of Open Access Journals (Sweden)

    Mamaghani Shadi

    2009-04-01

    Full Text Available Abstract Background Aberrant activation NF-kappaB has been proposed as a mechanism of drug resistance in pancreatic cancer. Recently, inhibition of glycogen synthase kinase-3 has been shown to exert anti-tumor effects on pancreatic cancer cells by suppressing NF-kappaB. Consequently, we investigated whether inhibition of GSK-3 sensitizes pancreatic cancer cells to the chemotherapeutic agent gemcitabine. Methods GSK-3 inhibition was achieved using the pharmacological agent AR-A014418 or siRNA against GSK-3 alpha and beta isoforms. Cytotoxicity was measured using a Sulphorhodamine B assay and clonogenic survival following exposure of six different pancreatic cancer cell lines to a range of doses of either gemcitabine, AR-A014418 or both for 24, 48 and 72 h. We measured protein expression levels by immunoblotting. Basal and TNF-alpha induced activity of NF-kappaB was assessed using a luciferase reporter assay in the presence or absence of GSK-3 inhibition. Results GSK-3 inhibition reduced both basal and TNF-alpha induced NF-kappaB luciferase activity. Knockdown of GSK-3 beta reduced nuclear factor kappa B luciferase activity to a greater extent than GSK-3 alpha, and the greatest effect was seen with dual knockdown of both GSK-3 isoforms. GSK-3 inhibition also resulted in reduction of the NF-kappaB target proteins XIAP, Bcl-XL, and cyclin D1, associated with growth inhibition and decreased clonogenic survival. In all cell lines, treatment with either AR-A014418, or gemcitabine led to growth inhibition in a dose- and time-dependent manner. However, with the exception of PANC-1 where drug synergy occurred with some dose schedules, the inhibitory effect of combined drug treatment was additive, sub-additive, or even antagonistic. Conclusion GSK-3 inhibition has anticancer effects against pancreatic cancer cells with a range of genetic backgrounds associated with disruption of NF-kappaB, but does not significantly sensitize these cells to the standard

  5. Inhibition of platelet activation by lachrymatory factor synthase (LFS)-silenced (tearless) onion juice.

    Science.gov (United States)

    Thomson, Susan J; Rippon, Paula; Butts, Chrissie; Olsen, Sarah; Shaw, Martin; Joyce, Nigel I; Eady, Colin C

    2013-11-06

    Onion and garlic are renowned for their roles as functional foods. The health benefits of garlic are attributed to di-2-propenyl thiosulfinate (allicin), a sulfur compound found in disrupted garlic but not found in disrupted onion. Recently, onions have been grown with repressed lachrymatory factor synthase (LFS) activity, which causes these onions to produce increased amounts of di-1-propenyl thiosulfinate, an isomer of allicin. This investigation into the key health attributes of LFS-silenced (tearless) onions demonstrates that they have some attributes more similar to garlic and that this is likely due to the production of novel thiosulfinate or metabolites. The key finding was that collagen-induced in vitro platelet aggregation was significantly reduced by tearless onion extract over normal onion extract. Thiosulfinate or derived compounds were shown not to be responsible for the observed changes in the inflammatory response of AGS (stomach adenocarcinoma) cells to tumor necrosis factor alpha (TNFα) when pretreated with model onion juices. A preliminary rat feeding trial indicated that the tearless onions may also play a key role in reducing weight gain.

  6. Monogalactosyldiacylglycerol: An abundant galactosyllipid of Cirsium brevicaule A. GRAY leaves inhibits the expression of gene encoding fatty acid synthase.

    Science.gov (United States)

    Inafuku, Masashi; Takara, Kensaku; Taira, Naoyuki; Nugara, Ruwani N; Kamiyama, Yasuo; Oku, Hirosuke

    2016-05-15

    The leaves of Cirsium brevicaule A. GRAY (CL) significantly decreased hepatic lipid accumulation and the expression of fatty acid synthase gene (FASN) in mice. We aimed to purify and identify the active compound(s) from CL and determine the inhibitory mechanism of expression of FASN. We purified monogalactosyldiacylglycerol (MGDG) from extracts of CL (CL-MGDG) and showed that it was the active CL component through analyses of its effects on the expression of genes of human breast cancer cell line, SKBR-3. The content and fatty acid composition of CL-MGDG are distinctly different from those of other vegetable-derived MGDGs. Treatment of SKBR-3 cells with MGDG decreased the level of FASN mRNA as well as the levels of mRNA encoding other protein involved in lipogenesis. Further, MGDG treatments significantly inhibited luciferase activities of constructs containing liver X receptor response element in FASN promoter region without altering the levels of mRNA encoding transcription factors. MGDG and the FASN inhibitor C75 decreased the viabilities of SKBR-3 cells in a concentration-dependent manner. CL-MGDG more potently inhibited cell viability than a commercial MGDG preparation. CL represents a good source of glycoglycerolipids with potential as functional ingredients of food. Copyright © 2016 Elsevier GmbH. All rights reserved.

  7. Broad resistance to acetohydroxyacid-synthase-inhibiting herbicides in feral radish (Raphanus sativus L.) populations from Argentina.

    Science.gov (United States)

    Pandolfo, Claudio E; Presotto, Alejandro; Moreno, Florencia; Dossou, Ida; Migasso, Juan P; Sakima, Ernesto; Cantamutto, Miguel

    2016-02-01

    Soon after the commercial release of sunflower cultivars resistant to imidazolinone herbicides, several uncontrolled feral radish (Raphanus sativus L.) populations were found in south-eastern Buenos Aires, Argentina. These populations were studied in field, glasshouse and laboratory experiments aiming to characterise their resistance profile and to develop management tools. Three feral radish accessions were highly resistant to ten active ingredients of five families of acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Sequence analysis of the AHAS gene detected a Trp574Leu mutation in all resistant accessions. One accession with an intermediate level of resistance was heterozygous for this mutation, probably owing to gene exchange with a susceptible subpopulation located in the field margin. Herbicide-resistant and herbicide-susceptible radish could be controlled in sunflower by alternative herbicides. This is the first report of feral radish with resistance to herbicides belonging to all the AHAS-inhibiting herbicide families, conferred by Trp574Leu mutation in the AHAS gene. An appropriate herbicide rotation with alternative herbicides such as fluorochloridone or aclonifen and an increase in the diversity of cropping systems are important for minimising the prevalence of these biotypes. © 2015 Society of Chemical Industry.

  8. Resistência de plantas aos herbicidas inibidores da acetolactato sintase Plant resistance to acetolactate synthase-inhibiting herbicides

    Directory of Open Access Journals (Sweden)

    M.A. Rizzardi

    2002-04-01

    inhibitors, its effects on weed ecophysiology and mutations which confer resistance to weeds, as well as the possibilities to develop resistant crops to ALS inhibitors. In weeds, resistance to ALS-inhibiting herbicides results from one or more mutations in the gene that codifies the ALS, which possesses dominant or semi-dominant characteristics when resistance is codified by one gene. Substitutions on a single nucleotide sequence cause alterations in the ALS, conferring resistance to herbicides inhibitors of this enzyme. Although the resistant biotype presents genetic and enzymatic alteration, when compared to the susceptible biotype, the ecophysiological behaviour of resistant and susceptible biotypes is similar. Resistant cultivars have already been developed in various crops, including rice and corn, which vary in their level of resistance to different chemical groups of ALS-inhibiting herbicides.

  9. Effects of nitric oxide synthase inhibition on cutaneous vasodilation in response to acupuncture stimulation in humans.

    Science.gov (United States)

    Kimura, Kenichi; Takeuchi, Hayato; Yuri, Kuniko; Wakayama, Ikuro

    2013-03-01

    The aim of the present study was to elucidate the mechanism of cutaneous vasodilation following acupuncture stimulation by investigating the roles of nitric oxide (NO) and axon reflex vasodilation. The subjects were 17 healthy male volunteers. The role of NO was investigated by administering N(G)-nitro-l-arginine methyl ester hydrochloride (L-NAME, 20 mM), an NO synthase inhibitor or Ringer's solution (control site), via intradermal microdialysis (protocol 1; n=7). The role of axon reflex vasodilation by local sensory neurones was investigated by comparing vasodilation at sites treated with 'eutectic mixture of local anaesthetics' (EMLA) cream (2.5% lidocaine and 2.5% prilocaine) with untreated sites (control site) (protocol 2; n=10). After 5 min of baseline recording, acupuncture was applied to PC4 and a control site in proximity to PC4 for 10 min and scanning was performed for 60 min after acupuncture stimulation. Skin blood flow (SkBF) was evaluated by laser Doppler perfusion imaging. Cutaneous vascular conductance (CVC) was calculated from the ratio of SkBF to mean arterial blood pressure. In the first protocol, sites administered L-NAME showed significant reductions in CVC responses following acupuncture stimulation compared to control sites (administered Ringer's solution) (pacupuncture stimulation did not differ significantly between treated sites with EMLA cream and untreated sites (p>0.05). These data suggest that cutaneous vasodilation in response to acupuncture stimulation may not occur through an axon reflex as previously reported. Rather, NO mechanisms appear to contribute to the vasodilator response.

  10. Oxidized lipoproteins induce long-lasting inhibition of nitric oxide synthase from a murine endothelioma cell line (bEnd.4).

    Science.gov (United States)

    Fogliatto, G; Musanti, R; Pirillo, A; Ghiselli, G

    1995-04-01

    The vascular endothelium produces nitric oxide, which has vasodilatory properties. It has been postulated that some lipoproteins may increase arterial vascular tone by decreasing the availability of endothelium-derived nitric oxide. The mechanism underlying this effect, however, is still poorly understood. We investigated the effect of native and oxidized human low- and high-density lipoproteins on the nitric oxide synthetic activity of an endothelioma cell line (bEnd.4). Oxidized lipoproteins were obtained by incubation with CuSO4. The production of nitric oxide by the cells was monitored by quantifying the nitrite concentration in the medium using Greiss reagent. The synthesis of nitric oxide by the bEnd.4 cell line was calcium-dependent and was abolished by a selective inhibitor of the constitutive nitric oxide synthase. Incubation with oxidized lipoproteins caused a time- and dose-dependent inhibition of nitric oxide synthetic activity. At a concentration of 100 micrograms/ml cholesterol, oxidized low- and high-density lipoproteins inhibited the production of nitric oxide by 27 and 51%, respectively, within 6h. The lipid fraction obtained from the native or the oxidized lipoproteins mimicked the effect of the intact lipoproteins. These results support the involvement of oxidized lipoproteins in the modulation of endothelial functions relevant to the pathogenesis of cardiovascular disease.

  11. Inhibition of an inducible nitric oxide synthase expression by a hexane extract from perilla frutescens cv. chookyoupjaso mutant induced by mutagenesis with gamma-ray

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yong Dae; Kang, Min Ah; Lee, Hyo Jung; Jin, Chang Hyun; Choi, Dae Seong; Kim, Dong Sub; Kang, Si Yong; Byun, Myung Woo; Jeong, Il Yun [Korea Atomic Energy Research Institte, Jeongeup (Korea, Republic of)

    2009-03-15

    In earlier investigations, seeds of Perilla frutescens(L.) Britt. cv. Chookyoupjaso were irradiated with 200 Gy gamma ray to generate mutagenesis. The aim of this study is to investigate the effects of a hexane extract from Perilla frutescens(L.) Britt. cv. Chookyoupjaso mutant 45 on the actions of anti-inflammatory activity on inducible nitric oxide synthase, and an identification of the major active compound. The hexane extract from P. frutescens exhibited activity of inhibition of a NO production (IC{sub 50}, 295.1{mu}g ml{sup -1}). The hexane extract was further divided into sub-fractions by silica-gel chromatogarphy. Inhibition of the NO production by various fractions was assayed in LPS-stimulated RAW 264.7 cells. Among the seven fractions, the 5th fraction was the most effective (IC{sub 50}, 19.5{mu}g ml{sup -1}). The 5th fraction suppressed the expression of protein of iNOS in LPS-induced RAW 264.7 cells, and GC/MS analyses showed that isoegomaketone is a major bio-active compound in the 5th fraction. The result indicated that isoegomaketone has a good potential to be developed as an anti-inflammation agent.

  12. The effects of nitric oxide synthase--versus lipoxygenase inhibition on coronary flow and nitrite outflow in isolated rat heart.

    Science.gov (United States)

    Jakovljevic, V Lj; Djuric, D M

    2005-06-01

    The aim of this study was to assess the changes of coronary flow (CF) and nitrite outflow under inhibition of nitric oxide synthase (NOS) by Nomega-nitro-L-arginine monomethyl ester (L-NAME) or lipoxygenase (LOX) induced by nordihydroguaiaretic acid (NDGA) in isolated rat heart. The hearts of male Wistar albino rats (n=18, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff's technique at gradually increased constant coronary perfusion pressure (CPP) conditions (40-120 cm H2O) which induced flow-dependent nitric oxide (NO) release (nitrite outflow). The experiments were performed during control conditions, in the presence of NO synthesis inhibitor L-NAME (30 micromol/l) or nonspecific LOX inhibitor (NDGA, 0.1 mmol/l) which were administered separately or in combination. CF varied in autoregulatory range from 4.12+/-0.26 ml/min/g wt at 50 cm H2O to 5.22+/-0.26 ml/min/g wt at 90 cm H2O. In autoregulatory range, nitrite outflow varied from 2.05+/-0.17 nmol/min/g wt at 50 cm H2O to 2.52+/-0.21 nmol/min/g wt at 90 cm H2O and was strictly parallel with CPP/CF curve. The autoregulatory range of CF was significantly extended (40-100 cm H2O, 2.22+/-0.12 ml/min/g wt and 2.90+/-0.25 ml/min/g wt, respectively) under the influence of L-NAME. Hemodynamic effects were accompanied by significant decrease in nitrite outflow after L-NAME administration (0.56+/-0.11 nmol/min/g wt at 40 cm H2O to 1.45+/-0.14 nmol/min/g wt at 100 cm H2O). NDGA affected CF in the range of CPP 40-70 cm H2O only (from 42% at 50 cm H2O to 12% at 90 cm H2O, respectively) with no significant changes in nitrite outflow. When L-NAME was applied in combination with NDGA vs. NDGA only, CF was significantly reduced (from 34% at 50 cm H2O to 50% at 90 cm H2O, respectively) with parallel changes in nitrite outflow (from 40% at 50 cm H2O to 51% at 90 cm H2O, respectively). The results showed that CF and nitrite outflow could be decreased under L-NAME administration. Nonselective

  13. Downy Brome (Bromus tectorum L. and Broadleaf Weed Control in Winter Wheat with Acetolactate Synthase-Inhibiting Herbicides

    Directory of Open Access Journals (Sweden)

    Patrick W. Geier

    2013-04-01

    Full Text Available A study was conducted for three seasons in northwest Kansas, USA to evaluate acetolactate synthase (ALS-inhibiting herbicides for downy brome (Bromus tectorum L. and winter annual broadleaf weed control in winter wheat. Herbicides included pyroxsulam at 18.4 g ai ha−1, propoxycarbazone-Na at 44 g ai ha−1, premixed propoxycarbazone-Na & mesosulfuron-methyl at 27 g ai ha−1, and sulfosulfuron at 35 g ai ha−1. The herbicides were applied postemergence in fall and spring seasons. Averaged over time of application, no herbicide controlled downy brome more than 78% in any year. When downy brome densities were high, control was less than 60%. Pyroxsulam controlled downy brome greater than or similar to other herbicides tested. Flixweed (Descurainia sophia L., blue mustard [Chorispora tenella (Pallas DC.], and henbit (Lamium amplexicaule L. control did not differ among herbicide treatments. All herbicides tested controlled flixweed and blue mustard at least 87% and 94%, respectively. However, none of the herbicides controlled henbit more than 73%. Fall herbicide applications improved weed control compared to early spring applications; improvement ranged from 3% to 31% depending on the weed species. Henbit control was greatly decreased by delaying herbicide applications until spring compared to fall applications (49% vs. 80% control. Herbicide injury was observed in only two instances. The injury was ≤13% with no difference between herbicides and the injury did not impact final plant height or grain yield.

  14. Jaspine B induces nonapoptotic cell death in gastric cancer cells independently of its inhibition of ceramide synthase.

    Science.gov (United States)

    Cingolani, Francesca; Simbari, Fabio; Abad, Jose Luis; Casasampere, Mireia; Fabrias, Gemma; Futerman, Anthony H; Casas, Josefina

    2017-08-01

    Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  15. Inducible nitric oxide synthase inhibition by 1400W limits pain hypersensitivity in a neuropathic pain rat model.

    Science.gov (United States)

    Staunton, C A; Barrett-Jolley, R; Djouhri, L; Thippeswamy, T

    2018-02-13

    What is the central question of this study? Can modulation of inducible NO synthase reduce pain behaviour and pro-inflammatory cytokine signalling in a rat model of neuropathic pain? What is the main finding and its importance? Nitric oxide synthase-based therapies could be effective for the treatment of peripheral neuropathic pain. Peripheral neuropathic pain (PNP), resulting from injury to or dysfunction of a peripheral nerve, is a major health problem that affects 7-8% of the population. It is inadequately controlled by current drugs and is characterized by pain hypersensitivity, which is believed to be attributable to sensitization of peripheral and CNS neurons by various inflammatory mediators. Here we examined, in a rat model of PNP: (i) whether reducing levels of nitric oxide (NO) with 1400W, a highly selective inhibitor of inducible NO synthase (iNOS), would prevent or attenuate pain hypersensitivity; and (ii) the effects of 1400W on plasma concentrations of several cytokines that are secreted after iNOS upregulation during chronic pain states. The L5 spinal nerve axotomy (SNA) model of PNP was used, and 1400W (20 mg kg -1 ) was administered i.p. at 8 h intervals for 3 days starting at 18 h post-SNA. Changes in plasma concentrations of 12 cytokines in SNA rats treated with 1400W were examined using multiplex enzyme-linked immunosorbent assay. The SNA rats developed behavioural signs of mechanical and heat hypersensitivity. Compared with the vehicle/control, 1400W significantly: (i) limited development of mechanical hypersensitivity at 66 h post-SNA and of heat hypersensitivity at 42 h and at several time points tested thereafter; and (ii) increased the plasma concentrations of interleukin (IL)-1α, IL-1β and IL-10 in the SNA rats. The findings suggest that 1400W might exert its analgesic effects by reducing iNOS and altering the balance between the pro-inflammatory (IL-1β and IL-1α) and anti-inflammatory (IL-10) cytokines and that therapies

  16. Nitric oxide synthase inhibition does not improve renal function in cirrhotic patients with ascites

    DEFF Research Database (Denmark)

    Thiesson, Helle C; Skøtt, Ole; Jespersen, Bente

    2003-01-01

    )Tc-diethylenetriamine-pentaacetate and (131)I-Hippuran. RESULTS: L-NMMA infusion resulted in an increased blood pressure, decreased heart rate, and dose-dependent suppression of renin of up to 42.1 +/- 7.1% (p

  17. Genetic knockout and pharmacologic inhibition of neuronal nitric oxide synthase attenuate nerve injury-induced mechanical hypersensitivity in mice

    Directory of Open Access Journals (Sweden)

    Tao Yuan-Xiang

    2007-10-01

    Full Text Available Abstract Neuronal nitric oxide synthase (nNOS is a key enzyme for nitric oxide production in neuronal tissues and contributes to the spinal central sensitization in inflammatory pain. However, the role of nNOS in neuropathic pain remains unclear. The present study combined a genetic strategy with a pharmacologic approach to examine the effects of genetic knockout and pharmacologic inhibition of nNOS on neuropathic pain induced by unilateral fifth lumbar spinal nerve injury in mice. In contrast to wildtype mice, nNOS knockout mice failed to display nerve injury-induced mechanical hypersensitivity. Furthermore, either intraperitoneal (100 mg/kg or intrathecal (30 μg/5 μl administration of L-NG-nitro-arginine methyl ester, a nonspecific NOS inhibitor, significantly reversed nerve injury-induced mechanical hypersensitivity on day 7 post-nerve injury in wildtype mice. Intrathecal injection of 7-nitroindazole (8.15 μg/5 μl, a selective nNOS inhibitor, also dramatically attenuated nerve injury-induced mechanical hypersensitivity. Western blot analysis showed that the expression of nNOS protein was significantly increased in ipsilateral L5 dorsal root ganglion but not in ipsilateral L5 lumbar spinal cord on day 7 post-nerve injury. The expression of inducible NOS and endothelial NOS proteins was not markedly altered after nerve injury in either the dorsal root ganglion or spinal cord. Our findings suggest that nNOS, especially in the dorsal root ganglion, may participate in the development and/or maintenance of mechanical hypersensitivity after nerve injury.

  18. Inhibition of microsomal PGE synthase-1 reduces human vascular tone by increasing PGI2 : a safer alternative to COX-2 inhibition.

    Science.gov (United States)

    Ozen, Gulsev; Gomez, Ingrid; Daci, Armond; Deschildre, Catherine; Boubaya, Lilia; Teskin, Onder; Uydeş-Doğan, B Sonmez; Jakobsson, Per-Johan; Longrois, Dan; Topal, Gokce; Norel, Xavier

    2017-11-01

    The side effects of cyclooxygenase-2 (COX-2) inhibitors on the cardiovascular system could be associated with reduced prostaglandin (PG)I 2 synthesis. Microsomal PGE synthase-1 (mPGES-1) catalyses the formation of PGE 2 from COX-derived PGH 2 . This enzyme is induced under inflammatory conditions and constitutes an attractive target for novel anti-inflammatory drugs. However, it is not known whether mPGES-1 inhibitors could be devoid of cardiovascular side effects. The aim of this study was to compare, in vitro, the effects of mPGES-1 and COX-2 inhibitors on vascular tone in human blood vessels. The vascular tone and prostanoid release from internal mammary artery (IMA) and saphenous vein (SV) incubated for 30 min with inhibitors of mPGES-1 or COX-2 were investigated under normal and inflammatory conditions. In inflammatory conditions, mPGES-1 and COX-2 proteins were more expressed, and increased levels of PGE 2 and PGI 2 were released. COX-2 and NOS inhibitors increased noradrenaline induced vascular contractions in IMA under inflammatory conditions while no effect was observed in SV. Interestingly, the mPGES-1 inhibitor significantly reduced (30-40%) noradrenaline-induced contractions in both vessels. This effect was reversed by an IP (PGI 2 receptor) antagonist but not modified by NOS inhibition. Moreover, PGI 2 release was increased with the mPGES-1 inhibitor and decreased with the COX-2 inhibitor, while both inhibitors reduced PGE 2 release. In contrast to COX-2 inhibition, inhibition of mPGES-1 reduced vasoconstriction by increasing PGI 2 synthesis. Targeting mPGES-1 could provide a lower risk of cardiovascular side effects, compared with those of the COX-2 inhibitors. This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc. © 2017

  19. Protective Effects of Kaempferol against Myocardial Ischemia/Reperfusion Injury in Isolated Rat Heart via Antioxidant Activity and Inhibition of Glycogen Synthase Kinase-3β

    Science.gov (United States)

    Zhou, Mingjie; Ren, Huanhuan; Wang, Wenjuan; Zheng, Qiusheng; Wang, Dong

    2015-01-01

    Objective. This study aimed to evaluate the protective effect of kaempferol against myocardial ischemia/reperfusion (I/R) injury in rats. Method. Left ventricular developed pressure (LVDP) and its maximum up/down rate (±dp/dt max) were recorded as myocardial function. Infarct size was detected with 2,3,5-triphenyltetrazolium chloride staining. Cardiomyocyte apoptosis was determined using terminal deoxynucleotidyl nick-end labeling (TUNEL). The levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione/glutathione disulfide (GSH/GSSG) ratio, and tumor necrosis factor-alpha (TNF-α) were determined using enzyme linked immunosorbent assay (ELISA). Moreover, total glycogen synthase kinase-3β (GSK-3β), phospho-GSK-3β (P-GSK-3β), precaspase-3, cleaved caspase-3, and cytoplasm cytochrome C were assayed using Western blot analysis. Results. Pretreatment with kaempferol significantly improved the recovery of LVDP and ±dp/dt max, as well as increased the levels of SOD and P-GSK-3β and GSH/GSSG ratio. However, the pretreatment reduced myocardial infarct size and TUNEL-positive cell rate, as well as decreased the levels of cleaved caspase-3, cytoplasm cytochrome C, CK, LDH, MDA, and TNF-α. Conclusion. These results suggested that kaempferol provides cardioprotection via antioxidant activity and inhibition of GSK-3β activity in rats with I/R. PMID:26265983

  20. Lithium Enhances Axonal Regeneration in Peripheral Nerve by Inhibiting Glycogen Synthase Kinase 3β Activation

    Directory of Open Access Journals (Sweden)

    Huanxing Su

    2014-01-01

    Full Text Available Brachial plexus injury often involves traumatic root avulsion resulting in permanent paralysis of the innervated muscles. The lack of sufficient regeneration from spinal motoneurons to the peripheral nerve (PN is considered to be one of the major causes of the unsatisfactory outcome of various surgical interventions for repair of the devastating injury. The present study was undertaken to investigate potential inhibitory signals which influence axonal regeneration after root avulsion injury. The results of the study showed that root avulsion triggered GSK-3β activation in the injured motoneurons and remaining axons in the ventral funiculus. Systemic application of a clinical dose of lithium suppressed activated GSK-3β in the lesioned spinal cord to the normal level and induced extensive axonal regeneration into replanted ventral roots. Our study suggests that GSK-3β activity is involved in negative regulation for axonal elongation and regeneration and lithium, the specific GSK-3β inhibitor, enhances motoneuron regeneration from CNS to PNS.

  1. Overexpression of fatty acid synthase in human gliomas correlates with the WHO tumor grade and inhibition with Orlistat reduces cell viability and triggers apoptosis.

    Science.gov (United States)

    Grube, Susanne; Dünisch, Pedro; Freitag, Diana; Klausnitzer, Maren; Sakr, Yasser; Walter, Jan; Kalff, Rolf; Ewald, Christian

    2014-06-01

    Fatty acid synthase (FASN), catalyzing the de novo synthesis of fatty acids, is known to be deregulated in several cancers. Inhibition of this enzyme reduces tumor cell proliferation. Unfortunately, adverse effects and chemical instability prevent the in vivo use of the best-known inhibitors, Cerulenin and C75. Orlistat, a drug used for obesity treatment, is also considered as a potential FASN inhibitor, but its impact on glioma cell biology has not yet been described. In this study, we analyzed FASN expression in human glioma samples and primary glioblastoma cell cultures and the effects of FASN inhibition with Orlistat, Cerulenin and C75. Immunohistochemistry followed by densitometric analysis of 20 glioma samples revealed overexpression of FASN that correlated with the WHO tumor grade. Treatment of glioblastoma cells with these inhibitors resulted in a significant, dose-dependent reduction in tumor cell viability and fatty acid synthesis. Compared to Cerulenin and C75, Orlistat was a more potent inhibitor in cell cultures and cell lines. In LN229, cell-growth was reduced by 63.9 ± 8.7 % after 48 h and 200 µM Orlistat compared to controls; in LT68, the reduction in cell growth was 76.3 ± 23.7 %. Nuclear fragmentation assay and Western blotting analysis after targeting FASN with Orlistat demonstrated autophagy and apoptosis. Organotypic slice cultures treated with Orlistat showed reduced proliferation after Ki67 staining and increased caspase-3 cleavage. Our results suggest that FASN may be a therapeutic target in malignant gliomas and identify Orlistat as a possible anti-tumor drug in this setting.

  2. Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase.

    Science.gov (United States)

    Zheng, Desen; Burr, Thomas J

    2016-02-01

    Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730(+)) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730(+) indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

  3. Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) and inhibits pro-survival function of Nrf1

    Energy Technology Data Exchange (ETDEWEB)

    Biswas, Madhurima; Kwong, Erick K.; Park, Eujean; Nagra, Parminder; Chan, Jefferson Y., E-mail: jchan@uci.edu

    2013-08-01

    Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF–Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 from phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A–Nrf1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation. Highlights: • The effect of GSK3 on Nrf1 expression was examined. • GSK3 destabilizes Nrf1 protein via Fbw7 ubiquitin ligase. • GSK3 binds and phosphorylates Nrf1. • Protection from stress-induced apoptosis by Nrf1 is inhibited by GSK3.

  4. Short communication: Effect of inhibition of fatty acid synthase on triglyceride accumulation and effect on lipid metabolism genes in goat mammary epithelial cells.

    Science.gov (United States)

    Zhu, J J; Luo, J; Sun, Y T; Shi, H B; Li, J; Wu, M; Yu, K; Haile, A B; Loor, J J

    2015-05-01

    The role of fatty acid synthase (FASN) on de novo fatty acid synthesis has been well established. In monogastrics, unlike acetyl-coenzyme A carboxylase, FASN is primarily controlled at the transcriptional level. However, no data exist on ruminant mammary cells evaluating effects of FASN knockdown on mRNA expression of lipogenic genes. Inhibition of FASN in mammary cells by C75-mediated interference, a synthetic inhibitor of FASN activity, and short hairpin RNA-mediated interference markedly reduced cellular triglyceride content at least in part by decreasing the expression of genes related to triglyceride synthesis (GPAT, AGPAT6, and DGAT2) and enhancing the expression of lipolysis-related genes (ATGL and HSL). Consistent with the markedly lower expression of genes related to lipid droplet formation and secretion (TIP47, ADFP, BTN1A1, and XDH), cellular lipid droplets also were reduced sharply after incubation with C75 or adenovirus-short-hairpin-RNA. The results underscored the essential role of FASN in the overall process of milk-fat formation in goat mammary epithelial cells. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Inhibition of cyclooxygenase-2 in hematopoietic cells results in salt-sensitive hypertension.

    Science.gov (United States)

    Zhang, Ming-Zhi; Yao, Bing; Wang, Yinqiu; Yang, Shilin; Wang, Suwan; Fan, Xiaofeng; Harris, Raymond C

    2015-11-02

    Inhibition of prostaglandin (PG) production with either nonselective or selective inhibitors of cyclooxygenase-2 (COX-2) activity can induce or exacerbate salt-sensitive hypertension. This effect has been previously attributed to inhibition of intrinsic renal COX-2 activity and subsequent increase in sodium retention by the kidney. Here, we found that macrophages isolated from kidneys of high-salt-treated WT mice have increased levels of COX-2 and microsomal PGE synthase-1 (mPGES-1). Furthermore, BM transplantation (BMT) from either COX-2-deficient or mPGES-1-deficient mice into WT mice or macrophage-specific deletion of the PGE2 type 4 (EP4) receptor induced salt-sensitive hypertension and increased phosphorylation of the renal sodium chloride cotransporter (NCC). Kidneys from high-salt-treated WT mice transplanted with Cox2-/- BM had increased macrophage and T cell infiltration and increased M1- and Th1-associated markers and cytokines. Skin macrophages from high-salt-treated mice with either genetic or pharmacologic inhibition of the COX-2 pathway expressed decreased M2 markers and VEGF-C production and exhibited aberrant lymphangiogenesis. Together, these studies demonstrate that COX-2-derived PGE2 in hematopoietic cells plays an important role in both kidney and skin in maintaining homeostasis in response to chronically increased dietary salt. Moreover, these results indicate that inhibiting COX-2 expression or activity in hematopoietic cells can result in a predisposition to salt-sensitive hypertension.

  6. Inhibition of Glycogen Synthase Kinase or the Apoptotic Protein p53 Lowers the Threshold of Helium Cardioprotection In Vivo: The Role of Mitochondrial Permeability Transition

    Science.gov (United States)

    Pagel, Paul S.; Krolikowski, John G.; Pratt, Phillip F.; Shim, Yon Hee; Amour, Julien; Warltier, David C.; Weihrauch, Dorothee

    2008-01-01

    BACKGROUND Prosurvival signaling kinases inhibit glycogen synthase kinase-3β (GSK-3β) activity and stimulate apoptotic protein p53 degradation. Helium produces cardioprotection by activating prosurvival kinases, but whether GSK and p53 inhibition mediate this process is unknown. We tested the hypothesis that inhibition of GSK or p53 lowers the threshold of helium cardioprotection via a mitochondrial permeability transition pore (mPTP)-dependent mechanism. METHODS Rabbits (n = 85) instrumented for hemodynamic measurement and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), or 1, 3, or 5 cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture (fraction of inspired oxygen concentration = 0.30) before LAD occlusion. Other rabbits received the GSK inhibitor SB 216763 (SB21; 0.2 or 0.6 mg/kg), the p53 inhibitor pifithrin-α (PIF; 1.5 or 3.0 mg/kg), or SB21 (0.2 mg/kg) or PIF (1.5 mg/kg) plus helium (1 cycle) before LAD occlusion in the presence or absence of the mPTP opener atractyloside (5 mg/kg). RESULTS Helium reduced (P < 0.05) myocardial infarct size (35 ± 6 [n = 7], 25 ± 4 [n = 7], and 20 ± 3% [n = 6] of area at risk, 1, 3, and 5 cycles, respectively) compared with control (44 ± 6% [n = 7]). SB21 (0.6 [n = 7] but not 0.2 mg/kg [n = 6]) and PIF (3.0 [n = 6] but not 1.5 mg/kg [n = 7]) also reduced necrosis. SB21 (0.2 mg/kg) or 1.5 mg/kg PIF (1.5 mg/kg) plus helium (1 cycle; n = 6 per group) decreased infarct size to an equivalent degree as three cycles of helium alone, and this cardioprotection was blocked by atractyloside (n = 7 per group). CONCLUSIONS Inhibition of GSK or p53 lowers the threshold of helium-induced preconditioning via a mPTP-dependent mechanism in vivo. PMID:18713881

  7. Lipoplex gene transfer of inducible nitric oxide synthase inhibits the reactive intimal hyperplasia after expanded polytetrafluoroethylene bypass grafting.

    Science.gov (United States)

    Pfeiffer, Tomas; Wallich, Martina; Sandmann, Wilhelm; Schrader, Jürgen; Gödecke, Axel

    2006-05-01

    Intimal hyperplasia (IH) is most commonly the cause of graft occlusion in infrainguinal bypass grafting for arterial occlusive disease. We investigated the influence of nitric oxide on the IH of the arterial vessel wall at the region of prosthetic bypass anastomoses. Experiments were performed in 10 Foxhound dogs. We used a technique of inducible nitric oxide synthase (iNOS) overexpression by a non-virus-mediated, liposome-based iNOS gene transfer. The plasmid pSCMV-iNOS, which drives the expression of iNOS under control of the cytomegalovirus promoter, was complexed with cationic liposomes (lipoplexes). Segments of both carotid arteries were pretreated by intramural injection of a lipoplex solution by using an infiltrator balloon catheter (Infiltrator Drug Delivery Balloon System). In each dog, iNOS was administered at one side, and a control vector (pSCMV2) was administered at the contralateral side. Carotid arteries were ligated, and bypass grafts (expanded polytetrafluoroethylene, 6-mm, ring enforced) were implanted on both sides. The proximal and distal anastomoses (end-to-side fashion; running nonabsorbable sutures) were placed in the pretreated regions. After 6 months, the prostheses were excised, and the intimal thicknesses of 50 cross sections (orcein staining) of each anastomosis were measured planimetrically. The average reduction of the neointima thickness of the iNOS side in proximal anastomoses at the prosthetic wall, suture region, and arterial wall was 43%, 52%, and 81%, respectively. In distal anastomoses, the average reduction was 40%, 47%, and 52%, respectively. All differences of neointima thickness between the iNOS and control sides were statistically significant (Wilcoxon test; P < or = .05). Inducible NOS expression is an efficient approach for inhibition of IH. In contrast to earlier studies, which investigated the efficacy of gene therapeutic NOS expression at 3 to 4 weeks after intervention, the novelty of our findings is that a single

  8. Nitric oxide synthase inhibition enhances the antitumor effect of radiation in the treatment of squamous carcinoma xenografts.

    Directory of Open Access Journals (Sweden)

    Robert J G Cardnell

    Full Text Available This study tests whether the nitric oxide synthase (NOS inhibitor, N(G-nitro-L-arginine (L-NNA, combines favorably with ionizing radiation (IR in controlling squamous carcinoma tumor growth. Animals bearing FaDu and A431 xenografts were treated with L-NNA in the drinking water. IR exposure was 10 Gy for tumor growth and survival studies and 4 Gy for ex vivo clonogenic assays. Cryosections were examined immunohistochemically for markers of apoptosis and hypoxia. Blood flow was assayed by fluorescent microscopy of tissue cryosections after i.v. injection of fluorospheres. Orally administered L-NNA for 24 hrs reduces tumor blood flow by 80% (p<0.01. Within 24 hrs L-NNA treatment stopped tumor growth for at least 10 days before tumor growth again ensued. The growth arrest was in part due to increased cell killing since a combination of L-NNA and a single 4 Gy IR caused 82% tumor cell killing measured by an ex vivo clonogenic assay compared to 49% by L-NNA or 29% by IR alone. A Kaplan-Meyer analysis of animal survival revealed a distinct survival advantage for the combined treatment. Combining L-NNA and IR was also found to be at least as effective as a single i.p. dose of cisplatin plus IR. In contrast to the in vivo studies, exposure of cells to L-NNA in vitro was without effect on clonogenicity with or without IR. Western and immunochemical analysis of expression of a number of proteins involved in NO signaling indicated that L-NNA treatment enhanced arginase-2 expression and that this may represent vasculature remodeling and escape from NOS inhibition. For tumors such as head and neck squamous carcinomas that show only modest responses to inhibitors of specific angiogenic pathways, targeting NO-dependent pro-survival and angiogenic mechanisms in both tumor and supporting stromal cells may present a potential new strategy for tumor control.

  9. Inhibition of Nitric Oxide Synthesis and Gene Knockout of Neuronal Nitric Oxide Synthase Impaired Adaptation of Mouse Optokinetic Response Eye Movements

    OpenAIRE

    Katoh, Akira; Kitazawa, Hiromasa; Itohara, Shigeyoshi; Nagao, Soichi

    2000-01-01

    Nitric oxide (NO) plays a key role in synaptic transmission efficiency in the central nervous system. To gain an insight on the role of NO in cerebellar functions, we, here, measured the dynamics of the horizontal optokinetic response (HOKR) and vestibulo-ocular reflex (HVOR), and the adaptation of HOKR in mice locally injected with NG-monomethyl-l-arginine (L-NMMA) that inhibits NO synthesis and in mice devoid of neuronal nitric oxide synthase (nNOS). Local application of L-NMMA into the cer...

  10. Glycogen synthase kinase-3β inactivation inhibits tumor necrosis factor-α production in microglia by modulating nuclear factor κB and MLK3/JNK signaling cascades

    Directory of Open Access Journals (Sweden)

    Chen Wu-Fu

    2010-12-01

    Full Text Available Abstract Background Deciphering the mechanisms that modulate the inflammatory response induced by microglial activation not only improves our insight into neuroinflammation but also provides avenues for designing novel therapies that could halt inflammation-induced neuronal degeneration. Decreasing glycogen synthase kinase-3β (GSK-3β activity has therapeutic benefits in inflammatory diseases. However, the exact molecular mechanisms underlying GSK-3β inactivation-mediated suppression of the inflammatory response induced by microglial activation have not been completely clarified. Tumor necrosis factor-α (TNF-α plays a central role in injury caused by neuroinflammation. We investigated the regulatory effect of GSK-3β on TNF-α production by microglia to discern the molecular mechanisms of this modulation. Methods Lipopolysaccharide (LPS was used to induce an inflammatory response in cultured primary microglia or murine BV-2 microglial cells. Release of TNF-α was measured by ELISA. Signaling molecules were analyzed by western blotting, and activation of NF-κB and AP-1 was measured by ELISA-based DNA binding analysis and luciferase reporter assay. Protein interaction was examined by coimmunoprecipitation. Results Inhibition of GSK-3β by selective GSK-3β inhibitors or by RNA interference attenuated LPS-induced TNF-α production in cultured microglia. Exploration of the mechanisms by which GSK-3β positively regulates inflammatory response showed that LPS-induced IκB-α degradation, NF-κBp65 nuclear translocation, and p65 DNA binding activity were not affected by inhibition of GSK-3β activity. However, GSK-3β inactivation inhibited transactivation activity of p65 by deacetylating p65 at lysine 310. Furthermore, we also demonstrated a functional interaction between mixed lineage kinase 3 (MLK3 and GSK-3β during LPS-induced TNF-α production in microglia. The phosphorylated levels of MLK3, MKK4, and JNK were increased upon LPS treatment

  11. Quinazoline antifolates inhibiting thymidylate synthase: synthesis of four oligo(L-gamma-glutamyl) conjugates of N10-propargyl-5,8-dideazafolic acid and their enzyme inhibition.

    Science.gov (United States)

    Pawelczak, K; Jones, T R; Kempny, M; Jackman, A L; Newell, D R; Krzyzanowski, L; Rzeszotarska, B

    1989-01-01

    The synthesis is described of four oligo(gamma-glutamyl) conjugates of N10-propargyl-5,8-dideazafolic acid containing a total of two, three, four, and five L-glutamic acid residues. The tert-butyl group was chosen as the carboxyl protecting group in order to obviate the use of alkali and thus the possibility of gamma----alpha transpeptidation. The starting material, di-tert-butyl glutamate, was coupled to N-(benzyloxycarbonyl)-L-glutamic acid alpha-tert-butyl ester via a mixed anhydride with isobutyl chloroformate. Hydrogenolysis of the benzyloxycarbonyl group in the product gave a carboxyl-protected diglutamate, which either was acylated with 4-[(benzyloxycarbonyl)amino] benzoyl chloride to give a protected aminobenzamide or was cycled further by using the above mixed anhydride/hydrogenolysis sequence into tri-, tetra-, and pentaglutamates. Each of the last named was also acylated, as above, to give a benzamide. The benzyloxycarbonyl group in the benzamides was removed by hydrogenolysis and the amino groups thus exposed were N-alkylated with propargyl bromide. The resulting proparglyamines were further alkylated with 2-amino-6-(bromomethyl)-4-hydroxyquinazoline hydrobromide to give the antifolate poly(t-Bu) esters. Deprotection with trifluoroacetic acid in the final step delivered the desired antifolates as their trifluoroacetate salts. The di- to pentaglutamates were, respectively, 31-, 97-, 171-, and 167-fold more inhibitory to WI-L2 human thymidylate synthase than the parent compound.

  12. Acyl carrier protein (ACP) inhibition and other differences between b-ketoacyl synthase (KAS) I and II

    DEFF Research Database (Denmark)

    McGuire, Kirsten Arnvig; McGuire, J.N.; Wettstein-Knowles, Penny von

    2000-01-01

    Escherichia coli b-ketoacyl synthases (KAS) I and II carry out the elongation steps in fatty acid synthesis. Analyses using the cross-linker BS3 [bis(sulphosuccinimidyl) suberate] and surface-enhanced laser desorption/ionization–time-of-flight MS disclosed only monomeric and dimeric forms of KAS ...

  13. Inhibition of Glycogen Synthase Kinase-3ß Enhances Cognitive Recovery after Stroke: The Role of TAK1

    Science.gov (United States)

    Venna, Venugopal Reddy; Benashski, Sharon E.; Chauhan, Anjali; McCullough, Louise D.

    2015-01-01

    Memory deficits are common among stroke survivors. Identifying neuroprotective agents that can prevent memory impairment or improve memory recovery is a vital area of research. Glycogen synthase kinase-3ß (GSK-3ß) is involved in several essential intracellular signaling pathways. Unlike many other kinases, GSK-3ß is active only when…

  14. Inhibition of muscle glycogen synthase activity and non-oxidative glucose disposal during hypoglycaemia in normal man

    DEFF Research Database (Denmark)

    Ørskov, Lotte; Bak, Jens Friis; Abildgaard, Ulrik

    1996-01-01

    The purpose of the present study was to evaluate the role of muscle glycogen synthase activity in the reduction of glucose uptake during hypoglycaemia. Six healthy young men were examined twice; during 120 min of hyperinsulinaemic (1.5 mU.kg-1. min-1) euglycaemia followed by: 1)240 min of graded ...

  15. Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

    Science.gov (United States)

    Borejsza-Wysocki, W.; Hrazdina, G.

    1996-03-01

    p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.

  16. Single-dose rosuvastatin ameliorates lung ischemia-reperfusion injury via upregulation of endothelial nitric oxide synthase and inhibition of macrophage infiltration in rats with pulmonary hypertension.

    Science.gov (United States)

    Matsuo, Satoshi; Saiki, Yuriko; Adachi, Osamu; Kawamoto, Shunsuke; Fukushige, Shinichi; Horii, Akira; Saiki, Yoshikatsu

    2015-03-01

    Lung ischemia-reperfusion (IR) injury during cardiopulmonary surgery is associated with postoperative morbidity and mortality, particularly in patients with pulmonary hypertension (PH). Using a rat model for monocrotaline-induced PH, we investigated the protective effect of rosuvastatin against IR injury in lungs affected by PH and attempted to elucidate its mechanism of action. Male Sprague-Dawley monocrotaline-treated rats were divided into 4 groups (n = 8-9): sham, control + IR, statin + IR, and statin + mevalonolactone + IR. Lung ischemia was induced by left pulmonary artery occlusion (1 hour), followed by reperfusion (4 hours). Rosuvastatin (2 mg/kg) was injected 18 hours before reperfusion and mevalonolactone (1 mg/kg) was injected immediately before reperfusion. The arterial oxygen tension/inspired oxygen fraction ratio was used as a measure of lung oxygenation. Left lung tissue was analyzed for the wet-to-dry lung weight ratio and protein expression of endothelial nitric oxide synthase (eNOS) and phospho-eNOS. Macrophage recruitment was assessed by CD68 immunostaining. Our results showed that rosuvastatin decreased IR lung injury (control + IR vs statin + IR) in terms of the arterial oxygen tension/inspired oxygen fraction ratio (272 ± 43 vs 442 ± 13), wet-to-dry ratio (5.7 ± 0.7 vs 4.8 ± 0.6), and macrophage infiltration (8.0 ± 0.6/field vs 4.0 ± 0.5/field) (P < .05 for all). eNOS and phospho-eNOS were downregulated by IR, which was blocked by rosuvastatin. Effects of rosuvastatin were blunted by mevalonolactone. Single-dose rosuvastatin decreased IR injury in lungs affected by PH via 2 anti-inflammatory mechanisms: preserving eNOS function and inhibiting macrophage infiltration. Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  17. Glycogen synthase kinase-3beta heterozygote knockout mice as a model of findings in postmortem schizophrenia brain or as a model of behaviors mimicking lithium action: negative results.

    Science.gov (United States)

    Bersudsky, Yuly; Shaldubina, Alona; Kozlovsky, Nitzan; Woodgett, James R; Agam, Galila; Belmaker, R H

    2008-05-01

    In mice glycogen synthase kinase (GSK)-3beta heterozygote knockout status was reported to cause reduced immobility in the Porsolt forced swim test and reduced amphetamine-induced hyperactivity, behaviors that mimic the effects of lithium. GSK-3beta protein and mRNA level and activity have been reported to be reduced in the postmortem brain of schizophrenia patients and this could suggest the involvement of GSK-3beta in the etiology of schizophrenia. However, apomorphine-induced stereotyping was reported to be unchanged in GSK-3beta heterozygote (HZ) knockout (KO) mice. As such behaviors are not always robust, study in another laboratory seemed indicated. Motor activity and coordination were assessed in the rotarod test. Behavior was studied in the following tests: pilocarpine-induced seizures model for lithium action, Porsolt forced swim test, tail suspension test, elevated plus-maze, large open field, startle response and prepulse inhibition of acoustic startle response, amphetamine-induced hyperactivity, and apomorphine-induced stereotypic climbing. We could not confirm the report that GSK-3beta HZ KO mice exhibit reduced immobility in the Porsolt forced swim or reduced amphetamine-induced hyperactivity in a manner mimicking the behavioral effects of lithium. We did not find increased apomorphine-induced stereotypic climbing or disruption of prepulse inhibition, suggesting that human postmortem findings regarding GSK-3beta in schizophrenia are not mediated by changes in dopamine receptors and are not the cause of prepulse inhibition deficits in schizophrenia. These data do not support the role of GSK-3beta in schizophrenia or in the mechanism of therapeutic action of lithium. Although differences in the genetic background of the GSK-3beta HZ KOs used in the present study compared with that of the previous study could be responsible, such results could suggest that the previously reported effects of GSK-3beta knockout on behavior are not robust.

  18. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3

    Energy Technology Data Exchange (ETDEWEB)

    Kultti, Anne, E-mail: anne.kultti@uku.fi [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Pasonen-Seppaenen, Sanna [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland); Jauhiainen, Marjo [Department of Pharmaceutical Chemistry, University of Kuopio, FIN-70211 Kuopio (Finland); Rilla, Kirsi J.; Kaernae, Riikka; Pyoeriae, Emma; Tammi, Raija H.; Tammi, Markku I. [Institute of Biomedicine, Anatomy, University of Kuopio, P.O.B. 1627, FIN-70211 Kuopio (Finland)

    2009-07-01

    Hyaluronan accumulation on cancer cells and their surrounding stroma predicts an unfavourable disease outcome, suggesting that hyaluronan enhances tumor growth and spreading. 4-Methylumbelliferone (4-MU) inhibits hyaluronan synthesis and retards cancer spreading in experimental animals through mechanisms not fully understood. These mechanisms were studied in A2058 melanoma cells, MCF-7 and MDA-MB-361 breast, SKOV-3 ovarian and UT-SCC118 squamous carcinoma cells by analysing hyaluronan synthesis, UDP-glucuronic acid (UDP-GlcUA) content, and hyaluronan synthase (HAS) mRNA levels. The maximal inhibition in hyaluronan synthesis ranged 22-80% in the cell lines tested. Active glucuronidation of 4-MU produced large quantities of 4-MU-glucuronide, depleting the cellular UDP-GlcUA pool. The maximal reduction varied between 38 and 95%. 4-MU also downregulated HAS mRNA levels: HAS3 was 84-60% lower in MDA-MB-361, A2058 and SKOV-3 cells. HAS2 was the major isoenzyme in MCF-7 cells and lowered by 81%, similar to 88% in A2058 cells. These data indicate that both HAS substrate and HAS2 and/or HAS3 mRNA are targeted by 4-MU. Despite different target point sensitivities, the reduction of hyaluronan caused by 4-MU was associated with a significant inhibition of cell migration, proliferation and invasion, supporting the importance of hyaluronan synthesis in cancer, and the therapeutic potential of hyaluronan synthesis inhibition.

  19. A Therapeutic Connection between Dietary Phytochemicals and ATP Synthase.

    Science.gov (United States)

    Ahmad, Zulfiqar; Hassan, Sherif S; Azim, Sofiya

    2017-11-20

    For centuries, phytochemicals have been used to prevent and cure multiple health ailments. Phytochemicals have been reported to have antioxidant, antidiabetic, antitussive, antiparasitic, anticancer, and antimicrobial properties. Generally, the therapeutic use of phytochemicals is based on tradition or word of mouth with few evidence-based studies. Moreover, molecular level interactions or molecular targets for the majority of phytochemicals are unknown. In recent years, antibiotic resistance by microbes has become a major healthcare concern. As such, the use of phytochemicals with antimicrobial properties has become pertinent. Natural compounds from plants, vegetables, herbs, and spices with strong antimicrobial properties present an excellent opportunity for preventing and combating antibiotic resistant microbial infections. ATP synthase is the fundamental means of cellular energy. Inhibition of ATP synthase may deprive cells of required energy leading to cell death, and a variety of dietary phytochemicals are known to inhibit ATP synthase. Structural modifications of phytochemicals have been shown to increase the inhibitory potency and extent of inhibition. Sitedirected mutagenic analysis has elucidated the binding site(s) for some phytochemicals on ATP synthase. Amino acid variations in and around the phytochemical binding sites can result in selective binding and inhibition of microbial ATP synthase. In this review, the therapeutic connection between dietary phytochemicals and ATP synthase is summarized based on the inhibition of ATP synthase by dietary phytochemicals. Research suggests selective targeting of ATP synthase is a valuable alternative molecular level approach to combat antibiotic resistant microbial infections. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Changes in angiotensin AT1 receptor mRNA levels in the rat brain after immobilization stress and inhibition of central nitric oxide synthase.

    Science.gov (United States)

    Kiss, A; Jurkovicova, D; Jezova, D; Krizanova, O

    2001-06-01

    To study functional interactions between angiotensin II AT1 receptors and nitric oxide (NO) activity in different brain areas in rats exposed to immobilization stress. Central inhibition of nitric oxide synthase (NOS) was provided by intracerebroventricular (i.c.v.) administration of (N-omega-nitro-L-arginine-methylester) L-NAME and analysis of AT1 receptor mRNA was performed using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The immobilization in prone position lasted 2 hrs and the rats were sacrificed 24 hr later. The hypothalamus, hippocampus, thalamus, and cortex were isolated from fresh brains. In the cortex, gene expression of AT1 receptors was unaffected either by L-NAME treatment, or by a single exposure to immobilization stress for 2 hours followed by 24 hours of rest. In the hippocampus, the repeated treatment with L-NAME increased mRNA levels of AT1 receptors approximately 9-times compared to those in the control (untreated) group. Immobilization also increased AT1 receptor mRNA levels in the hippocampus which was similar to that induced by the L-NAME. The increase of AT1 receptor mRNA levels in the hippocampus of immobilized rats was not further altered when the animals were pretreated with L-NAME. In control rats, exposure to immobilization resulted in a significant rise in mRNA levels coding for AT1 receptors in the hypothalamus, but not in the thalamus. L-NAME treatment showed a tendency of increase in AT1 receptor mRNA levels in the hypothalamus. Moreover, when animals treated with L-NAME were subjected to immobilization, a further increase in AT1 receptor mRNA levels was observed in the hypothalamus in comparison with corresponding controls. The present data indicate that a single immobilization stress results in increased gene expression of AT1 receptors in the hypothalamus and hippocampus. The rise in AT1 mRNA levels in the same brain structures after repeated treatment with L-NAME allow to suggest an

  1. Inhibition of G-protein-coupled Receptor Kinase 2 Prevents the Dysfunctional Cardiac Substrate Metabolism in Fatty Acid Synthase Transgenic Mice*♦

    Science.gov (United States)

    Abd Alla, Joshua; Graemer, Muriel; Fu, Xuebin; Quitterer, Ursula

    2016-01-01

    Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart. PMID:26670611

  2. Inhibition of G-protein-coupled Receptor Kinase 2 Prevents the Dysfunctional Cardiac Substrate Metabolism in Fatty Acid Synthase Transgenic Mice.

    Science.gov (United States)

    Abd Alla, Joshua; Graemer, Muriel; Fu, Xuebin; Quitterer, Ursula

    2016-02-05

    Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

    Science.gov (United States)

    Muhitch, Michael J.

    1988-01-01

    Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis. PMID:16665871

  4. Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling.

    Science.gov (United States)

    Dave, Bhuvanesh; Granados-Principal, Sergio; Zhu, Rui; Benz, Stephen; Rabizadeh, Shahrooz; Soon-Shiong, Patrick; Yu, Ke-Da; Shao, Zhimin; Li, Xiaoxian; Gilcrease, Michael; Lai, Zhao; Chen, Yidong; Huang, Tim H-M; Shen, Haifa; Liu, Xuewu; Ferrari, Mauro; Zhan, Ming; Wong, Stephen T C; Kumaraswami, Muthiah; Mittal, Vivek; Chen, Xi; Gross, Steven S; Chang, Jenny C

    2014-06-17

    We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.

  5. 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...

  6. Chemical control of California arrowhead (Sagittaria montevidensis resistant to acetolactate synthase and photosystem II inhibiting herbicides in irrigated rice

    Directory of Open Access Journals (Sweden)

    Diogo da Silva Moura

    Full Text Available ABSTRACT: California arrowhead is one of the primary weeds infesting paddy rice fields in the Brazilian states of Santa Catarina and Rio Grande do Sul, where the system of pre-germinated seeding is used. The objective of this study was to evaluate the selectivity and effectiveness of saflufenacil application in irrigated rice, either singly or in combination with other herbicides in the same application or sequentially, for the control of Sagittaria montevidensis biotype that is resistant to ALSand PSII-inhibiting herbicides. In the first experiment carried out in a greenhouse, saflufenacil was applied, either singly or in combination with penoxsulam, bispyribac-sodium, pyrazosulfuron-ethyl, bentazon, or propanil to the S. montevidensis (SAGMO 32 biotype and the irrigated rice variety Epagri 108. In the second experiment, single or combined (including sequential applications of saflufenacil, bentazon, and cyhalofop-butyl were applied to Epagri 108 in open field conditions. Saflufenacil combined with propanil showed a high degree of phytotoxicity and a reduction in the accumulation of dry mass in Epagri 108. Application of saflufenacil, bentazon, and cyhalofop-butyl in combination or sequentially resulted in an increase in phytotoxicity in Epagri 108 compared to when applied singly. A pplication of saflufenacil singly or in combination with penoxsulam, bispyribac-sodium, bentazon, or pyrazosulfuron-ethyl did not adequately control SAGMO 32.

  7. A novel amino acid substitution Trp574Arg in acetolactate synthase (ALS) confers broad resistance to ALS-inhibiting herbicides in crabgrass (Digitaria sanguinalis).

    Science.gov (United States)

    Li, Jian; Li, Mei; Gao, Xingxiang; Fang, Feng

    2017-12-01

    Crabgrass (Digitaria sanguinalis) is an annual monocotyledonous weed. In recent years, field applications of nicosulfuron have been ineffective in controlling crabgrass populations in Shandong Province, China. To investigate the mechanisms of resistance to nicosulfuron in crabgrass populations, the acetolactate synthase (ALS) gene fragment covering known resistance-confering mutation sites was amplified and sequenced. Dose-response experiments suggested that the resistant population SD13 (R) was highly resistant to nicosulfuron (resistance index R/S = 43.7) compared with the sensitive population SD22 (S). ALS gene sequencing revealed a Trp574Arg substitution in the SD13 population, and no other known resistance-conferring mutations were found. In vitro ALS enzyme assays further confirmed that the SD13 population was resistant to all tested ALS-inhibiting herbicides. The resistance pattern experiments revealed that, compared with SD22, the SD13 population exhibited broad-spectrum resistance to nicosulfuron (43.7-fold), imazethapyr (11.4-fold) and flumetsulam (16.1-fold); however, it did not develop resistance to atrazine, mesotrione and topramezone. This study demonstrated that Trp574Arg substitution was the main reason for crabgrass resistance to ALS-inhibiting herbicides. To our knowledge, this is the first report of Trp574Arg substitution in a weed species, and is the first report of target-site mechanisms of herbicide resistance for crabgrass. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  8. Cross-resistance patterns to acetolactate synthase (ALS)-inhibiting herbicides of flixweed (Descurainia sophia L.) conferred by different combinations of ALS isozymes with a Pro-197-Thr mutation or a novel Trp-574-Leu mutation.

    Science.gov (United States)

    Deng, Wei; Yang, Qian; Zhang, Yongzhi; Jiao, Hongtao; Mei, Yu; Li, Xuefeng; Zheng, Mingqi

    2017-03-01

    Acetolactate synthase (ALS) is the common target of ALS-inhibiting herbicides, and target-site ALS mutations are the main mechanism of resistance to ALS-inhibiting herbicides. In this study, ALS1 and ALS2 genes with full lengths of 2004bp and 1998bp respectively were cloned in individual plants of susceptible (S) or resistant (R) flixweed (Descurainia sophia L.) populations. Two ALS mutations of Pro-197-Thr and/or Trp-574-Leu were identified in plants of three R biotypes (HB24, HB30 and HB42). In order to investigate the function of ALS isozymes in ALS-inhibiting herbicide resistance, pHB24 (a Pro-197-Thr mutation in ALS1 and a wild type ALS2), pHB42 (a Trp-574-Leu mutation in ALS1 and a wild type ALS2) and pHB30 (a Trp-574-Leu mutation in ALS1 and a Pro-197-Thr mutation in ALS2) subpopulations individually homozygous for different ALS mutations were generated. Individuals of pHB30 had mutations in each isozyme of ALS and had higher resistance than pHB24 and pHB42 populations containing mutations in only one ALS isozyme. Moreover, the pHB24 had resistance to SU, TP and SCT herbicides, whereas pHB24 and pHB42 had resistance to these classes of herbicides as well as IMI and PTB herbicides. The sensitivity of isolated ALS enzyme to inhibition by herbicides in these populations correlated with whole plant resistance levels. Therefore, reduced ALS sensitivity resulting from the mutations in ALS was responsible for resistance to ALS-inhibiting herbicides in flixweed. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Inibição da atividade da citrato sintase cerebral em um modelo animal de sepse Inhibition of brain citrate synthase activity in an animal model of sepsis

    Directory of Open Access Journals (Sweden)

    Giselli Scaini

    2011-06-01

    fisiopatologia desta doença.OBJECTIVE: An extensive body of evidence from experimental studies indicates that sepsis is associated with increased reactive oxygen species production, depletion of antioxidants, and accumulation of markers of oxidative stress. Moreover, mitochondrial dysfunction has been implicated in the pathogenesis of multiple organ dysfunction syndrome (MODS. Citrate synthase is an enzyme localized in the mitochondrial matrix and an important component of the Krebs cycle; consequently, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase activity in the brains of rats submitted to a cecal ligation puncture model of sepsis. METHODS: At several times points (3, 6, 12, 24 and 48 hours after the cecal ligation puncture operation, six rats were killed by decapitation. Their brains were removed, and the hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used to determine citrate synthase activity. RESULTS: We found that citrate synthase activity in the prefrontal cortex was inhibited 12, 24 and 48 hours after cecal ligation puncture. In the cerebral cortex, citrate synthase activity was inhibited 3, 12, 24 and 48 hours after cecal ligation puncture. Citrate synthase was not affected in the hippocampus, striatum or cerebellum up to 48 hours after cecal ligation puncture. CONCLUSION: Considering that energy impairment due to mitochondrial dysfunction in sepsis has been well described and that oxidative stress plays a crucial role in sepsis development, we believe that energy impairment may also be involved in these processes. If citrate synthase inhibition also occurs in a sepsis model, it is tempting to speculate that a reduction in brain metabolism may be related to the pathophysiology of this disease.

  10. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans.

    Science.gov (United States)

    Bito, Tomohiro; Yabuta, Yukinori; Ichiyanagi, Tsuyoshi; Kawano, Tsuyoshi; Watanabe, Fumio

    2014-01-01

    In this study, we showed that cyanocobalamin dodecylamine, a ribose 5'-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), methylmalonic aciduria cblC type (cblc-1), and methionine synthase reductase (mtrr-1). In contrast, the level of the mRNAs encoding cob(I)alamin adenosyltransferase (mmab-1) was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  11. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Tomohiro Bito

    2014-01-01

    Full Text Available In this study, we showed that cyanocobalamin dodecylamine, a ribose 5′-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1, methylmalonic acidemia cobalamin A complementation group (mmaa-1, methylmalonic aciduria cblC type (cblc-1, and methionine synthase reductase (mtrr-1. In contrast, the level of the mRNAs encoding cob(Ialamin adenosyltransferase (mmab-1 was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  12. The effects of folic acid and nitric oxide synthase inhibition on coronary flow and oxidative stress markers in isolated rat heart.

    Science.gov (United States)

    Djurić, Dragan; Vusanović, Ana; Jakovljević, Vladimir

    2007-06-01

    The aim of this study was to assess the effects of folic acid on coronary flow and oxidative stress markers with or without non-specific inhibition of nitric oxide synthase by L-NAME in isolated rat hearts. The hearts of male Wistar albino rats (n = 12, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure (40-120 cmH2O). Coronary flow and markers of oxidative stress: nitrite outflow, superoxide anion production, and index of lipid peroxidation (by measuring thiobarbituric acid reactive substances) in coronary effluent were calculated. The experiments were performed during control conditions and in presence of folic acid (100 microM) alone or folic acid (100 microM) plus L-NAME (30 microM). Control values of coronary flow varied in range from 4.37 +/- 0.10 ml/min/g wt at 40 cmH2O to 12.05 +/- 0.42 ml/min/g wt at 120 cmH2O. Nitrite outflow varied from 1.68 +/- 0.17 nmol/min/g wt at 40 cmH2O to 3.56 +/- 0.17 nmol/min/g wt at 120 cmH2O and was parallel with coronary perfusion pressure-coronary flow curve. Folic acid significantly increased coronary flow (40-120 cmH2O, 5.63 +/- 0.10 ml/min/g wt and 15.2 +/- 0.42 ml/min/g wt, respectively) and was accompanied by significant increase in nitrite outflow (2.28 +/- 0.29 nmol/min/g wt at 40 cmH2O to 6.66 +/- 0.50 nmol/min/g wt at 120 cmH2O). In addition, folic acid significantly decreased superoxide anion production especially at upper coronary perfusion pressure values (60% at 120 cmH2O) and increased index of lipid peroxidation (37.16% at 120 cmH2O), respectively. Folic acid plus L-NAME did not change control values of coronary flow significantly. However, folic acid plus L-NAME increased nitrite outflow especially at upper coronary perfusion pressure values (43.05% at 120 cmH2O) and did not change significantly superoxide anion production or index of lipid peroxidation versus control values, respectively. The results clearly

  13. 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...

  14. The guanylhydrazone CNI-1493: an inhibitor with dual activity against malaria-inhibition of host cell pro-inflammatory cytokine release and parasitic deoxyhypusine synthase.

    Science.gov (United States)

    Specht, Sabine; Sarite, Salem Ramadan; Hauber, Ilona; Hauber, Joachim; Görbig, Ulf F; Meier, Chris; Bevec, Dorian; Hoerauf, Achim; Kaiser, Annette

    2008-05-01

    Malaria is still a major cause of death in the tropics. There is an urgent need for new anti-malarial drugs because drug-resistant plasmodia frequently occur. Over recent years, we elucidated the biosynthesis of hypusine, a novel amino acid contained in eukaryotic initiation factor 5A (eIF-5A) in Plasmodium. Hypusine biosynthesis involves catalysis of deoxyhypusine synthase (DHS) in the first step of post-translational modification. In a screen for new inhibitors of purified plasmodium DHS, CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohn's disease, inhibited the enzyme of the parasite 3-fold at a concentration of 2 microM. In vitro experiments with 200 microM CNI-1493 in Plasmodium-infected erythrocytes, which lack nuclei and DHS protein, showed a parasite clearance within 2 days. This can presumably be attributed to an anti-proliferating effect because of the inhibition of DHS by the parasite. The determined IC50 of CNI-1493 was 135.79 microM after 72 h. In vivo application of this substance in Plasmodium berghei ANKA-infected C57BL/6 mice significantly reduced parasitemia after dosage of 1 mg/kg or 4 mg/kg/body weight and prevented death of mice with cerebral malaria. This effect was paralleled by a decrease in serum TNF levels of the mice. We suggest that the new mechanism of CNI-1493 is caused by a decrease in modified eIF-5A biosynthesis with a downstream effect on the TNF synthesis of the host. From the current data, we consider CNI-1493 to be a promising drug for anti-malarial therapy because of its combined action, i.e., the decrease in eIF-5A biosynthesis of the parasite and host cell TNF biosynthesis.

  15. An innovative strategy for dual inhibitor design and its application in dual inhibition of human thymidylate synthase and dihydrofolate reductase enzymes.

    Directory of Open Access Journals (Sweden)

    Mahreen Arooj

    Full Text Available Due to the diligence of inherent redundancy and robustness in many biological networks and pathways, multitarget inhibitors present a new prospect in the pharmaceutical industry for treatment of complex diseases. Nevertheless, to design multitarget inhibitors is concurrently a great challenge for medicinal chemists. We have developed a novel computational approach by integrating the affinity predictions from structure-based virtual screening with dual ligand-based pharmacophore to discover potential dual inhibitors of human Thymidylate synthase (hTS and human dihydrofolate reductase (hDHFR. These are the key enzymes in folate metabolic pathway that is necessary for the biosynthesis of RNA, DNA, and protein. Their inhibition has found clinical utility as antitumor, antimicrobial, and antiprotozoal agents. A druglike database was utilized to perform dual-target docking studies. Hits identified through docking experiments were mapped over a dual pharmacophore which was developed from experimentally known dual inhibitors of hTS and hDHFR. Pharmacophore mapping procedure helped us in eliminating the compounds which do not possess basic chemical features necessary for dual inhibition. Finally, three structurally diverse hit compounds that showed key interactions at both active sites, mapped well upon the dual pharmacophore, and exhibited lowest binding energies were regarded as possible dual inhibitors of hTS and hDHFR. Furthermore, optimization studies were performed for final dual hit compound and eight optimized dual hits demonstrating excellent binding features at target systems were also regarded as possible dual inhibitors of hTS and hDHFR. In general, the strategy used in the current study could be a promising computational approach and may be generally applicable to other dual target drug designs.

  16. Roles of the β subunit hinge domain in ATP synthase F1 sector: Hydrophobic network formed by introduced βPhe174 inhibits subunit rotation

    International Nuclear Information System (INIS)

    Nakanishi-Matsui, Mayumi; Kashiwagi, Sachiko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu

    2010-01-01

    The ATP synthase β subunit hinge domain (βPhe148 ∼ βGly186, P-loop/α-helixB/loop/β-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F 1 with the βSer174 to Phe mutation in the domain lowered the γ subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F 1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the βMet159, βIle163, and βAla167 residues of the β subunit are involved together with the mutant βPhe174. The network is expected to stabilize the conformation of β DP (nucleotide-bound form of the β subunit), resulting in increased activation energy for transition to β E (empty β subunit). The modeling further predicts that replacement of βMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of βS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the β subunit hinge domain is pertinent for the rotational catalysis.

  17. Endotoxin and interferon-γ inhibit translation in skeletal muscle cells by stimulating nitric oxide synthase activity

    Science.gov (United States)

    Frost, Robert A.; Nystrom, Gerald J.; Lang, Charles H.

    2012-01-01

    The purpose of the present study was to test the hypothesis that endogenous nitric oxide (NO) negatively affects translation in skeletal muscle cells after exposure to a combination of endotoxin (LPS) and interferon (IFN)γ. Individually LPS and IFNγ did not alter protein synthesis but in combination they inhibited protein synthesis by 80% in C2C12 myotubes. The combination of LPS and IFNγ dramatically down regulated the auto-phosphorylation of the mammalian target of rapamycin (mTOR) and its substrates S6K1 and 4EBP-1. The phosphorylation of ribosomal protein S6 was decreased whereas phosphorylation of elongation factor-2 (eEF-2) and raptor was enhanced consistent with defects in both translation initiation and elongation. Reduced S6 phosphorylation occurred 8–18 h after LPS/IFNγ and coincided with a prolonged upregulation of NOS2 mRNA and protein. NOS2 protein expression and the LPS/IFNγ –induced fall in phosphorylated S6 were prevented by the proteasome inhibitor MG132. The general NOS inhibitor L-NAME and the specific NOS2 inhibitor 1400W also prevented the LPS/IFNγ-induced decrease in protein synthesis and restored translational signaling. LPS/IFNγ down regulated the phosphorylation of multiple Akt substrates including the proline rich Akt substrate-40 (PRAS40) while enhancing the phosphorylation of raptor on an AMPK regulated site. The negative effects of LPS/IFNγ were blunted by the AMPK inhibitor compound C. The data suggest that in combination LPS and IFNγ induce a prolonged expression of NOS2 and excessive production of NO that reciprocally alters Akt and AMPK activity and consequently down regulates translation via reduced mTOR signaling. PMID:19295495

  18. 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

  19. Glycogen synthase kinase-3β inhibition in the medial prefrontal cortex mediates paradoxical amphetamine action in a mouse model of ADHD

    Directory of Open Access Journals (Sweden)

    Yi-Chun eYen

    2015-03-01

    Full Text Available Psychostimulants show therapeutic efficacy in the treatment of attention-deficit hyperactivity disorder (ADHD. It is generally assumed that they ameliorate ADHD symptoms via interfering with monoaminergic signaling. We combined behavioral pharmacology, neurochemistry and molecular analyses to identify mechanisms underlying the paradoxical calming effect of amphetamine in low trait anxiety behavior (LAB mice, a novel multigenetic animal model of ADHD. Amphetamine (1 mg/kg and methylphenidate (10 mg/kg elicited similar dopamine and norepinephrine release in the medial prefrontal cortex (mPFC and in the striatum of LAB mice. In contrast, amphetamine decreased, while methylphenidate increased locomotor activity. This argues against changes in dopamine and/or norepinephrine release as mediators of amphetamine paradoxical effects. Instead, the calming activity of amphetamine corresponded to the inhibition of glycogen synthase kinase3β (GSK3β activity, specifically in the mPFC. Accordingly, not only systemic administration of the GSK3β inhibitor TDZD-8 (20 mg/kg, but also local microinjections of TDZD-8 and amphetamine into the mPFC, but not into the striatum, decreased locomotor activity in LAB mice. Amphetamine effects seem to depend on NMDA receptor signaling, since pre- or co-treatment with MK-801 (0.3 mg/kg abolished the effects of amphetamine (1 mg/kg on the locomotion and on the phosphorylation of GSK3β at the level of the mPFC. Taken together, the paradoxical calming effect of amphetamine in hyperactive LAB mice concurs with a decreased GSK3β activity in the mPFC. This effect appears to be independent of dopamine or norepinephrine release, but contingent on NMDA receptor signaling.

  20. Gastrodin inhibits expression of inducible NO synthase, cyclooxygenase-2 and proinflammatory cytokines in cultured LPS-stimulated microglia via MAPK pathways.

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    Ji-Nan Dai

    Full Text Available Microglial activation plays an important role in neurodegenerative diseases by producing several proinflammatory enzymes and proinflammatory cytokines. The phenolic glucoside gastrodin, a main constituent of a Chinese herbal medicine, has been known to display anti-inflammatory properties. The current study investigates the potential mechanisms whereby gastrodin affects the expression of potentially pro-inflammatory proteins by cultured murine microglial BV-2 cells stimulated with lipopolysaccharide (LPS.BV-2 cells were pretreated with gastrodin (30, 40, and 60 µM for 1 h and then stimulated with LPS (1 µg/ml for another 4 h. The effects on proinflammatory enzymes, inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2, and proinflammatory cytokines, tumor necrosis factor-α (TNF-α, and interleukin-1β (IL-1β, are analysed by double-immunofluorescence labeling and RT-PCR assay. To reveal the mechanisms of action of gastrodin we investigated the involvement of mitogen-activated protein kinases (MAPKs cascades and their downstream transcription factors, nuclear factor-κB (NF-κB and cyclic AMP-responsive element (CRE-binding protein (CREB. Gastrodin significantly reduced the LPS-induced protein and mRNA expression levels of iNOS, COX-2, TNF-α, IL-1β and NF-κB. LPS (1 µg/ml, 30 min-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2, c-Jun N-terminal protein kinase (JNK and p38 mitogen-activated protein kinase (p38 MAPK and this was inhibited by pretreatment of BV-2 cells with different concentrations of gastrodin (30, 40, and 60 µM. In addition, gastrodin blocked LPS-induced phosphorylation of inhibitor κB-α (IκB-α (and hence the activation of NF-κB and of CREB, respectively.This study indicates that gastrodin significantly attenuate levels of neurotoxic proinflammatory mediators and proinflammatory cytokines by inhibition of the NF-κB signaling pathway and phosphorylation of MAPKs in LPS

  1. Glycogen synthase kinase-3 inhibitors suppress the AR-V7-mediated transcription and selectively inhibit cell growth in AR-V7-positive prostate cancer cells.

    Science.gov (United States)

    Nakata, Daisuke; Koyama, Ryokichi; Nakayama, Kazuhide; Kitazawa, Satoshi; Watanabe, Tatsuya; Hara, Takahito

    2017-06-01

    Recent evidence suggests that androgen receptor (AR) splice variants, including AR-V7, play a pivotal role in resistance to androgen blockade in prostate cancer treatment. The development of new therapeutic agents that can suppress the transcriptional activities of AR splice variants has been anticipated as the next generation treatment of castration-resistant prostate cancer. High-throughput screening of AR-V7 signaling inhibitors was performed using an AR-V7 reporter system. The effects of a glycogen synthase kinase-3 (GSK3) inhibitor, LY-2090314, on endogenous AR-V7 signaling were evaluated in an AR-V7-positive cell line, JDCaP-hr, by quantitative reverse transcription polymerase chain reaction. The relationship between AR-V7 signaling and β-catenin signaling was assessed using RNA interference. The effect of LY-2090314 on cell growth in various prostate cancer cell lines was also evaluated. We identified GSK3 inhibitors as transcriptional suppressors of AR-V7 using a high-throughput screen with an AR-V7 reporter system. LY-2090314 suppressed the reporter activity and endogenous AR-V7 activity in JDCaP-hr cells. Because silencing of β-catenin partly rescued the suppression, it was evident that the suppression was mediated, at least partially, via the activation of β-catenin signaling. AR-V7 signaling and β-catenin signaling reciprocally regulate each other in JDCaP-hr cells, and therefore, GSK3 inhibition can repress AR-V7 transcriptional activity by accumulating intracellular β-catenin. Notably, LY-2090314 selectively inhibited the growth of AR-V7-positive prostate cancer cells in vitro. Our findings demonstrate the potential of GSK3 inhibitors in treating advanced prostate cancer driven by AR splice variants. In vivo evaluation of AR splice variant-positive prostate cancer models will help illustrate the overall significance of GSK3 inhibitors in treating prostate cancer. © 2017 Wiley Periodicals, Inc.

  2. Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers.

    OpenAIRE

    Landschütze, V; Willmitzer, L; Müller-Röber, B

    1995-01-01

    The tricarboxylic acid (TCA) cycle constitutes a major component of the mitochondrial metabolism of eucaryotes, including higher plants. To analyze the importance of this pathway, we down-regulated mitochondrial citrate synthase (mCS; EC 4.1.3.7), the first enzyme of the TCA cycle, in transgenic potato plants using an antisense RNA approach. Several transformants were identified with reduced citrate synthase activity (down to approximately 6% of wild-type activity). These plants were indistin...

  3. Nitric oxide synthase (NOS inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

    Directory of Open Access Journals (Sweden)

    Jon Romero-Aguirregomezcorta

    Full Text Available Nitric oxide (NO is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME, NG-monomethyl-L-arginine (L-NMMA and aminoguanidine (AG. A NO donor, S-nitrosoglutathione (GSNO, was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt and, finally, on in vitro fertilization (IVF parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05. Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

  4. Nitric oxide synthase in ferret brain: localization and characterization.

    Science.gov (United States)

    Matsumoto, T.; Mitchell, J. A.; Schmidt, H. H.; Kohlhaas, K. L.; Warner, T. D.; Förstermann, U.; Murad, F.

    1992-01-01

    1. In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide synthase was determined biochemically and immunochemically. 2. In the rat brain, the highest nitric oxide synthase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it was dependent on NADPH and Ca2+, and inhibited by NG-nitro-L-arginine or NG-methyl-L-arginine. 3. Western blot analysis revealed that all regions of the ferret brain contained a 160 kD protein crossreacting with an antibody to nitric oxide synthase purified from the rat cerebellum, and the levels of relative intensity of staining by the antibody correlated with the distribution of nitric oxide synthase activity. 4. These results indicate that the ferret brain contains a nitric oxide synthase similar to the rat brain, but the distribution of enzymatic activity in the ferret brain differs markedly from the rat brain. Images Figure 1 PMID:1282076

  5. Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclo-oxygenase-2 expression in RAW 264.7 macrophages through the inactivation of NF-κB

    Science.gov (United States)

    Whan Han, Jeung; Gon Lee, Byeong; Kee Kim, Yong; Woo Yoon, Jong; Kyoung Jin, Hye; Hong, Sungyoul; Young Lee, Hoi; Ro Lee, Kang; Woo Lee, Hyang

    2001-01-01

    We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica.iNOS activity in cell-free extract of LPS/IFN-γ-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-γ-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS.Ergolide markedly decreased the production of prostaglandin E2 (PGE2) in cell-free extract of LPS/IFN-γ-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself.Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-γ in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively.Ergolide inhibited nuclear factor-κB (NF-κB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-γ. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-κB as well as IκB-α degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-κB itself or an upstream molecule of NF-κB.Ergolide also directly inhibited the DNA-binding activity of active NF-κB in LPS/IFN-γ-pretreated RAW 264.7 macrophages.These results demonstrate that the suppression of NF-κB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-κB resulted from blockade of the degradation of IκB and the direct modification of active NF-κB, leading to the suppression of the expression of iNOS and COX-2, which play important

  6. Ergolide, sesquiterpene lactone from Inula britannica, inhibits inducible nitric oxide synthase and cyclo-oxygenase-2 expression in RAW 264.7 macrophages through the inactivation of NF-kappaB.

    Science.gov (United States)

    Whan Han, J; Gon Lee, B; Kee Kim, Y; Woo Yoon, J; Kyoung Jin, H; Hong, S; Young Lee, H; Ro Lee, K; Woo Lee, H

    2001-06-01

    We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E(2) (PGE(2)) in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of IkappaB and the direct modification of active NF-kappaB, leading to the

  7. [Gly14]-Humanin offers neuroprotection through glycogen synthase kinase-3β inhibition in a mouse model of intracerebral hemorrhage.

    Science.gov (United States)

    Wang, Tao; Huang, Ya; Zhang, Mingyang; Wang, Long; Wang, Yaoqi; Zhang, Lu; Dong, Wenwen; Chang, Pan; Wang, Zufeng; Chen, Xiping; Tao, Luyang

    2013-06-15

    Perihematomal brain edema formation and consequent cell death contribute to second brain injury resulting in severe neurological deficits and sometimes delayed fatality after intracerebral hemorrhage (ICH). [Gly14]-Humanin (HNG), a variant of Humanin (HN) in which the 14th amino acid serine is replaced with glycine, reduced Alzheimer's disease-relevant insults and improved neurological deficits in an ischemia stroke model. In the study, we aimed to evaluate whether HNG posttreatment attenuated early brain injury after ICH and whether the protective effect was associated with regulation of apoptosis via phosphatidylinositol 3-kinase (PI3K)-Akt/GSK-3β signaling. Male ICR mice were subjected to infusion of Type IV collagenase (to induce ICH) of saline (for shams) into the left striatum. ICH animals received vehicle, HNG (1 or 2.5 μg in 100 μl saline) administration intraperitoneally 1h post injury. Compared with vehicle, HNG-2.5 μg treatment improved neurological outcome and reduced brain edema at 24 and 72 h after surgery (P<0.05), but wortmannin (15 μg/kg, 90 min before HNG-2.5 μg, intravenously) obliterated the effect. HNG-2.5 μg also reduced cell insults and injury volume at 24 and 72 h after surgery (P<0.05, vs. vehicle). Furthermore, HNG-2.5 μg treatment increased p-Akt and Bcl-2 and decreased p-GSK-3β, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase expressions in the ipsilateral hemisphere (P<0.05, vs. vehicle), however, the effect was reversed by wortmannin. In conclusion, HNG treatment improved functional and morphological outcomes after experimental ICH in mice and the protective effect was associated with suppressing apoptosis through PI3K-Akt/GSK-3β signaling pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. 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.

  9. Studying the mechanism that enables paullones to selectively inhibit glycogen synthase kinase 3 rather than cyclin-dependent kinase 5 by molecular dynamics simulations and free-energy calculations.

    Science.gov (United States)

    Chen, Quan; Cui, Wei; Cheng, Yuanhua; Zhang, Fushi; Ji, Mingjuan

    2011-04-01

    Glycogen synthase kinase 3 (GSK-3) is an attractive target for the treatment of diabetes, and paullones have been reported to be effective inhibitors of GSK-3. However, it is still a challenging task to improve selectivity among protein kinases, especially cyclin-dependent kinases (CDKs). Here we investigated the mechanism that enables paullones to selectively inhibit GSK-3 rather than cyclin-dependent kinase 5 (CDK5) using sequence alignment, molecular dynamics simulations, free-energy calculations and free-energy decomposition analysis. The results indicate that the interaction between paullones and Val135 of GSK-3 is obviously stronger than that between paullones and Cys83 of CDK5, suggesting that paullones could be utilized as potent selective inhibitors. Meanwhile, we observed that the decrease in the interaction between paullones and the Asp86 of CDK5 favors their selectivity towards GSK-3 rather than CDK5, as demonstrated using 1-azakenpaullone as an example. Although substitution at position 9 and replacement at position 2 may influence the activity of GSK-3, they only have a minor effect on the selectivity. We expect that the information obtained here could prove useful for developing specific paullone inhibitors of GSK-3.

  10. Algal growth inhibition test results of 425 organic chemical substances

    DEFF Research Database (Denmark)

    Kusk, Kresten Ole; Christensen, Anne Munch; Nyholm, Niels

    2018-01-01

    values were corrected accordingly. The model helped to identify substances, where the calculated water concentration was too uncertain. Substances covering a wide range of physical-chemical properties and different modes of action were tested. Median effect concentrations (EC50) lower than 1000 mg/L were......The toxicity towards the algal species Pseudokirchneriella subcapitata of 425 organic chemical substances was tested in a growth inhibition test. Precautions were taken to prevent loss of the compounds from the water phase and the test system (closed test system, low biomass, shorter test duration......, silanized glass) and to keep pH constant by applying a higher alkalinity. Chemical phase distribution was modelled taking ionization, volatilisation, and adsorption to glass and biomass into consideration. If the modelled water concentration was below 90% of the nominal concentration the calculated EC...

  11. Diverse mechanisms of growth inhibition by luteolin, resveratrol, and quercetin in MIA PaCa-2 cells: a comparative glucose tracer study with the fatty acid synthase inhibitor C75.

    Science.gov (United States)

    Harris, Diane M; Li, Luyi; Chen, Monica; Lagunero, F Tracy; Go, Vay Liang W; Boros, Laszlo G

    2012-04-01

    The rationale of this dose matching/dose escalating study was to compare a panel of flavonoids-luteolin, resveratrol, and quercetin-against the metabolite flux-controlling properties of a synthetic targeted fatty acid synthase inhibitor drug C75 on multiple macromolecule synthesis pathways in pancreatic tumor cells using [1,2-(13)C(2)]-d-glucose as the single precursor metabolic tracer. MIA PaCa-2 pancreatic adenocarcinoma cells were cultured for 48 h in the presence of 0.1% DMSO (control), or 50 or 100 μM of each test compound, while intracellular glycogen, RNA ribose, palmitate and cholesterol as well as extra cellular (13)CO(2), lactate and glutamate production patterns were measured using gas chromatography/mass spectrometry (GC/MS) and stable isotope-based dynamic metabolic profiling (SiDMAP). The use of 50% [1,2-(13)C(2)]-d-glucose as tracer resulted in an average of 24 excess (13)CO(2) molecules for each 1,000 CO(2) molecule in the culture media, which was decreased by 29 and 33% (P tracer glucose-derived (13)C-labeled lactate fractions (Σm) were between 45.52 and 47.49% in all cultures with a molar ratio of 2.47% M + 1/Σm lactate produced indirectly by direct oxidation of glucose in the pentose cycle in control cultures; treatment with 100 μM C75 and luteolin decreased this figure to 1.80 and 1.67%. The tracer glucose-derived (13)C labeled fraction (Σm) of ribonucleotide ribose was 34.73% in controls, which was decreased to 20.58 and 8.45% with C75, 16.15 and 6.86% with luteolin, 27.66 and 19.25% with resveratrol, and 30.09 and 25.67% with quercetin, respectively. Luteolin effectively decreased nucleotide precursor synthesis pentose cycle flux primarily via the oxidative branch, where we observed a 41.74% flux (M + 1/Σm) in control cells, in comparison with only a 37.19%, 32.74%, or a 26.57%, 25.47% M + 1/Σm flux (P tracer glucose-derived (13)C-labeled fractions, respectively. On the other hand there was a significant 192 and 159% (P tracer glucose

  12. Endogenous post-transcriptional gene silencing of flavone synthase resulting in high accumulation of anthocyanins in black dahlia cultivars.

    Science.gov (United States)

    Deguchi, Ayumi; Ohno, Sho; Hosokawa, Munetaka; Tatsuzawa, Fumi; Doi, Motoaki

    2013-05-01

    Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar 'Kokucho' and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping.

  13. Evidence that the "waxy" protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase.

    Science.gov (United States)

    Smith, A M

    1990-11-01

    The aim of this work was to identify the starch-granule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with α-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of thewaxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starch-synthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between "waxy" proteins and starch-granule-bound starch synthases generally.

  14. Plasmodium falciparum spermidine synthase inhibition results in unique perturbation-specific effects observed on transcript, protein and metabolite levels

    CSIR Research Space (South Africa)

    Becker, JVW

    2010-01-01

    Full Text Available for the homeostatic control of AdoMet levels in Plasmodia is the downregulation of its synthesis, effec- tively by downregulating the synthesis of AdoMet syn- thetase [39]. This downregulation was also observed on both transcript and protein levels in this study.... Similar amounts of labelled probe (typically 100 picomoles) were hybridized to the microarray slides. Slides were prehybridized with a solution containing 5× SSC (from 20× SSC: 3 M NaCl, 1.5 M sodium citrate, pH 7.0), 0.1% SDS and 0.1 mg/mL BSA...

  15. Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers.

    Science.gov (United States)

    Landschütze, V; Willmitzer, L; Müller-Röber, B

    1995-02-15

    The tricarboxylic acid (TCA) cycle constitutes a major component of the mitochondrial metabolism of eucaryotes, including higher plants. To analyze the importance of this pathway, we down-regulated mitochondrial citrate synthase (mCS; EC 4.1.3.7), the first enzyme of the TCA cycle, in transgenic potato plants using an antisense RNA approach. Several transformants were identified with reduced citrate synthase activity (down to approximately 6% of wild-type activity). These plants were indistinguishable from wild-type plants in the greenhouse during vegetative growth. A major change, however, was seen upon initiation of the generative phase (flower formation). In the case of transgenic plants with a strong reduction in citrate synthase activity ( 2 weeks later as compared with wild-type plants. Furthermore, flower buds from these plants did not develop into mature flowers but rather were aborted at an early stage of development. Microscopic analysis showed that in these cases ovaries disintegrated during flower development. We conclude that the TCA cycle is of major importance during the transition from the vegetative to the generative phase.

  16. Pharmacological blockade of fatty acid synthase (FASN) reverses acquired autoresistance to trastuzumab (Herceptin by transcriptionally inhibiting 'HER2 super-expression' occurring in high-dose trastuzumab-conditioned SKBR3/Tzb100 breast cancer cells.

    Science.gov (United States)

    Vazquez-Martin, Alejandro; Colomer, Ramon; Brunet, Joan; Menendez, Javier A

    2007-10-01

    Elucidating the mechanisms underlying resistance to the human epidermal growth factor receptor 2 (HER2)-targeted antibody trastuzumab (Tzb; Herceptin) is a major challenge that is beginning to be addressed. This dilemma is becoming increasingly important as recent studies strongly support a role for Tzb in the adjuvant setting for HER2-overexpressing early-stage breast cancers. We previously reported that pharmacological and RNA interference-induced inhibition of tumor-associated fatty acid synthase (FASN; Oncogenic antigen-519), a key metabolic enzyme catalyzing the synthesis of long-chain saturated fatty acids, drastically down-regulates HER2 expression in human breast cancer cells bearing HER2 gene amplification. Given that FASN blockade was found to suppress HER2 overexpression by attenuating the promoter activity of the HER2 gene, we here envisioned that this mechanism of action may represent a valuable strategy in breast cancers that have progressed while under Tzb. We created a preclinical model of Tzb resistance by continuously growing HER2-overexpressing SKBR3 breast cancer cells in the presence of clinically relevant concentrations of Tzb (20-185 microg/ml Tzb). This pool of Tzb-conditioned SKBR3 cells, which optimally grows now in the presence of 100 microg/ml trastuzumab (SKBR3/Tzb100 cells), exhibited HER2 levels notably higher (approximately 2-fold) than those found in SKBR3 parental cells. Real-time polymerase chain reaction studies showed that up-regulation of HER2 mRNA levels closely correlated with HER2 protein up-regulation in SKBR3/Tzb100 cells, thus suggesting that 'HER2 super-expression' upon acquisition of autoresistance to Tzb resulted, at least in part, from up-regulatory effects in the transcriptional rate of the HER2 gene. SKBR3/Tzb100 cells did not exhibit cross-resistance to C75, a small-compound specifically inhibiting FASN activity. On the contrary, SKBR3/Tzb100 cells showed a remarkably increased sensitivity (approximately 3-fold) to

  17. Inhibition of inducible nitric oxide synthase expression and nitric oxide production in plateau pika (Ochotona curzoniae) at high altitude on Qinghai-Tibet Plateau.

    Science.gov (United States)

    Xie, Ling; Zhang, Xuze; Qi, Delin; Guo, Xinyi; Pang, Bo; Du, Yurong; Zou, Xiaoyan; Guo, Songchang; Zhao, Xinquan

    2014-04-30

    Nitric oxide (NO), a potent vasodilator, plays an important role in preventing hypoxia induced pulmonary hypertension. Endogenous NO is synthesized by nitric oxide synthases (NOSs) from l-arginine. In mammals, three different NOSs have been identified, including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Plateau pika (Ochotona curzoniae) is a typical hypoxia tolerant mammal that lives at 3000-5000 m above sea level on the Qinghai-Tibet Plateau. The aim of this study was to investigate whether NOS expression and NO production are regulated by chronic hypoxia in plateau pika. Quantitative real-time PCR and western blot analyses were conducted to quantify relative abundances of iNOS and eNOS transcripts and proteins in the lung tissues of plateau pikas at different altitudes (4550, 3950 and 3200 m). Plasma NO metabolites, nitrite/nitrate (NO(x)⁻) levels were also examined by Ion chromatography to determine the correlation between NO production and altitude level. The results revealed that iNOS transcript levels were significantly lower in animals at high altitudes (decreased by 53% and 57% at altitude of 3950 and 4550 m compared with that at 3200 m). Similar trends in iNOS protein abundances were observed (26% and 41% at 3950 and 4550 m comparing with at 3200 m). There were no significant differences in eNOS mRNA and protein levels in the pika lungs among different altitudes. The plasma NO(x)⁻ levels of the plateau pikas at high altitudes significantly decreased (1.65±0.19 μg/mL at 3200 m to 0.44±0.03 μg/mL at 3950 m and 0.24±0.01 μg/mL at 4550 m). This is the first evidence describing the effects of chronic hypoxia on NOS expression and NO levels in the plateau pika in high altitude adaptation. We conclude that iNOS expression and NO production are suppressed at high altitudes, and the lower NO concentration at high altitudes may serve crucial roles for helping the plateau pika to survive at hypoxic environment. Copyright © 2014

  18. Inhibition of melanosome transfer results in skin lightening.

    Science.gov (United States)

    Seiberg, M; Paine, C; Sharlow, E; Andrade-Gordon, P; Costanzo, M; Eisinger, M; Shapiro, S S

    2000-08-01

    The chemical basis of melanogenesis is well documented, but the mechanism of melanosome transfer and the regulation of pigmentation by keratinocyte-melanocyte interactions are not well understood. Therefore we examined the effects of serine protease inhibitors on skin pigmentation and found that the protease-activated receptor 2, expressed on keratinocytes, may regulate pigmentation via keratinocyte-melanocyte interactions. Here we show that modulation of protease-activated receptor 2 activation affects melanosome transfer into keratinocytes, resulting in changes in pigment production and deposition. SLIGRL, the protease-activated receptor 2 activating peptide, enhanced melanosome ingestion by keratinocytes, thus increasing pigment deposition. RWJ-50353, a serine protease inhibitor, led to reduced pigment deposition in melanocytes and depigmentation. Electron microscopy studies illustrated an accumulation of immature melanosomes inside melanocytes and abnormal dendrite dynamics in RWJ-50353-treated epidermal equivalents. RWJ-50353 induced a visible and dose-dependent skin lightening effect in the dark-skinned Yucatan swine. Examinations by electron microscopy indicated that the in vivo transfer of melanosomes from melanocytes to keratinocytes was affected. Our data suggest that modulation of keratinocyte-melanocyte interactions via the protease-activated receptor 2 pathway affects melanosome transfer. The use of RWJ-50353 to modulate protease-activated receptor 2 activation could lead to a new class of depigmenting agents.

  19. The activity of inducible nitric oxide synthase in rejected skin xenografts is selectively inhibited by a factor produced by grafted cells

    Czech Academy of Sciences Publication Activity Database

    Holáň, Vladimír; Pindjáková, Jana; Zajícová, Alena; Krulová, Magdalena; Železná, Blanka; Matoušek, Petr; Svoboda, Petr

    2005-01-01

    Roč. 12, č. 3 (2005), s. 227-234 ISSN 0908-665X R&D Projects: GA MZd(CZ) NR7816; GA ČR(CZ) GP310/02/D162; GA ČR(CZ) GD310/03/H147; GA MŠk(CZ) ME 300; GA AV ČR KSK5020115 Institutional research plan: CEZ:AV0Z5052915; CEZ:AV0Z50110509 Keywords : inducible nitric oxide synthase production * nitric oxide * suppressive molecule Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.114, year: 2005

  20. Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3{beta}

    Energy Technology Data Exchange (ETDEWEB)

    Zeadin, Melec G.; Butcher, Martin K.; Shaughnessy, Stephen G. [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada); Werstuck, Geoff H., E-mail: Geoff.Werstuck@taari.ca [Department of Medicine, McMaster University, Hamilton, ON (Canada); Thrombosis and Atherosclerosis Research Institute, Hamilton, ON (Canada)

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Leptin promotes osteoblast differentiation of primary smooth muscle cells. Black-Right-Pointing-Pointer Leptin regulates the expression of genes involved in osteoblast differentiation. Black-Right-Pointing-Pointer Constitutively active GSK-3{beta} attenuates leptin-induced osteoblast differentiation. Black-Right-Pointing-Pointer This suggests that leptin signals through GSK-3{beta} to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 {mu}g/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3{beta} on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of {beta}-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3{beta} (Ad-GSK-3{beta} S9A) resulted in a >2-fold increase in GSK-3{beta} activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3{beta} activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.

  1. 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....

  2. Activation of Nrf2/HO-1 Pathway by Glycogen Synthase Kinase-3β Inhibition Attenuates Renal Ischemia/Reperfusion Injury in Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Xiaohua Shen

    2017-06-01

    Full Text Available Background/Aims: Diabetes mellitus can exacerbate renal ischemia-reperfusion (I/R injury (RI/RI. The aim of the present study was to evaluate the protective effect of GSK-3β inhibition (TDZD-8 on I/R-induced renal injury through the Nrf2/HO-1 pathway in a streptozocin (STZ-induced diabetic rat model. Methods: STZ-induced diabetic rats preconditioned with TDZD-8 and ZnPP were subjected to renal I/R. The extent of renal morphologic lesions. Renal function was assessed from blood urea nitrogen (BUN and serum creatinine (Scr, as determined utlizing commercial kits. Oxidative stress and inflammatory activity in the kidney tissue was estimated from levels of malondialdehyde (MDA, interleukin-10 (IL-10, tumor necrosis factor-α (TNF-α, and nitric oxide (NO, as well as the activities of superoxide dismutase (SOD and glutathione (GSH using qRT-PCR and ELISA. The expressions of Nrf2, HO-1, Bcl-2 and NF-κB in the renal tissue were measured by qRT-PCR and western blotting. Results: I/R-induced renal inflammation was reduced significantly by TDZD-8 pretreatment. Preconditioning with TDZD-8 suppressed NF-κB expression and enhanced Bcl-2 expression in the renal tissue. The upregulated level of malondialdehyde (MDA, and reduced activities of superoxide dismutase (SOD and glutathione (GSH in I/R-shocked rats were markedly restored by TDZD-8 pretreatment. Furthermore, pretreatment with TDZD-8 enhanced activation of the Nrf2/HO-1 pathway in the renal tissue of diabetic RI/RI rats. Conclusion: These findings suggest that preconditioning with TDZD-8 may protect the kidney from I/R-induced damage via the activation of the Nrf2/HO-1 pathway in STZ-induced diabetic rats. Further detailed studies are needed to further clarify the underlying mechanisms.

  3. Expression of Clarkia S-linalool synthase in transgenic petunia plant results in the accumulation of S-linalyl-b-D-glucopyranoside

    NARCIS (Netherlands)

    Lücker, J.; Bouwmeester, H.J.; Schwab, W.; Blaas, J.; Plas, van der L.H.W.; Verhoeven, H.A.

    2001-01-01

    Petunia hybrida W115 was transformed with a Clarkia breweri S-linalool synthase cDNA (lis). Lis was expressed in all tissues analysed, and linalool was detected in leaves, sepals, corolla, stem and ovary, but not in nectaries, roots, pollen and style. However, the S-linalool produced by the plant in

  4. A transcriptomic analysis for identifying the unintended effects of introducing a heterologous glyphosate-tolerant EPSP synthase into Escherichia coli.

    Science.gov (United States)

    Li, Liang; Zhou, Zhengfu; Jin, Wujun; Wan, Yusong; Lu, Wei

    2015-03-01

    Glyphosate is one of the most commonly used broad-spectrum herbicides with little to no hazard to animals, human beings, or the environment. Some microbial 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase variants are not inhibited by glyphosate, and they provide a powerful tool to engineer glyphosate-tolerant plants. However, the unintended effects of EPSP synthase expression patterns on microbes are not yet clear. Here, we use an Affymetrix GeneChip analysis to study how introduction of a heterologous glyphosate-tolerant EPSP synthase into a model microorganism Escherichia coli (E. coli) affects the global gene expression profile. The profile showed that 161 of 4071 genes were differentially expressed after the introduction of the synthase: 19 (0.47%) were up-regulated and 143 (3.49%) were down-regulated. The microarray results, in combination with BiOLOG substrate utilization and amino acid composition assays, suggested that heterologous EPSP synthase expression had very minor effects on E. coli. Although a small number of genes and metabolites were affected by EPSP synthase expression, no functional correlations were identified among the dataset. This study may shed light on the effect of EPSP synthase expression on microbes, which should help in the assessment of environmental safety.

  5. Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia.

    Science.gov (United States)

    Ohno, Sho; Hosokawa, Munetaka; Kojima, Misa; Kitamura, Yoshikuni; Hoshino, Atsushi; Tatsuzawa, Fumi; Doi, Motoaki; Yazawa, Susumu

    2011-11-01

    Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar 'Yuino' and the two seedling cultivars 'OriW1' and 'OriW2' borne from a red-white bicolor cultivar, 'Orihime', indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in 'Yuino' petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.

  6. U-Bang-Haequi Tang: A Herbal Prescription that Prevents Acute Inflammation through Inhibition of NF-κB-Mediated Inducible Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Min Hwangbo

    2014-01-01

    Full Text Available Since antiquity, medical herbs have been prescribed for both treatment and preventative purposes. Herbal formulas are used to reduce toxicity as well as increase efficacy in traditional Korean medicine. U-bang-haequi tang (UBT is a herbal prescription containing Arctii fructus and Forsythia suspensa as its main components and has treated many human diseases in traditional Korean medicine. This research investigated the effects of UBT against an acute phase of inflammation. For this, we measured induction of nitric oxide (NO and related proteins in macrophage cell line stimulated by lipopolysaccharide (LPS. Further, paw swelling was measured in carrageenan-treated rats. Carrageenan significantly induced activation of inflammatory cells and increases in paw volume, whereas oral administration of 0.3 or 1 g/kg/day of UBT inhibited the acute inflammatory response. In RAW264.7 cells, UBT inhibited mRNA and protein expression levels of iNOS. UBT treatment also blocked elevation of NO production, nuclear translocation of NF-κB, phosphorylation of Iκ-Bα induced by LPS. Moreover, UBT treatment significantly blocked the phosphorylation of p38 and c-Jun NH2-terminal kinases by LPS. In conclusion, UBT prevented both acute inflammation in rats as well as LPS-induced NO and iNOS gene expression through inhibition of NF-κB in RAW264.7 cells.

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

    Science.gov (United States)

    Lather, Amit; Khatkar, Anurag; Sharma, Sunil

    2018-03-29

    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

  8. Hypouricaemic action of mangiferin results from metabolite norathyriol via inhibiting xanthine oxidase activity.

    Science.gov (United States)

    Niu, Yanfen; Liu, Jia; Liu, Hai-Yang; Gao, Li-Hui; Feng, Guo-Hua; Liu, Xu; Li, Ling

    2016-09-01

    Context Mangiferin has been reported to possess a potential hypouricaemic effect. However, the pharmacokinetic studies in rats showed that its oral bioavailability was only 1.2%, suggesting that mangiferin metabolites might exert the action. Objective The hypouricaemic effect and the xanthine oxidase inhibition of mangiferin and norathyriol, a mangiferin metabolite, were investigated. Inhibition of norathyriol analogues (compounds 3-9) toward xanthine oxidase was also evaluated. Materials and methods For a dose-dependent study, mangiferin (1.5-6.0 mg/kg) and norathyriol (0.92-3.7 mg/kg) were administered intragastrically to mice twice daily for five times. For a time-course study, mice received mangiferin and norathyriol both at a single dose of 7.1 μmol/kg. In vitro, inhibition of test compounds (2.4-2.4 mM) against xanthine oxidase activity was evaluated by the spectrophotometrical method. The inhibition type was identified from Lineweaver-Burk plots. Results Norathyriol (0.92, 1.85 and 3.7 mg/kg) dose dependently decreased the serum urate levels by 27.0, 33.6 and 37.4%, respectively. The action was more potent than that of mangiferin at the low dose, but was equivalent at the higher doses. Additionally, the hypouricaemic action of them exhibited a time dependence. In vitro, norathyriol markedly inhibited the xanthine oxidase activities, with the IC50 value of 44.6 μM, but mangiferin did not. The kinetic studies showed that norathyriol was an uncompetitive inhibitor by Lineweaver-Burk plots. The structure-activity relationships exhibited that three hydroxyl groups in norathyriol at the C-1, C-3 and C-6 positions were essential for maintaining xanthine oxidase inhibition. Discussion and conclusion Norathyriol was responsible for the hypouricaemic effect of mangiferin via inhibiting xanthine oxidase activity.

  9. Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase

    Directory of Open Access Journals (Sweden)

    Gila Idelman

    2015-08-01

    Full Text Available It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4 signaling. LPS-stimulated iNOS and NADPH oxidase (Nox activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide (O2− production, respectively. The generation of both nitrate and O2− in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD and bilirubin effectively abolished LPS-mediated O2− production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1α (HIF-1α, an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1α-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1α mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-β, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-β release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin.

  10. Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase.

    Science.gov (United States)

    Idelman, Gila; Smith, Darcey L H; Zucker, Stephen D

    2015-08-01

    It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4) signaling. LPS-stimulated iNOS and NADPH oxidase (Nox) activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide ( [Formula: see text] ) production, respectively. The generation of both nitrate and [Formula: see text] in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD) and bilirubin effectively abolished LPS-mediated [Formula: see text] production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1α (HIF-1α), an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1α-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1α mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-β, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-β release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin. Copyright © 2015. Published by Elsevier B.V.

  11. Endothelial nitric oxide synthase-enhancing G-protein coupled receptor antagonist inhibits pulmonary artery hypertension by endothelin-1-dependent and endothelin-1-independent pathways in a monocrotaline model

    Directory of Open Access Journals (Sweden)

    Chung-Pin Liu

    2014-06-01

    Full Text Available This study investigates whether endothelin-1 (ET-1 mediates monocrotaline (MCT-induced pulmonary artery hypertension (PAH and right ventricular hypertrophy (RVH, and if so, whether the G-protein coupled receptor antagonist KMUP-1 (7-{2-[4-(2-chlorobenzenepiperazinyl]ethyl}-1,3-dimethylxanthine inhibits ET-1-mediated PA constriction and the aforementioned pathological changes. In a chronic rat model, intraperitoneal MCT (60 mg/kg induced PAH and increased PA medial wall thickening and RV/left ventricle + septum weight ratio on Day 21 after MCT injection. Treatment with sublingual KMUP-1 (2.5 mg/kg/day for 21 days prevented these changes and restored vascular endothelial nitric oxide synthase (eNOS immunohistochemical staining of lung tissues. Western blotting analysis demonstrated that KMUP-1 enhanced eNOS, soluble guanylate cyclase, and protein kinase G levels, and reduced ET-1 expression and inactivated Rho kinase II (ROCKII in MCT-treated lung tissue over long-term administration. In MCT-treated rats, KMUP-1 decreased plasma ET-1 on Day 21. KMUP-1 (3.6 mg/kg maximally appeared at 0.25 hours in the plasma and declined to basal levels within 24 hours after sublingual administration. In isolated PA of MCT-treated rats, compared with control and pretreatment with l-NG-nitroarginine methyl ester (100 μM, KMUP-1 (0.1–100 μM inhibited ET-1 (0.01 μM-induced vasoconstriction. Endothelium-denuded PA sustained higher contractility in the presence of KMUP-1. In a 24-hour culture of smooth muscle cells (i.e., PA smooth muscle cells or PASMCs, KMUP-1 (0.1–10 μM inhibited RhoA- and ET-1-induced RhoA activation. KMUP-1 prevented MCT-induced PAH, PA wall thickening, and RVH by enhancing eNOS and suppressing ET-1/ROCKII expression. In vitro, KMUP-1 inhibited ET-1-induced PA constriction and ET-1-dependent/independent RhoA activation of PASMCs. In summary, KMUP-1 attenuates ET-1-induced/ET-1-mediated PA constriction, and could thus aid in

  12. Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate.

    Science.gov (United States)

    Burke, Charles; Croteau, Rodney

    2002-02-01

    Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C(5) units, of the respective C(10), C(15), and C(20) precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56-75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C(10) chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.

  13. 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.

  14. Ablation of periostin inhibits post-infarction myocardial regeneration in neonatal mice mediated by the phosphatidylinositol 3 kinase/glycogen synthase kinase 3β/cyclin D1 signalling pathway.

    Science.gov (United States)

    Chen, Zhenhuan; Xie, Jiahe; Hao, Huixin; Lin, Hairuo; Wang, Long; Zhang, Yingxue; Chen, Lin; Cao, Shiping; Huang, Xiaobo; Liao, Wangjun; Bin, Jianping; Liao, Yulin

    2017-05-01

    To resolve the controversy as to whether periostin plays a role in myocardial regeneration after myocardial infarction (MI), we created a neonatal mouse model of MI to investigate the influence of periostin ablation on myocardial regeneration and clarify the underlying mechanisms. Neonatal periostin-knockout mice and their wildtype littermates were subjected to MI or sham surgery. In the wildtype mice after MI, fibrosis was detectable at 3 days and fibrotic tissue was completely replaced by regenerated myocardium at 21 days. In contrast, in the knockout mice, significant fibrosis in the infarcted area was present at even 3 weeks after MI. Levels of phosphorylated-histone 3 and aurora B in the myocardium, detected by immunofluorescence and western blotting, were significantly lower in knockout than in wildtype mice at 7 days after MI. Similarly, angiogenesis was decreased in the knockout mice after MI. Expression of both the endothelial marker CD-31 and α-smooth muscle actin was markedly lower in the knockout than in wildtype mice at 7 days after MI. The knockout MI group had elevated levels of glycogen synthase kinase (GSK) 3β and decreased phosphatidylinositol 3-kinase (PI3K), phosphorylated serine/threonine protein kinase B (p-Akt), and cyclin D1, compared with the wildtype MI group. Similar effects were observed in experiments using cultured cardiomyocytes from neonatal wildtype or periostin knockout mice. Administration of SB216763, a GSK3β inhibitor, to knockout neonatal mice decreased myocardial fibrosis and increased angiogenesis in the infarcted area after MI. Ablation of periostin suppresses post-infarction myocardial regeneration by inhibiting the PI3K/GSK3β/cyclin D1 signalling pathway, indicating that periostin is essential for myocardial regeneration. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Cardiology

  15. Inhibition of STAT-3 results in radiosensitization of human squamous cell carcinoma

    International Nuclear Information System (INIS)

    Bonner, James A.; Trummell, Hoa Q.; Willey, Christopher D.; Plants, Brian A.; Raisch, Kevin P.

    2009-01-01

    Background: Signal transducer and activator of transcription-3 (STAT-3) is a downstream component of the Epidermal Growth Factor Receptor (EGFr) signaling process that may facilitate the resistance of tumor cells to conventional cancer treatments. Studies were performed to determine if inhibition of this downstream protein produces radiosensitization. Methods/Results: A431 cells (human squamous cell carcinoma cells with EGFr overexpression) were found to be sensitized to radiation after treatment with STAT-3 small interfering RNA (siRNA). Therefore, a short hairpin RNA (shRNA) against STAT-3 was designed and cloned into a pBABE vector system modified for shRNA expression. Following transfection, clone 2.1 was selected for further study as it showed a dramatic reduction of STAT-3 protein (and mRNA) when compared to A431 parental cells or a negative control shRNA cell line (transfected with STAT-3 shRNA with 2 base pairs mutated). A431 2.1 showed doubling times of 25-31 h as compared to 18-24 h for the parental cell line. The A431 shRNA knockdown STAT-3 cells A431 were more sensitive to radiation than A431 parental or negative STAT-3 control cells. Conclusion: A431 cells stably transfected with shRNA against STAT-3 resulted in enhanced radiosensitivity. Further work will be necessary to determine whether the inhibition of STAT-3 phosphorylation is a necessary step for the radiosensitization that is induced by the inhibition of EGFr.

  16. Ketoconazole Inhibits Ovulation as a Result of Arrest of Follicular Steroidogenesis in the Rat Ovary

    Directory of Open Access Journals (Sweden)

    Michael Gal

    2014-01-01

    Full Text Available Objective Ketoconazole (KCZ is a known inhibitor of steroidogenic P450 enzymes in the adrenal cortex and the gonads. Previous studies examined the potential clinical use of KCZ for attenuation of ovarian response to gonadotropin treatments. This study aimed to use the superovuating rat model to explore the effect of KCZ on ovarian steroidogenesis, follicular function, and development toward ovulation. Methods Prepubertal rats were treated with equine chorionic gonadotropin (eCG/human CG (hCG resulting in multiple follicular development and ovulation. The effect of KCZ on this model was examined by administration of KCZ-gel formula and subsequent analyses of ovarian steroidogenesis, rate of ovulation, morphometric assessments of follicular parameters, and cell-specific steroidogenic maturation of the treated ovaries. Results When applied shortly before gonadotropin stimulation, KCZ markedly reduced ovarian progesterone, androstenedione, and estradiol levels down to 18.7, 36.5, and 19.0%, respectively ( P < 0.001. A single KCZ-gel administration of 6, 12, and 24 mg/rat resulted in reduction of ovulated ova/ovary down to 8.6 ± 4.9, 5.1 ± 4.3, and 2.4 ± 3.2, respectively, as compared to 13.6 ± 4.4 ova found in the oviduct of control-gel-injected animals ( P < 0.001. An alternative protocol made use of small KCZ doses injected in non-gel formula (5 mg/dose/8 hours, commenced with the eCG administration and terminated 24 hours later; this treatment readily inhibited the ovulation rates to 6.6 ± 6.6 as compared to 16.5 ± 4.1 ova/ovary in the control group ( P < 0.01. By contrast, KCZ failed to inhibit ovulation if administered 24 hours after eCG injection. Anovulation by KCZ resulted from arrest of follicular development at the stage of 800-840 μm Graafian follicles as compared to 920 μm of peri-ovulatory follicles (OFs observed in the control group, P = 0.029. In addition, absence of CYP11A1 expression was evident in the granulosa cell layers

  17. Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro.

    Science.gov (United States)

    Wang, Tao Fang; Wang, Heng; Peng, Ai Fen; Luo, Qing Feng; Liu, Zhi Li; Zhou, Rong Ping; Gao, Song; Zhou, Yang; Chen, Wen Zhao

    2013-10-18

    FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the "HER2/PI3K/AKT" axis in vitro. FASN blocker may be a new therapeutic strategy in OS management. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. The inhibition of PARP but not EGFR results in the radiosensitization of HPV/p16-positive HNSCC cell lines

    International Nuclear Information System (INIS)

    Güster, Julian David; Weissleder, Stephanie Valerie; Busch, Chia-Jung; Kriegs, Malte; Petersen, Cordula; Knecht, Rainald; Dikomey, Ekkehard; Rieckmann, Thorsten

    2014-01-01

    Background and purpose: HPV-negative and HPV-positive HNSCC comprise distinct tumor entities with different biological characteristics. Specific regimens for the comparably well curable HPV-positive entity that reduce side effects without compromising outcome have yet to be established. Therefore, we tested here whether the inhibition of EGFR or PARP may be used to specifically enhance the radiosensitivity of HPV-positive HNSCC cells. Materials and methods: Experiments were performed with five HPV/p16-positive HNSCC cell lines. Inhibitors used were cetuximab, olaparib and PF-00477736. The respective inhibition of EGFR, PARP and Chk1 was evaluated by Western blot, immunofluorescence analysis and assessment of cell cycle distribution. Cell survival was assessed by colony formation assay. Results: Inhibition of EGFR by cetuximab failed to radiosensitize any of the HPV-positive HNSCC cell lines tested. In contrast, PARP-inhibition resulted in a substantial radiosensitization of all strains, with the sensitization being further enhanced by the additional inhibition of Chk1. Conclusions: PARP-inhibition effectively radiosensitizes HPV-positive HNSCC cells and may therefore represent a viable alternative to chemotherapy possibly even allowing for a reduction in radiation dose. For the latter, PARP-inhibition may be combined with the inhibition of Chk1. In contrast, the inhibition of EGFR cannot be expected to radiosensitize HPV-positive HNSCC through the modulation of cellular radiosensitivity

  19. Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Tao Fang; Wang, Heng [Department of Orthopedics, First Affiliated Hospital of Nanchang University, Jiangxi (China); Peng, Ai Fen [Jiangxi University of Traditional Chinese Medicine, Jiangxi (China); Luo, Qing Feng [Department of Pathology, Cancer Hospital of Jiangxi Province, Jiangxi (China); Liu, Zhi Li, E-mail: zgm7977@163.com [Department of Orthopedics, First Affiliated Hospital of Nanchang University, Jiangxi (China); Zhou, Rong Ping [Department of Orthopedics, Second Affiliated Hospital of Nanchang University, Jiangxi (China); Gao, Song; Zhou, Yang; Chen, Wen Zhao [Department of Orthopedics, First Affiliated Hospital of Nanchang University, Jiangxi (China)

    2013-10-18

    Highlights: •We investigate the relationship between FASN and HER2 or p-HER2 by IHC in OS tissues. •We construct FASN-specific RNAi plasmid. •Inhibiting FASN down-regulates HER2/PI3K/AKT cell signaling in U-2 OS. •Inhibiting FASN blocks U-2 OS cell invasion and migration. -- Abstract: FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the “HER2/PI3K/AKT” axis in vitro. FASN blocker may be a new therapeutic strategy in OS management.

  20. Functional plasticity of paralogous diterpene synthases involved in conifer defense

    OpenAIRE

    Keeling, Christopher I.; Weisshaar, Sabrina; Lin, Roy P. C.; Bohlmann, Jörg

    2008-01-01

    The diversity of terpenoid compounds produced by plants plays an important role in mediating various plant–herbivore, plant–pollinator, and plant–pathogen interactions. This diversity has resulted from gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes. Two diterpene synthases in Norway spruce (Picea abies), isopimaradiene synthase and levopimaradiene/abietadiene synthase, provide the hydrocarbon precursors for most of the diterpene resin...

  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...... in the sarcolemma as well as the cytoplasm of type I muscle fibres. NADPH diaphorase activity confirmed a higher level of NO synthase activity in the sarcolemma as well as the cytoplasm of type I muscle fibers. Histochemical staining for cytochrome oxidase showed a staining pattern similar to that observed for type...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...

  2. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). 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 geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  3. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). 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 geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  4. 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

  5. Polyketide synthase from Fusarium

    DEFF Research Database (Denmark)

    Kvesel, Kasper; Wimmer, Reinhard; Sørensen, Jens Laurids

    Fungi produce a wide array of secondary metabolites, with interesting bioactivities by help of a number of enzyme complexes. Polyketide synthases (PKS) are a class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been...

  6. Sub-MIC tylosin inhibits Streptococcus suis biofilm formation and results in differential protein expression

    Directory of Open Access Journals (Sweden)

    Shuai eWang

    2016-03-01

    Full Text Available Streptococcus suis (S. suis is a crucial zoonotic pathogen which causes severe diseases in humans and pigs. Biofilms of S. suis can induce persistent infections which are harder to treat. Sub-minimal inhibitory concentration (sub-MIC of tylosin can inhibit biofilm formation in bacteria. By using iTRAQ strategy, we compared the protein expression profiles of S. suis grown with sub-MIC tylosin treatment or no treatement. The result showed that 96 proteins expression were changed with 77 up-regulated and 19 down-regulated proteins. Several metabolism proteins (such as phosphoglycerate kinase, as well as cell surface proteins (such as ABC transporter proteins, were found to be involved in biofilm formation. Overall, our results indicated that S. suis metabolic regulation, cell surface proteins, and virulence proteins appear to be of importance in biofilm growth by sub-MIC tylosin treated. Thus, our data analyzed rough regulation of biofilm formation that lay the foundation for the future research of mechanism and targets.

  7. Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (-)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries.

    Science.gov (United States)

    Lücker, Joost; Bowen, Pat; Bohlmann, Jörg

    2004-10-01

    Valencene is a volatile sesquiterpene emitted from flowers of grapevine, Vitis vinifera L. A full-length cDNA from the cultivar Gewürztraminer was functionally expressed in Escherichia coli and found to encode valencene synthase (VvVal). The two major products formed by recombinant VvVal enzyme activity with farnesyl diphosphate (FPP) as substrate are (+)-valencene and (-)-7-epi-alpha-selinene. Grapevine valencene synthase is closely related to a second sesquiterpene synthase from this species, (-)-germacrene D synthase (VvGerD). VvVal and VvGerD cDNA probes revealed strong signals in Northern hybridizations with RNA isolated from grapevine flower buds. Transcript levels were lower in open pre-anthesis flowers, flowers after anthesis, or at early onset of fruit development. Similar results were obtained using a third probe, (-)-alpha-terpineol synthase, a monoterpenol synthase. Sesquiterpene synthase and monoterpene synthase transcripts were not detected in the mesocarp and exocarp during early stages of fruit development, but transcripts hybridizing with VvVal appeared during late ripening of the berries. Sesquiterpene synthase transcripts were also detected in young seeds.

  8. Cleaning up polyketide synthases.

    Science.gov (United States)

    Kwan, Jason C; Schmidt, Eric W

    2012-03-23

    Complex biosynthetic enzymes such as polyketide synthases make mistakes. In this issue of Chemistry & Biology, Jensen et al. report that a discrete family of acyltransferases is responsible for error correction, hydrolyzing key biosynthetic intermediates from a multi-enzyme complex. This activity might find use in understanding polyketide biosynthesis, particularly in uncultivated organisms and in tailoring the synthesis of small molecules. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. 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.

  10. Radiosensitization of NSCLC cells by EGFR inhibition is the result of an enhanced p53-dependent G1 arrest

    International Nuclear Information System (INIS)

    Kriegs, Malte; Gurtner, Kristin; Can, Yildiz; Brammer, Ingo; Rieckmann, Thorsten; Oertel, Reinhard; Wysocki, Marek; Dorniok, Franziska; Gal, Andreas; Grob, Tobias J.; Laban, Simon; Kasten-Pisula, Ulla; Petersen, Cordula; Baumann, Michael; Krause, Mechthild; Dikomey, Ekkehard

    2015-01-01

    Purpose: How EGF receptor (EGFR) inhibition induces cellular radiosensitization and with that increase in tumor control is still a matter of discussion. Since EGFR predominantly regulates cell cycle and proliferation, we studied whether a G1-arrest caused by EGFR inhibition may contribute to these effects. Materials and methods: We analyzed human non-small cell lung cancer (NSCLC) cell lines either wild type (wt) or mutated in p53 (A549, H460, vs. H1299, H3122) and HCT116 cells (p21 wt and negative). EGFR was inhibited by BIBX1382BS, erlotinib or cetuximab; p21 was knocked down by siRNA. Functional endpoints analyzed were cell signaling, proliferation, G1-arrest, cell survival as well as tumor control using an A549 tumor model. Results: When combined with IR, EGFR inhibition enhances the radiation-induced permanent G1 arrest, though solely in cells with intact p53/p21 signaling. This increase in G1-arrest was always associated with enhanced cellular radiosensitivity. Strikingly, this effect was abrogated when cells were re-stimulated, suggesting the initiation of dormancy. In line with this, only a small non-significant increase in tumor control was observed for A549 tumors treated with fractionated RT and EGFR inhibition. Conclusion: For NSCLC cells increase in radiosensitivity by EGFR inhibition results from enhanced G1-arrest. However, this effect does not lead to improved tumor control because cells can be released from this arrest by re-stimulation

  11. Genetic ablation and short-duration inhibition of lipoxygenase results in increased macroautophagy

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Insook; Park, Sujin; Cho, Jin Won [Department of Integrated OMICS for Biomedical Science, WCU Program of Graduate School, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Yigitkanli, Kazim; Leyen, Klaus van [Neuroprotection Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129 (United States); Roth, Jürgen, E-mail: jurgen.roth@bluewin.ch [Department of Integrated OMICS for Biomedical Science, WCU Program of Graduate School, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of)

    2014-02-15

    12/15-lipoxygenase (12/15-LOX) is involved in organelle homeostasis by degrading mitochondria in maturing red blood cells and by eliminating excess peroxisomes in liver. Furthermore, 12/15-LOX contributes to diseases by exacerbating oxidative stress-related injury, notably in stroke. Nonetheless, it is unclear what the consequences are of abolishing 12/15-LOX activity. Mice in which the alox15 gene has been ablated do not show an obvious phenotype, and LOX enzyme inhibition is not overtly detrimental. We show here that liver histology is also unremarkable. However, electron microscopy demonstrated that 12/15-LOX knockout surprisingly leads to increased macroautophagy in the liver. Not only macroautophagy but also mitophagy and pexophagy were increased in hepatocytes, which otherwise showed unaltered fine structure and organelle morphology. These findings were substantiated by immunofluorescence showing significantly increased number of LC3 puncta and by Western blotting demonstrating a significant increase for LC3-II protein in both liver and brain homogenates of 12/15-LOX knockout mice. Inhibition of 12/15-LOX activity by treatment with four structurally different inhibitors had similar effects in cultured HepG2 hepatoma cells and SH-SY5Y neuroblastoma cells with significantly increased autophagy discernable already after 2 hours. Hence, our study reveals a link between ablation or inhibition of 12/15-LOX and stimulation of macroautophagy. The enhanced macroautophagy may be related to the known tissue-protective effects of LOX ablation or inhibition under various diseased conditions caused by oxidative stress and ischemia. This could provide an important cleaning mechanism of cells and tissues to prevent accumulation of damaged mitochondria and other cellular components. - Highlights: • A relationship between lipoxygenases and autophagy is disclosed. • 12/15-lipoxygenase knockout increases autophagy in mice liver and brain. • Lipoxygenase inhibition boosts

  12. [Advances in isoprene synthase research].

    Science.gov (United States)

    Gou, Yan; Liu, Zhongchuan; Wang, Ganggang

    2017-11-25

    Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

  13. Structure of the ATP Synthase Catalytic Complex (F1) from Escherichia coli in an Autoinhibited conformation

    Energy Technology Data Exchange (ETDEWEB)

    G Cingolani; T Duncan

    2011-12-31

    ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F{sub 1}) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit {var_epsilon} adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F{sub 1} structures.

  14. Chelerythrine inhibits the sarco/endoplasmic reticulum Ca(2+)-ATPase and results in cell Ca(2+) imbalance.

    Science.gov (United States)

    Vieira, Saulo Martins; de Oliveira, Vanessa Honorato; Valente, Raphael do Carmo; Moreira, Otacílio da Cruz; Fontes, Carlos Frederico Leite; Mignaco, Julio Alberto

    2015-03-15

    The isoquinoline alkaloid chelerythrine is described as an inhibitor of SERCA. The ATPase inhibition presented two non-competitive components, Ki1=1, 2 μM and Ki2=26 μM. Conversely, chelerythrine presented a dual effect on the p-nitrophenylphosphatase (pNPPase) of SERCA. Ca(2+)-dependent pNPPase was activated up to ∼5 μM chelerythrine with inhibition thereafter. Ca(2+)-independent pNPPase was solely inhibited. The phosphorylation of SERCA with ATP reached half-inhibition with 10 μM chelerythrine and did not parallel the decrease of ATPase activity. In contrast, chelerythrine up to 50 μM increased the phosphorylation by Pi. Cross-linking of SERCA with glutaraldehyde was counteracted by high concentrations of chelerythrine. The controlled tryptic digestion of SERCA shows that the low-affinity binding of chelerythrine evoked an E2-like pattern. Our data indicate a non-competitive inhibition of ATP hydrolysis that favors buildup of the E2-conformers of the enzyme. Chelerythrine as low as 0.5-1.5 μM resulted in an increase of intracellular Ca(2+) on cultured PBMC cells. The inhibition of SERCA and the loss of cell Ca(2+) homeostasis could in part be responsible for some described cytotoxic effects of the alkaloid. Thus, the choice of chelerythrine as a PKC-inhibitor should consider its potential cytotoxicity due to the alkaloid's effects on SERCA. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. ACE inhibition with perindopril and biomarkers of atherosclerosis and thrombosis : Results from the PERTINENT study

    NARCIS (Netherlands)

    Ceconi, C.; Fox, K.M.; Remme, W.J.; Simoons, M.L.; Deckers, J.W.; Bertrand, M.; Parrinello, G.; Kluft, C.; Blann, A.; Cokkinos, D.; Ferrari, R.

    2009-01-01

    The PERTINENT study measured biomarkers of atherosclerosis and thrombosis in a stable coronary artery disease population from EUROPA receiving ACE inhibition with perindopril 8 mg/day or placebo. Biomarkers of inflammation, C-reactive protein (CRP), fibrinogen, and tumor necrosis factor-alpha

  16. A Molecular Dynamics Investigation of Mycobacterium tuberculosis Prenyl Synthases: Conformational Flexibility and Implications for Computer-aided Drug Discovery.

    Science.gov (United States)

    Kim, Meekyum Olivia; Feng, Xinxin; Feixas, Ferran; Zhu, Wei; Lindert, Steffen; Bogue, Shannon; Sinko, William; de Oliveira, César; Rao, Guodong; Oldfield, Eric; McCammon, James Andrew

    2015-06-01

    With the rise in antibiotic resistance, there is interest in discovering new drugs active against new targets. Here, we investigate the dynamic structures of three isoprenoid synthases from Mycobacterium tuberculosis using molecular dynamics (MD) methods with a view to discovering new drug leads. Two of the enzymes, cis-farnesyl diphosphate synthase (cis-FPPS) and cis-decaprenyl diphosphate synthase (cis-DPPS), are involved in bacterial cell wall biosynthesis, while the third, tuberculosinyl adenosine synthase (Rv3378c), is involved in virulence factor formation. The MD results for these three enzymes were then compared with previous results on undecaprenyl diphosphate synthase (UPPS) by means of active site volume fluctuation and principal component analyses. In addition, an analysis of the binding of prenyl diphosphates to cis-FPPS, cis-DPPS, and UPPS utilizing the new MD results is reported. We also screened libraries of inhibitors against cis-DPPS, finding ~1 μm inhibitors, and used the receiver operating characteristic-area under the curve (ROC-AUC) method to test the predictive power of X-ray and MD-derived cis-DPPS receptors. We found that one compound with potent M. tuberculosis cell growth inhibition activity was an IC(50) ~0.5- to 20-μm inhibitor (depending on substrate) of cis-DPPS, a ~660-nm inhibitor of Rv3378c as well as a 4.8-μm inhibitor of cis-FPPS, opening up the possibility of multitarget inhibition involving both cell wall biosynthesis and virulence factor formation. © 2014 John Wiley & Sons A/S.

  17. Catalysis and Sulfa Drug Resistance in Dihydropteroate Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Yun, Mi-Kyung; Wu, Yinan; Li, Zhenmei; Zhao, Ying; Waddell, M. Brett; Ferreira, Antonio M.; Lee, Richard E.; Bashford, Donald; White, Stephen W. (SJCH)

    2013-04-08

    The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. We report structural, computational, and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an S{sub N}1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active-site residues and reveals how sulfonamide resistance arises.

  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. Engineering cotton (+)-delta-cadinene synthase to an altered function: germacrene D-4-ol synthase.

    Science.gov (United States)

    Yoshikuni, Yasuo; Martin, Vincent J J; Ferrin, Thomas E; Keasling, Jay D

    2006-01-01

    The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-cadinene synthase suggested that the G helix plays a very important role in (+)-delta-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).

  20. Inhibitors of Fatty Acid Synthase for Prostate Cancer

    Science.gov (United States)

    2010-05-31

    inhibitors into the clinic. fatty acid synthase, thioesterase, inhibitors, drug development U U U UU 44 USAMRMC Table of Contents ...targeting. Ursolic acid , a pentacyclic triterpenoid acid , as well as the tea polyphenols, epigallocatechin gallate (EGCG) and epicatechin gallate...2007,  6(7), 2120‐2126.  73.  Liu, Y., Tian, W., Ma, X., and Ding, W. Evaluation of  inhibition of  fatty  acid  synthase by  ursolic   acid : positive

  1. Inhibition of endogenous heat shock protein 70 attenuates inducible nitric oxide synthase induction via disruption of heat shock protein 70/Na(+) /H(+) exchanger 1-Ca(2+) -calcium-calmodulin-dependent protein kinase II/transforming growth factor β-activated kinase 1-nuclear factor-κB signals in BV-2 microglia.

    Science.gov (United States)

    Huang, Chao; Lu, Xu; Wang, Jia; Tong, Lijuan; Jiang, Bo; Zhang, Wei

    2015-08-01

    Inducible nitric oxide synthase (iNOS) critically contributes to inflammation and host defense. The inhibition of heat shock protein 70 (Hsp70) prevents iNOS induction in lipopolysaccharide (LPS)-stimulated macrophages. However, the role and mechanism of endogenous Hsp70 in iNOS induction in microglia remains unclear. This study addresses this issue in BV-2 microglia, showing that Hsp70 inhibition or knockdown prevents LPS-induced iNOS protein expression and nitric oxide production. Real-time PCR experiments showed that LPS-induced iNOS mRNA transcription was blocked by Hsp70 inhibition. Further studies revealed that the inhibition of Hsp70 attenuated LPS-stimulated nuclear translocation and phosphorylation of nuclear factor (NF)-κB as well as the degradation of inhibitor of κB (IκB)-α and phosphorylation of IκB kinase β (IKKβ). This prevention effect of Hsp70 inhibition on IKKβ-NF-κB activation was found to be dependent on the Ca(2+) /calcium-calmodulin-dependent protein kinase II (CaMKII)/transforming growth factor β-activated kinase 1 (TAK1) signals based on the following observations: 1) chelation of intracellular Ca(2+) or inhibition of CaMKII reduced LPS-induced increases in TAK1 phosphorylation and 2) Hsp70 inhibition reduced LPS-induced increases in CaMKII/TAK1 phosphorylation, intracellular pH value, [Ca(2+) ]i , and CaMKII/TAK1 association. Mechanistic studies showed that Hsp70 inhibition disrupted the association between Hsp70 and Na(+) /H(+) exchanger 1 (NHE1), which is an important exchanger responsible for Ca(2+) influx in LPS-stimulated cells. These studies demonstrate that the inhibition of endogenous Hsp70 attenuates the induction of iNOS, which likely occurs through the disruption of NHE1/Hsp70-Ca(2+) -CaMKII/TAK1-NF-κB signals in BV-2 microglia, providing further insight into the functions of Hsp70 in the CNS. © 2015 Wiley Periodicals, Inc.

  2. Enzymatic Assays to Investigate Acyl-Homoserine Lactone Autoinducer Synthases.

    Science.gov (United States)

    Shin, Daniel; Nagarajan, Rajesh

    2018-01-01

    Bacteria use chemical molecules called autoinducers as votes to poll their numerical strength in a colony. This polling mechanism, commonly referred to as quorum sensing, enables bacteria to build a social network and provide a collective response for fighting off common threats. In Gram-negative bacteria, AHL synthases synthesize acyl-homoserine lactone (AHL) autoinducers to turn on the expression of several virulent genes including biofilm formation, protease secretion, and toxin production. Therefore, inhibiting AHL signal synthase would limit quorum sensing and virulence. In this chapter, we describe four enzymatic methods that could be adopted to investigate a broad array of AHL synthases. The enzymatic assays described here should accelerate our mechanistic understanding of quorum-sensing signal synthesis that could pave the way for discovery of potent antivirulence compounds.

  3. Inhibition of Matriptase Activity Results in Decreased Intestinal Epithelial Monolayer Integrity In Vitro.

    Directory of Open Access Journals (Sweden)

    E Pászti-Gere

    Full Text Available Barrier dysfunction in inflammatory bowel diseases implies enhanced paracellular flux and lowered transepithelial electrical resistance (TER causing effective invasion of enteropathogens or altered intestinal absorption of toxins and drug compounds. To elucidate the role of matriptase-driven cell surface proteolysis in the maintenance of intestinal barrier function, the 3-amidinophenylalanine-derived matriptase inhibitor, MI-432 was used on porcine IPEC-J2 cell monolayer. Studies with two fluorescent probes revealed that short (2 h treatment with MI-432 caused an altered distribution of oxidative species between intracellular and extracellular spaces in IPEC-J2 cells. This perturbation was partially compensated when administration of inhibitor continued for up to 48 h. Significant decrease in TER between apical and basolateral compartments of MI-432-treated IPEC-J2 cell monolayers proved that matriptase is one of the key effectors in the maintenance of barrier integrity. Changes in staining pattern of matriptase and in localization of the junctional protein occludin were observed suggesting that inhibition of matriptase by MI-432 can also exert an effect on paracellular gate opening via modulation of tight junctional protein assembly. This study confirms that non-tumorigenic IPEC-J2 cells can be used as an appropriate small intestinal model for the in vitro characterization of matriptase-related effects on intestinal epithelium. These findings demonstrate indirectly that matriptase plays a pivotal role in the development of barrier integrity; thus matriptase dysfunction can facilitate the occurence of leaky gut syndrome observed in intestinal inflammatory diseases.

  4. Japanese Encephalitis Virus Infection Results in Transient Dysfunction of Memory Learning and Cholinesterase Inhibition.

    Science.gov (United States)

    Chauhan, Prashant Singh; Khanna, Vinay Kumar; Kalita, Jayantee; Misra, Usha Kant

    2017-08-01

    Cholinergic system has an important role in memory and learning. Abnormal cognitive and behavioral changes have been reported in Japanese encephalitis (JE), but their basis has not been comprehensively evaluated. In this study, we report memory and learning and its association with acetylcholinesterase (AChE) activity, JE virus titer, and with histopathological observations in a rat model of JE. Wistar rats were intracerebrally inoculated on 12th day with 3 × 10 6  pfu/ml of JE virus. Memory and learning were assessed by the active and passive avoidance tests on 10, 33, and 48 days post inoculation (dpi). After 10, 33, and 48 dpi AChE activity, Japanese encephalitis virus (JEV) titer and histopathological changes were studied in the frontal cortex, thalamus, midbrain, cerebellum, and hippocampus. There was significant impairment in memory and learning on 10 dpi which started improving from 33 dpi to 48 dpi by active avoidance test. Passive avoidance test showed decrease in transfer latency time of retention trial compared to acquisition on first, second, and third retention day trial compared to controls. AChE inhibition was more marked in the hippocampus, frontal cortex, and cerebellum on 10 dpi. However, AChE activity started improving from 33 dpi to 48 dpi. AChE activity in the thalamus and midbrain correlated with active avoidance test on 10 dpi and 33 dpi. Histopathological studies also revealed improvement on 33 and 48 compared to 10 dpi. The present study demonstrates transient memory and learning impairment which was associated with reduction in AChE, JEV titer, and damage in different brain regions of JEV infected rats.

  5. Structural study and thermodynamic characterization of inhibitor binding to lumazine synthase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Morgunova, Ekaterina [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden); Illarionov, Boris; Saller, Sabine [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Popov, Aleksander [European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble CEDEX 09 (France); Sambaiah, Thota [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Bacher, Adelbert [Chemistry Department, Technical University of Munich, 85747 Garching (Germany); Cushman, Mark [Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University (United States); Fischer, Markus [Institut für Lebensmittelchemie, Universität Hamburg, Grindelallee 117, 20146 Hamburg (Germany); Ladenstein, Rudolf, E-mail: rudolf.ladenstein@ki.se [Karolinska Institutet NOVUM, Center of Structural Biochemistry, Hälsovägen 7-9, 141 57 Huddinge (Sweden)

    2010-09-01

    Crystallographic studies of lumazine synthase, the penultimate enzyme of the riboflavin-biosynthetic pathway in B. anthracis, provide a structural framework for the design of antibiotic inhibitors, together with calorimetric and kinetic investigations of inhibitor binding. The crystal structure of lumazine synthase from Bacillus anthracis was solved by molecular replacement and refined to R{sub cryst} = 23.7% (R{sub free} = 28.4%) at a resolution of 3.5 Å. The structure reveals the icosahedral symmetry of the enzyme and specific features of the active site that are unique in comparison with previously determined orthologues. The application of isothermal titration calorimetry in combination with enzyme kinetics showed that three designed pyrimidine derivatives bind to lumazine synthase with micromolar dissociation constants and competitively inhibit the catalytic reaction. Structure-based modelling suggested the binding modes of the inhibitors in the active site and allowed an estimation of the possible contacts formed upon binding. The results provide a structural framework for the design of antibiotics active against B. anthracis.

  6. Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

    Directory of Open Access Journals (Sweden)

    Yi-Hsuan Wu

    Full Text Available ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

  7. Spermine Synthase Deficiency Leads to Deafness and a Profound Sensitivity to α-Difluoromethylornithine*♦

    Science.gov (United States)

    Wang, Xiaojing; Levic, Snezana; Gratton, Michael Anne; Doyle, Karen Jo; Yamoah, Ebenezer N.; Pegg, Anthony E.

    2009-01-01

    Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the SpmS and Phex genes, were found to be profoundly hearing impaired. This defect was due to alteration in polyamine content due to the absence of spermine synthase, the product of the SpmS gene. It was reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that ubiquitously expresses spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost complete loss of the endocochlear potential in the Gy mice, which parallels the hearing deficiency, and this was also reversed by the production of spermine from the spermine synthase transgene. Gy mice showed a striking toxic response to treatment with the ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor function resulting in death within 5 days. This effect was due to an inability to maintain normal balance and was also prevented by the transgenic expression of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no effect on balance. The loss of balance in Gy mice treated with DFMO was due to inhibition of polyamine synthesis because it was prevented by administration of putrescine. Our results are consistent with a critical role for polyamines in regulation of Kir channels that maintain the endocochlear potential and emphasize the importance of normal spermidine:spermine ratio in the hearing and balance functions of the inner ear. PMID:19001365

  8. Inhibition of Curcumin on ZAKα Activity Resultant in Apoptosis and Anchorage-Independent Growth in Cancer Cells.

    Science.gov (United States)

    Lee, Jin-Sun; Wang, Tsu-Shing; Lin, Ming Cheng; Lin, Wei-Wen; Yang, Jaw-Ji

    2017-10-31

    Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.

  9. Regulation of CDP-diacylglycerol synthase activity in Saccharomyces cerevisiae.

    OpenAIRE

    Homann, M J; Henry, S A; Carman, G M

    1985-01-01

    The addition of ethanolamine or choline to inositol-containing growth medium resulted in a reduction of CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) activity in Saccharomyces cerevisiae. The reduction of activity did not occur in the absence of inositol. CDP-diacylglycerol synthase activity was not regulated in a S. cerevisiae mutant strain (opi1; an inositol biosynthesis regulatory mutant) by the addition of phospholipid precursors to the growth medium.

  10. Inhibition of the Pim1 oncogene results in diminished visual function.

    Directory of Open Access Journals (Sweden)

    Jun Yin

    Full Text Available Our objective was to profile genetic pathways whose differential expression correlates with maturation of visual function in zebrafish. Bioinformatic analysis of transcriptomic data revealed Jak-Stat signalling as the pathway most enriched in the eye, as visual function develops. Real-time PCR, western blotting, immunohistochemistry and in situ hybridization data confirm that multiple Jak-Stat pathway genes are up-regulated in the zebrafish eye between 3-5 days post-fertilisation, times associated with significant maturation of vision. One of the most up-regulated Jak-Stat genes is the proto-oncogene Pim1 kinase, previously associated with haematological malignancies and cancer. Loss of function experiments using Pim1 morpholinos or Pim1 inhibitors result in significant diminishment of visual behaviour and function. In summary, we have identified that enhanced expression of Jak-Stat pathway genes correlates with maturation of visual function and that the Pim1 oncogene is required for normal visual function.

  11. The Rice Terpene Synthase Gene OsTPS19 Functions as an (S)-Limonene Synthase in planta and its Overexpression Leads to Enhanced Resistance to the Blast Fungus Magnaporthe oryzae.

    Science.gov (United States)

    Chen, Xujun; Chen, Hao; Yuan, Joshua S; Köllner, Tobias G; Chen, Yuying; Guo, Yufen; Zhuang, Xiaofeng; Chen, Xinlu; Zhang, Yong-Jun; Fu, Jianyu; Nebenführ, Andreas; Guo, Zejian; Chen, Feng

    2018-03-06

    Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defense mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defense gene. Here, we report the functional characterization of OsTPS19, which is upregulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)-limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)-limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)-limonene synthase in rice and plays a role in defense against M. oryzae, at least partly, by inhibiting spore germination. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  12. Dienogest, a synthetic progestin, inhibits prostaglandin E2 production and aromatase expression by human endometrial epithelial cells in a spheroid culture system.

    Science.gov (United States)

    Shimizu, Yutaka; Mita, Shizuka; Takeuchi, Takashi; Notsu, Tatsuto; Mizuguchi, Kiyoshi; Kyo, Satoru

    2011-01-01

    Prostaglandin E(2) (PGE(2)) is a major mediator in the pathophysiology, and pathogenesis of gynecological diseases associated with abnormal endometrial disease with proliferation and inflammation, such as endometriosis. In this study, we investigated the effect of dienogest, a selective progesterone receptor agonist, on PGE(2) production and the expression of aromatase, an estrogen synthase, in human immortalized endometrial epithelial cells. Compared with monolayer culture, the cells showed enhanced PGE(2) production and expression of the PGE(2) synthases cyclooxygenase-2 (COX-2), and microsomal prostaglandin E(2) synthase-1 (mPGES-1) in a spheroid culture system. Dienogest inhibited PGE(2) production and this effect was reversed by RU486, a progesterone receptor antagonist. Dienogest inhibited the PGE(2) synthases mRNA and protein expression, and the nuclear factor-κB activation. Moreover, the suppressive effect of dienogest on PGE(2) production was sustained 24h after the drug was withdrawn. Dienogest but not COX inhibitors inhibited aromatase expression. These results suggest that progesterone receptor activation reduces the gene expressions of COX-2, mPGES-1, and aromatase. Our findings suggest that the pharmacological mechanism of dienogest includes the direct inhibition of PGE(2) synthase and aromatase expression and may contribute to the therapeutic effect on the progression of endometriosis. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. The Peptidylarginine Deiminase Inhibitor Cl-Amidine Suppresses Inducible Nitric Oxide Synthase Expression in Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Byungki Jang

    2017-10-01

    Full Text Available The conversion of peptidylarginine into peptidylcitrulline by calcium-dependent peptidylarginine deiminases (PADs has been implicated in the pathogenesis of a number of diseases, identifying PADs as therapeutic targets for various diseases. The PAD inhibitor Cl-amidine ameliorates the disease course, severity, and clinical manifestation in multiple disease models, and it also modulates dendritic cell (DC functions such as cytokine production, antigen presentation, and T cell proliferation. The beneficial effects of Cl-amidine make it an attractive compound for PAD-targeting therapeutic strategies in inflammatory diseases. Here, we found that Cl-amidine inhibited nitric oxide (NO generation in a time- and dose-dependent manner in maturing DCs activated by lipopolysaccharide (LPS. This suppression of NO generation was independent of changes in NO synthase (NOS enzyme activity levels but was instead dependent on changes in inducible NO synthase (iNOS transcription and expression levels. Several upstream signaling pathways for iNOS expression, including the mitogen-activated protein kinase, nuclear factor-κB p65 (NF-κB p65, and hypoxia-inducible factor 1 pathways, were not affected by Cl-amidine. By contrast, the LPS-induced signal transducer and the activator of transcription (STAT phosphorylation and activator protein-1 (AP-1 transcriptional activities (c-Fos, JunD, and phosphorylated c-Jun were decreased in Cl-amidine-treated DCs. Inhibition of Janus kinase/STAT signaling dramatically suppressed iNOS expression and NO production, whereas AP-1 inhibition had no effect. These results indicate that Cl-amidine-inhibited STAT activation may suppress iNOS expression. Additionally, we found mildly reduced cyclooxygenase-2 expression and prostaglandin E2 production in Cl-amidine-treated DCs. Our findings indicate that Cl-amidine acts as a novel suppressor of iNOS expression, suggesting that Cl-amidine has the potential to ameliorate the effects of

  14. Venting of a Water/Inhibited Propylene Glycol Mixture in a Vacuum Environment-Characterization and Representative Test Results

    Science.gov (United States)

    Ungar, Eugene K.; Erickson, Lisa R.

    2011-01-01

    A planned use of the Orion space vehicle involves its residence at the International Space Station for six months at a time. One concept of operations involves temporarily venting portions of the idle Orion active thermal control system (ATCS) during the docked phase, preventing freezing. The venting would have to be reasonably complete with few, if any, completely filled pockets of frozen liquid. Even if pockets of frozen liquid did not damage the hardware during the freezing process, they could prevent the system from filling completely prior to its reactivation. The venting of single component systems in a space environment has been performed numerous times and is well understood. Local nucleation occurs at warm, relatively massive parts of the system, which creates vapor and forces the bulk liquid out of the system. The remnants of the liquid will freeze, then evaporate over time through local heating. Because the Orion ATCS working fluid is a 50/50 mixture of water and inhibited propylene glycol, its boiling behavior was expected to differ from that of a pure fluid. It was thought that the relatively high vapor pressure water might evaporate preferentially, leaving behind a mixture enriched with the low vapor pressure propylene glycol, which would be vaporization ]resistant. Owing to this concern, a test was developed to compare the evaporation behavior of pure water, a 50/50 mixture of water and inhibited propylene glycol, and inhibited propylene glycol. The test was performed using room temperature fluids in an instrumented thin walled stainless steel vertical tube. The 1 in x 0.035 in wall tube was instrumented with surface thermocouples and encased in closed cell polyurethane foam. Reticulated polyurethane foam was placed inside the tube to reduce the convection currents. A vacuum system connected to the top of the tube set the pressure boundary condition. Tests were run for the three fluids at back pressures ranging from 1 to 18 torr. During each test

  15. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

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

    2016-01-01

    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...

  16. STRUCTURAL ENZYMOLOGY OF POLYKETIDE SYNTHASES

    OpenAIRE

    Tsai, Shiou-Chuan (Sheryl); Ames, Brian Douglas

    2009-01-01

    This chapter describes structural and associated enzymological studies of polyketide synthases, including isolated single domains and multidomain fragments. The sequence–structure–function relationship of polyketide biosynthesis, compared with homologous fatty acid synthesis, is discussed in detail. Structural enzymology sheds light on sequence and structural motifs that are important for the precise timing, substrate recognition, enzyme catalysis, and protein–protein interactions leading to ...

  17. 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.

  18. Inhibition of ethylene production by putrescine alleviates aluminium-induced root inhibition in wheat plants

    Science.gov (United States)

    Yu, Yan; Jin, Chongwei; Sun, Chengliang; Wang, Jinghong; Ye, Yiquan; Zhou, Weiwei; Lu, Lingli; Lin, Xianyong

    2016-01-01

    Inhibition of root elongation is one of the most distinct symptoms of aluminium (Al) toxicity. Although putrescine (Put) has been identified as an important signaling molecule involved in Al tolerance, it is yet unknown how Put mitigates Al-induced root inhibition. Here, the possible mechanism was investigated by using two wheat genotypes differing in Al resistance: Al-tolerant Xi Aimai-1 and Al-sensitive Yangmai-5. Aluminium caused more root inhibition in Yangmai-5 and increased ethylene production at the root apices compared to Xi Aimai-1, whereas the effects were significantly reversed by ethylene biosynthesis inhibitors. The simultaneous exposure of wheat seedlings to Al and ethylene donor, ethephon, or ethylene biosynthesis precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), increased ethylene production and aggravated root inhibition, which was more pronounced in Xi Aimai-1. In contrast, Put treatment decreased ethylene production and alleviated Al-induced root inhibition in both genotypes, and the effects were more conspicuous in Yangmai-5. Furthermore, our results indicated that Al-induced ethylene production was mediated by ACC synthase (ACS) and ACC oxidase, and that Put decreased ethylene production by inhibiting ACS. Altogether, these findings indicate that ethylene is involved in Al-induced root inhibition and this process could be alleviated by Put through inhibiting ACS activity. PMID:26744061

  19. Inhibition of ethylene production by putrescine alleviates aluminium-induced root inhibition in wheat plants.

    Science.gov (United States)

    Yu, Yan; Jin, Chongwei; Sun, Chengliang; Wang, Jinghong; Ye, Yiquan; Zhou, Weiwei; Lu, Lingli; Lin, Xianyong

    2016-01-08

    Inhibition of root elongation is one of the most distinct symptoms of aluminium (Al) toxicity. Although putrescine (Put) has been identified as an important signaling molecule involved in Al tolerance, it is yet unknown how Put mitigates Al-induced root inhibition. Here, the possible mechanism was investigated by using two wheat genotypes differing in Al resistance: Al-tolerant Xi Aimai-1 and Al-sensitive Yangmai-5. Aluminium caused more root inhibition in Yangmai-5 and increased ethylene production at the root apices compared to Xi Aimai-1, whereas the effects were significantly reversed by ethylene biosynthesis inhibitors. The simultaneous exposure of wheat seedlings to Al and ethylene donor, ethephon, or ethylene biosynthesis precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), increased ethylene production and aggravated root inhibition, which was more pronounced in Xi Aimai-1. In contrast, Put treatment decreased ethylene production and alleviated Al-induced root inhibition in both genotypes, and the effects were more conspicuous in Yangmai-5. Furthermore, our results indicated that Al-induced ethylene production was mediated by ACC synthase (ACS) and ACC oxidase, and that Put decreased ethylene production by inhibiting ACS. Altogether, these findings indicate that ethylene is involved in Al-induced root inhibition and this process could be alleviated by Put through inhibiting ACS activity.

  20. Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors.

    Science.gov (United States)

    Warrilow, Andrew G S; Hawkesford, Malcolm J

    2002-03-01

    The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model. Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.

  1. Regulation of expression, activity and localization of fungal chitin synthases

    Science.gov (United States)

    Rogg, Luise E.; Fortwendel, Jarrod R.; Juvvadi, Praveen R.; Steinbach, William J.

    2013-01-01

    The fungal cell wall represents an attractive target for pharmacologic inhibition, as many of the components are fungal-specific. Though targeted inhibition of β-glucan synthesis is effective treatment for certain fungal infections, the ability of the cell wall to dynamically compensate via the cell wall integrity pathway may limit overall efficacy. To date, chitin synthesis inhibitors have not been successfully deployed in the clinical setting. Fungal chitin synthesis is a complex and highly regulated process. Regulation of chitin synthesis occurs on multiple levels, thus targeting of these regulatory pathways may represent an exciting alternative approach. A variety of signaling pathways have been implicated in chitin synthase regulation, at both transcriptional and post-transcriptional levels. Recent research suggests that localization of chitin synthases likely represents a major regulatory mechanism. However, much of the regulatory machinery is not necessarily shared among different chitin synthases. Thus, an in depth understanding of the precise roles of each protein in cell wall maintenance and repair will be essential to identifying the most likely therapeutic targets. PMID:21526913

  2. [Reduction of obesity and improvement in metabolic parameters by inhibition of intestinal lipases: current results with orlistat].

    Science.gov (United States)

    Toplak, H; Marhardt, K

    1998-01-01

    Until recently, obesity did not play a major role in considerations of physicians and public health authorities. The impact of health-threatening overweight was so far considered only as a risk factor for various other serious illnesses, such as hypertension, diabetes mellitus, hyperuricemia, elevated blood lipid levels and of vascular diseases of the heart, the brain and the kidneys. Recently however, obesity has been rated by the WHO as an unique disease, resulting in elevated morbidity and mortality. It is of constantly increasing importance because of the raising number of obese individuals in all industrial countries. In Austria an incidence of 8.5% of the adult population is estimated to be obese with a BMI > 30. Though the established concept for treatment of overweight consists of reduction of the caloric intake by diet, there is an obvious need for drugs making dieting easier acceptable to obese patients for prolonged periods. Orlistat is the first representative of a new class of such drugs, inhibiting intestinal acting lipase thus reducing the intestinal absorption of triglycerides; it contributes, therefore, to a reduced calorie intake. Preliminary results of treatment studies with Orlistat are presented, demonstrating its efficacy in inducing weight loss and improving metabolic parameters with tolerable intestinal side effects. After finalization of international studies, demonstrating efficacy and tolerability, orlistat has been registered in Austria in September 1998.

  3. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB α–β-subunit interface and affects multiple steps in the enzyme's overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  4. A small-molecule allosteric inhibitor of Mycobacterium tuberculosis tryptophan synthase

    Energy Technology Data Exchange (ETDEWEB)

    Wellington, Samantha; Nag, Partha P.; Michalska, Karolina; Johnston, Stephen E.; Jedrzejczak, Robert P.; Kaushik, Virendar K.; Clatworthy, Anne E.; Siddiqi, Noman; McCarren, Patrick; Bajrami, Besnik; Maltseva, Natalia I.; Combs, Senya; Fisher, Stewart L.; Joachimiak, Andrzej; Schreiber, Stuart L.; Hung, Deborah T.

    2017-07-03

    New antibiotics with novel targets are greatly needed. Bacteria have numerous essential functions, but only a small fraction of such processes—primarily those involved in macromolecular synthesis—are inhibited by current drugs. Targeting metabolic enzymes has been the focus of recent interest, but effective inhibitors have been difficult to identify. We describe a synthetic azetidine derivative, BRD4592, that kills Mycobacterium tuberculosis (Mtb) through allosteric inhibition of tryptophan synthase (TrpAB), a previously untargeted, highly allosterically regulated enzyme. BRD4592 binds at the TrpAB a–b-subunit interface and affects multiple steps in the enzyme’s overall reaction, resulting in inhibition not easily overcome by changes in metabolic environment. We show that TrpAB is required for the survival of Mtb and Mycobacterium marinum in vivo and that this requirement may be independent of an adaptive immune response. This work highlights the effectiveness of allosteric inhibition for targeting proteins that are naturally highly dynamic and that are essential in vivo, despite their apparent dispensability under in vitro conditions, and suggests a framework for the discovery of a next generation of allosteric inhibitors.

  5. 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 Brandt; 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...

  6. Platensimycin activity against mycobacterial beta-ketoacyl-ACP synthases.

    Directory of Open Access Journals (Sweden)

    Alistair K Brown

    2009-07-01

    Full Text Available There is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin.We have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 microg/ml and against Mycobacterium tuberculosis (MIC = 12 microg/ml. Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against beta-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 microg/ml, to 30 and 124 microg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH.Our results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues.

  7. Effect of a selective thromboxane synthase inhibitor on arterial graft patency and platelet deposition in dogs

    International Nuclear Information System (INIS)

    McDaniel, M.D.; Huntsman, W.T.; Miett, T.O.; Cronenwett, J.L.

    1987-01-01

    This study examined the effect of selective thromboxane synthase inhibition and nonselective cyclooxygenase inhibition on vascular graft patency and indium 111-labeled platelet deposition in 35 mongrel dogs undergoing carotid artery replacement with 4 mm X 4 cm polytetrafluoroethylene (PTFE) (one side) and Dacron (opposite side) end-to-end grafts. Aspirin-dipyridamole therapy improved one-week graft patency, from 46% in untreated dogs to 93% in treated dogs. Thromboxane synthase inhibition (U-63557A) improved graft patency in these dogs to 81%. Both drug treatments reduced platelet deposition on Dacron and PTFE grafts by 48% to 68% compared with control dogs. Dacron grafts accumulated significantly more platelets than PTFE grafts but had comparable patency rates. Low-dose aspirin therapy had no significant effect on either graft patency or platelet deposition. All treatment groups showed a 60% to 76% reduction in serum thromboxane B2, but only thromboxane synthase inhibitor treatment increased plasma 6-keto-prostaglandin F1 alpha by 100%. Selective thromboxane synthase inhibition improved small-caliber prosthetic graft patency to the same extent as did conventional cyclooxygenase inhibition in this preliminary study

  8. 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.

  9. 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)...

  10. Anticonvulsion effect of acupuncture might be related to the decrease of neuronal and inducible nitric oxide synthases.

    Science.gov (United States)

    Yang, R; Huang, Z N; Cheng, J S

    1999-01-01

    To measure the levels of hippocampal nitric oxide synthase isoforms in penicillin induced epilepsy and to test the effect of electroacupuncture (EA) on changes of these levels during epilepsy, we injected penicillin into rat hippocampus to make an epilepsy model and performed electroacupuncture treatment on "Feng Fu" (DU 16) and "Jin Suo" (DU 8) points in Wistar rats. Nitric oxide synthase (NOS) mRNA levels of rat hippocampus were determined by reverse transcription-polymerase chain reaction (RT-PCR). The neuronal nitric oxide synthase (nNOS) mRNA markedly increased (pepilepsy, whereas no significant change in epithelial nitric oxide synthase (eNOS) mRNA was observed. EA inhibited the epilepsy and decreased nNOS (pepilepsy caused an increase in nNOS and iNOS, and the EA anticonvulsant effect might be related to the decrease of these nitric oxide synthases.

  11. Quantification of the glycogen cascade system: the ultrasensitive responses of liver glycogen synthase and muscle phosphorylase are due to distinctive regulatory designs

    Directory of Open Access Journals (Sweden)

    Venkatesh KV

    2005-05-01

    Full Text Available Abstract Background Signaling pathways include intricate networks of reversible covalent modification cycles. Such multicyclic enzyme cascades amplify the input stimulus, cause integration of multiple signals and exhibit sensitive output responses. Regulation of glycogen synthase and phosphorylase by reversible covalent modification cycles exemplifies signal transduction by enzyme cascades. Although this system for regulating glycogen synthesis and breakdown appears similar in all tissues, subtle differences have been identified. For example, phosphatase-1, a dephosphorylating enzyme of the system, is regulated quite differently in muscle and liver. Do these small differences in regulatory architecture affect the overall performance of the glycogen cascade in a specific tissue? We address this question by analyzing the regulatory structure of the glycogen cascade system in liver and muscle cells at steady state. Results The glycogen cascade system in liver and muscle cells was analyzed at steady state and the results were compared with literature data. We found that the cascade system exhibits highly sensitive switch-like responses to changes in cyclic AMP concentration and the outputs are surprisingly different in the two tissues. In muscle, glycogen phosphorylase is more sensitive than glycogen synthase to cyclic AMP, while the opposite is observed in liver. Furthermore, when the liver undergoes a transition from starved to fed-state, the futile cycle of simultaneous glycogen synthesis and degradation switches to reciprocal regulation. Under such a transition, different proportions of active glycogen synthase and phosphorylase can coexist due to the varying inhibition of glycogen-synthase phosphatase by active phosphorylase. Conclusion The highly sensitive responses of glycogen synthase in liver and phosphorylase in muscle to primary stimuli can be attributed to distinctive regulatory designs in the glycogen cascade system. The different

  12. Engineering Isoprene Synthase Expression and Activity in Cyanobacteria.

    Science.gov (United States)

    Chaves, Julie E; Rueda-Romero, Paloma; Kirst, Henning; Melis, Anastasios

    2017-12-15

    Efforts to heterologously produce quantities of isoprene hydrocarbons (C 5 H 8 ) renewably from CO 2 and H 2 O through the photosynthesis of cyanobacteria face barriers, including low levels of recombinant enzyme accumulation compounded by their slow innate catalytic activity. The present work sought to alleviate the "expression level" barrier upon placing the isoprene synthase (IspS) enzyme in different fusion configurations with the cpcB protein, the highly expressed β-subunit of phycocyanin. Different cpcB*IspS fusion constructs were made, distinguished by the absence or presence of linker amino acids between the two proteins. Composition of linker amino acids was variable with lengths of 7, 10, 16, and 65 amino acids designed to test for optimal activity of the IspS through spatial positioning between the cpcB and IspS. Results showed that fusion constructs with the highly expressed cpcB gene, as the leader sequence, improved transgene expression in the range of 61 to 275-fold over what was measured with the unfused IspS control. However, the specific activity of the IspS enzyme was attenuated in all fusion transformants, possibly because of allosteric effects exerted by the leader cpcB fusion protein. This inhibition varied depending on the nature of the linker amino acids between the cpcB and IspS proteins. In terms of isoprene production, the results further showed a trade-off between specific activity and transgenic enzyme accumulation. For example, the cpcB*L7*IspS strain showed only about 10% the isoprene synthase specific-activity of the unfused cpcB-IspS control, but it accumulated 254-fold more IspS enzyme. The latter more than countered the slower specific activity and made the cpcB*L7*IspS transformant the best isoprene producing strain in this work. Isoprene to biomass yield ratios improved from 0.2 mg g -1 in the unfused cpcB-IspS control to 5.4 mg g -1 in the cpcB*L7*IspS strain, a 27-fold improvement.

  13. Examination of thromboxane synthase as a prognostic factor and therapeutic target in non-small cell lung cancer

    LENUS (Irish Health Repository)

    Cathcart, Mary-Clare

    2011-03-09

    Abstract Background Thromboxane synthase (TXS) metabolises prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with a poor prognosis. TXS inhibition induces cell death in-vitro, providing a rationale for therapeutic intervention. We aimed to determine the expression profile of TXS in NSCLC and if it is prognostic and\\/or a survival factor in the disease. Methods TXS expression was examined in human NSCLC and matched controls by western analysis and IHC. TXS metabolite (TXB2) levels were measured by EIA. A 204-patient NSCLC TMA was stained for COX-2 and downstream TXS expression. TXS tissue expression was correlated with clinical parameters, including overall survival. Cell proliferation\\/survival and invasion was examined in NSCLC cells following both selective TXS inhibition and stable TXS over-expression. Results TXS was over-expressed in human NSCLC samples, relative to matched normal controls. TXS and TXB2 levels were increased in protein (p < 0.05) and plasma (p < 0.01) NSCLC samples respectively. TXS tissue expression was higher in adenocarcinoma (p < 0.001) and female patients (p < 0.05). No significant correlation with patient survival was observed. Selective TXS inhibition significantly reduced tumour cell growth and increased apoptosis, while TXS over-expression stimulated cell proliferation and invasiveness, and was protective against apoptosis. Conclusion TXS is over-expressed in NSCLC, particularly in the adenocarcinoma subtype. Inhibition of this enzyme inhibits proliferation and induces apoptosis. Targeting thromboxane synthase alone, or in combination with conventional chemotherapy is a potential therapeutic strategy for NSCLC.

  14. Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, J.; Xue, Z.; Du, Z.; Melese, T.; Boyer, P.D.

    1988-07-12

    Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F/sub 1/ ATPase (CF/sub 1/) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. The authors have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg/sup 2 +/ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF/sub 1/ that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF/sub 1/. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (P/sub i/) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with (/sup 32/P)P/sub i/, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. They also report the occurrence of a 1-2-min delay in the onset of the Mg/sup 2 +/-induced inhibition after addition of CF/sub 1/ to solutions containing Mg/sup 2 +/ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of P/sub i/ formation is followed by a much lower, constant steady-state rate. The burst is not observed with GTP as a substrate or with Ca/sup 2 +/ as the activating cation.

  15. Inhibition of catalase by aminotriazole in vivo results in reduction of glucose-6-phosphate dehydrogenase activity in Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Bayliak, M; Gospodaryov, D; Semchyshyn, H; Lushchak, V

    2008-04-01

    The inhibitor of catalase 3-amino-1,2,4-triazole (AMT) was used to study the physiological role of catalase in the yeast Saccharomyces cerevisiae under starvation. It was shown that AMT at the concentration of 10 mM did not affect the growth of the yeast. In vivo and in vitro the degree of catalase inhibition by AMT was concentration- and time-dependent. Peroxisomal catalase in bakers' yeast was more sensitive to AMT than the cytosolic one. In vivo inhibition of catalase by AMT in S. cerevisiae caused a simultaneous decrease in glucose-6-phosphate dehydrogenase activity and an increase in glutathione reductase activity. At the same time, the level of protein carbonyls, a marker of oxidative modification, was not affected. Possible mechanisms compensating the negative effects caused by AMT inhibition of catalase are discussed.

  16. Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

    Science.gov (United States)

    Burke, Russell T; Marcus, Joshua M; Orth, James D

    2017-06-13

    Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers.

  17. Spermidine Synthase is Required for Growth of Synechococcus sp. PCC 7942 Under Osmotic Stress.

    Science.gov (United States)

    Pothipongsa, Apiradee; Jantaro, Saowarath; Incharoensakdi, Aran

    2016-11-01

    The Synechococcus sp. PCC 7942 spermidine synthase encoded by spds gene (Synpcc7942_0628) is responsible for spermidine biosynthesis. Two Synechococcus strains, the overexpressing spds (OX-spds) and the spds knockout (Δspds), were constructed and characterized for their growth and photosynthetic efficiency under osmotic stress imposed by sorbitol. The growth of Δspds was completely inhibited when cells were grown in the presence of 400 mM sorbitol. Under the same condition, the OX-spds showed a slightly higher growth than the wild type. The OX-spds under osmotic stress also had a significant increase of spermidine level in conjunction with the up-regulation of the genes involved in spermidine biosynthesis. A higher ratio of spermidine to putrescine, an index for stress tolerance, under osmotic stress was found in the OX-spds strain than in the wild type. Overall results indicated that the spermidine synthase enzyme plays an essential role in the survival of Synechococcus sp. PCC 7942 under osmotic stress.

  18. Efeito da inibição da óxido nítrico sintase induzível na capacitação in vitro de espermatozoides bovinos Effect of inhibition of inducible nitric oxide synthase on in vitro capacitation of bovine spermatozoa

    Directory of Open Access Journals (Sweden)

    J.B.P. Ferreira-Berbari

    2010-06-01

    Full Text Available Avaliaram-se o papel do óxido nítrico (NO por meio da inibição da enzima óxido nítrico sintase induzível (iNOS, após a adição da aminoguanidina (AG, na motilidade, no vigor e na integridade da membrana plasmática nos tempos de 15, 60, 120, 180, 240 e 300min e a atividade mitocondrial e a capacitação de espermatozoides bovinos após 300min de cultivo. Adicionaram-se diferentes concentrações (0,001, 0,01 e 0,1M de AG durante a capacitação induzida pela heparina e 500μM de nitroprussiato de sódio (SNP, doador de NO à concentração deletéria. A adição de 0,1M de AG diminuiu a motilidade e o vigor espermático e a integridade da membrana (PThe role of nitric oxide (NO was evaluated by inhibition of inducible nitric oxide synthase (iNOS, with aminoguanidine (AG on motility, vigor, and plasmatic membrane integrity of bovine spermatozoa culture after 15, 60, 120, 180, 240, and 300min and on mitochondrial activity and capacitation after 300min, respectively. Different concentrations, 0.001, 0.01, and 0.1M of AG were added during the heparin induced capacitation and sodium nitroprusside (SNP, NO donor-500μM to the deleterious concentration. The addition of 0.1M of AG diminished progressive motility, spermatic vigor, and membrane integrity (P<0.05. SNP addition to the 0.1M of AG did revert only plasmatic membrane integrity after 300min. Mitochondrial activity was not influenced by addition of AG. Percentage of penetrated oocytes after addition of 0.01 and 0.1M of AG diminished, 20.3 and 100%, respectively, in relation to the control oocytes (P<0.05. However, an increase of 15% was observed when denuded oocytes were used with 0.1M AG treated sperm (P<0.05. It was concluded that the inhibition of NO synthesis with aminoguanidine diminished sperm quality during in vitro capacitation of bovine spermatozoa, except the mitochondrial activity. Only membrane integrity was reverted with the addition of NO to culture medium, suggesting

  19. Inhibition of the mammalian target of rapamycin (mTOR in advanced pancreatic cancer: results of two phase II studies

    Directory of Open Access Journals (Sweden)

    Zhang Yujian

    2010-07-01

    & rash (n = 2. Four patients were hospitalized. Progressive disease occurred in 15 and 1 was non-evaluable. Pretreatment biopsies revealed a higher pAkt/Akt ratio in tumor specimens that in nonmalignant pancreatic tissue. No such trends were noted for the other biomarkers. Conclusions Neither study with mTOR inhibitors demonstrated objective responses or disease stability. The negative feedback loop resulting from mTOR inhibition may account for the disease progression and toxicity noted in these studies. Future strategies should aim for a broader targeting of the PI3K pathway in pancreatic cancer. Trial Registration Trial registration: Study A: NCT 0075647. Study B: NCT00640978

  20. 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.

  1. 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

  2. Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase.

    Science.gov (United States)

    Wang, Yang; Desai, Janish; Zhang, Yonghui; Malwal, Satish R; Shin, Christopher J; Feng, Xinxin; Sun, Hong; Liu, Guizhi; Guo, Rey-Ting; Oldfield, Eric

    2016-10-19

    We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED 50 ∼0.15 μg mL -1 ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Adiponectin inhibits insulin function in primary trophoblasts by PPARα-mediated ceramide synthesis.

    Science.gov (United States)

    Aye, Irving L M H; Gao, Xiaoli; Weintraub, Susan T; Jansson, Thomas; Powell, Theresa L

    2014-04-01

    Maternal adiponectin (ADN) levels are inversely correlated with birth weight, and ADN infusion in pregnant mice down-regulates placental nutrient transporters and decreases fetal growth. In contrast to the insulin-sensitizing effects in adipose tissue and muscle, ADN inhibits insulin signaling in the placenta. However, the molecular mechanisms involved are unknown. We hypothesized that ADN inhibits insulin signaling and insulin-stimulated amino acid transport in primary human trophoblasts by peroxisome proliferator-activated receptor-α (PPARα)-mediated ceramide synthesis. Primary human term trophoblast cells were treated with ADN and/or insulin. ADN increased the phosphorylation of p38 MAPK and PPARα. ADN inhibited insulin signaling and insulin-stimulated amino acid transport. This effect was dependent on PPARα, because activation of PPARα with an agonist (GW7647) inhibited insulin signaling and function, whereas PPARα-small interfering RNA reversed the effects of ADN on the insulin response. ADN increased ceramide synthase expression and stimulated ceramide production. C2-ceramide inhibited insulin signaling and function, whereas inhibition of ceramide synthase (with Fumonisin B1) reversed the effects of ADN on insulin signaling and amino acid transport. These findings are consistent with the model that maternal ADN limits fetal growth mediated by activation of placental PPARα and ceramide synthesis, which inhibits placental insulin signaling and amino acid transport, resulting in reduced fetal nutrient availability.

  4. Plasticity and evolution of (+)-3-carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.

    Science.gov (United States)

    Roach, Christopher R; Hall, Dawn E; Zerbe, Philipp; Bohlmann, Jörg

    2014-08-22

    The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multiproduct (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by (+)-3-carene in PsTPS-3car1, PsTPS-3car2, and PsTPS-3car3, or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against insects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The role of NO synthase isoforms in PDT-induced injury of neurons and glial cells

    Science.gov (United States)

    Kovaleva, V. D.; Berezhnaya, E. V.; Uzdensky, A. B.

    2015-03-01

    Nitric oxide (NO) is an important second messenger, involved in the implementation of various cell functions. It regulates various physiological and pathological processes such as neurotransmission, cell responses to stress, and neurodegeneration. NO synthase is a family of enzymes that synthesize NO from L-arginine. The activity of different NOS isoforms depends both on endogenous and exogenous factors. In particular, it is modulated by oxidative stress, induced by photodynamic therapy (PDT). We have studied the possible role of NOS in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Antinecrotic and proapoptotic effects of NO on the glial cells were found using inhibitory analysis. We have shown the role of inducible NO synthase in photoinduced apoptosis and involvement of neuronal NO synthase in photoinduced necrosis of glial cells in the isolated crayfish stretch receptor. The activation of NO synthase was evaluated using NADPH-diaphorase histochemistry, a marker of neurons expressing the enzyme. The activation of NO synthase in the isolated crayfish stretch receptor was evaluated as a function of time after PDT. Photodynamic treatment induced transient increase in NO synthase activity and then slowly inhibited this enzyme.

  6. Angiotensin-converting enzyme inhibition and novel cardiovascular risk biomarkers: results from the Trial of Angiotensin Converting Enzyme Inhibition and Novel Cardiovascular Risk Factors (TRAIN) study

    NARCIS (Netherlands)

    Cesari, M.; Kritchevsky, S.B.; Atkinson, H.H.; Penninx, B.W.J.H.; Bari, M. Di; Tracy, R.P.; Pahor, M.

    2009-01-01

    .009) were performed to compare biomarkers modifications after fosinopril/placebo interventions. RESULTS: Mean age of the sample (n = 290, women 43.4%) was 66.0 years old. No significant differences were reported for C-reactive protein, interleukin 6, plasminogen activator inhibitor 1, vascular cell

  7. 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.

  8. Functional plasticity of paralogous diterpene synthases involved in conifer defense.

    Science.gov (United States)

    Keeling, Christopher I; Weisshaar, Sabrina; Lin, Roy P C; Bohlmann, Jörg

    2008-01-22

    The diversity of terpenoid compounds produced by plants plays an important role in mediating various plant-herbivore, plant-pollinator, and plant-pathogen interactions. This diversity has resulted from gene duplication and neofunctionalization of the enzymes that synthesize and subsequently modify terpenes. Two diterpene synthases in Norway spruce (Picea abies), isopimaradiene synthase and levopimaradiene/abietadiene synthase, provide the hydrocarbon precursors for most of the diterpene resin acids found in the defensive oleoresin of conifers. Although these paralogous enzymes are 91% identical at the amino acid level, one is a single-product enzyme, whereas the other is a multiproduct enzyme that forms completely different products. We used a rational approach of homology modeling, protein sequence comparison, domain swapping, and a series of reciprocal site-directed mutagenesis to identify the specific residues that direct the different product outcomes. A one-amino acid mutation switched the levopimaradiene/abietadiene synthase into producing isopimaradiene and sandaracopimaradiene and none of its normal products. Four mutations were sufficient to reciprocally reverse the product profiles for both of these paralogous enzymes while maintaining catalytic efficiencies similar to the wild-type enzymes. This study illustrates how neofunctionalization can result from relatively minor changes in protein sequence, increasing the diversity of secondary metabolites important for conifer defense.

  9. 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...

  10. 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)

  11. Cryo-EM structure of the yeast ATP synthase.

    Science.gov (United States)

    Lau, Wilson C Y; Baker, Lindsay A; Rubinstein, John L

    2008-10-24

    We have used electron cryomicroscopy of single particles to determine the structure of the ATP synthase from Saccharomyces cerevisiae. The resulting map at 24 A resolution can accommodate atomic models of the F(1)-c(10) subcomplex, the peripheral stalk subcomplex, and the N-terminal domain of the oligomycin sensitivity conferral protein. The map is similar to an earlier electron cryomicroscopy structure of bovine mitochondrial ATP synthase but with important differences. It resolves the internal structure of the membrane region of the complex, especially the membrane embedded subunits b, c, and a. Comparison of the yeast ATP synthase map, which lacks density from the dimer-specific subunits e and g, with a map of the bovine enzyme that included e and g indicates where these subunits are located in the intact complex. This new map has allowed construction of a model of subunit arrangement in the F(O) motor of ATP synthase that dictates how dimerization of the complex via subunits e and g might occur.

  12. Understanding plant cellulose synthases through a comprehensive investigation of the cellulose synthase family sequences.

    Directory of Open Access Journals (Sweden)

    Andrew eCarroll

    2011-03-01

    Full Text Available The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized subfamilies and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair cellulose synthase function, and compare these sites to those observed in the closest cellulose synthase-like (Csl families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

  13. 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.

  14. 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.

  15. Decoding the substrate supply to human neuronal nitric oxide synthase.

    Directory of Open Access Journals (Sweden)

    Alexandra Simon

    Full Text Available Nitric oxide, produced by the neuronal nitric oxide synthase (nNOS from L-arginine is an important second messenger molecule in the central nervous system: It influences the synthesis and release of neurotransmitters and plays an important role in long-term potentiation, long-term depression and neuroendocrine secretion. However, under certain pathological conditions such as Alzheimer's or Parkinson's disease, stroke and multiple sclerosis, excessive NO production can lead to tissue damage. It is thus desirable to control NO production in these situations. So far, little is known about the substrate supply to human nNOS as a determinant of its activity. Measuring bioactive NO via cGMP formation in reporter cells, we demonstrate here that nNOS in both, human A673 neuroepithelioma and TGW-nu-I neuroblastoma cells can be fast and efficiently nourished by extracellular arginine that enters the cells via membrane transporters (pool I that is freely exchangeable with the extracellular space. When this pool was depleted, NO synthesis was partially sustained by intracellular arginine sources not freely exchangeable with the extracellular space (pool II. Protein breakdown made up by far the largest part of pool II in both cell types. In contrast, citrulline to arginine conversion maintained NO synthesis only in TGW-nu-I neuroblastoma, but not A673 neuroepithelioma cells. Histidine mimicked the effect of protease inhibitors causing an almost complete nNOS inhibition in cells incubated additionally in lysine that depletes the exchangeable arginine pool. Our results identify new ways to modulate nNOS activity by modifying its substrate supply.

  16. 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.

  17. 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.

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

    Science.gov (United States)

    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), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids. PMID:28966840

  19. The personality trait of behavioral inhibition modulates perceptions of moral character and performance during the trust game: behavioral results and computational modeling

    Directory of Open Access Journals (Sweden)

    Milen L. Radell

    2016-02-01

    against the neutral partner. Overall, the results suggest inhibited individuals may be predisposed to interpret neutral or ambiguous information more negatively which could, at least in part, account for the tendency to avoid unfamiliar people characteristic of behaviorally inhibited temperament, as well as its relationship to anxiety disorders.

  20. The personality trait of behavioral inhibition modulates perceptions of moral character and performance during the trust game: behavioral results and computational modeling.

    Science.gov (United States)

    Radell, Milen L; Sanchez, Rosanna; Weinflash, Noah; Myers, Catherine E

    2016-01-01

    neutral partner. Overall, the results suggest inhibited individuals may be predisposed to interpret neutral or ambiguous information more negatively which could, at least in part, account for the tendency to avoid unfamiliar people characteristic of behaviorally inhibited temperament, as well as its relationship to anxiety disorders.

  1. 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.

  2. Interaction between Mitochondrial Reactive Oxygen Species, Heme Oxygenase, and Nitric Oxide Synthase Stimulates Phagocytosis in Macrophages

    Directory of Open Access Journals (Sweden)

    Andrea Müllebner

    2018-01-01

    Full Text Available BackgroundMacrophages are cells of the innate immune system that populate every organ. They are required not only for defense against invading pathogens and tissue repair but also for maintenance of tissue homeostasis and iron homeostasis.AimThe aim of this study is to understand whether heme oxygenase (HO and nitric oxide synthase (NOS contribute to the regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX activity and phagocytosis, two key components of macrophage function.MethodsThis study was carried out using resting J774A.1 macrophages treated with hemin or vehicle. Activity of NOS, HO, or NOX was inhibited using specific inhibitors. Reactive oxygen species (ROS formation was determined by Amplex® red assay, and phagocytosis was measured using fluorescein isothiocyanate-labeled bacteria. In addition, we analyzed the fate of the intracellular heme by using electron spin resonance.ResultsWe show that both enzymes NOS and HO are essential for phagocytic activity of macrophages. NOS does not directly affect phagocytosis, but stimulates NOX activity via nitric oxide-triggered ROS production of mitochondria. Treatment of macrophages with hemin results in intracellular accumulation of ferrous heme and an inhibition of phagocytosis. In contrast to NOS, HO products, including carbon monoxide, neither clearly affect NOX activity nor clearly affect phagocytosis, but phagocytosis is accelerated by HO-mediated degradation of heme.ConclusionBoth enzymes contribute to the bactericidal activity of macrophages independently, by controlling different pathways.

  3. 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.

  4. Fungal type III polyketide synthases.

    Science.gov (United States)

    Hashimoto, Makoto; Nonaka, Takamasa; Fujii, Isao

    2014-10-01

    This article covers the literature on fungal type III polyketide synthases (PKSs) published from 2005 to 2014. Since the first discovery of fungal type III PKS genes in Aspergillus oryzae, reported in 2005, putative genes for type III PKSs have been discovered in fungal genomes. Compared with type I PKSs, type III PKSs are much less abundant in fungi. However, type III PKSs could have some critical roles in fungi. This article summarizes the studies on fungal type III PKS functional analysis, including Neurospora crassa ORAS, Aspergillus niger AnPKS, Botrytis cinerea BPKS and Aspergillus oryzae CsyA and CsyB. It is mostly in vitro analysis using their recombinant enzymes that has revealed their starter and product specificities. Of these, CsyB was found to be a new kind of type III PKS that catalyses the coupling of two β-keto fatty acyl CoAs. Homology modelling reported in this article supports the importance of the capacity of the acyl binding tunnel and active site cavity in fungal type III PKSs.

  5. 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.

  6. 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...... 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...

  7. Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

    Directory of Open Access Journals (Sweden)

    Marquez Rodolfo

    2004-09-01

    Full Text Available Abstract Background Hepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK, glucose-6-phosphatase (G6Pase and insulin-like growth factor binding protein-1 (IGFBP-1, is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE. The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase. However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3 is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase, whose products are required for gluconeogenesis. Results In this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression. Conclusions These results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.

  8. Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods

    Directory of Open Access Journals (Sweden)

    Cabrera-Luque Juan

    2008-09-01

    Full Text Available Abstract Background The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG, an essential allosteric activator of carbamylphosphate synthetase I (CPSI in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS, which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS. Results Information from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occur, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine. Conclusion Difference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine

  9. Triterpenoid modulates the salt tolerance of lanosterol synthase deficient Saccharomyces cerevisiae, GIL77

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    Masashi Inafuku

    2018-01-01

    Full Text Available This study examined the effect of triterpenoid on the salt tolerance of lanosterol synthase deficient yeast mutant GIL77. The expression of the triterpenoid synthase gene under GAL1 promoter in GIL77 increased the triterpenoid concentration of both whole cell and plasma membrane fractions. Without the induction of the genes, the growth curve of BgbAS or RsM1 transformant depicted patterns similar to control cells in both the presence and absence of salt with growth inhibition at 500 mM NaCl. The induction of BgbAS and RsM1 gene expression slightly repressed growth compared with control cells in the absence of NaCl. The growth of GIL77 was significantly suppressed by the expression of BgbAS or RsM1 under salinity conditions. Of the triterpenoid synthase genes, BgbAS rather than RsM1 was found to strongly inhibit the growth of GIL77 cells under salt stressed conditions. The expression of the triterpenoid synthase gene in GIL77 also influenced their tolerance to other abiotic stresses. In contrast to the endogenous synthesis, the exogenous supply of triterpenoid in the culture medium appeared to occur in the plasma membrane fraction and enhanced the salt tolerance of GIL77. This study thus discussed the physiological significance of triterpenoid in relation to its possible role in modulating salt tolerance.

  10. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms.

    Science.gov (United States)

    Barouch, Lili A; Harrison, Robert W; Skaf, Michel W; Rosas, Gisele O; Cappola, Thomas P; Kobeissi, Zoulficar A; Hobai, Ion A; Lemmon, Christopher A; Burnett, Arthur L; O'Rourke, Brian; Rodriguez, E Rene; Huang, Paul L; Lima, João A C; Berkowitz, Dan E; Hare, Joshua M

    2002-03-21

    Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.

  11. A novel mechanism of V-type zinc inhibition of glutamate dehydrogenase results from disruption of subunit interactions necessary for efficient catalysis.

    Science.gov (United States)

    Bailey, Jaclyn; Powell, Lakeila; Sinanan, Leander; Neal, Jacob; Li, Ming; Smith, Thomas; Bell, Ellis

    2011-09-01

    Bovine glutamate dehydrogenase is potently inhibited by zinc and the major impact is on V(max) suggesting a V-type effect on catalysis or product release. Zinc inhibition decreases as glutamate concentrations decrease suggesting a role for subunit interactions. With the monocarboxylic amino acid norvaline, which gives no evidence of subunit interactions, zinc does not inhibit. Zinc significantly decreases the size of the pre-steady state burst in the reaction but does not affect NADPH binding in the enzyme-NADPH-glutamate complex that governs the steady state turnover, again suggesting that zinc disrupts subunit interactions required for catalytic competence. While differential scanning calorimetry suggests zinc binds and induces a slightly conformationally more rigid state of the protein, limited proteolysis indicates that regions in the vicinity of the antennae regions and the trimer-trimer interface become more flexible. The structures of glutamate dehydrogenase bound with zinc and europium show that zinc binds between the three dimers of subunits in the hexamer, a region shown to bind novel inhibitors that block catalytic turnover, which is consistent with the above findings. In contrast, europium binds to the base of the antenna region and appears to abrogate the inhibitory effect of zinc. Structures of various states of the enzyme have shown that both regions are heavily involved in the conformational changes associated with catalytic turnover. These results suggest that the V-type inhibition produced with glutamate as the substrate results from disruption of subunit interactions necessary for efficient catalysis rather than by a direct effect on the active site conformation. © 2011 The Authors Journal compilation © 2011 FEBS.

  12. A Novel Mechanism of V Type Zinc Inhibition of Glutamate Dehydrogenase Results from Disruption of Subunit Interactions Necessary for Efficient Catalysis

    Science.gov (United States)

    Bailey, Jaclyn; Powell, Lakeila; Sinanan, Leander; Neal, Jacob; Li, Ming; Smith, Thomas; Bell, Ellis

    2011-01-01

    SUMMARY Bovine Glutamate Dehydrogenase is potently inhibited by zinc and the major impact is on Vmax suggesting a V-type effect on catalysis or product release. Zinc inhibition decreases as glutamate concentrations decrease suggesting a role for subunit interactions. With the monocarboxylic amino acid, norvaline, which gives no evidence of subunit interactions zinc does not inhibit. Zinc significantly decreases the size of the pre-steady state burst in the reaction but does not affect NADPH binding in the Enzyme-NADPH-glutamate complex that governs the steady state turn-over again suggesting that zinc disrupts subunit interactions required for catalytic competence. While differential scanning calorimetry suggests zinc binds and induces a slightly conformationally more rigid state of the protein, limited proteolysis indicate regions in the vicinity of the antennae regions and the trimer-trimer interface become more flexible. The structures of GDH bound with zinc and europium show that zinc binds between the three dimers of subunits in the hexamer, a region shown to bind novel inhibitors that block catalytic turnover and is consistent with the above findings. In contrast, europium binds to the base of the antenna region and appears to abrogate the inhibitory effect of zinc. Structures of various states of the enzyme have shown that both regions are heavily involved in the conformational changes associated with catalytic turnover. These results suggest that the V-type inhibition produced with glutamate as the substrate results from disruption of subunit interactions necessary for efficient catalysis rather than by a direct effect on the active site conformation. PMID:21749647

  13. Prostaglandin H synthase-mediated bioactivation of the amino acid pyrolysate product Trp P-2

    Energy Technology Data Exchange (ETDEWEB)

    Petry, T.W.; Krauss, R.S.; Eling, T.E.

    1986-08-01

    We report evidence that the mutagen and carcinogen 3-amino-1-methyl-5H pyrido(4,3b)indole (Trp P-2) is a substrate for co-oxidation by prostaglandin H synthase (PHS) in ram seminal vesicle (RSV) microsomes. Trp P-2 serves as a reducing cofactor for the hydroperoxidase activity of PHS as shown by the concentration-dependent inhibition of the hydroperoxidase catalyzed incorporation of molecular oxygen into phenylbutazone. Spectral data suggest that this metabolism results in disruption of the double bond conjugation within the nucleus of the molecule. A single metabolite peak which was dependent upon arachidonic acid and substrate concentration was separated from the parent compound by h.p.l.c. following incubation with RSV microsomes. Co-oxidation of Trp P-2 produced reactive intermediates which bound covalently to microsomal protein (9 nmol/mg) and to calf thymus DNA (475 pmol/mg). Binding was inhibited by indomethacin, and supported by substitution of hydrogen peroxide for arachidonic acid. These data suggest a possible role for PHS in the in situ activation of Trp P-2 to its ultimate carcinogenic form in tissues which contain PHS.

  14. 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

  15. Microangiopathy triggers, and inducible nitric oxide synthase exacerbates dextran sulfate sodium-induced colitis.

    Science.gov (United States)

    Saijo, Hiroki; Tatsumi, Norifumi; Arihiro, Seiji; Kato, Tomohiro; Okabe, Masataka; Tajiri, Hisao; Hashimoto, Hisashi

    2015-07-01

    Ulcerative colitis (UC) is a representative clinical manifestation of inflammatory bowel disease that causes chronic gastrointestinal tract inflammation. Dextran sulfate sodium (DSS)-induced colitis mice have been used to investigate UC pathogenesis, and in this UC model, disturbance and impairment of the mucosal epithelium have been reported to cause colitis. However, how DSS sporadically breaks down the epithelium remains unclear. In this study, we focused on the colonic microcirculation and myenteric neurons of DSS-induced colitis. Moreover, we examined the potential of myenteric neurons as a target to prevent exacerbation of colitis. Fluorescent angiographic and histopathological studies revealed that DSS administration elicited blood vessel disruption before epithelial disorders appeared. Ischemic conditions in the lamina propria induced inducible nitric oxide synthase (iNOS) expression in myenteric neurons as colitis aggravated. When neuronal activity was inhibited with butylscopolamine, neuronal iNOS expression decreased, and the exacerbation of colitis was prevented. These results suggested that DSS-induced colitis was triggered by microcirculatory disturbance in the mucosa, and that excessive neuronal excitation aggravated colitis. During remission periods of human UC, endoscopic inspection of the colonic microcirculation may enable the early detection of disease recurrence, and inhibition of neuronal iNOS expression may prevent the disease from worsening.

  16. A domain swapping approach to elucidate differential regiospecific hydroxylation by geraniol and linalool synthases from perilla.

    Science.gov (United States)

    Sato-Masumoto, Naoko; Ito, Michiho

    2014-06-01

    Geraniol and linalool are acyclic monoterpenes found in plant essential oils that have attracted much attention for their commercial use and in pharmaceutical studies. They are synthesized from geranyl diphosphate (GDP) by geraniol and linalool synthases, respectively. Both synthases are very similar at the amino acid level and share the same substrate; however, the position of the GDP to which they introduce hydroxyl groups is different. In this study, the mechanisms underlying the regiospecific hydroxylation of geraniol and linalool synthases were investigated using a domain swapping approach and site-directed mutagenesis in perilla. Sequences of the synthases were divided into ten domains (domains I to IV-4), and each corresponding domain was exchanged between both enzymes. It was shown that different regions were important for the formation of geraniol and linalool, namely, domains IV-1 and -4 for geraniol, and domains III-b, III-d, and IV-4 for linalool. These results suggested that the conformation of carbocation intermediates and their electron localization were seemingly to be different between geraniol and linalool synthases. Further, five amino acids in domain IV-4 were apparently indispensable for the formation of geraniol and linalool. According to three-dimensional structural models of the synthases, these five residues seemed to be responsible for the different spatial arrangement of the amino acid at H524 in the case of geraniol synthase, while N526 is the corresponding residue in linalool synthase. These results suggested that the side-chains of these five amino acids, in combination with several relevant domains, localized the positive charge in the carbocation intermediate to determine the position of the introduced hydroxyl group. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Vasorelaxing Activity of Ulmus davidiana Ethanol Extracts in Rats: Activation of Endothelial Nitric Oxide Synthase

    Science.gov (United States)

    Cho, Eun Jung; Park, Myoung Soo; Kim, Sahng Seop; Kang, Gun; Choi, Sunga; Lee, Yoo Rhan; Chang, Seok Jong; Lee, Kwon Ho; Lee, Sang Do; Park, Jin Bong

    2011-01-01

    Ulmus davidiana var. japonica Rehder (Urticales: Ulmaceae) (UD) is a tree widespread in northeast Asia. It is traditionally used for anticancer and anti-inflammatory therapy. The present study investigated the effect of an ethanol extract of UD on vascular tension and its underlying mechanism in rats. The dried root bark of UD was ground and extracted with 80% ethanol. The prepared UD extract was used in further analysis. The effect of UD on the cell viability, vasoreactivity and hemodynamics were investigated using propidium iodide staining in cultured cells, isometric tension recording and blood pressure analysis, respectively. Low dose of UD (10~100µg/ml) did not affect endothelial cell viability, but high dose of UD reduced cell viability. UD induced vasorelaxation in the range of 0.1~10µg/ml with an ED50 value of 2µg/ml. UD-induced vasorelaxation was completely abolished by removal of the endothelium or by pre-treatment with L-NAME, an inhibitor of nitric oxide synthase. UD inhibited calcium influx induced by phenylephrine and high K+ and also completely abolished the effect of L-NAME. Intravenous injection of UD extracts (10~100 mg/kg) decreased arterial and ventricular pressure in a dose-dependent manner. Moreover, UD extracts reduced the ventricular contractility (+dP/dt) in anesthetized rats. However, UD-induced hypotensive actions were minimized in L-NAME-treated rats. Taken together, out results showed that UD induced vasorelaxation and has antihypertensive properties, which may be due the activation of nitric oxide synthase in endothelium. PMID:22359471

  18. results

    Directory of Open Access Journals (Sweden)

    Salabura Piotr

    2017-01-01

    Full Text Available HADES experiment at GSI is the only high precision experiment probing nuclear matter in the beam energy range of a few AGeV. Pion, proton and ion beams are used to study rare dielectron and strangeness probes to diagnose properties of strongly interacting matter in this energy regime. Selected results from p + A and A + A collisions are presented and discussed.

  19. Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme.

    Science.gov (United States)

    Liebergesell, M; Rahalkar, S; Steinbüchel, A

    2000-08-01

    The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (Mr 40,950) and PhaC (Mr 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed.

  20. Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne; Larsen, Sine

    2000-01-01

    A steady state kinetic investigation of the Pi activation of 5-phospho-D-ribosyl α-1-diphosphate synthase from Escherichia coli suggests that Pi can bind randomly to the enzyme either before or after an ordered addition of free Mg2+ and substrates. Unsaturation with ribose 5-phosphate increased...... the apparent cooperativity of Pi activation. At unsaturating Pi concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with Pi directs the subsequent ordered binding of Mg2+ and substrates via a fast pathway, whereas...... saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pi binds to the enzyme last. The random mechanism for Pi binding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when...

  1. The cellulose synthase superfamily in fully sequenced plants and algae

    Directory of Open Access Journals (Sweden)

    Xu Ying

    2009-07-01

    Full Text Available Abstract Background The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl families and one cellulose synthase (CesA family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses. Results A single-copy gene is found in the six chlorophyte green algae, which is most closely related to the CslA and CslC families that are present in the seven land plants investigated in our analyses. Six proteins from poplar, grape and sorghum form a distinct family (CslJ, providing further support for the conclusions from two recent studies. CslB/E/G/H/J families have evolved significantly more rapidly than their widely distributed relatives, and tend to have intragenomic duplications, in particular in the grape genome. Conclusion Our data suggest that the CslA and CslC families originated through an ancient gene duplication event in land plants. We speculate that the single-copy Csl gene in green algae may encode a mannan synthase. We confirm that the rest of the Csl families have a different evolutionary origin than CslA and CslC, and have proposed a model for the divergence order among them. Our study provides new insights about the evolution of this important gene family in plants.

  2. The Structural Basis of Erwinia rhapontici Isomaltulose Synthase

    Science.gov (United States)

    Xu, Zheng; Li, Sha; Li, Jie; Li, Yan; Feng, Xiaohai; Wang, Renxiao; Xu, Hong; Zhou, Jiahai

    2013-01-01

    Sucrose isomerase NX-5 from Erwiniarhapontici efficiently catalyzes the isomerization of sucrose to isomaltulose (main product) and trehalulose (by-product). To investigate the molecular mechanism controlling sucrose isomer formation, we determined the crystal structures of native NX-5 and its mutant complexes E295Q/sucrose and D241A/glucose at 1.70 Å, 1.70 Å and 2.00 Å, respectively. The overall structure and active site architecture of NX-5 resemble those of other reported sucrose isomerases. Strikingly, the substrate binding mode of NX-5 is also similar to that of trehalulose synthase from Pseudomonasmesoacidophila MX-45 (MutB). Detailed structural analysis revealed the catalytic RXDRX motif and the adjacent 10-residue loop of NX-5 and isomaltulose synthase PalI from Klebsiella sp. LX3 adopt a distinct orientation from those of trehalulose synthases. Mutations of the loop region of NX-5 resulted in significant changes of the product ratio between isomaltulose and trehalulose. The molecular dynamics simulation data supported the product specificity of NX-5 towards isomaltulose and the role of the loop330-339 in NX-5 catalysis. This work should prove useful for the engineering of sucrose isomerase for industrial carbohydrate biotransformations. PMID:24069347

  3. Radiolabeling of a wound-inducible pyridoxal phosphate utilizing protein from tomato: evidence for its identification as ACC synthase

    International Nuclear Information System (INIS)

    Privalle, L.S.; Graham, J.S.; Caughey, P.A.

    1986-01-01

    Aminocyclopropane 1-carboxylic acid (ACC) synthase, a pyridoxal phosphate utilizing enzyme, catalyzes the conversion of S-adenosylmethionine to ACC, the rate limiting step in the biosynthesis of the plant hormone, ethylene. Ethylene, besides being involved in normal plant growth processes, is also produced in response to stress, e.g. wounding, pathogen infection, etc. The authors report the partial purification (400 fold) of ACC synthase from wounded pink tomato pericarp by classical techniques including ammonium sulfate precipitation, ion exchange and phenyl sepharose chromatography. Further purification results in a decrease in specific activity apparently due to the instability of the enzyme and the low levels present in plant tissue. Radiolabeling of a pyridoxal phosphate-utilizing protein in the ACC synthase enriched fraction was achieved. Evidence that this radiolabeled protein is ACC synthase will be presented. Amino acid sequence determination of putative ACC synthase-derived peptides is underway

  4. 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.

  5. Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes

    DEFF Research Database (Denmark)

    Højlund, Kurt; Staehr, Peter; Hansen, Bo Falck

    2003-01-01

    -hyperinsulinemic clamps. Analysis using phospho-specific antibodies revealed that insulin decreases phosphorylation of sites 3a + 3b in human muscle, and this was accompanied by activation of Akt and inhibition of glycogen synthase kinase-3alpha. In type 2 diabetic subjects these effects of insulin were fully intact...

  6. Lipophilic Bisphosphonates as Dual Farnesyl/Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMR Investigation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Y.; Cao, R; Yin, F; Hudock, M; Guo, R; Song, Y; No, J; Bergan, K; Leon, A; et al,

    2009-01-01

    Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.

  7. Noncovalent Intermediate of Thymidylate Synthase: Fact or Fiction?

    Science.gov (United States)

    Kholodar, Svetlana A; Kohen, Amnon

    2016-07-06

    Thymidylate synthase is an attractive target for antibiotic and anticancer drugs due to its essential role in the de novo biosynthesis of the DNA nucleotide thymine. The enzymatic reaction is initiated by a nucleophilic activation of the substrate via formation of a covalent bond to an active site cysteine. The traditionally accepted mechanism is then followed by a series of covalently bound intermediates, where that bond is only cleaved upon product release. Recent computational and experimental studies suggest that the covalent bond between the protein and substrate is actually quite labile. Importantly, these findings predict the existence of a noncovalently bound bisubstrate intermediate, not previously anticipated, which could be the target of a novel class of drugs inhibiting DNA biosynthesis. Here we report the synthesis of the proposed intermediate and findings supporting its chemical and kinetic competence. These findings substantiate the predicted nontraditional mechanism and the potential of this intermediate as a new drug lead.

  8. Linalool and linalool nerolidol synthases in roses, several genes for little scent.

    Science.gov (United States)

    Magnard, Jean-Louis; Bony, Aurélie Rius; Bettini, Fabienne; Campanaro, Ausilia; Blerot, Bernard; Baudino, Sylvie; Jullien, Frédéric

    2018-03-11

    Roses are widely appreciated for the appearance of their flowers and for their fragrance. This latter character results from the combination of different odorant molecules among which monoterpenes are often prevalent constituents. In this study, we report the cloning and characterization of three rose monoterpene synthases. In vitro functional characterization of these enzymes showed that one is a (-)-(3R)-linalool synthase whereas the others have a dual (+)-(3S)-linalool nerolidol synthase activity. However, given that the characterized rose cultivars were only able to produce the (-)-(3R)-linalool stereoisomer, the linalool nerolidol synthases are probably not active in planta. Furthermore, these three enzymes were also characterized by a weak expression level as assessed by RT-qPCR and by the low abundance of the corresponding sequences in an EST library. This characteristic is likely to explain why linalool is generally a minor constituent in rose flowers' scents. On this basis, we propose that in roses the monoterpene biosynthesis effort is focused on the production of acyclic monoterpenes derived from geraniol through the recently characterized Nudix biosynthesis pathway, at the expense of conventional monoterpene biosynthesis via terpene synthases such as linalool or linalool nerolidol synthases. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  9. PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries.

    Science.gov (United States)

    Mauban, Joseph R H; Zacharia, Joseph; Fairfax, Seth; Wier, Withrow Gil

    2015-06-15

    Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K(+) concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca(2+) release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca(2+) waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca(2+) concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca(2+) entry and promote vasoconstriction. Copyright © 2015 the American Physiological Society.

  10. 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...

  11. Understanding Plant Cellulose Synthases through a Comprehensive Investigation of the Cellulose Synthase Family Sequences.

    Science.gov (United States)

    Carroll, Andrew; Specht, Chelsea D

    2011-01-01

    The development of cellulose as an organizing structure in the plant cell wall was a key event in both the initial colonization and the subsequent domination of the terrestrial ecosystem by vascular plants. A wealth of experimental data has demonstrated the complicated genetic interactions required to form the large synthetic complex that synthesizes cellulose. However, these results are lacking an extensive analysis of the evolution, specialization, and regulation of the proteins that compose this complex. Here we perform an in-depth analysis of the sequences in the cellulose synthase (CesA) family. We investigate the phylogeny of the CesA family, with emphasis on evolutionary specialization. We define specialized clades and identify the class-specific regions within the CesA sequence that may explain this specialization. We investigate changes in regulation of CesAs by looking at the conservation of proposed phosphorylation sites. We investigate the conservation of sites where mutations have been documented that impair CesA function, and compare these sites to those observed in the closest cellulose synthase-like (Csl) families to better understand what regions may separate the CesAs from other Csls. Finally we identify two positions with strong conservation of the aromatic trait, but lacking conservation of amino acid identity, which may represent residues important for positioning the sugar substrate for catalysis. These analyses provide useful tools for understanding characterized mutations and post-translational modifications, and for informing further experiments to probe CesA assembly, regulation, and function through site-directed mutagenesis or domain swapping experiments.

  12. Defective mitochondrial gene expression results in reactive oxygen species-mediated inhibition of respiration and reduction of yeast life span.

    Science.gov (United States)

    Bonawitz, Nicholas D; Rodeheffer, Matthew S; Shadel, Gerald S

    2006-07-01

    Mitochondrial dysfunction causes numerous human diseases and is widely believed to be involved in aging. However, mechanisms through which compromised mitochondrial gene expression elicits the reported variety of cellular defects remain unclear. The amino-terminal domain (ATD) of yeast mitochondrial RNA polymerase is required to couple transcription to translation during expression of mitochondrial DNA-encoded oxidative phosphorylation subunits. Here we report that several ATD mutants exhibit reduced chronological life span. The most severe of these (harboring the rpo41-R129D mutation) displays imbalanced mitochondrial translation, conditional inactivation of respiration, elevated production of reactive oxygen species (ROS), and increased oxidative stress. Reduction of ROS, via overexpression of superoxide dismutase (SOD1 or SOD2 product), not only greatly extends the life span of this mutant but also increases its ability to respire. Another ATD mutant with similarly reduced respiration (rpo41-D152A/D154A) accumulates only intermediate levels of ROS and has a less severe life span defect that is not rescued by SOD. Altogether, our results provide compelling evidence for the "vicious cycle" of mitochondrial ROS production and lead us to propose that the amount of ROS generated depends on the precise nature of the mitochondrial gene expression defect and initiates a downward spiral of oxidative stress only if a critical threshold is crossed.

  13. Evolutionary and mechanistic insights from the reconstruction of (+)-humulene synthases from a modern (+)-Germacrene A Synthase

    OpenAIRE

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

    2014-01-01

    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

  14. 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 ...

  15. Glutamate synthase: An archaeal horizontal gene transfer?

    Indian Academy of Sciences (India)

    (GOGAT) which is a key enzyme in ammonia assimilation in bacteria, algae and plants. It catalyzes the reductive transamidation of amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate (Temple et al 1998). Glutamate synthases differ according to their molecular weights, subunit compositions, ...

  16. Relationship between endothelial nitric oxide synthase gene ...

    African Journals Online (AJOL)

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  17. Producing alpha-olefins using polyketide synthases

    Energy Technology Data Exchange (ETDEWEB)

    Fortman, Jeffrey L.; Katz, Leonard; Steen, Eric J.; Keasling, Jay D.

    2018-01-02

    The present invention provides for a polyketide synthase (PKS) capable of synthesizing an .alpha.-olefin, such as 1-hexene or butadiene. The present invention also provides for a host cell comprising the PKS and when cultured produces the .alpha.-olefin.

  18. Expression of Deinococcus geothermalis trehalose synthase gene ...

    African Journals Online (AJOL)

    A novel trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) containing 1692 bp reading-frame encoding 564 amino acids was amplified using polymerase chain reaction (PCR). The gene was ligated into pET30Ek/LIC vector and expressed after isopropyl β-D-thiogalactopyranoside induction in ...

  19. 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 ...

  20. Synthesis of isoprenoid bisphosphonate ethers through C–P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    2014-07-01

    Full Text Available A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS. Five of the new compounds show IC50 values of less than 1 μM against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS. The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 μM concentration.

  1. 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.

  2. Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

    Science.gov (United States)

    Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

    1996-01-01

    Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

  3. Characteristics of inositol phosphorylceramide synthase and effects of aureobasidin A on growth and pathogenicity of Botrytis cinerea.

    Science.gov (United States)

    Wang, Xin-hui; Guo, Xing-Jun; Li, Hong-Ye; Gou, Ping

    2015-01-01

    Inositol phosphorylceramide (IPC) synthase is the key enzyme with highly conserved sequences, which is involved in fungal sphingolipid biosynthesis. The antibiotic aureobasidin A (AbA) induces the death of fungi through inhibiting IPC synthase activity. The mutations of AUR1 gene coding IPC synthase in fungi and protozoa causes a resistance to AbA. However, the mechanism of AbA resistance is still elusive. In this paper, we generated two mutants of Botrytis cinerea with AbA-resistance, BcAUR1a and BcAUR1b, through UV irradiation. BcAUR1a lost an intron and BcAUR1b had three amino acid mutations (L197P, F288S and T323A) in the AUR1 gene. AbA strongly inhibits the activity of IPC synthase in wild-type B. cinerea, which leads to distinct changes in cell morphology, including the delay in conidial germination, excessive branching near the tip of the germ tube and mycelium, and the inhibition of the mycelium growth. Further, AbA prevents the infection of wild-type B. cinerea in tomato fruits via reducing oxalic acid secretion and the activity of cellulase and pectinase. On the contrary, AbA has no effect on the growth and pathogenicity of the two mutants. Although both mutants show a similar AbA resistance, the molecular mechanisms might be different between the two mutants.

  4. Modeling and optimization of acid dye manufacturing wastewater treatment with Fenton's reagent: comparison with electrocoagulation treatment results and effects on activated sludge inhibition.

    Science.gov (United States)

    Arslan-Alaton, Idil; Gursoy, B Hande; Akyol, Abdurahman; Kobya, Mehmet; Bayramoglu, Mahmut

    2010-01-01

    In the present study, Fenton's oxidation of a chromium complex disazo dye (Acid Blue 193) synthesis wastewater was evaluated, modeled and optimized by employing Central Composite Design. Within this context, the individual and interactive effects of critical process parameters such as Fe(2 + ), H(2)O(2) concentrations, initial chemical oxygen demand (COD) and reaction time was assessed. The process response (output) variables were chosen as percent color, COD and total organic carbon (TOC) removal efficiencies. Optimum working conditions in terms of color and organic carbon removals were established to be Fe(2 + )=3 mM; H(2)O(2)=25 mM; reaction time = 10 min at pH 3 and an initial COD content of 245 mg/L. Under these conditions, 96% color, 82% COD and 51% TOC removals were obtained. The established polynomial regression models describing color, COD and TOC removals satisfactorily fitted the experimental data and could be used to predict Fenton's treatment results at statistically significant rates. Optimized treatment results were compared with those obtained via electrocoagulation treatment under optimized conditions (applied current = 50 A/m(2); reaction time = 15 min; initial pH = 7 for an initial COD content of 245 mg/L). The relative inhibition of heterotrophic oxygen uptake rate was measured to examine the inhibitory effect of azo dye synthesis effluent before and after Fenton's oxidation and electrocoagulation with respect to synthetic domestic wastewater. Untreated azo dye production wastewater exhibited a slightly inhibitory effect that was appreciably reduced but not entirely removed after Fenton's oxidation, whereas no inhibition of mixed bioculture was observed for azo dye synthesis effluent subjected to electrocoagulation treatment.

  5. NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3β resulting in the stabilization of MYCN in human neuroblastomas.

    Directory of Open Access Journals (Sweden)

    Yusuke Suenaga

    2014-01-01

    Full Text Available The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

  6. 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)

  7. 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)

  8. Association of a Soybean Raffinose Synthase Gene with Low Raffinose and Stachyose Seed Phenotype

    Directory of Open Access Journals (Sweden)

    Emily C. Dierking

    2008-11-01

    Full Text Available Oligosaccharides are an important component of soybean [ (L. Merr.] meal in terms of metabolizable energy for monogastric animals. Sucrose, raffinose, and stachyose are the three main oligosaccharides present in soybean meal. Of the three, only sucrose is nutritionally useful. When raffinose and stachyose are fermented by microbes present in the gut, the results are flatulence and discomfort, which ultimately lead to poor weight gain. The long term objective of this research is ultimately to increase the nutritional value of soybean meal by elevating the metabolizable energy at the expense of raffinose and stachyose through the manipulation of soybean raffinose synthase, the key enzyme for raffinose and stachyose biosynthesis. The objectives of this work were to develop molecular genetic information about soybean raffinose synthases and to evaluate the candidate raffinose synthase genes in a soybean germplasm accession (PI 200508 that contains low levels of raffinose and stachyose. Our results indicate the soybean genome contains at least two expressed genes similar to other characterized raffinose synthases. A novel allele of one of these putative soybean raffinose synthase genes was discovered from the PI 200508 that completely associates with the low raffinose and stachyose phenotype. Molecular marker assays specific for the PI 200508 allele were developed to allow direct selection for the low raffinose and low stachyose phenotype.

  9. Squalene synthase as a target for Chagas disease therapeutics.

    Directory of Open Access Journals (Sweden)

    Na Shang

    2014-05-01

    Full Text Available Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

  10. Identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene (crtE) and phytoene synthase gene (crtB) for carotenoid biosynthesis in Euglena gracilis.

    Science.gov (United States)

    Kato, Shota; Takaichi, Shinichi; Ishikawa, Takahiro; Asahina, Masashi; Takahashi, Senji; Shinomura, Tomoko

    2016-01-05

    Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated. To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 μmol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 % and 13-91 %, respectively, relative to the control light intensity (55 μmol m(-2) s(-1)). When grown under continuous light at 920 μmol m(-2) s(-1), the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the light-stress treatments examined. We identified genes

  11. Fatty acid synthase inhibitors from the hulls of Nephelium lappaceum L.

    Science.gov (United States)

    Zhao, You-Xing; Liang, Wen-Juan; Fan, Hui-Jin; Ma, Qing-Yun; Tian, Wei-Xi; Dai, Hao-Fu; Jiang, He-Zhong; Li, Ning; Ma, Xiao-Feng

    2011-08-16

    Natural products inhibiting fatty acid synthase (FAS) are appearing as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Nephelium lappaceum L. resulted in the isolation of ten compounds (1-10) mainly including flavonoids and oleane-type triterpene oligoglycosides, in which all of the compounds were isolated from this plant for the first time. Additionally, compounds 8 and 9 were new hederagenin derivatives and were elucidated as hederagenin 3-O-(2,3-di-O-acetyl-α-l-arabinofuranosyl)-(1→3)-[α-l-rhamnopyranosyl(1→2)]-β-l-arabinopyranoside and hederagenin 3-O-(3-O-acetyl-α-l-arabinofuranosyl)-(1→3)-[α-l-rhamnopyranosyl-(1→2)]-β-l-arabinopyranoside, respectively. All these isolates were evaluated for inhibitory activities of FAS, which showed these isolates had inhibitory activity against FAS with IC(50) values ranging from 6.69 to 204.40 μM, comparable to the known FAS inhibitor EGCG (IC(50)=51.97 μM). The study indicates that the hulls of Nephelium lappaceum L. could be considered as potential sources of promising FAS inhibitors and the oleane-type triterpene oligoglycosides could be considered as another type of natural FAS inhibitors. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Potent Inhibitory Effect of Chinese Dietary Spices on Fatty Acid Synthase.

    Science.gov (United States)

    Jiang, Bing; Liang, Yan; Sun, Xuebing; Liu, Xiaoxin; Tian, Weixi; Ma, Xiaofeng

    2015-09-01

    Dietary spices have been adopted in cooking since ancient times to enhance flavor and also as food preservatives and disease remedies. In China, the use of spices and other aromatic plants as food flavoring is an integral part of dietary behavior, but relatively little is known about their functions. Fatty acid synthase (FAS) has been recognized as a remedy target, and its inhibitors might be applied in disease treatment. The present work was designed to assess the inhibitory activities on FAS of spices extracts in Chinese menu. The in vitro inhibitory activities on FAS of 22 extracts of spices were assessed by spectrophotometrically monitoring oxidation of NADPH at 340 nm. Results showed that 20 spices extracts (90.9 %) exhibited inhibitory activities on FAS, with half inhibition concentration (IC(50)) values ranging from 1.72 to 810.7 μg/ml. Among them, seven spices showed strong inhibitory effect with IC(50) values lower than 10 μg/ml. These findings suggest that a large proportion of the dietary spices studied possess promising inhibitory activities on FAS, and subsequently might be applied in the treatment of obesity and obesity-related human diseases.

  13. Glycogen Synthase Kinase-3 is involved in glycogen metabolism control and embryogenesis of Rhodnius prolixus.

    Science.gov (United States)

    Mury, Flávia B; Lugon, Magda D; DA Fonseca, Rodrigo Nunes; Silva, Jose R; Berni, Mateus; Araujo, Helena M; Fontenele, Marcio Ribeiro; Abreu, Leonardo Araujo DE; Dansa, Marílvia; Braz, Glória; Masuda, Hatisaburo; Logullo, Carlos

    2016-10-01

    Rhodnius prolixus is a blood-feeding insect that transmits Trypanosoma cruzi and Trypanosoma rangeli to vertebrate hosts. Rhodnius prolixus is also a classical model in insect physiology, and the recent availability of R. prolixus genome has opened new avenues on triatomine research. Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism, also acting as a downstream component of the Wnt pathway during embryogenesis. GSK-3 has been shown to be highly conserved among several organisms, mainly in the catalytic domain region. Meanwhile, the role of GSK-3 during R. prolixus embryogenesis or glycogen metabolism has not been investigated. Here we show that chemical inhibition of GSK-3 by alsterpaullone, an ATP-competitive inhibitor of GSK3, does not affect adult survival rate, though it alters oviposition and egg hatching. Specific GSK-3 gene silencing by dsRNA injection in adult females showed a similar phenotype. Furthermore, bright field and 4'-6-diamidino-2-phenylindole (DAPI) staining analysis revealed that ovaries and eggs from dsGSK-3 injected females exhibited specific morphological defects. We also demonstrate that glycogen content was inversely related to activity and transcription levels of GSK-3 during embryogenesis. Lastly, after GSK-3 knockdown, we observed changes in the expression of the Wingless (Wnt) downstream target β-catenin as well as in members of other pathways such as the receptor Notch. Taken together, our results show that GSK-3 regulation is essential for R. prolixus oogenesis and embryogenesis.

  14. Impairment of Cellulose Synthases Required for Arabidopsis Secondary Cell Wall Formation Enhances Disease Resistance[W

    Science.gov (United States)

    Hernández-Blanco, Camilo; Feng, Dong Xin; Hu, Jian; Sánchez-Vallet, Andrea; Deslandes, Laurent; Llorente, Francisco; Berrocal-Lobo, Marta; Keller, Harald; Barlet, Xavier; Sánchez-Rodríguez, Clara; Anderson, Lisa K.; Somerville, Shauna; Marco, Yves; Molina, Antonio

    2007-01-01

    Cellulose is synthesized by cellulose synthases (CESAs) contained in plasma membrane–localized complexes. In Arabidopsis thaliana, three types of CESA subunits (CESA4/IRREGULAR XYLEM5 [IRX5], CESA7/IRX3, and CESA8/IRX1) are required for secondary cell wall formation. We report that mutations in these proteins conferred enhanced resistance to the soil-borne bacterium Ralstonia solanacearum and the necrotrophic fungus Plectosphaerella cucumerina. By contrast, susceptibility to these pathogens was not altered in cell wall mutants of primary wall CESA subunits (CESA1, CESA3/ISOXABEN RESISTANT1 [IXR1], and CESA6/IXR2) or POWDERY MILDEW–RESISTANT5 (PMR5) and PMR6 genes. Double mutants indicated that irx-mediated resistance was independent of salicylic acid, ethylene, and jasmonate signaling. Comparative transcriptomic analyses identified a set of common irx upregulated genes, including a number of abscisic acid (ABA)–responsive, defense-related genes encoding antibiotic peptides and enzymes involved in the synthesis and activation of antimicrobial secondary metabolites. These data as well as the increased susceptibility of ABA mutants (abi1-1, abi2-1, and aba1-6) to R. solanacearum support a direct role of ABA in resistance to this pathogen. Our results also indicate that alteration of secondary cell wall integrity by inhibiting cellulose synthesis leads to specific activation of novel defense pathways that contribute to the generation of an antimicrobial-enriched environment hostile to pathogens. PMID:17351116

  15. Molecular cloning and expression of a novel trehalose synthase gene from Enterobacter hormaechei

    Directory of Open Access Journals (Sweden)

    Yue Ming

    2009-06-01

    Full Text Available Abstract Background Trehalose synthase (TreS which converts maltose to trehalose is considered to be a potential biocatalyst for trehalose production. This enzymatic process has the advantage of simple reaction and employs an inexpensive substrate. Therefore, new TreS producing bacteria with suitable enzyme properties are expected to be isolated from extreme environment. Results Six TreS producing strains were isolated from a specimen obtained from soil of the Tibetan Plateau using degenerate PCR. A novel treS gene from Enterobacter hormaechei was amplified using thermal asymmetric interlaced PCR. The gene contained a 1626 bp open reading frame encoding 541 amino acids. The gene was expressed in Escherichia coli, and the recombinant TreS was purified and characterized. The purified TreS had a molecular mass of 65 kDa and an activity of 18.5 U/mg. The optimum temperature and pH for the converting reaction were 37°C and 6, respectively. Hg2+, Zn2+, Cu2+and SDS inhibited the enzyme activity at different levels whereas Mn2+ showed an enhancing effect by 10%. Conclusion In this study, several TreS producing strains were screened from a source of soil bacteria. The characterization of the recombinant TreS of Enterobacter hormaechei suggested its potential application. Consequently, a strategy for isolation of TreS producing strains and cloning of novel treS genes from natural sources was demonstrated.

  16. Discovery of a compound that acts as a bacterial PyrG (CTP synthase) inhibitor.

    Science.gov (United States)

    Yoshida, Tatsuhiko; Nasu, Hatsumi; Namba, Eiko; Ubukata, Osamu; Yamashita, Makoto

    2012-09-01

    PyrG (CTP synthase) catalyses the conversion of UTP to CTP, an essential step in the pyrimidine metabolic pathway in a variety of bacteria, including those causing community-acquired respiratory tract infections (RTIs). In this study, a luminescence-based ATPase assay of PyrG was developed and used to evaluate the inhibitory activity of 2-(3-[3-oxo-1,2-benzisothiazol-2(3H)-yl]phenylsulfonylamino) benzoic acid (compound G1). Compound G1 inhibited PyrG derived from Streptococcus pneumoniae with a 50 % inhibitory concentration value of 0.091 µM, and the inhibitory activity of compound G1 was 13 times higher than that of acivicin (1.2 µM), an established PyrG inhibitor. The results of saturation transfer difference analysis using nuclear magnetic resonance spectroscopy suggested that these compounds compete with ATP and/or UTP for binding to Strep. pneumoniae PyrG. Finally, compound G1 was shown to have antimicrobial activity against several different bacteria causing RTIs, such as Staphylococcus aureus and Haemophilus influenzae, suggesting that it is a prototype chemical compound that could be harnessed as an antimicrobial drug with a novel structure to target bacterial PyrG.

  17. The Polymorphisms in Methylenetetrahydrofolate Reductase, Methionine Synthase, Methionine Synthase Reductase, and the Risk of Colorectal Cancer

    Science.gov (United States)

    Zhou, Daijun; Mei, Qiang; Luo, Han; Tang, Bo; Yu, Peiwu

    2012-01-01

    Polymorphisms in genes involved in folate metabolism may modulate the risk of colorectal cancer (CRC), but data from published studies are conflicting. The current meta-analysis was performed to address a more accurate estimation. A total of 41 (17,552 cases and 26,238 controls), 24(8,263 cases and 12,033 controls), 12(3,758 cases and 5,646 controls), and 13 (5,511 cases and 7,265 controls) studies were finally included for the association between methylenetetrahydrofolate reductase (MTHFR) C677T and A1289C, methione synthase reductase (MTRR) A66G, methionine synthase (MTR) A2756G polymorphisms and the risk of CRC, respectively. The data showed that the MTHFR 677T allele was significantly associated with reduced risk of CRC (OR = 0.93, 95%CI 0.90-0.96), while the MTRR 66G allele was significantly associated with increased risk of CRC (OR = 1.11, 95%CI 1.01-1.18). Sub-group analysis by ethnicity revealed that MTHFR C677T polymorphism was significantly associated with reduced risk of CRC in Asians (OR = 0.80, 95%CI 0.72-0.89) and Caucasians (OR = 0.84, 95%CI 0.76-0.93) in recessive genetic model, while the MTRR 66GG genotype was found to significantly increase the risk of CRC in Caucasians (GG vs. AA: OR = 1.18, 95%CI 1.03-1.36). No significant association was found between MTHFR A1298C and MTR A2756G polymorphisms and the risk of CRC. Cumulative meta-analysis showed no particular time trend existed in the summary estimate. Probability of publication bias was low across all comparisons illustrated by the funnel plots and Egger's test. Collectively, this meta-analysis suggested that MTHFR 677T allele might provide protection against CRC in worldwide populations, while MTRR 66G allele might increase the risk of CRC in Caucasians. Since potential confounders could not be ruled out completely, further studies were needed to confirm these results. PMID:22719222

  18. Engineering of plant type III polyketide synthases.

    Science.gov (United States)

    Wakimoto, Toshiyuki; Morita, Hiroyuki; Abe, Ikuro

    2012-01-01

    Members of the chalcone synthase superfamily of type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkable structural diversity and biological activities. The homodimeric type III PKSs share a common three-dimensional overall fold with a conserved Cys-His-Asn catalytic triad; notably, only a slight modification of the active site dramatically expands the catalytic repertoire of the enzymes. In addition, the enzymes exhibit extremely promiscuous substrate specificities, and accept a variety of nonphysiological substrates, making the type III PKSs an excellent platform for the further production of unnatural, novel polyketide scaffolds with promising biological activities. This chapter summarizes recent advances in the engineering of plant type III PKS enzymes in our laboratories, using approaches combining structure-based enzyme engineering and precursor-directed biosynthesis with rationally designed substrate analogs. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. 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.

  20. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. (Vanderbilt Univ., Nashville, TN (United States))

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  1. Inhibition of hydroxycinnamoyl-CoA thioesterases in ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.) by lipase inhibitors.

    Science.gov (United States)

    Flores-Sanchez, Isvett Josefina; Gang, David Roger

    2013-11-01

    Ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.), members of the Zingiberaceae, are widely used in traditional Asian cuisines and herbal medicine. Gingerols and diarylheptanoids, important compounds from these plants, appear to be produced by enzymes of the type III polyketide synthase class. Previous efforts to detect activity of such enzymes in tissues from these plants were only marginally successful in turmeric and completely unsuccessful in ginger because of very rapid hydrolysis of the hydroxycinnamoyl-CoA substrates (p-coumaroyl-CoA, feruloyl-CoA and caffeoyl-CoA) in these assays, presumably due to the presence of thioesterases in these tissues. In order to determine whether such thioesterase activities were specific and could be reduced so that the polyketide synthase activities could be better characterized, three inhibitors of the thioesterase domain of fatty acid synthase were tested in assays with leaf and rhizome crude protein extracts from these plants: orlistat, a reduced form of lipstatin, and peptide 1 and peptide 2 from hydrolysates of soybean β-conglycinin. Results of these analyses indicated that specific thioesterases do exist in these plants and that they could indeed be inhibited, with highest inhibition occurring with a mixture of these three compounds, leading for example to a reduction of caffeoyl-CoA hydrolysis in leaves and rhizomes of ginger by 40-fold and 27-fold, respectively. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  2. Ganoderic acid T inhibits tumor invasion in vitro and in vivo through inhibition of MMP expression.

    Science.gov (United States)

    Chen, Nian-Hong; Liu, Jian-Wen; Zhong, Jian-Jiang

    2010-01-01

    The traditional Chinese medicinal mushroom, Ganoderma lucidum, has been used in Asia for several thousand years for the prevention and treatment of a variety of diseases, including cancer. In previous work, we purified ganoderic acid T (GA-T) from G. lucidum [28]. In the present study, we investigate the functions of GA-T in terms of its effects on invasion in vitro and metastasis in vivo. A trypan blue dye exclusion assay indicates that GA-T inhibits proliferation of HCT-116 cells, a human colon carcinoma cell line. Cell aggregation and adhesion assays show that GA-T promotes homotypic aggregation and simultaneously inhibits the adhesion of HCT-116 cells to the extracellular matrix (ECM) in a dose-dependent manner.Wound healing assays indicate that GA-T also inhibits the migration of HCT-116 cells in a dose-dependent manner, and it suppresses the migration of 95-D cells, a highly metastatic human lung tumor cell line, in a dose- and time-dependent manner. In addition, GA-T inhibits the nuclear translocation of nuclear factor-kappaB (NF-kappaB) and the degradation of inhibitor of kappaB-alpha (IkappaBalpha), which leads to down-regulated expression of matrix metalloproteinase-9 (MMP-9), inducible nitric oxide synthase (iNOS), and urokinase-type plasminogen activator (uPA). Animal and Lewis Lung Carcinoma (LLC) model experiments demonstrate that GA-T suppresses tumor growth and LLC metastasis and down-regulates MMP-2 and MMP-9 mRNA expression in vivo. Taken together, these results demonstrate that GA-T effectively inhibits cancer cell invasion in vitro and metastasis in vivo, and thus it may act as a potential drug for treating cancer.

  3. 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.

  4. New Role for L-Arginine in Regulation of Inducible Nitric-Oxide-Synthase-Derived Superoxide Anion Production in Raw 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Michaela Pekarova

    2011-01-01

    Full Text Available Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O2∙- formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O2∙- and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O2∙- soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O2∙- formation in inflammatory macrophages.

  5. New role for L-arginine in regulation of inducible nitric-oxide-synthase-derived superoxide anion production in raw 264.7 macrophages.

    Science.gov (United States)

    Pekarova, Michaela; Lojek, Antonin; Martiskova, Hana; Vasicek, Ondrej; Bino, Lucia; Klinke, A; Lau, D; Kuchta, Radek; Kadlec, Jaroslav; Vrba, Radimir; Kubala, Lukas

    2011-01-01

    Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O(2)(∙-) formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O(2)(∙-) and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O(2)(∙-) soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O(2) (∙-) formation in inflammatory macrophages.

  6. New Role for L-Arginine in Regulation of Inducible Nitric-Oxide-Synthase-Derived Superoxide Anion Production in Raw 264.7 Macrophages

    Science.gov (United States)

    Pekarova, Michaela; Lojek, Antonin; Martiskova, Hana; Vasicek, Ondrej; Bino, Lucia; Klinke, A.; Lau, D.; Kuchta, Radek; Kadlec, Jaroslav; Vrba, Radimir; Kubala, Lukas

    2011-01-01

    Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O2∙− formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O2∙− and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O2∙− soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O2∙− formation in inflammatory macrophages. PMID:22219714

  7. 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.

  8. Proposed mechanism for diterpene synthases in the formation of phomactatriene and taxadiene.

    Science.gov (United States)

    Tokiwano, Tetsuo; Endo, Taeko; Tsukagoshi, Tae; Goto, Hitoshi; Fukushi, Eri; Oikawa, Hideaki

    2005-08-07

    To obtain insight into how the cyclization pathway is controlled, the mechanism of diterpene synthase reactions (the putative phomactatriene synthase and taxadiene synthases) involving the same intermediate was investigated in detail. The mechanism of the initial transformation of GGDP to verticillen-12-yl cation (A+) was proposed based on the labelling pattern of phomactatriene (9a) obtained in the feeding experiments with 13C-labelled acetates. To obtain information on the reaction pathway of A+ to 9a and taxadiene, reactions of verticillol with various acids were conducted. Structural determination of products allowed us to propose a reaction pathway via cations A+, D+, E+, F+ and G+. Identification of hydrocarbons in mycelial extracts of phomactin-producing fungus supported the proposed reaction mechanism. Based on the results of ab initio calculations for highly flexible cation intermediates, a mechanism is proposed.

  9. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

    The R and S enantionmers of S-adenosyl-3-(/sup 2/H)3-(methylthio)-1-propylamine (decarboxylated S-adenosylmethionine), previously synthesized in this laboratory, were incubated with (1,4-/sup 2/H/sub 4/)-putrescine in the presence of spermidine synthase from E. coli. The resulting chiral (/sup 2/H/sub 5/)spermidines were isolated and converted to their N/sub 1/,N/sub 7/-dibocspermidine-N/sub 4/-(1S,4R)-camphanamides. The derivatives were analyzed by 500 MHz /sup 1/H-NMR and the configuration of the chiral center assigned by correlation with the spectra of synthetic chiral (/sup 2/H/sub 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/sub 3/) in all cases studied to date.

  11. Tomato linalool synthase is induced in trichomes by jasmonic acid

    Science.gov (United States)

    van Schie, Chris C. N.; Haring, Michel A.

    2007-01-01

    Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato. PMID:17440821

  12. Effects of small interfering RNA targeting thymidylate synthase on survival of ACC3 cells from salivary adenoid cystic carcinoma

    International Nuclear Information System (INIS)

    Shirasaki, Takashi; Maruya, Shin-ichiro; Mizukami, Hiroki; Kakehata, Seiji; Kurotaki, Hidekachi; Yagihashi, Soroku; Shinkawa, Hideichi

    2008-01-01

    Thymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer and high expression of TS has been associated with poor prognosis or refractory disease in several cancers including colorectal and head and neck cancer. Although TS is known to regulate cell cycles and transcription factors, its potency as a therapeutic target has not been fully explored in adenoid cystic carcinoma (ACC). An ACC cell line (ACC3) was transfected with siRNA targeting the TS gene and inhibition of cell growth and induction of apoptosis-associated molecules were evaluated in vitro. In addition, the in vivo effect of TS siRNA on tumor progression was assessed using a xenograft model. Our results demonstrated that ACC3 cells showed significantly higher TS expression than non-cancer cell lines and the induction of TS siRNA led to inhibition of cell proliferation. The effect was associated with an increase in p53, p21, and active caspase-3 and S-phase accumulation. We also found up-regulation of spermidine/spermine N1-acetyltransferase (SSAT), a polyamine metabolic enzyme. Furthermore, treatment with TS siRNA delivered by atelocollagen showed a significant cytostatic effect through the induction of apoptosis in a xenograft model. TS may be an important therapeutic target and siRNA targeting TS may be of potential therapeutic value in ACC

  13. Molecular and phenotypic characterization of Als1 and Als2 mutations conferring tolerance to acetolactate synthase herbicides in soybean

    Science.gov (United States)

    Walter, Kay L; Strachan, Stephen D; Ferry, Nancy M; Albert, Henrik H; Castle, Linda A; Sebastian, Scott A

    2014-01-01

    BACKGROUND Sulfonylurea (SU) herbicides are effective because they inhibit acetolactate synthase (ALS), a key enzyme in branched-chain amino acid synthesis required for plant growth. A soybean line known as W4-4 was developed through rounds of seed mutagenesis and was demonstrated to have a high degree of ALS-based resistance to both post-emergence and pre-emergence applications of a variety of SU herbicides. This report describes the molecular and phenotypic characterization of the Als1 and Als2 mutations that confer herbicide resistance to SUs and other ALS inhibitors. RESULTS The mutations are shown to occur in two different ALS genes that reside on different chromosomes: Als1 (P178S) on chromosome 4 and Als2 (W560L) on chromosome 6 (P197S and W574L in Arabidopsis thaliana). CONCLUSION Although the Als1 and Als2 genes are unlinked, the combination of these two mutations is synergistic for improved tolerance of soybeans to ALS-inhibiting herbicides. © 2014 DuPont Pioneer. Pest Management Science published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:24425499

  14. (+)-(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.

  15. Momilactone B Inhibits Ketosis In Vitro by Regulating the ANGPTL3-LPL Pathway and Inhibiting HMGCS2.

    Science.gov (United States)

    Kang, Dong Young; S P, Nipin; Darvin, Pramod; Joung, Youn Hee; Byun, Hyo Joo; Do, Chang Hee; Park, Kyung Do; Park, Mi Na; Cho, Kwang Hyun; Yang, Young Mok

    2017-07-03

    Ketogenesis is the production of ketone bodies, which provide energy when the body lacks glucose. Under ketogenic conditions, the body switches from primarily carbohydrate to fat metabolism to maintain energy balance. However, accumulation of high levels of ketone bodies in the blood results in ketosis. Treating ketosis with natural substances is preferable, because they are unlikely to cause side-effects. Momilactone B is an active compound isolated from Korean rice. Based on previous studies, we hypothesized that momilactone B could inhibit ketosis. We constructed an in vitro ketosis model by glucose starvation. We used this model to test the anti-ketosis effects of momilactone B. A primary target for treating ketosis is angiopoietin-like-3 (ANGPTL3), which modulates lipoprotein metabolism by inhibiting lipoprotein lipase (LPL), a multifunctional enzyme that breaks down stored fat to produce triglycerides. We showed that momilactone B could regulate the ANGPTL3-LPL pathway. However, a strong anti-ketosis candidate drug should also inhibit ketogenesis. Ketogenesis can be suppressed by inhibiting the expression of 3-hydroxy-3-methylglutaryl-CoA synthase-2 (HMGCS2), a mitochondrial enzyme that converts acetyl-CoA to ketone bodies. We found that momilactone B suppressed the expression of HMGCS2 through the increased expression of STAT5b. We also elucidated the relationship of STAT5b to ANGPTL3 and LPL expression.

  16. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney Bruce [Pullman, WA; Burke, Charles Cullen [Moscow, ID

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  17. Assessment of acetolactate synthase (ALS) tolerance to imazethapyr in red rice ecotypes (Oryza spp) and imidazolinone tolerant/ resistant rice (Oryza sativa) varieties.

    Science.gov (United States)

    Avila, Luis A; Lee, Do-Jin; Senseman, Scott A; McCauley, Garry N; Chandler, James M; Cothren, J Tom

    2005-02-01

    Three red rice ecotypes (Oryza spp), including LA 5, MS 5 and TX 4, were evaluated for acetolactate synthase resistance/tolerance to imazethapyr. The red rice ecotypes were compared with a tolerant line (CL-121), a resistant line (CL-161) and a conventional rice variety (Cypress). Based on enzymatic activity, the mean I(50) values were 1.5, 1.1, 1.5, 1.6, 20.8 and 590.6 microM imazethapyr, respectively, for LA 5, MS 5, TX 4, Cypress, CL-121 and CL-161. CL-161 was 32 times more resistant than CL-121 and at least 420 times more resistant than the average of the red rice ecotypes and Cypress. Results from the acetolactate synthase (ALS) assay showed that red rice ecotypes and Cypress had high susceptibility to imazethapyr when compared with the tolerant CL-121 and the resistant CL-161. Measurable enzymatic tolerance to ALS-inhibiting herbicides has not yet developed in these red rice ecotypes. Copyright (c) 2005 Society of Chemical Industry.

  18. Suppression by Ghrelin of Porphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells

    Directory of Open Access Journals (Sweden)

    Bronislaw L. Slomiany

    2010-01-01

    Full Text Available Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS. We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

  19. Prenatal Nicotine Exposure Results in the Inhibition of Baroreflex Sensitivity Induced by Intravenous Injection Angiotensin II in the Adult Male Offspring Rats.

    Science.gov (United States)

    Yu, Feng; Li, Yuexia; Yang, Jian; Qian, Jin; Li, Xining; Liu, Chongbin

    2017-04-01

    Epidemiological studies show that maternal cigarette smoking is associated with an increased risk of cardiovascular diseases in postnatal life. Baroreflex sensitivity (BRS) is an important index for evaluating the homeostasis of the cardiovascular system. This experiment was designed to investigate the possible mechanism of prenatal nicotine on the adult male offspring's heart rate (HR) increase due to BRS. Pregnant rats received the 0.3 ml of saline or nicotine (1.5 mg kg -1 ) by subcutaneous injection from gestational days 3 to 21. The male offsprings of saline injected dams were the control group, and the male offsprings of the nicotine injected dams were the nicotine group. The 90-day-old male offsprings' funny current (I f ) of their sinoatrial node (SAN) cells, BRS induced by intravenous injection of angiotensin (Ang) II in the presence or absence of the L-nitro-arginine methylester (L-NAME), cervical vagal activity, c-fos protein levels of the cervical spinal cord-8 to the thoracic spinal cord-5 (C 8 -T 5 ) lateral horn neuron, and blood hormones were tested. The results showed that prenatal nicotine exposure had no effect on the offsprings' I f of their SAN cells, but it significantly decreased the offsprings' BRS. The c-fos protein levels of the C 8 -T 5 lateral horn neurons and the blood catecholamine levels were increased in the nicotine group, but the cervical vagal activity was not changed. After intraventricular injection of L-NAME, the nicotine exposed offsprings' BRS was partly recovered. These data suggest that prenatal nicotine exposure results in hyper reactivity of the spinal sympathetic nerve center and a higher peripheral catecholamine hormone state of 90-day-old male offsprings, and these may be the reason for the BRS inhibition and HR increase. Nitric oxide (NO) may participate in the process acting as an important neurotransmitter.

  20. 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.

  1. 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

  2. Divinyl ether synthase gene and protein, and uses thereof

    Science.gov (United States)

    Howe, Gregg A [East Lansing, MI; Itoh, Aya [Tsuruoka, JP

    2011-09-13

    The present invention relates to divinyl ether synthase genes, proteins, and methods of their use. The present invention encompasses both native and recombinant wild-type forms of the synthase, as well as mutants and variant forms, some of which possess altered characteristics relative to the wild-type synthase. The present invention also relates to methods of using divinyl ether synthase genes and proteins, including in their expression in transgenic organisms and in the production of divinyl ether fatty acids, and to methods of suing divinyl ether fatty acids, including in the protection of plants from pathogens.

  3. Kinetics of reversible reductive carbonylation of heme in human cystathionine β-synthase.

    Science.gov (United States)

    Carballal, Sebastián; Cuevasanta, Ernesto; Marmisolle, Inés; Kabil, Omer; Gherasim, Carmen; Ballou, David P; Banerjee, Ruma; Alvarez, Beatriz

    2013-07-02

    Cystathionine β-synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. In addition to pyridoxal phosphate, human CBS has a heme cofactor with cysteine and histidine as ligands. While Fe(III)-CBS is inert to exogenous ligands, Fe(II)-CBS can be reversibly inhibited by carbon monoxide (CO) and reoxidized by O2 to yield superoxide radical. In this study, we have examined the kinetics of Fe(II)CO-CBS formation and reoxidation. Reduction of Fe(III)-CBS by dithionite showed a square root dependence on concentration, indicating that the reductant species was the sulfur dioxide radical anion (SO2(•-)) that exists in rapid equilibrium with S2O4(2-). Formation of Fe(II)CO-CBS from Fe(II)-CBS and 1 mM CO occurred with a rate constant of (3.1 ± 0.4) × 10(-3) s(-1) (pH 7.4, 25 °C). The reaction of Fe(III)-CBS with the reduced form of the flavoprotein methionine synthase reductase in the presence of CO and NADPH resulted in its reduction and carbonylation to form Fe(II)CO-CBS. Fe(II)-CBS was formed as an intermediate with a rate constant of (9.3 ± 2.5) × 10(2) M(-1) s(-1). Reoxidation of Fe(II)CO-CBS by O2 was multiphasic. The major phase showed a hyperbolic dependence on O2 concentration. Although H2S is a product of the CBS reaction and a potential heme ligand, we did not find evidence of an effect of exogenous H2S on activity or heme binding. Reversible reduction of CBS by a physiologically relevant oxidoreductase is consistent with a regulatory role for the heme and could constitute a mechanism for cross talk among the CO, H2S, and superoxide signaling pathways.

  4. Expression pattern and biochemical properties of zebrafish N-acetylglutamate synthase.

    Directory of Open Access Journals (Sweden)

    Ljubica Caldovic

    Full Text Available The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS, carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1 catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C. Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Km(app for acetyl coenzyme A increased while the Km(app for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under

  5. 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 ...

  6. 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...

  7. Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene.

    NARCIS (Netherlands)

    Nijtmans, L.G.J.; Henderson, N.S.; Attardi, G.; Holt, L.J.

    2001-01-01

    Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome.

  8. Studies on the expression of sesquiterpene synthases using promoter-β-glucuronidase fusions in transgenic Artemisia annua L.

    Directory of Open Access Journals (Sweden)

    Hongzhen Wang

    Full Text Available In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS, epi-cedrol (ECS and β-farnesene (FS synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS, a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

  9. Studies on the expression of sesquiterpene synthases using promoter-β-glucuronidase fusions in transgenic Artemisia annua L.

    Science.gov (United States)

    Wang, Hongzhen; Han, Junli; Kanagarajan, Selvaraju; Lundgren, Anneli; Brodelius, Peter E

    2013-01-01

    In order to better understand the influence of sesquiterpene synthases on artemisinin yield in Artemisia annua, the expression of some sesquiterpene synthases has been studied using transgenic plants expressing promoter-GUS fusions. The cloned promoter sequences were 923, 1182 and 1510 bp for β-caryophyllene (CPS), epi-cedrol (ECS) and β-farnesene (FS) synthase, respectively. Prediction of cis-acting regulatory elements showed that the promoters are involved in complex regulation of expression. Transgenic A. annua plants carrying promoter-GUS fusions were studied to elucidate the expression pattern of the three sesquiterpene synthases and compared to the previously studied promoter of amorpha-4,11-diene synthase (ADS), a key enzyme of artemisinin biosynthesis. The CPS and ECS promoters were active in T-shaped trichomes of leaves and stems, basal bracts of flower buds and also in some florets cells but not in glandular secretory trichome while FS promoter activity was only observed in leaf cells and trichomes of transgenic shoots. ADS, CPS, ECS and FS transcripts were induced by wounding in a time depended manner. The four sesquiterpene synthases may be involved in responsiveness of A. annua to herbivory. Methyl jasmonate treatment triggered activation of the promoters of all four sesquiterpene synthases in a time depended manner. Southern blot result showed that the GUS gene was inserted into genomic DNA of transgenic lines as a single copy or two copies. The relative amounts of CPS and ECS as well as germacrene A synthase (GAS) transcripts are much lower than that of ADS transcript. Consequently, down-regulation of the expression of the CPS, ECS or GAS gene may not improve artemsinin yield. However, blocking the expression of FS may have effects on artemisinin production.

  10. Antioxidant biomarkers from Vanda coerulea stems reduce irradiated HaCaT PGE-2 production as a result of COX-2 inhibition.

    Science.gov (United States)

    Simmler, Charlotte; Antheaume, Cyril; Lobstein, Annelise

    2010-10-28

    In our investigations towards the isolation of potentially biologically active constituents from Orchidaceae, we carried out phytochemical and biological analyses of Vanda species. A preliminary biological screening revealed that Vanda coerulea (Griff. ex. Lindl) crude hydro-alcoholic stem extract displayed the best DPPH /(•)OH radical scavenging activity and in vitro inhibition of type 2 prostaglandin (PGE-2) release from UV(B) (60 mJ/cm(2)) irradiated HaCaT keratinocytes. Bio-guided fractionation and phytochemical analysis led to the isolation of five stilbenoids: imbricatin (1) methoxycoelonin (2) gigantol (3) flavidin (4) and coelonin (5). Stilbenoids (1-3) were the most concentrated in crude hydro-alcoholic stem extract and were considered as Vanda coerulea stem biomarkers. Dihydro-phenanthropyran (1) and dihydro-phenanthrene (2) displayed the best DPPH/(•)OH radical scavenging activities as well as HaCaT intracellular antioxidant properties (using DCFH-DA probe: IC(50) 8.8 µM and 9.4 µM, respectively) compared to bibenzyle (3) (IC(50) 20.6 µM). In turn, the latter showed a constant inhibition of PGE-2 production, stronger than stilbenoids (1) and (2) (IC(50) 12.2 µM and 19.3 µM, respectively). Western blot analysis revealed that stilbenoids (1-3) inhibited COX-2 expression at 23 µM. Interestingly, stilbenoids (1) and (2) but not (3) were able to inhibit human recombinant COX-2 activity. Major antioxidant stilbenoids (1-3) from Vanda coerulea stems displayed an inhibition of UV(B)-induced COX-2 expression. Imbricatin (1) and methoxycoelonin (2) were also able to inhibit COX-2 activity in a concentration-dependent manner thereby reducing PGE-2 production from irradiated HaCaT cells. Our studies suggest that stilbenoids (1-3) could be potentially used for skin protection against the damage caused by UV(B) exposure.

  11. Primary clear cell renal carcinoma cells display minimal mitochondrial respiratory capacity resulting in pronounced sensitivity to glycolytic inhibition by 3-Bromopyruvate.

    Science.gov (United States)

    Nilsson, H; Lindgren, D; Mandahl Forsberg, A; Mulder, H; Axelson, H; Johansson, M E

    2015-01-08

    Changes of cellular metabolism are an integral property of the malignant potential of most cancer cells. Already in the 1930s, Otto Warburg observed that tumor cells preferably utilize glycolysis and lactate fermentation for energy production, rather than the mitochondrial oxidative phosphorylation dominating in normal cells, a phenomenon today known as the Warburg effect. Even though many tumor types display a high degree of aerobic glycolysis, they still retain the activity of other energy-producing metabolic pathways. One exception seems to be the clear cell variant of renal cell carcinoma, ccRCC, where the activity of most other pathways than that of glycolysis has been shown to be reduced. This makes ccRCC a promising candidate for the use of glycolytic inhibitors in treatment of the disease. However, few studies have so far addressed this issue. In this report, we show a strikingly reduced mitochondrial respiratory capacity of primary human ccRCC cells, resulting in enhanced sensitivity to glycolytic inhibition by 3-Bromopyruvate (3BrPA). This effect was largely absent in established ccRCC cell lines, a finding that highlights the importance of using biologically relevant models in the search for new candidate cancer therapies. 3BrPA markedly reduced ATP production in primary ccRCC cells, followed by cell death. Our data suggest that glycolytic inhibitors such as 3BrPA, that has been shown to be well tolerated in vivo, should be further analyzed for the possible development of selective treatment strategies for patients with ccRCC.

  12. 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

  13. Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts...

  14. Modified cellulose synthase gene from 'Arabidopsis thaliana' confers herbicide resistance to plants

    Energy Technology Data Exchange (ETDEWEB)

    Somerville, Chris R.; Scieble, Wolf

    2000-10-11

    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.

  15. Examination of thromboxane synthase as a prognostic factor and therapeutic target in non-small cell lung cancer.

    LENUS (Irish Health Repository)

    Cathcart, Mary-Clare

    2011-03-01

    Thromboxane synthase (TXS) metabolises prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with a poor prognosis. TXS inhibition induces cell death in-vitro, providing a rationale for therapeutic intervention. We aimed to determine the expression profile of TXS in NSCLC and if it is prognostic and\\/or a survival factor in the disease.

  16. Salinomycin enhances cisplatin-induced cytotoxicity in human lung cancer cells via down-regulation of AKT-dependent thymidylate synthase expression.

    Science.gov (United States)

    Ko, Jen-Chung; Zheng, Hao-Yu; Chen, Wen-Ching; Peng, Yi-Shuan; Wu, Chia-Hung; Wei, Chia-Li; Chen, Jyh-Cheng; Lin, Yun-Wei

    2016-12-15

    Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2μg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction.

    Science.gov (United States)

    Haagsma, Anna C; Driessen, Nicole N; Hahn, Marc-Manuel; Lill, Holger; Bald, Dirk

    2010-12-01

    ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ɛ and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  18. 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.

  19. Phosphorylation Regulates myo-Inositol-3-phosphate Synthase

    Science.gov (United States)

    Deranieh, Rania M.; He, Quan; Caruso, Joseph A.; Greenberg, Miriam L.

    2013-01-01

    myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using 32Pi labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS. PMID:23902760

  20. Treadmill exercise decreases incidence of Alzheimer’s disease by suppressing glycogen synthase kinase-3β expression in streptozotocin-induced diabetic rats

    Science.gov (United States)

    Kim, Dae-Young; Jung, Sun-Young; Kim, Tae-Woon; Lee, Kwang-Sik; Kim, Kijeong

    2015-01-01

    Diabetes is a metabolic disorder, and it is considered as a major risk factor for Alzheimer’s disease (AD). In the present study, we evaluated whether treadmill exercise ameliorates progression of AD in relation with glycogen synthase kinase-3β (GSK-3β) activity using streptozotocin (STZ)-induced diabetic rats. For this study, step-down avoidance task, immunohistochemistry for glycogen synthase kinase-3β (GSK-3β) and tau, and western blot for phosphor-phosphoinositide 3 kinase (p-PI3K)/PI3K and phosphor-Akt (p-Akt)/Akt were performed. Diabetes mellitus was induced by intraperitoneal injection of STZ. The rats in the exercise groups were made to run on the treadmill for 30 min per one day, five times a week, during 12 weeks. The present results showed that short-term and long-term latencies in the step-down avoidance task were decreased by induction of diabetes, and treadmill exercise inhibited these latencies in the diabetic rats. Induction of diabetes suppressed the ratio of p-PI3K to PI3K and the ratio of p-Akt to Akt, and treadmill exercise increased these ratios in the diabetic rats. The numbers of GSK-3β-positive and tau-positive cells in the hippocampal dentate gyrus was higher in the diabetes-induction group than that in the control group, and treadmill exercise inhibited these numbers in the diabetic rats. In the present study, treadmill exercise suppressed hyperphosphorylation of tau in the hippocampus by decreased GSK-3β activity through PI3K/Akt pathway activation in the diabetic rats. Based on the present results, treadmill exercise may helpful to prevent diabetes-associated AD occurrence. PMID:25960981

  1. Inhibition of Intestinal OATP2B1 by the Calcium Receptor Antagonist Ronacaleret Results in a Significant Drug-Drug Interaction by Causing a 2-Fold Decrease in Exposure of Rosuvastatin.

    Science.gov (United States)

    Johnson, Marta; Patel, Dipal; Matheny, Christopher; Ho, May; Chen, Liangfu; Ellens, Harma

    2017-01-01

    Rosuvastatin is a widely prescribed antihyperlipidemic which undergoes limited metabolism, but is an in vitro substrate of multiple transporters [organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP1A2, OATP2B1, sodium-taurocholate cotransporting polypeptide, breast cancer resistance protein (BCRP), multidrug resistance protein 2 (MRP2), MRP4, organic anion transporter 3]. It is therefore frequently used as a probe substrate in clinical drug-drug interaction (DDI) studies to investigate transporter inhibition. Although each of these transporters is believed to play a role in rosuvastatin disposition, multiple pharmacogenetic studies confirm that OATP1B1 and BCRP play an important role in vivo. Ronacaleret, a drug-development candidate for treatment of osteoporosis (now terminated), was shown to inhibit OATP1B1 in vitro (IC 50 = 11 µM), whereas it did not inhibit BCRP. Since a DDI risk through inhibition of OATP1B1 could not be discharged, a clinical DDI study was performed with rosuvastatin before initiation of phase II trials. Unexpectedly, coadministration with ronacaleret decreased rosuvastatin exposure by approximately 50%, whereas time of maximal plasma concentration and terminal half-life remained unchanged, suggesting decreased absorption and/or enhanced first-pass elimination of rosuvastatin. Of the potential in vivo rosuvastatin transporter pathways, two might explain the observed results: intestinal OATP2B1 and hepatic MRP4. Further investigations revealed that ronacaleret inhibited OATP2B1 (in vitro IC 50 = 12 µM), indicating a DDI risk through inhibition of absorption. Ronacaleret did not inhibit MRP4, discharging the possibility of enhanced first-pass elimination of rosuvastatin (reduced basolateral secretion from hepatocytes into blood). Therefore, a likely mechanism of the observed DDI is inhibition of intestinal OATP2B1, demonstrating the in vivo importance of this transporter in rosuvastatin absorption in humans. Copyright © 2016

  2. Process for inhibiting the growth of a culture of lactic acid bacteria, and optionally lysing the bacterial cells, and uses of the resulting lysed culture

    NARCIS (Netherlands)

    Nauta, Arjen; Venema, Gerard; Kok, Jan; Ledeboer, Aat M.

    1995-01-01

    The invention provides a process for inhibiting the growth of a culture of lactic acid bacteria, or a product containing such culture e.g. a cheese product, in which in the cells of the lactic acid bacteria a holin obtainable from bacteriophages of Gram-positive bacteria, esp. from bacteriophages of

  3. Uncovering the structures of modular polyketide synthases.

    Science.gov (United States)

    Weissman, Kira J

    2015-03-01

    The modular polyketide synthases (PKSs) are multienzyme proteins responsible for the assembly of diverse secondary metabolites of high economic and therapeutic importance. These molecular 'assembly lines' consist of repeated functional units called 'modules' organized into gigantic polypeptides. For several decades, concerted efforts have been made to understand in detail the structure and function of PKSs in order to facilitate genetic engineering of the systems towards the production of polyketide analogues for evaluation as drug leads. Despite this intense activity, it has not yet been possible to solve the crystal structure of a single module, let alone a multimodular subunit. Nonetheless, on the basis of analysis of the structures of modular fragments and the study of the related multienzyme of animal fatty acid synthase (FAS), several models of modular PKS architecture have been proposed. This year, however, the situation has changed - three modular structures have been characterized, not by X-ray crystallography, but by the complementary methods of single-particle cryo-electron microscopy and small-angle X-ray scattering. This review aims to compare the cryo-EM structures and SAXS-derived structural models, and to interpret them in the context of previously obtained data and existing architectural proposals. The consequences for genetic engineering of the systems will also be discussed, as well as unresolved questions and future directions.

  4. Pharmacological inhibition of Polo Like Kinase 2 (PLK2) does not cause chromosomal damage or result in the formation of micronuclei

    Energy Technology Data Exchange (ETDEWEB)

    Fitzgerald, Kent, E-mail: Kent.fitzgerald@elan.com [Pharmacological Sciences, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Bergeron, Marcelle, E-mail: Marcelle.bergeron@elan.com [Pharmacological Sciences, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Willits, Christopher, E-mail: Chris.willits@elan.com [Pharmacological Sciences, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Bowers, Simeon, E-mail: Simeon.bowers@elan.com [Chemistry, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Aubele, Danielle L., E-mail: Danielle.aubele@elan.com [Chemistry, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Goldbach, Erich, E-mail: Erich.goldbach@elan.com [Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Tonn, George, E-mail: George.tonn@elan.com [Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Ness, Daniel, E-mail: Dan.ness@elan.com [Nonclinical Safety Evaluation, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States); Olaharski, Andrew, E-mail: andrew.olaharski@agios.com [Nonclinical Safety Evaluation, Elan Pharmaceuticals Inc., 180 Oyster Point Boulevard, South San Francisco, CA 94080 (United States)

    2013-05-15

    Polo Like Kinase 2 (PLK2) phosphorylates α-synuclein and is considered a putative therapeutic target for Parkinson's disease. Several lines of evidence indicate that PLK2 is involved with proper centriole duplication and cell cycle regulation, inhibition of which could impact chromosomal integrity during mitosis. The objectives of the series of experiments presented herein were to assess whether specific inhibition of PLK2 is genotoxic and determine if PLK2 could be considered a tractable pharmacological target for Parkinson's disease. Several selective PLK2 inhibitors, ELN 582175 and ELN 582646, and their inactive enantiomers, ELN 582176 and ELN 582647, did not significantly increase the number of micronuclei in the in vitro micronucleus assay. ELN 582646 was administered to male Sprague Dawley rats in an exploratory 14-day study where flow cytometric analysis of peripheral blood identified a dose-dependent increase in the number of micronucleated reticulocytes. A follow-up investigative study demonstrated that ELN 582646 administered to PLK2 deficient and wildtype mice significantly increased the number of peripheral micronucleated reticulocytes in both genotypes, suggesting that ELN 582646-induced genotoxicity is not through the inhibition of PLK2. Furthermore, significant reduction of retinal phosphorylated α-synuclein levels was observed at three non-genotoxic doses, additional data to suggest that pharmacological inhibition of PLK2 is not the cause of the observed genotoxicity. These data, in aggregate, indicate that PLK2 inhibition is a tractable CNS pharmacological target that does not cause genotoxicity at doses and exposures that engage the target in the sensory retina. - Highlights: • Active and inactive enantiomers test negative in the in vitro micronucleus test. • ELN 582646 significantly increased micronuclei at 100 and 300 mg/kg/day doses. • ELN 582646 significantly increased micronuclei in PLK2 knockout mice. • ELN 582646

  5. Pharmacological inhibition of Polo Like Kinase 2 (PLK2) does not cause chromosomal damage or result in the formation of micronuclei

    International Nuclear Information System (INIS)

    Fitzgerald, Kent; Bergeron, Marcelle; Willits, Christopher; Bowers, Simeon; Aubele, Danielle L.; Goldbach, Erich; Tonn, George; Ness, Daniel; Olaharski, Andrew

    2013-01-01

    Polo Like Kinase 2 (PLK2) phosphorylates α-synuclein and is considered a putative therapeutic target for Parkinson's disease. Several lines of evidence indicate that PLK2 is involved with proper centriole duplication and cell cycle regulation, inhibition of which could impact chromosomal integrity during mitosis. The objectives of the series of experiments presented herein were to assess whether specific inhibition of PLK2 is genotoxic and determine if PLK2 could be considered a tractable pharmacological target for Parkinson's disease. Several selective PLK2 inhibitors, ELN 582175 and ELN 582646, and their inactive enantiomers, ELN 582176 and ELN 582647, did not significantly increase the number of micronuclei in the in vitro micronucleus assay. ELN 582646 was administered to male Sprague Dawley rats in an exploratory 14-day study where flow cytometric analysis of peripheral blood identified a dose-dependent increase in the number of micronucleated reticulocytes. A follow-up investigative study demonstrated that ELN 582646 administered to PLK2 deficient and wildtype mice significantly increased the number of peripheral micronucleated reticulocytes in both genotypes, suggesting that ELN 582646-induced genotoxicity is not through the inhibition of PLK2. Furthermore, significant reduction of retinal phosphorylated α-synuclein levels was observed at three non-genotoxic doses, additional data to suggest that pharmacological inhibition of PLK2 is not the cause of the observed genotoxicity. These data, in aggregate, indicate that PLK2 inhibition is a tractable CNS pharmacological target that does not cause genotoxicity at doses and exposures that engage the target in the sensory retina. - Highlights: • Active and inactive enantiomers test negative in the in vitro micronucleus test. • ELN 582646 significantly increased micronuclei at 100 and 300 mg/kg/day doses. • ELN 582646 significantly increased micronuclei in PLK2 knockout mice. • ELN 582646 decreased

  6. 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.

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

    DEFF Research Database (Denmark)

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

    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...

  8. 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 ...

  9. 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.

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

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i...

  11. Characterization of α-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Palittapongarnpim Prasit

    2009-06-01

    Full Text Available Abstract Background Alpha-isopropylmalate synthase (α-IPMS is the key enzyme that catalyzes the first committed step in the leucine biosynthetic pathway. The gene encoding α-IPMS in Mycobacterium tuberculosis, leuA, is polymorphic due to the insertion of 57-bp repeat units referred to as Variable Number of Tandem Repeats (VNTR. The role of the VNTR found within the M. tuberculosis genome is unclear. To investigate the role of the VNTR in leuA, we compared two α-IPMS proteins with different numbers of amino acid repeats, one with two copies and the other with 14 copies. We have cloned leuA with 14 copies of the repeat units into the pET15b expression vector with a His6-tag at the N-terminus, as was previously done for the leuA gene with two copies of the repeat units. Results The recombinant His6-α-IPMS proteins with two and 14 copies (α-IPMS-2CR and α-IPMS-14CR, respectively of the repeat units were purified by immobilized metal ion affinity chromatography and gel filtration. Both enzymes were found to be dimers by gel filtration. Both enzymes work well at pH values of 7–8.5 and temperatures of 37–42°C. However, α-IPMS-14CR tolerates pH values and temperatures outside of this range better than α-IPMS-2CR does. α-IPMS-14CR has higher affinity than α-IPMS-2CR for the two substrates, α-ketoisovalerate and acetyl CoA. Furthermore, α-IPMS-2CR was feedback inhibited by the end product l-leucine, whereas α-IPMS-14CR was not. Conclusion The differences in the kinetic properties and the l-leucine feedback inhibition between the two M. tuberculosis α-IPMS proteins containing low and high numbers of VNTR indicate that a large VNTR insertion affects protein structure and function. Demonstration of l-leucine binding to α-IPMS-14CR would confirm whether or not α-IPMS-14CR responds to end-product feedback inhibition.

  12. Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs.

    Science.gov (United States)

    Grasemann, Hartmut; Dhaliwal, Rupinder; Ivanovska, Julijana; Kantores, Crystal; McNamara, Patrick J; Scott, Jeremy A; Belik, Jaques; Jankov, Robert P

    2015-03-15

    Arginase is an enzyme that limits substrate L-arginine bioavailability for the production of nitric oxide by the nitric oxide synthases and produces L-ornithine, which is a precursor for collagen formation and tissue remodeling. We studied the pulmonary vascular effects of arginase inhibition in an established model of repeated systemic bleomycin sulfate administration in neonatal rats that results in pulmonary hypertension and lung injury mimicking the characteristics typical of bronchopulmonary dysplasia. We report that arginase expression is increased in the lungs of bleomycin-exposed neonatal rats and that treatment with the arginase inhibitor amino-2-borono-6-hexanoic acid prevented the bleomycin-induced development of pulmonary hypertension and deposition of collagen. Arginase inhibition resulted in increased L-arginine and L-arginine bioavailability and increased pulmonary nitric oxide production. Arginase inhibition also normalized the expression of inducible nitric oxide synthase, and reduced bleomycin-induced nitrative stress while having no effect on bleomycin-induced inflammation. Our data suggest that arginase is a promising target for therapeutic interventions in neonates aimed at preventing lung vascular remodeling and pulmonary hypertension. Copyright © 2015 the American Physiological Society.

  13. Neuronal nitric oxide synthase mediates insulin- and oxidative stress-induced glucose uptake in skeletal muscle myotubes.

    Science.gov (United States)

    Kellogg, Dean L; McCammon, Karen M; Hinchee-Rodriguez, Kathryn S; Adamo, Martin L; Roman, Linda J

    2017-09-01

    Previously published studies strongly suggested that insulin- and exercise-induced skeletal muscle glucose uptake require nitric oxide (NO) production. However, the signal transduction mechanisms by which insulin and contraction regulated NO production and subsequent glucose transport are not known. In the present study, we utilized the myotube cell lines treated with insulin or hydrogen peroxide, the latter to mimic contraction-induced oxidative stress, to characterize these mechanisms. We found that insulin stimulation of neuronal nitric oxide synthase (nNOS) phosphorylation, NO production, and GLUT4 translocation were all significantly reduced by inhibition of either nNOS or Akt2. Hydrogen peroxide (H 2 O 2 ) induced phosphorylation of nNOS at the same residue as did insulin, and also stimulated NO production and GLUT4 translocation. nNOS inhibition prevented H 2 O 2 -induced GLUT4 translocation. AMP activated protein kinase (AMPK) inhibition prevented H 2 O 2 activation and phosphorylation of nNOS, leading to reduced NO production and significantly attenuated GLUT4 translocation. We conclude that nNOS phosphorylation and subsequently increased NO production are required for both insulin- and H 2 O 2 -stimulated glucose transport. Although the two stimuli result in phosphorylation of the same residue on nNOS, they do so through distinct protein kinases. Thus, insulin and H 2 O 2 -activated signaling pathways converge on nNOS, which is a common mediator of glucose uptake in both pathways. However, the fact that different kinases are utilized provides a basis for the use of exercise to activate glucose transport in the face of insulin resistance. Copyright © 2017. Published by Elsevier Inc.

  14. Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Malaviya, Rama; Venosa, Alessandro [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Hall, LeRoy [Drug Safety Sciences, Johnson and Johnson, Raritan, NJ 08869 (United States); Gow, Andrew J. [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Sinko, Patrick J. [Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854 (United States); Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States)

    2012-12-15

    Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125 mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1 d–28 d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS{sup +} and cyclooxygenase-2{sup +}) and alternatively activated profibrotic (YM-1{sup +} and galectin-3{sup +}) macrophages appeared in the lung following NM administration; this was evident within 1 d, and persisted for 28 d. AG administration (50 mg/kg, 2 ×/day, 1 d–3 d) abrogated NM-induced injury, oxidative stress and inflammation at 1 d and 3 d post exposure, with no effects at 7 d or 28 d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis. -- Highlights: ► Nitrogen mustard (NM) induces acute lung injury and fibrosis. ► Pulmonary toxicity is associated with increased expression of iNOS. ► Transient inhibition of iNOS attenuates acute

  15. Cytotoxicity of VEGF121/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2

    Directory of Open Access Journals (Sweden)

    Hittelman Walter N

    2011-08-01

    Full Text Available Abstract Background The fusion protein VEGF121/rGel composed of the growth factor VEGF121 and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF121/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo. Methods We investigated the binding, cytotoxicity and internalization profile of VEGF121/rGel on endothelial cells expressing VEGFR-1 or VEGFR-2, identified its effects on angiogenesis models in vitro and ex vivo, and explored its intracellular effects on a number of molecular pathways using microarray analysis. Results Incubation of PAE/VEGFR-2 and PAE/VEGFR-1 cells with 125I-VEGF121/rGel demonstrated binding specificity that was competed with unlabeled VEGF121/rGel but not with unlabeled gelonin. Assessment of the effect of VEGF121/rGel on blocking tube formation in vitro revealed a 100-fold difference in IC50 levels between PAE/VEGFR-2 (1 nM and PAE/VEGFR-1 (100 nM cells. VEGF121/rGel entered PAE/VEGFR-2 cells within one hour of treatment but was not detected in PAE/VEGFR-1 cells up to 24 hours after treatment. In vascularization studies using chicken chorioallantoic membranes, 1 nM VEGF121/rGel completely inhibited bFGF-stimulated neovascular growth. The cytotoxic effects of VEGF121/rGel were not apoptotic since treated cells were TUNEL-negative with no evidence of PARP cleavage or alteration in the protein levels of select apoptotic markers. Microarray analysis of VEGF121/rGel-treated HUVECs revealed the upregulation of a unique "fingerprint" profile of 22 genes that control cell adhesion, apoptosis, transcription regulation, chemotaxis, and inflammatory response. Conclusions Taken together, these data confirm the selectivity of VEGF121/rGel for VEGFR-2

  16. Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

    Directory of Open Access Journals (Sweden)

    Tomishima Yoshiro

    2013-01-01

    Full Text Available Abstract Background Overdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2 synthase inhibitor, on liver injury induced by APAP overdose in mice. Methods Hepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg. The effects of ozagrel (200 mg/kg treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL on cytochrome P450 2E1 (CYP2E1 activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI, a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1–100 muM were evaluated by the WST-1 cell viability assay. Results Ozagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos and C/EBP homologous protein (chop, but did not suppress B-cell lymphoma 2-like protein11 (bim expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16. Conclusions We demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest

  17. The assembly of the plasmodial PLP synthase complex follows a defined course.

    Directory of Open Access Journals (Sweden)

    Ingrid B Müller

    Full Text Available BACKGROUND: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP. For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers. METHODOLOGY/PRINCIPAL FINDINGS: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK, amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.

  18. Lack of Thromboxane Synthase Prevents Hypertension and Fetal Growth Restriction after High Salt Treatment during Pregnancy.

    Directory of Open Access Journals (Sweden)

    Chen-Hsueh Pai

    Full Text Available Preeclampsia (PE is a potentially fatal pregnancy-related hypertensive disorder characterized by poor placenta development that can cause fetal growth restriction. PE-associated pathologies, including thrombosis, hypertension, and impaired placental development, may result from imbalances between thromboxane A2 (TXA2 and prostacyclin. Low-dose aspirin, which selectively inhibits TXA2 production, is used to prevent high-risk PE. However, the role of TXA2 in aspirin-mediated protective effects in women with PE is not understood fully. In this study, we examined the role of prostanoids in PE using human samples and an induced PE mouse model. We demonstrated that the administration of salted drinking water (2.7% NaCl to wild-type mice resulted in elevated placental TXA2 synthase (TXAS and plasma TXA2, but not prostacyclin, levels, which was also found in our clinical PE placenta samples. The high salt-treated wild-type pregnant mice had shown unchanged maternal body weight, hypertension (MAP increase 15 mmHg, and decreased pup weight (~50% and size (~24%, but these adverse effects were ameliorated in TXAS knockout (KO mice. Moreover, increased expression of interleukin-1β and downstream phosphorylated-p38-mitogen-activated protein kinase were concordant with apoptosis induction in the placentas of salt water-treated wild-type mice. These alterations were not observed in TXAS KO mice. Together, our data suggest that TXA2 depletion has anti-PE effects due to the prevention of hypertension and placental damage through downregulation of the interleukin-1β pathway.

  19. Falcarindiol inhibits nitric oxide-mediated neuronal death in lipopolysaccharide-treated organotypic hippocampal cultures.

    Science.gov (United States)

    Kim, Jeong Min; Lee, Pyeongjae; Son, Dongwook; Kim, Hocheol; Kim, Sun Yeou

    2003-10-27

    Excessive nitric oxide (NO) release from activated microglia has a predominant role in neuronal death. This study investigated the effect of falcarindiol, which was isolated from Cnidium officinale Makino, on the NO-mediated neuronal death in lipopolysaccharide (LPS)-treated organotypic hippocampal cultures. Falcarindiol dose-dependently reduced inducible NO synthase (iNOS)-mediated NO production without cytotoxic effects on LPS-activated BV-2 and microglia. Predictably, falcarindiol inhibited neuronal death by reducing NO production in the LPS-treated organotypic hippocampal cultures. N-monomethyl-L-arginine (NMMA), an iNOS inhibitor, also inhibited neuronal death at 500 microM. In contrast, massive neuronal death was induced by excessive NO production in the LPS-treated alone cultures. These results suggest that excessive NO production plays an important role in the neurotoxic effect, and falcarindiol is a potential inhibitor in NO-mediated neuronal death.

  20. 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.

  1. 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.

  2. A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex

    International Nuclear Information System (INIS)

    Grum, Daniel; Boom, Johannes van den; Neumann, Daniel; Matena, Anja; Link, Nina M.; Mueller, Jonathan W.

    2010-01-01

    3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Foerster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

  3. 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

  4. Novel applications of plant polyketide synthases.

    Science.gov (United States)

    Abe, Ikuro

    2012-04-01

    The structurally and mechanistically simple type III polyketide synthases (PKSs) catalyze iterative condensations of CoA thioesters to produce a variety of polyketide scaffolds with remarkably diverse structures and biological activities. By exploiting the enzymes, we combined precursor-directed biosynthesis with nitrogen-containing substrates and structure-based enzyme engineering and generated unnatural, novel polyketide-alkaloid scaffolds with promising biological activities. The nucleophilic nitrogen atom and the engineered enzymes thus facilitated the formation of additional CC and CN bonds during the enzymatic transformations. The methodology will contribute to the further production of chemically and structurally divergent, unnatural natural products, as well as the rational design of novel biocatalysts with unprecedented catalytic functions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Tyrosine nitration affects thymidylate synthase properties.

    Science.gov (United States)

    Dąbrowska-Maś, Elżbieta; Frączyk, Tomasz; Ruman, Tomasz; Radziszewska, Karolina; Wilk, Piotr; Cieśla, Joanna; Zieliński, Zbigniew; Jurkiewicz, Agata; Gołos, Barbara; Wińska, Patrycja; Wałajtys-Rode, Elżbieta; Leś, Andrzej; Nizioł, Joanna; Jarmuła, Adam; Stefanowicz, Piotr; Szewczuk, Zbigniew; Rode, Wojciech

    2012-01-14

    Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

  6. 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.

  7. CLYBL is a polymorphic human enzyme with malate synthase and β-methylmalate synthase activity

    Science.gov (United States)

    Strittmatter, Laura; Li, Yang; Nakatsuka, Nathan J.; Calvo, Sarah E.; Grabarek, Zenon; Mootha, Vamsi K.

    2014-01-01

    CLYBL is a human mitochondrial enzyme of unknown function that is found in multiple eukaryotic taxa and conserved to bacteria. The protein is expressed in the mitochondria of all mammalian organs, with highest expression in brown fat and kidney. Approximately 5% of all humans harbor a premature stop polymorphism in CLYBL that has been associated with reduced levels of circulating vitamin B12. Using comparative genomics, we now show that CLYBL is strongly co-expressed with and co-evolved specifically with other components of the mitochondrial B12 pathway. We confirm that the premature stop polymorphism in CLYBL leads to a loss of protein expression. To elucidate the molecular function of CLYBL, we used comparative operon analysis, structural modeling and enzyme kinetics. We report that CLYBL encodes a malate/β-methylmalate synthase, converting glyoxylate and acetyl-CoA to malate, or glyoxylate and propionyl-CoA to β-methylmalate. Malate synthases are best known for their established role in the glyoxylate shunt of plants and lower organisms and are traditionally described as not occurring in humans. The broader role of a malate/β-methylmalate synthase in human physiology and its mechanistic link to vitamin B12 metabolism remain unknown. PMID:24334609

  8. Endothelium-dependent relaxation of rat aorta to a histamine H3 agonist is reduced by inhibitors of nitric oxide synthase, guanylate cyclase and Na+,K+-ATPase

    Directory of Open Access Journals (Sweden)

    D. M. Djuric

    1996-01-01

    Full Text Available The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, β-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na+,K+-ATPase was evaluated in a histamine H3 receptor agonist-induced ((Rα-methylhistamine, (Rα-MeHA endothelium-dependent rat aorta relaxation assay. (Rα-MeHA (0.1 nM – 0.01 mM relaxed endothelium-dependent rat aorta, with a pD2 value of 8.22 ± 0.06, compared with a pD2 value of 7.98 ± 0.02 caused by histamine (50% and 70% relaxation, respectively. The effect of (Rα-MeHA (0.1 nM – 0.01 mM was competitively antagonized by thioperamide (1, 10 and 30 nM (pA2 = 9.21 ± 0.40; slope = 1.03 ± 0.35 but it was unaffected by pyrilamine (100 nM, cimetidine (1 μM, atropine (10 μM, propranolol (1 μM, indomethacin (10 μM or nordthydroguaiaretic acid (0.1 mM. Inhibitors of nitric oxide synthase, L-NG-monomethylarginine (L-NMMA, 10 μM and NG-nitro-L-arginine methylester (L-NOARG, 10 μM inhibited the relaxation effect of (Rα-MeHA, by approximately 52% and 70%, respectively. This inhibitory effect of L-NMMA was partially reversed by L-arginine (10 μM. Methylene blue (10 μM and ouabain (10 μM inhibited relaxation (Rα-MeHA-induced by approximately 50% and 90%, respectively. The products of cyclooxygenase and lipoxygenase are not involved in (Rα-MeHA-induced endothelium-dependent rat aorta relaxation nor are the muscarinic cholinergic and β-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na+,K+-ATPase in (Rα-MeHA-induced endothelium-dependent rat aorta relaxation.

  9. 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.

  10. 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.

  11. Nuclear glycogen synthase kinase-3 {beta} (GSK-3) in Rhipicephalus (Boophilus) microplus tick embryogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Mentzingen, Leticia; Andrade, Josiana G. de; Logullo, Carlos [Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ (Brazil). Centro de Biociencias e Biotecnologia. Lab. de Quimica e Funcao de Proteinas e Peptideos (LQFPP); Andrade, Caroline P. de; Vaz Junior, Itabajara [Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS (Brazil). Centro de Biotecnologia

    2008-07-01

    Full text: Glycogen synthase kinase-3 (GSK3) is recognized as a key component of a large number of cellular processes and diseases. Several mechanisms play a part in controlling the actions of GSK3, including phosphorylation, protein complex formation, and subcellular distribution. Recent observations point to functions for phosphorylases several transcription factors in the nucleus. Also, GSK3b participate of the canonical W nt signalling pathway, which has been studied intensively in embryonic and cancer cells. Like in many other signaling pathways, most components in W nt signal transduction were highly conserved during the evolution. More than 40 proteins have been reported to be phosphorylated by GSK3, including over a dozen transcription factors. Although the mechanisms regulating GSK3 are not fully understood, precise control appears to be achieved by a combination of phosphorylation, localization, and interactions with GSK3-binding proteins. Although GSK3 is traditionally considered a cytosolic protein, it is also present in nuclei. Nuclear GSK3 is particularly interesting because of the many transcription factors that it regulates enabling GSK3 to influence many signaling pathways that converge on these transcription factors, thereby regulating the expression of many genes. Our group identified that GSK-3 {beta} could be detected in different stage eggs of R. micro plus. In this work we detected the GSK-3 in isolated nuclear fraction from the egg homogenates of R. micro plus by western-blot analysis, using anti-GSK- 3 {beta} antibodies. The enzyme activity was also detected radiochemically throughout embryogenesis in same fraction. The GSK-3 activity was inhibiting by using SB 216763 (selective molecule inhibitors of GSK-3). Taken together our results suggest that GSK-3 {beta} isoform probably is involved in gene transcription factors during R. micro plus embryo development.

  12. 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.

  13. 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

  14. Antenatal insults modify newborn olfactory function by nitric oxide produced from neuronal nitric oxide synthase.

    Science.gov (United States)

    Drobyshevsky, Alexander; Yu, Lei; Yang, Yirong; Khalid, Syed; Luo, Kehuan; Jiang, Rugang; Ji, Haitao; Derrick, Matthew; Kay, Leslie; Silverman, Richard B; Tan, Sidhartha

    2012-10-01

    Newborn feeding, maternal, bonding, growth and wellbeing depend upon intact odor recognition in the early postnatal period. Antenatal stress may affect postnatal odor recognition. We investigated the exact role of a neurotransmitter, nitric oxide (NO), in newborn olfactory function. We hypothesized that olfactory neuron activity depended on NO generated by neuronal NO synthase (NOS). Utilizing in vivo functional manganese enhanced MRI (MEMRI) in a rabbit model of cerebral palsy we had shown previously that in utero hypoxia-ischemia (H-I) at E22 (70% gestation) resulted in impaired postnatal response to odorants and poor feeding. With the same antenatal insult, we manipulated NO levels in the olfactory neuron in postnatal day 1 (P1) kits by administration of intranasal NO donors or a highly selective nNOS inhibitor. Olfactory function was quantitatively measured by the response to amyl acetate stimulation by MEMRI. The relevance of nNOS to normal olfactory development was confirmed by the increase of nNOS gene expression from fetal ages to P1 in olfactory epithelium and bulbs. In control kits, nNOS inhibition decreased NO production in the olfactory system and increased MEMRI slope enhancement. In H-I kits the MEMRI slope did not increase, implicating modification of endogenous NO-mediated olfactory function by the antenatal insult. NO donors as a source of exogenous NO did not significantly change function in either group. In conclusion, olfactory epithelium nNOS in newborn rabbits probably modulates olfactory signal transduction. Antenatal H-I injury remote from delivery may affect early functional development of the olfactory system by decreasing NO-dependent signal transduction. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    Energy Technology Data Exchange (ETDEWEB)

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara (Psychogenics); (Purdue); (UIC); (UTSMC)

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  16. 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.

  17. Inhibiting DPP4 in a mouse model of HHT1 results in a shift towards regenerative macrophages and reduces fibrosis after myocardial infarction.

    Directory of Open Access Journals (Sweden)

    Calinda K E Dingenouts

    Full Text Available Hereditary Hemorrhagic Telangiectasia type-1 (HHT1 is a genetic vascular disorder caused by haploinsufficiency of the TGFβ co-receptor endoglin. Dysfunctional homing of HHT1 mononuclear cells (MNCs towards the infarcted myocardium hampers cardiac recovery. HHT1-MNCs have elevated expression of dipeptidyl peptidase-4 (DPP4/CD26, which inhibits recruitment of CXCR4-expressing MNCs by inactivation of stromal cell-derived factor 1 (SDF1. We hypothesize that inhibiting DPP4 will restore homing of HHT1-MNCs to the infarcted heart and improve cardiac recovery.After inducing myocardial infarction (MI, wild type (WT and endoglin heterozygous (Eng+/- mice were treated for 5 days with the DPP4 inhibitor Diprotin A (DipA. DipA increased the number of CXCR4+ MNCs residing in the infarcted Eng+/- hearts (Eng+/- 73.17±12.67 vs. Eng+/- treated 157.00±11.61, P = 0.0003 and significantly reduced infarct size (Eng+/- 46.60±9.33% vs. Eng+/- treated 27.02±3.04%, P = 0.03. Echocardiography demonstrated that DipA treatment slightly deteriorated heart function in Eng+/- mice. An increased number of capillaries (Eng+/- 61.63±1.43 vs. Eng+/- treated 74.30±1.74, P = 0.001 were detected in the infarct border zone whereas the number of arteries was reduced (Eng+/- 11.88±0.63 vs. Eng+/- treated 6.38±0.97, P = 0.003. Interestingly, while less M2 regenerative macrophages were present in Eng+/- hearts prior to DipA treatment, (WT 29.88±1.52% vs. Eng+/- 12.34±1.64%, P<0.0001, DPP4 inhibition restored the number of M2 macrophages to wild type levels.In this study, we demonstrate that systemic DPP4 inhibition restores the impaired MNC homing in Eng+/- animals post-MI, and enhances cardiac repair, which might be explained by restoring the balance between the inflammatory and regenerative macrophages present in the heart.

  18. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Yong, E-mail: drbiany@126.com [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China); Yu, Yun [College of Pharmacy, Nanjing University of Chinese Medicine, 210023 (China); Wang, Shanshan; Li, Lin [Department of Science and Technology, Nanjing University of Chinese Medicine, 210023 (China)

    2015-08-07

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression.

  19. Solid-State NMR, Crystallographic, and Computational Investigation of Bisphosphonates and Farnesyl Diphosphate Synthase-Bisphosphonate Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Mao,J.; Mukherjee, S.; Zhang, Y.; Cao, R.; Sanders, J.; Song, Y.; Zhang, Y.; Meints, G.; Gao, Y.; et al.

    2006-01-01

    Bisphosphonates are a class of molecules in widespread use in treating bone resorption diseases and are also of interest as immunomodulators and anti-infectives. They function by inhibiting the enzyme farnesyl diphosphate synthase (FPPS), but the details of how these molecules bind are not fully understood. Here, we report the results of a solid-state {sup 13}C, {sup 15}N, and {sup 31}P magic-angle sample spinning (MAS) NMR and quantum chemical investigation of several bisphosphonates, both as pure compounds and when bound to FPPS, to provide information about side-chain and phosphonate backbone protonation states when bound to the enzyme. We then used computational docking methods (with the charges assigned by NMR) to predict how several bisphosphonates bind to FPPS. Finally, we used X-ray crystallography to determine the structures of two potent bisphosphonate inhibitors, finding good agreement with the computational results, opening up the possibility of using the combination of NMR, quantum chemistry and molecular docking to facilitate the design of other, novel prenytransferase inhibitors.

  20. Up-regulation of fatty acid synthase induced by EGFR/ERK activation promotes tumor growth in pancreatic cancer

    International Nuclear Information System (INIS)

    Bian, Yong; Yu, Yun; Wang, Shanshan; Li, Lin

    2015-01-01

    Lipid metabolism is dysregulated in many human diseases including atherosclerosis, type 2 diabetes and cancers. Fatty acid synthase (FASN), a key lipogenic enzyme involved in de novo lipid biosynthesis, is significantly upregulated in multiple types of human cancers and associates with tumor progression. However, limited data is available to understand underlying biological functions and clinical significance of overexpressed FASN in pancreatic ductal adenocarcinoma (PDAC). Here, upregulated FASN was more frequently observed in PDAC tissues compared with normal pancreas in a tissue microarray. Kaplan–Meier survival analysis revealed that high expression level of FASN resulted in a significantly poor prognosis of PDAC patients. Knockdown or inhibition of endogenous FASN decreased cell proliferation and increased cell apoptosis in HPAC and AsPC-1 cells. Furthermore, we demonstrated that EGFR/ERK signaling accounts for elevated FASN expression in PDAC as ascertained by performing siRNA assays and using specific pharmacological inhibitors. Collectively, our results indicate that FASN exhibits important roles in tumor growth and EGFR/ERK pathway is responsible for upregulated expression of FASN in PDAC. - Highlights: • Increased expression of FASN indicates a poor prognosis in PDAC. • Elevated FASN favors tumor growth in PDAC in vitro. • Activation of EGFR signaling contributes to elevated FASN expression

  1. The affinity purification and characterization of ATP synthase complexes from mitochondria.

    Science.gov (United States)

    Runswick, Michael J; Bason, John V; Montgomery, Martin G; Robinson, Graham C; Fearnley, Ian M; Walker, John E

    2013-02-13

    The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1-60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.

  2. Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes.

    Science.gov (United States)

    Wendisch, V F; Spies, M; Reinscheid, D J; Schnicke, S; Sahm, H; Eikmanns, B J

    1997-10-01

    In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes (aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum.

  3. Inducible and neuronal nitric oxide synthases exert contrasting effects during rat intestinal recovery following fasting.

    Science.gov (United States)

    Ito, Junta; Uchida, Hiroyuki; Machida, Naomi; Ohtake, Kazuo; Saito, Yuki; Kobayashi, Jun

    2017-04-01

    We investigated the effects of endogenous inducible (iNOS) and neuronal nitric oxide synthase on recovery from intestinal mucosal atrophy caused by fasting-induced apoptosis and decreased cell proliferation during refeeding in rats. Rats were divided into five groups, one of which was fed ad libitum, and four of which underwent 72 h of fasting, followed by refeeding for 0, 6, 24, and 48 h, respectively. iNOS and neuronal nitric oxide synthase mRNA and protein levels in jejunal tissues were measured, and mucosal height was histologically evaluated. Apoptotic indices, interferon-γ (IFN-γ) transcription levels, nitrite levels (as a measure of nitric oxide [NO] production),8-hydroxydeoxyguanosine formation (indicating reactive oxygen species [ROS] levels), crypt cell proliferation, and the motility indices (MI) were also estimated. Associations between mucosal height and NOS protein levels were determined using Spearman's rank correlation test. Notably, we observed significant increases in mucosal height and in neuronal nitric oxide synthase mRNA and protein expression as refeeding time increased. Indeed, there was a significant positive correlation between neuronal nitric oxide synthase protein level and mucosal height during the 48-h refeeding period ( r = 0.725, P fasting. Our finding suggests that refeeding likely repairs fasting-induced jejunal atrophy by suppressing iNOS expression and subsequently inhibiting NO, ROS, and IFN-γ as apoptosis mediators, and by promoting neuronal nitric oxide synthase production and inducing crypt cell proliferation via mechanical stimulation. Impact statement Besides providing new data confirming the involvement of iNOS and nNOS in intestinal mucosal atrophy caused by fasting, this study details their expression and function during recovery from this condition following refeeding. We demonstrate a significant negative correlation between iNOS and nNOS levels during refeeding, and associate this with cell proliferation

  4. Wild-type phosphoribosylpyrophosphate synthase (PRS from Mycobacterium tuberculosis: a bacterial class II PRS?

    Directory of Open Access Journals (Sweden)

    Ardala Breda

    Full Text Available The 5-phospho-α-D-ribose 1-diphosphate (PRPP metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS. Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure

  5. Berberine improves glucose metabolism in diabetic rats by inhibition of hepatic gluconeogenesis.

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    Xuan Xia

    2011-02-01

    Full Text Available Berberine (BBR is a compound originally identified in a Chinese herbal medicine Huanglian (Coptis chinensis French. It improves glucose metabolism in type 2 diabetic patients. The mechanisms involve in activation of adenosine monophosphate activated protein kinase (AMPK and improvement of insulin sensitivity. However, it is not clear if BBR reduces blood glucose through other mechanism. In this study, we addressed this issue by examining liver response to BBR in diabetic rats, in which hyperglycemia was induced in Sprague-Dawley rats by high fat diet. We observed that BBR decreased fasting glucose significantly. Gluconeogenic genes, Phosphoenolpyruvate carboxykinase (PEPCK and Glucose-6-phosphatase (G6Pase, were decreased in liver by BBR. Hepatic steatosis was also reduced by BBR and expression of fatty acid synthase (FAS was inhibited in liver. Activities of transcription factors including Forkhead transcription factor O1 (FoxO1, sterol regulatory element-binding protein 1c (SREBP1 and carbohydrate responsive element-binding protein (ChREBP were decreased. Insulin signaling pathway was not altered in the liver. In cultured hepatocytes, BBR inhibited oxygen consumption and reduced intracellular adenosine triphosphate (ATP level. The data suggest that BBR improves fasting blood glucose by direct inhibition of gluconeogenesis in liver. This activity is not dependent on insulin action. The gluconeogenic inhibition is likely a result of mitochondria inhibition by BBR. The observation supports that BBR improves glucose metabolism through an insulin-independent pathway.

  6. Differentially expressed galactinol synthase(s) in chickpea are implicated in seed vigor and longevity by limiting the age induced ROS accumulation

    Science.gov (United States)

    Salvi, Prafull; Saxena, Saurabh Chandra; Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kaur, Harmeet; Verma, Pooja; Rao, Venkateswara; Ghosh, Shraboni; Majee, Manoj

    2016-01-01

    Galactinol synthase (GolS) catalyzes the first and rate limiting step of Raffinose Family Oligosaccharide (RFO) biosynthetic pathway, which is a highly specialized metabolic event in plants. Increased accumulation of galactinol and RFOs in seeds have been reported in few plant species, however their precise role in seed vigor and longevity remain elusive. In present study, we have shown that galactinol synthase activity as well as galactinol and raffinose content progressively increase as seed development proceeds and become highly abundant in pod and mature dry seeds, which gradually decline as seed germination progresses in chickpea (Cicer arietinum). Furthermore, artificial aging also stimulates galactinol synthase activity and consequent galactinol and raffinose accumulation in seed. Molecular analysis revealed that GolS in chickpea are encoded by two divergent genes (CaGolS1 and CaGolS2) which potentially encode five CaGolS isoforms through alternative splicing. Biochemical analysis showed that only two isoforms (CaGolS1 and CaGolS2) are biochemically active with similar yet distinct biochemical properties. CaGolS1 and CaGolS2 are differentially regulated in different organs, during seed development and germination however exhibit similar subcellular localization. Furthermore, seed-specific overexpression of CaGolS1 and CaGolS2 in Arabidopsis results improved seed vigor and longevity through limiting the age induced excess ROS and consequent lipid peroxidation. PMID:27725707

  7. Arctigenin inhibits the activation of the mTOR pathway, resulting in autophagic cell death and decreased ER expression in ER-positive human breast cancer cells.

    Science.gov (United States)

    Maxwell, Thressi; Lee, Kyu Shik; Kim, Soyoung; Nam, Kyung-Soo

    2018-04-01

    Arctigenin, a member of the Asteraceae family, is a biologically active lignan that is consumed worldwide due to its several health benefits. However, its use may pose a problem for patients with estrogen receptor (ER)α-positive breast cancer, since studies have shown that arctigenin is a phytoestrogen that exerts a proliferative effect by binding to the ER. Thus, in this study, we examined the effect of arctigenin on ERα-positive MCF-7 human breast cancer cells to determine whether the consumption of arctigenin is safe for patients with breast cancer. First, we found that arctigenin inhibited the viability of the MCF-7 cells, and colony formation assay confirmed that this effect was cytotoxic rather than cytostatic. The cytotoxic effects were not mediated by cell cycle arrest, apoptosis, or necroptosis, despite DNA damage, as indicated by poly(ADP-ribose) polymerase (PARP) cleavage and phosphorylated H2A.X. An increase in lipidated LC3, a marker of autophagosome formation, was observed, indicating that autophagy was induced by arctigenin, which was found to be triggered by the inhibition of the mechanistic target of rapamycin (mTOR) pathway. We then examined the effects of arctigenin on ERα expression and determined whether it affects the sensitivity of the cells to tamoxifen, as tamoxifen is commonly used against hormone-responsive cancers and is known to act via the ERα. We found that treatment with arctigenin effectively downregulated ERα expression, which was found to be a consequence of the inhibition of the mTOR pathway. However, treatment with arctigenin in combination with tamoxifen did not affect the sensitivity of the cells to tamoxifen, but instead, exerted a synergistic effect. On the whole, our data indicate that the phytoestrogen, arctigenin, mainly targeted the mTOR pathway in ERα-positive MCF-7 human breast cancer cells, leading to autophagy-induced cell death and the downregulation of ERα expression. Furthermore, the synergistic effects

  8. Inhibition of Neuroinflammation in LPS-Activated Microglia by Cryptolepine

    Directory of Open Access Journals (Sweden)

    Olumayokun A. Olajide

    2013-01-01

    Full Text Available Cryptolepine, an indoloquinoline alkaloid in Cryptolepis sanguinolenta, has anti-inflammatory property. In this study, we aimed to evaluate the effects of cryptolepine on lipopolysaccharide (LPS- induced neuroinflammation in rat microglia and its potential mechanisms. Microglial activation was induced by stimulation with LPS, and the effects of cryptolepine pretreatment on microglial activation and production of proinflammatory mediators, PGE2/COX-2, microsomal prostaglandin E2 synthase and nitric oxide/iNOS were investigated. We further elucidated the role of Nuclear Factor-kappa B (NF-κB and the mitogen-activated protein kinases in the antiinflammatory actions of cryptolepine in LPS-stimulated microglia. Our results showed that cryptolepine significantly inhibited LPS-induced production of tumour necrosis factor-alpha (TNFα, interleukin-6 (IL-6, interleukin-1beta (IL-1β, nitric oxide, and PGE2. Protein and mRNA levels of COX-2 and iNOS were also attenuated by cryptolepine. Further experiments on intracellular signalling mechanisms show that IκB-independent inhibition of NF-κB nuclear translocation contributes to the anti-neuroinflammatory actions of cryptolepine. Results also show that cryptolepine inhibited LPS-induced p38 and MAPKAPK2 phosphorylation in the microglia. Cell viability experiments revealed that cryptolepine (2.5 and 5 μM did not produce cytotoxicity in microglia. Taken together, our results suggest that cryptolepine inhibits LPS-induced microglial inflammation by partial targeting of NF-κB signalling and attenuation of p38/MAPKAPK2.

  9. Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif.

    Science.gov (United States)

    Hurd, Matthew C; Kwon, Moonhyuk; Ro, Dae-Kyun

    2017-08-26

    Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX 8 W) motifs, and enzyme assays showed that the presence of both RRX 8 W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX 8 W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Distinct Molecular Regulation of Glycogen Synthase Kinase-3α Isozyme Controlled by Its N-terminal Region

    Science.gov (United States)

    Azoulay-Alfaguter, Inbar; Yaffe, Yakey; Licht-Murava, Avital; Urbanska, Malgorzata; Jaworski, Jacek; Pietrokovski, Shmuel; Hirschberg, Koret; Eldar-Finkelman, Hagit

    2011-01-01

    Glycogen synthase kinase-3 (GSK-3) is expressed as two isozymes α and β. They share high similarity in their catalytic domains but differ in their N- and C-terminal regions, with GSK-3α having an extended glycine-rich N terminus. Here, we undertook live cell imaging combined with molecular and bioinformatic studies to understand the distinct functions of the GSK-3 isozymes focusing on GSK-3α N-terminal region. We found that unlike GSK-3β, which shuttles between the nucleus and cytoplasm, GSK-3α was excluded from the nucleus. Deletion of the N-terminal region of GSK-3α resulted in nuclear localization, and treatment with leptomycin B resulted in GSK-3α accumulation in the nucleus. GSK-3α rapidly accumulated in the nucleus in response to calcium or serum deprivation, and accumulation was strongly inhibited by the calpain inhibitor calpeptin. This nuclear accumulation was not mediated by cleavage of the N-terminal region or phosphorylation of GSK-3α. Rather, we show that calcium-induced GSK-3α nuclear accumulation was governed by GSK-3α binding with as yet unknown calpain-sensitive protein or proteins; this binding was mediated by the N-terminal region. Bioinformatic and experimental analyses indicated that nuclear exclusion of GSK-3α was likely an exclusive characteristic of mammalian GSK-3α. Finally, we show that nuclear localization of GSK-3α reduced the nuclear pool of β-catenin and its target cyclin D1. Taken together, these data suggest that the N-terminal region of GSK-3α is responsible for its nuclear exclusion and that binding with a calcium/calpain-sensitive product enables GSK-3α nuclear retention. We further uncovered a novel link between calcium and nuclear GSK-3α-mediated inhibition of the canonical Wnt/β-catenin pathway. PMID:21266584

  11. UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6

    Energy Technology Data Exchange (ETDEWEB)

    Miyata, Maiko [Department of Life and Medical Sciences, Chubu University Faculty of Life and Health Sciences, Matsumoto, Kasugai 487-8501 (Japan); Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Ichihara, Masatoshi; Tajima, Orie; Sobue, Sayaka; Kambe, Mariko [Department of Life and Medical Sciences, Chubu University Faculty of Life and Health Sciences, Matsumoto, Kasugai 487-8501 (Japan); Sugiura, Kazumitsu [Department of Dermatology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Furukawa, Koichi, E-mail: koichi@med.nagoya-u.ac.jp [Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan); Furukawa, Keiko [Department of Life and Medical Sciences, Chubu University Faculty of Life and Health Sciences, Matsumoto, Kasugai 487-8501 (Japan); Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065 (Japan)

    2014-03-07

    Highlights: • Melanocytes showed low ST8SIA1 and high B3GALT4 levels in contrast with melanomas. • Direct UVB irradiation of melanocytes did not induce ganglioside synthase genes. • Culture supernatants of UVB-irradiated keratinocytes induced ST8SIA1 in melanocytes. • TNFα and IL-6 secreted from keratinocytes enhanced ST8SIA1 expression in melanocytes. • Inflammatory cytokines induced melanoma-related ST8SIA1 in melanocytes. - Abstract: Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.

  12. Regulation of glycogen synthase kinase-3{beta} (GSK-3{beta}) after ionizing radiation; Regulation der Glykogen Synthase Kinase-3{beta} (GSK-3{beta}) nach ionisierender Strahlung

    Energy Technology Data Exchange (ETDEWEB)

    Boehme, K.A.

    2006-12-15

    Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3{beta} by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3{beta} at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3{beta} serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKB{beta}, is required for phosphorylation of GSK- 3{beta} at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3{beta} at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3{beta} in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer

  13. 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.

  14. Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells.

    Science.gov (United States)

    Wang, J F; Komarov, P; Sies, H; de Groot, H

    1991-01-01

    Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2. PMID:1718262

  15. A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants.

    Directory of Open Access Journals (Sweden)

    Gaoyi Cao

    Full Text Available A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops.

  16. Fo Shou San, an ancient Chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity.

    Directory of Open Access Journals (Sweden)

    Cathy W C Bi

    Full Text Available Fo Shou San (FSS is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong and Angelicae Sinensis Radix (ASR; Danggui in a ratio of 2:3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs, and it also stimulated the production of nitric oxide (NO as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca(2+ levels in HUVECs, and the Ca(2+ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca(2+ and a reduction of ROS.

  17. Synthesis and biological evaluation of novel 3-substituted amino-4-hydroxylcoumarin derivatives as chitin synthase inhibitors and antifungal agents.

    Science.gov (United States)

    Ge, Zhiqiang; Ji, Qinggang; Chen, Chunyan; Liao, Qin; Wu, Hualong; Liu, Xiaofei; Huang, Yanrong; Yuan, Lvjiang; Liao, Fei

    2016-01-01

    A series of novel 3-substituted amino-4-hydroxycoumarin derivatives have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their CHS inhibition activity and antimicrobial activity in vitro. The enzymatic assay indicated that most of the compounds have good inhibitory activity against CHS, in which compound 6o with IC50 of 0.10 mmol/L had stronger activity than that of polyoxins B, which acts as control drug with IC50 of 0.18 mmol/L. As far as the antifungal activity is concerned, most of the compounds possessed moderate to excellent activity against some representative pathogenic fungi. Especially, compound 6b was found to be the most potent agent against Cryptococcus neoformans with minimal inhibitory concentration (MIC) of 4 μg/mL. Moreover, the results of antibacterial screening showed that these compounds have negligible actions to some tested bacteria. Therefore, these compounds would be promising to develop selective antifungal agents.

  18. Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae.

    Science.gov (United States)

    Keszei, Andras; Brubaker, Curt L; Carter, Richard; Köllner, Tobias; Degenhardt, Jörg; Foley, William J

    2010-06-01

    Myrtaceae is one of the chemically most variable and most significant essential oil yielding plant families. Despite an abundance of chemical information, very little work has focussed on the biochemistry of terpene production in these plants. We describe 70 unique partial terpene synthase transcripts and eight full-length cDNA clones from 21 myrtaceous species, and compare phylogenetic relationships and leaf oil composition to reveal clades defined by common function. We provide further support for the correlation between function and phylogenetic relationships by the first functional characterisation of terpene synthases from Myrtaceae: a 1,8-cineole synthase from Eucalyptus sideroxylon and a caryophyllene synthase from Eucalyptusdives. Copyright 2010 Elsevier Ltd. All rights reserved.

  19. Chalcone synthase genes from milk thistle (Silybum marianum ...

    Indian Academy of Sciences (India)

    Leyva et al. 1995), UV treatments and blue light (Hartmann et al. 1998; Wade et al. 2001; Zhou et al. 2007), elicitor treatments such as salicylic acid and. Keywords. chalcone synthase; real-time PCR; silymarin; anthocyanin; Silybum marianum.

  20. 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.

  1. Dynamic 1-aminocyclopropane-1-carboxylate-synthase and -oxidase transcript accumulation patterns during pollen tube growth in tobacco styles.

    Science.gov (United States)

    Weterings, Koen; Pezzotti, Mario; Cornelissen, Marc; Mariani, Celestina

    2002-11-01

    In flowering plants, pollination of the stigma sets off a cascade of responses in the distal flower organs. Ethylene and its biosynthetic precursor 1-aminocyclopropane-1-carboxylate (ACC) play an important role in regulating these responses. Because exogenous application of ethylene or ACC does not invoke the full postpollination syndrome, the pollination signal probably consists of a more complex set of stimuli. We set out to study how and when the pollination signal moves through the style of tobacco (Nicotiana tabacum) by analyzing the expression patterns of pistil-expressed ACC-synthase and -oxidase genes. Results from this analysis showed that pollination induces high ACC-oxidase transcript levels in all cells of the transmitting tissue. ACC-synthase mRNA accumulated only in a subset of transmitting tract cells and to lower levels as compared with ACC-oxidase. More significantly, we found that although ACC-oxidase transcripts accumulate to uniform high levels, the ACC-synthase transcripts accumulate in a wave-like pattern in which the peak coincides with the front of the ingrowing pollen tube tips. This wave of ACC-synthase expression can also be induced by incongruous pollination and (partially) by wounding. This indicates that wounding-like features of pollen tube invasion might be part of the stimuli evoking the postpollination response and that these stimuli are interpreted differently by the regulatory mechanisms of the ACC-synthase and -oxidase genes.

  2. Engineering fatty acid synthases for directed polyketide production.

    Science.gov (United States)

    Gajewski, Jan; Buelens, Floris; Serdjukow, Sascha; Janßen, Melanie; Cortina, Niña; Grubmüller, Helmut; Grininger, Martin

    2017-04-01

    In this study, we engineered fatty acid synthases (FAS) for the biosynthesis of short-chain fatty acids and polyketides, guided by a combined in vitro and in silico approach. Along with exploring the synthetic capability of FAS, we aim to build a foundation for efficient protein engineering, with the specific goal of harnessing evolutionarily related megadalton-scale polyketide synthases (PKS) for the tailored production of bioactive natural compounds.

  3. Kinetic characterization and phosphoregulation of the Francisella tularensis 1-deoxy-D-xylulose 5-phosphate reductoisomerase (MEP synthase.

    Directory of Open Access Journals (Sweden)

    Safdar Jawaid

    Full Text Available Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (K(M(DXP = 104 microM, K(M(NADPH = 13 microM, k(cat(DXP = 2 s(-1, k(cat(NADPH = 1.3 s(-1, an IC(50 for fosmidomycin of 247 nM, and a K(i for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg(+2 as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis.

  4. 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.

  5. Type III polyketide synthases in microorganisms.

    Science.gov (United States)

    Katsuyama, Yohei; Ohnishi, Yasuo

    2012-01-01

    Type III polyketide synthases (PKSs) are simple homodimers of ketosynthases which catalyze the condensation of one to several molecules of extender substrate onto a starter substrate through iterative decarboxylative Claisen condensation reactions. Type III PKSs have been found in bacteria and fungi, as well as plants. Microbial type III PKSs, which are involved in the biosynthesis of some lipidic compounds and various secondary metabolites, have several interesting characteristics that are not shared by plant type III PKSs. Further, many compounds produced by microbial type III PKSs have significant biological functions and/or important pharmaceutical activities. Thus, studies on this class of enzymes will expand our knowledge of the biosynthetic machineries that generate natural products and generate new findings about microbial physiology. The recent development of next-generation DNA sequencing has allowed for an increase in the number of microbial genomes sequenced and the discovery of many microbial type III PKS genes. Here, we describe basic methods to study microbial type III PKSs whose genes are easy to clone. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    Science.gov (United States)

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism.

  7. 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

    BACKGROUND: Microsatellite instability (MSI) refers to mutations in short motifs of tandemly repeated nucleotides resulting from replication errors and deficient mismatch repair (MMR). Colorectal cancer with MSI has characteristic biology and chemosensitivity, however the molecular basis remains...... 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......) and dihydropyrimidine dehydrogenase (DPD) expression were assessed in paraffin embedded tumor specimens, and associated with outcome in 340 consecutive patients completely resected for colorectal cancer stages II-IV and subsequently receiving adjuvant 5-fluorouracil therapy. RESULTS: MSI was found in 43 (13.8%) tumors...

  8. An unusual plant triterpene synthase with predominant α-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica.

    Science.gov (United States)

    Brendolise, Cyril; Yauk, Yar-Khing; Eberhard, Ellen D; Wang, Mindy; Chagne, David; Andre, Christelle; Greenwood, David R; Beuning, Lesley L

    2011-07-01

    The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. α-Amyrin and β-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α-amyrin to β-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α-amyrin produced is > 80% of the total product and is, therefore, primarily an α-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either

  9. Glycogen synthase kinase 3β in the basolateral amygdala is critical for the reconsolidation of cocaine reward memory.

    Science.gov (United States)

    Wu, Ping; Xue, Yan-Xue; Ding, Zeng-Bo; Xue, Li-Fen; Xu, Chun-Mei; Lu, Lin

    2011-07-01

    Exposure to cocaine-associated conditioned stimuli elicits craving and increases the probability of cocaine relapse in cocaine users even after extended periods of abstinence. Recent evidence indicates that cocaine seeking can be inhibited by disrupting the reconsolidation of the cocaine cue memories and that basolateral amygdala (BLA) neuronal activity plays a role in this effect. Previous studies demonstrated that glycogen synthase kinase 3β (GSK-3β) plays a role in the reconsolidation of fear memory. Here, we used a conditioned place preference procedure to examine the role of GSK-3β in the BLA in the reconsolidation of cocaine cue memories. GSK-3β activity in the BLA, but not central amygdala (CeA), in rats that acquired cocaine (10 mg/kg)-induced conditioned place preference increased after re-exposure to a previously cocaine-paired chamber (i.e., a memory reactivation procedure). Systemic injections of the GSK-3β inhibitor lithium chloride after memory reactivation impaired the reconsolidation of cocaine cue memories and inhibited subsequent cue-induced GSK-3β activity in the BLA. Basolateral amygdala, but not central amygdala, injections of SB216763, a selective inhibitor of GSK-3β, immediately after the reactivation of cocaine cue memories also disrupted cocaine cue memory reconsolidation and prevented cue-induced increases in GSK-3β activity in the BLA. The effect of SB216763 on the reconsolidation of cocaine cue memories lasted at least 2 weeks and was not recovered by a cocaine priming injection. These results indicate that GSK-3β activity in the BLA mediates the reconsolidation of cocaine cue memories. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  10. First experimental application of bevacizumab-eluting PC coated stent for inhibition of vasa vasorum of atherosclerotic plaque: angiographic results in a rabbit atheromatic model.

    Science.gov (United States)

    Stefanadis, Christodoulos; Toutouzas, Konstantinos; Stefanadi, Elli; Kolodgie, Frank; Virmani, Renu; Kipshidze, Nicholas

    2006-01-01

    Atheromatosis is associated with angiogenesis, through the development of a dense net of vasa vasorum. The role of vascular endothelial growth factor (VEGF) is important in this process. Bevacizumab, an antibody specific for VEGF, has recently been applied in the clinical field. We hypothesized that local delivery of bevacizumab by stent would inhibit the development of vasa vasorum at the stented arterial segment in an atheromatic rabbit model. We used 10 New Zealand rabbits under atherogenic diet for 3 weeks. We immersed a BiodivYsio stent into a solution of 4 ml bevacizumab as in previous studies. Both eluting stents and non-eluting BiodivYsio stents were implanted in the middle segment of the 2 iliac arteries of the animals, with the same procedural characteristics. Sacrifice following repeat angiogram was scheduled after 28 days. In all animals the procedure of stent loading with bevacizumab and stent implantation was successful. There was no acute or subacute thrombosis. Iliac artery lumen diameters before and immediately after stent placement were similar in all stent treatment groups. At euthanasia stent lumen diameters were similar in all groups. All stents were angiographically patent at the time of euthanasia without aneurysm formation. Moreover, gross pathologic analysis did not show any evidence of vascular necrosis. Bevacizumab-eluting stent implantation in atheromatic rabbit iliac arteries is feasible and safe. This new approach for the treatment of stable and vulnerable plaques needs to be further investigated.

  11. Fatty Acid Synthase Inhibitors Engage the Cell Death Program Through the Endoplasmic Reticulum

    Science.gov (United States)

    2007-12-01

    Saccharomyces cerevisiae , J Biol Chem 268 (1993) 27269-27276. Epstein, J. I., Carmichael, M. and Partin, A. W., OA-519 (fatty acid synthase) as an independent...547cholesterol lowering drugs is bile acid sequestrants , which 548prevent reabsorption of bile acids by the intestine and result 549in excretion through the...and measured for protein content using a standard copper reduction/bicinchoninic acid assay (BCA; Pierce Biotech- nology), with bovine serum albumin as

  12. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  13. Prostaglandin D2 generation by rat peritoneal mast cells stimulated with Datura stramonium agglutinin and its inhibition by haptenic sugar and wheat germ agglutinin.

    Science.gov (United States)

    Suzuki-Nishimura, Tamiko; Uchida, Masaatsu K

    2002-09-01

    The production of prostaglandin D2 (PGD2) by rat peritoneal mast cells incubated with N-acetyl glucosamine (GlcNAc) oligomer-specific Datura stramonium agglutinin (DSA) for 10 min in the presence of 0.3 mM Ca2+ was examined. Previously, our group reported that the incubation of rat mast cells with DSA (5 - 100 microg/ml) under similar conditions resulted in a calcium influx and histamine release via a pertussis toxin-sensitive G-protein pathway of the mast cells, and the histamine release was inhibited by haptenic sugar chitooligosaccharides or GlcNAc-specific lectin wheat germ agglutinin (WGA) (K. Matsuda et al., Jpn J Pharmacol 66, 195 - 204 (1994)). DSA (5 - 100 microg/ml) dose-dependently stimulated the mast cells to generate PGD2. Chitooligosaccharides (1% w/v) and WGA (100 microg/ml) inhibited the production of PGD2 induced by 100 microg/ml of DSA, suggesting that the effect of DSA is sugar-specific. A prostaglandin G/H synthase inhibitor NS-398 (N-[cyclohexyloxy-4-nitrophenyl] methanesulfonamide) (10 microM) inhibited the formation of PGD2 induced by DSA (20 microg/ml). These results suggest that the binding of DSA to the corresponding sugar residues on the mast cell surface mediates the signaling of the prostaglandin G/H synthase pathway.

  14. Ethylene Inhibits Root Elongation during Alkaline Stress through AUXIN1 and Associated Changes in Auxin Accumulation.

    Science.gov (United States)

    Li, Juan; Xu, Heng-Hao; Liu, Wen-Cheng; Zhang, Xiao-Wei; Lu, Ying-Tang

    2015-08-01

    Soil alkalinity causes major reductions in yield and quality of crops worldwide. The plant root is the first organ sensing soil alkalinity, which results in shorter primary roots. However, the mechanism underlying alkaline stress-mediated inhibition of root elongation remains to be further elucidated. Here, we report that alkaline conditions inhibit primary root elongation of Arabidopsis (Arabidopsis thaliana) seedlings by reducing cell division potential in the meristem zones and that ethylene signaling affects this process. The ethylene perception antagonist silver (Ag(+)) alleviated the inhibition of root elongation by alkaline stress. Moreover, the ethylene signaling mutants ethylene response1-3 (etr1-3), ethylene insensitive2 (ein2), and ein3-1 showed less reduction in root length under alkaline conditions, indicating a reduced sensitivity to alkalinity. Ethylene biosynthesis also was found to play a role in alkaline stress-mediated root inhibition; the ethylene overproducer1-1 mutant, which overproduces ethylene because of increased stability of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE5, was hypersensitive to alkaline stress. In addition, the ethylene biosynthesis inhibitor cobalt (Co(2+)) suppressed alkaline stress-mediated inhibition of root elongation. We further found that alkaline stress caused an increase in auxin levels by promoting expression of auxin biosynthesis-related genes, but the increase in auxin levels was reduced in the roots of the etr1-3 and ein3-1 mutants and in Ag(+)/Co(2+)-treated wild-type plants. Additional genetic and physiological data showed that AUXIN1 (AUX1) was involved in alkaline stress-mediated inhibition of root elongation. Taken together, our results reveal that ethylene modulates alkaline stress-mediated inhibition of root growth by increasing auxin accumulation by stimulating the expression of AUX1 and auxin biosynthesis-related genes. © 2015 American Society of Plant Biologists. All Rights Reserved.

  15. 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.

  16. Drug-Induced Inhibition of Angiotensin Converting Enzyme and Dipeptidyl Peptidase 4 Results in Nearly Therapy Resistant Bradykinin Induced Angioedema: A Case Report.

    Science.gov (United States)

    Hahn, Janina; Trainotti, Susanne; Hoffmann, Thomas K; Greve, Jens

    2017-05-25

    BACKGROUND Bradykinin is an underestimated mediator of angioedema. One subgroup of bradykinin induced angioedema is angioedema triggered by treatment with angiotensin converting enzyme (ACE) inhibitors. Due to its localization in the head and neck region and its unpredictable course, it is a possibly life-threatening condition. There is not an officially approved treatment for ACE inhibitor induced angioedema. CASE REPORT We present a case of an 83-year-old woman, who presented to our ENT department because of acute swelling of the tongue. On admission, there was no pharyngeal or laryngeal edema and no dyspnea. Treatment with glucocorticoids and antihistamines had no response. The patient had ramipril as regular medication, so we assumed ACE inhibitor induced angioedema and treated consequently with C1-inhibitor (human) 1,500 IU. Nevertheless, swelling was progressive and required intubation. Even after the second specific treatment with icatibant, her angioedema subsided extremely slowly. The patient also had regular treatment with saxagliptin, a dipeptidyl peptidase 4 inhibitor, so we assumed that the simultaneous inhibition of two bradykinin degrading enzymes led to a treatment-refractory course of angioedema. CONCLUSIONS General awareness for bradykinin induced angioedema due to regular medication is limited. Our case demonstrated the importance of improving awareness and knowledge about this side effect. We need a better understanding of the pathomechanism to aid in more precise clinical diagnosis. Securing the patient's airway as well as administration of an officially approved therapy is of utmost importance. As the number of patients simultaneously treated with antihypertensive and antidiabetic drugs is likely to increase, the incidence of bradykinin mediated drug induced angioedema is likely to increase as well.

  17. TNF blockers inhibit spinal radiographic progression in ankylosing spondylitis by reducing disease activity: results from the Swiss Clinical Quality Management cohort.

    Science.gov (United States)

    Molnar, Christoph; Scherer, Almut; Baraliakos, Xenofon; de Hooge, Manouk; Micheroli, Raphael; Exer, Pascale; Kissling, Rudolf O; Tamborrini, Giorgio; Wildi, Lukas M; Nissen, Michael J; Zufferey, Pascal; Bernhard, Jürg; Weber, Ulrich; Landewé, Robert B M; van der Heijde, Désirée; Ciurea, Adrian

    2018-01-01

    To analyse the impact of tumour necrosis factor inhibitors (TNFis) on spinal radiographic progression in ankylosing spondylitis (AS). Patients with AS in the Swiss Clinical Quality Management cohort with up to 10 years of follow-up and radiographic assessments every 2 years were included. Radiographs were scored by two readers according to the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) with known chronology. The relationship between TNFi use before a 2-year radiographic interval and progression within the interval was investigated using binomial generalised estimating equation models with adjustment for potential confounding and multiple imputation of missing values. Ankylosing Spondylitis Disease Activity Score (ASDAS) was regarded as mediating the effect of TNFi on progression and added to the model in a sensitivity analysis. A total of 432 patients with AS contributed to data for 616 radiographic intervals. Radiographic progression was defined as an increase in ≥2 mSASSS units in 2 years. Mean (SD) mSASSS increase was 0.9 (2.6) units in 2 years. Prior use of TNFi reduced the odds of progression by 50% (OR 0.50, 95% CI 0.28 to 0.88) in the multivariable analysis. While no direct effect of TNFi on progression was present in an analysis including time-varying ASDAS (OR 0.61, 95% CI 0.34 to 1.08), the indirect effect, via a reduction in ASDAS, was statistically significant (OR 0.75, 95% CI 0.59 to 0.97). TNFis are associated with a reduction of spinal radiographic progression in patients with AS. This effect seems mediated through the inhibiting effect of TNFi on disease activity. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  18. Alternatively Spliced Methionine Synthase in SH-SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal

    Directory of Open Access Journals (Sweden)

    Mostafa Waly

    2016-01-01

    Full Text Available The folate and cobalamin (Cbl- dependent enzyme methionine synthase (MS is highly sensitive to oxidation and its activity affects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient-derived fibroblasts. Here we show that MS mRNA in SH-SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl or the combination of hydroxocobalamin (OHCbl and S-adenosylmethionine (SAM. OHCbl-based activity was eliminated by depletion of the antioxidant glutathione (GSH but could be rescued by provision of either glutathionylcobalamin (GSCbl or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury-containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate-limiting for GSH synthesis. Thimerosal treatment decreased cellular levels of GSCbl and MeCbl. These findings indicate that the alternatively spliced form of MS expressed in SH-SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action.

  19. In vitro and in vivo evidences that antioxidant action contributes to the neuroprotective effects of the neuronal nitric oxide synthase and monoamine oxidase-B inhibitor, 7-nitroindazole.

    Science.gov (United States)

    Thomas, Bobby; Saravanan, Karuppagounder S; Mohanakumar, Kochupurackal P

    2008-05-01

    The neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI) is neuroprotective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Monoamine oxidase (MAO)-B inhibitory action partially contributes to this effect. We tested the hypothesis that 7-NI could be a powerful hydroxyl radical (OH) scavenger, and interferes with oxidative stress caused by MPTP. We measured OH, reduced glutathione (GSH), as well as superoxide dismutase (SOD) and catalase activities in the nucleus caudatus putamen and substantia nigra of Balb/c mice following MPTP and/or 7-NI administration. The nNOS inhibitor caused dose-dependent inhibition in the production of OH in (i) Fenton-like reaction employing ferrous citrate in a cell-free system in test tubes, (ii) in isolated mitochondrial preparation in presence of MPP+, and (iii) in the striatum of mice systemically treated with MPTP. An MPTP-induced depletion of GSH in both the nuclei was blocked by 7-NI, which was dose-dependent (10-50mg/kg), but independent of MAO-B inhibition. The nNOS-mediated recovery of GSH paralleled attenuation of MPTP-induced depletion of striatal dopamine. MPTP-induced increase in the activities of striatal or nigral SOD and catalase were significantly attenuated by 7-NI treatment. These results suggest potent antioxidant action of 7-NI in its neuroprotective effects against MPTP-induced neurotoxicity.

  20. β-Carboline harmine reverses the effects induced by stress on behaviour and citrate synthase activity in the rat prefrontal cortex.

    Science.gov (United States)

    Abelaira, Helena Mendes; Réus, Gislaine Zilli; Scaini, Giselli; Streck, Emilio Luiz; Crippa, José Alexandre; Quevedo, João

    2013-12-01

    The present study was aimed at evaluating the effects of the administration of β-carboline harmine on behaviour and citrate synthase activity in the brain of rats exposed to chronic mild stress (CMS) procedure. To this aim, after 40 days of exposure to CMS procedure, rats were treated with harmine (15 mg/kg/day) for 7 days, then memory, anhedonia and citrate synthase activity were assessed. Result Our findings demonstrated that stressed rats treated with saline increased the sucrose intake, and the stressed rats treated with harmine reversed this effect. Neither stress nor harmine treatment altered memory performance in rats. In addition, chronic stressful situations induced increase in citrate synthase activity in the prefrontal cortex, but not in the hippocampus and striatum. Treatment with harmine reversed the increase in citrate synthase activity in the prefrontal cortex. These findings support the hypothesis that harmine could be involved in controlling the energy metabolism.

  1. Clinicopathological and biological significance of aberrant activation of glycogen synthase kinase-3 in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Fu Y

    2014-06-01

    Full Text Available Yunfeng Fu,1 Xinyu Wang,1 Xiaodong Cheng,1 Feng Ye,2 Xing Xie,1,2 Weiguo Lu1,2 1Department of Gynecologic Oncology, Women's Hospital, School of Medicine, Zhejiang University, 2Women's Reproduction and Health Laboratory of Zhejiang Province, Hangzhou, People's Republic of China Background: Glycogen synthase kinase-3 (GSK-3 plays an important role in human cancer. The aim of this study is to evaluate the clinicopathological significance of expression of GSK-3α/β and pGSK-3α/βTyr279/216 in patients with epithelial ovarian cancer and to investigate whether GSK-3 inhibition can influence cell viability and tumor growth of ovarian cancer. Methods: Immunohistochemistry was used to examine expression of GSK-3α/β and pGSK-3α/βTyr279/216 in 71 human epithelial ovarian cancer tissues and correlations between protein expression, and clinicopathological factors were analyzed. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay following exposure of ovarian carcinoma cells to pharmacological inhibitors of GSK-3 or GSK-3 small interfering RNA. In vivo validation of tumor growth inhibition was performed with xenograft mice. Results: The expression levels of GSK-3α/β and pGSK-3α/βTyr279/216 in ovarian cancers were significantly higher than those in benign tumors. High expression of GSK-3α/β was more likely to be found in patients with advanced International Federation of Gynecology and Obstetrics (FIGO stages and high serum cancer antigen 125. Higher expression of pGSK-3α/βTyr279/216 was associated with advanced FIGO stages, residual tumor mass, high serum cancer antigen 125, and poor chemoresponse. Worse overall survival was revealed by Kaplan–Meier survival curves in patients with high expression of GSK-3α/β or pGSK-3α/βTyr279/216. Multivariate analysis indicated that FIGO stage, GSK-3α/β expression, and pGSK-3α/βTyr279/216 expression were independent prognostic factors for overall

  2. 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.

  3. IDENTIFICATION AND CHARACTERIZATION OF THE SUCROSE SYNTHASE 2 GENE (Sus2 IN DURUM WHEAT

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    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.

  4. 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.

  5. Expanding the chemical space of polyketides through structure-guided mutagenesis of Vitis vinifera stilbene synthase.

    Science.gov (United States)

    Bhan, Namita; Cress, Brady F; Linhardt, Robert J; Koffas, Mattheos

    2015-08-01

    Several natural polyketides (PKs) have been associated with important pharmaceutical properties. Type III polyketide synthases (PKS) that generate aromatic PK polyketides have been studied extensively for their substrate promiscuity and product diversity. Stilbene synthase-like (STS) enzymes are unique in the type III PKS class as they possess a hydrogen bonding network, furnishing them with thioesterase-like properties, resulting in aldol condensation of the polyketide intermediates formed. Chalcone synthases (CHS) in contrast, lack this hydrogen-bonding network, resulting primarily in the Claisen condensation of the polyketide intermediates formed. We have attempted to expand the chemical space of this interesting class of compounds generated by creating structure-guided mutants of Vitis vinifera STS. Further, we have utilized a previously established workflow to quickly compare the wild-type reaction products to those generated by the mutants and identify novel PKs formed by using XCMS analysis of LC-MS and LC-MS/MS data. Based on this approach, we were able to generate 15 previously unreported PK molecules by exploring the substrate promiscuity of the wild-type enzyme and all mutants using unnatural substrates. These structures were specific to STSs and cannot be formed by their closely related CHS-like counterparts. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  6. Mutation of Cellulose Synthase Gene Improves the Nutritive Value of Rice Straw

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    Yanjing Su

    2012-06-01

    Full Text Available Rice straw is an important roughage resource for ruminants in many rice-producing countries. In this study, a rice brittle mutant (BM, mutation in OsCesA4, encoding cellulose synthase and its wild type (WT were employed to investigate the effects of a cellulose synthase gene mutation on rice straw morphological fractions, chemical composition, stem histological structure and in situ digestibility. The morphological fractions investigation showed that BM had a higher leaf sheath proportion (43.70% vs 38.21%, p0.05 was detected in neutral detergent fiber (NDFom and ADL contents for both strains. Histological structure observation indicated that BM stems had fewer sclerenchyma cells and a thinner sclerenchyma cell wall than WT. The results of in situ digestion showed that BM had higher DM, NDFom, cellulose and hemicellulose disappearance at 24 or 48 h of incubation (p<0.05. The effective digestibility of BM rice straw DM and NDFom was greater than that of WT (31.4% vs 26.7% for DM, 29.1% vs 24.3% for NDFom, p<0.05, but the rate of digestion of the slowly digested fraction of BM rice straw DM and NDF was decreased. These results indicated that the mutation in the cellulose synthase gene could improve the nutritive value of rice straw for ruminants.

  7. Exogenous ethylene inhibits sprout growth in onion bulbs

    Science.gov (United States)

    Bufler, Gebhard

    2009-01-01

    Background and Aims Exogenous ethylene has recently gained commercial interest as a sprouting inhibitor of onion bulbs. The role of ethylene in dormancy and sprouting of onions, however, is not known. Methods A cultivar (Allium cepa ‘Copra’) with a true period of dormancy was used. Dormant and sprouting states of onion bulbs were treated with supposedly saturating doses of ethylene or with the ethylene-action inhibitor 1-methylcyclopropene (1-MCP). Initial sprouting was determined during storage at 18 °C by monitoring leaf blade elongation in a specific size class of leaf sheaths. Changes in ATP content and sucrose synthase activity in the sprout leaves, indicators of the sprouting state, were determined. CO2 and ethylene production of onion bulbs during storage were recorded. Key results Exogenous ethylene suppressed sprout growth of both dormant and already sprouting onion bulbs by inhibiting leaf blade elongation. In contrast to this growth-inhibiting effect, ethylene stimulated CO2 production by the bulbs about 2-fold. The duration of dormancy was not significantly affected by exogenous ethylene. However, treatment of dormant bulbs with 1-MCP caused premature sprouting. Conclusions Exogenous ethylene proved to be a powerful inhibitor of sprout growth in onion bulbs. The dormancy breaking effect of 1-MCP indicates a regulatory role of endogenous ethylene in onion bulb dormancy. PMID:18940850

  8. Luteolin inhibits microglial inflammation and improves neuron survival against inflammation.

    Science.gov (United States)

    Zhu, Li-Hong; Bi, Wei; Qi, Ren-bin; Wang, Hua-dong; Lu, Da-xiang

    2011-06-01

    Microglia activation is one of the causative factors for neuroinflammation, which results in brain damage during neurodegenerative disease. Accumulating evidence has shown that the flavonoid luteolin (Lut) possesses potent anti-inflammatory properties; however, its effect on microglia inhibition is currently unknown. Moreover, it is not clear whether Lut also has indirect neuroprotective effects by reducing inflammatory mediators and suppressing microglia activation. In this study, we examined the effects of Lut on lipopolysaccharide (LPS)-induced proinflammatory mediator production and signaling pathways in murine BV2 microglia. In addition, we cocultured microglia and neurons to observe the indirect neuroprotective effects of Lut. Lut inhibited the LPS-stimulated expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) as well as the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). Moreover, Lut blocked LPS-induced nuclear factor kappa B (NF-κB) activation. Preincubation of microglia with Lut diminished the neurotoxic effects, owing to the direct anti-inflammatory effects of the compound. Taken together, our findings suggest that Lut may have a potential therapeutic application in the treatment of neuroinflammatory disorders. Copyright © 2011 Informa Healthcare USA, Inc.

  9. Post-irradiation inactivation, protection, and repair of the sulfhydryl enzyme malate synthase

    International Nuclear Information System (INIS)

    Durchschlag, H.; Zipper, P.

    1985-01-01

    Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0,30,60 h after irradiation. To elucidate the role of OH - , O 2 , and H 2 O 2 in the X-ray inactivation of the enzyme, experiments were performed in the absence of presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results: 1. Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Asub(r)=3% (corresponding to a D 37 =0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated at t=0 led to Asub(r)=21%; repairs started later on resulted in somewhat lower activities. The decay of reparability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly. 2. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., Asub(r)=72% and D 37 =6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate. (orig.)

  10. Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia.

    Science.gov (United States)

    Motta-Mejia, Carolina; Kandzija, Neva; Zhang, Wei; Mhlomi, Vuyane; Cerdeira, Ana Sofia; Burdujan, Alexandra; Tannetta, Dionne; Dragovic, Rebecca; Sargent, Ian L; Redman, Christopher W; Kishore, Uday; Vatish, Manu

    2017-08-01

    Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N   G -nitro-l-arginine methyl ester (eNOS inhibitor; P preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P preeclampsia women had lower STBEV-eNOS expression compared with that from NP women ( P preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease. © 2017 The Authors.

  11. Structure and Mechanism of the Farnesyl Diphosphate Synthase from Trypanosoma cruzi: Implications for Drug Design

    Energy Technology Data Exchange (ETDEWEB)

    Gabelli,S.; McLellan, J.; Montalvetti, A.; Oldfield, E.; Docampo, R.; Amzel, L.

    2006-01-01

    Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C{sub 5} alcohols (isopentenyl and dimethylallyl) to form C{sub 10} and C{sub 15} diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.

  12. Hyperbaric oxygen upregulates cochlear constitutive nitric oxide synthase

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    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.

  13. An Indomethacin Analogue, N-(4-Chlorobenzoyl)-melatonin, is a Selective Inhibitor of Aldo-keto Reductase 1C3 (Type 2 3α-HSD, Type 5 17β-HSD, and Prostaglandin F Synthase), a Potential Target for the Treatment of Hormone Dependent and Hormone Independent Malignancies

    Science.gov (United States)

    Byrns, Michael C.; Steckelbroeck, Stephan; Penning, Trevor M.

    2008-01-01

    Aldo-keto reductase (AKR) 1C3 (type 2 3α-HSD, type 5 17β-HSD, and prostaglandin F synthase) regulates ligand access to steroid hormone and prostaglandin receptors and may stimulate proliferation of prostate and breast cancer cells. NSAIDs are known inhibitors of AKR1C enzymes. An NSAID analogue that inhibits AKR1C3 but is inactive against the cyclooxygenases and the o