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Sample records for acc synthase expression

  1. Ozone stress induces the expression of ACC synthase in potato plants

    Schlagnhaufer, C.D.; Arteca, R.N.; Pell, E.J. (Pennsylvania State Univ., University Park (United States))

    1993-05-01

    When potato plants (Solanum tuberosum L. cv Norland) are subjected to oxone stress ethylene is emitted. Increases in ethylene production are often the result of increased expression of the enzyme ACC synthase. We used the polymerase chain reaction (PCR) to clone a cDNA encoding an ozone-induced ACC synthase. After treating potato plants with 300 ppb ozone for 4 h, RNA was extracted using a guanidinium isothiocyanate method. Using degenerate oligonucleotides corresponding to several conserved regions of ACC synthase sequences reported from different plant tissues as primers, we were able to reverse transcribe the RNA and amplify a cDNA for ACC synthase. The clone is 1098 bp in length encoding for 386 amino acids comprising [approximately]80% of the protein. Computer analysis of the deduced amino acid sequence showed that our clone is 50-70% homologous with ACC synthase genes cloned from other plant tissues. Using the cDNA as a probe in northern analysis we found that there is little or no expression in control tissue: however there is a large increase in the expression of the ACC synthase message in response to ozone treatment.

  2. Ethylene Production and 1-Aminocyclopropane-1-Carboxylate (ACC) Synthase Gene Expression in Tomato(Lycopsicon esculentum Mill.) Leaves Under Enhanced UV-B Radiation

    Lizhe An; Xunling Wang; Xiaofeng Xu; Hongguan Tang; Manxiao Zhang; Zongdong Hou; Yanhong Liu; Zhiguang Zhao; Huyuan Feng; Shijian Xu

    2006-01-01

    Tomato (Lycopsicon esculentum Mill.) plants grown in a greenhouse were irradiated with two different levels of UV-B, namely 8.82 (T1) and 12.6 kJ/m2 per day (T2). Ethylene production, 1-aminocyclopropane-1carboxylate (ACC) content, 1-(malonylamino) cyclopvopane-1-carboxylic acid (MACC) content, gene expression of ACC synthase (EC 4.4.1.14), and ACC oxidase activity in tomato leaves were determined. The results indicated that ACC content, the activity of ACC synthase and ACC oxidase, and ethylene production increased continuously under low doses of UV-B radiation, whereas at high doses of radiation these parameters increased during the first 12 d and then started to decrease. The MACC content increased continuously over 18 d under both doses of UV-B irradiation. The changes in ACC content, ACC synthase activity,ACC oxidase activity, the transcriptional level of the ACC synthase gene, and ethylene production were consistent with each other, suggesting that ACC synthase was the key enzyme in ethylene biosynthesis and that ethylene production in tomato leaf tissues under UV-B radiation could be regulated by the expression of the ACC synthase gene. The results also indicate that the change in ethylene metabolism may be an adaptive mechanism to enhanced UV-B radiation.

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

    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. C-terminal phosphorylation is essential for regulation of ethylene synthesizing ACC synthase enzyme.

    Choudhury, Swarup Roy; Roy, Sujit; Sengupta, Dibyendu N

    2013-02-01

    The genetic and molecular biological studies mainly in Arabidopsis and in some other plants have begun to uncover the various components of ripening signaling pathway in plants. Although transcriptional regulation of major ripening genes have been studied in detail, information on role of phosphorylation in regulating the activity and stability of core ripening pathway associated proteins in relation to ethylene biosynthesis during fruit ripening is still limited. Recently we have demonstrated the evidence for post-translational regulation of MA-ACS1 (Musa acuminata ACC synthase 1), the rate limiting step enzyme regulating ripening ethylene production in banana, through phosphorylation at the C-terminal Ser 476 and 479 residues by a 41-kDa Ser/Thr protein kinase. (1) Here we have further discussed role of protein phosphorylation in regulation of stability and activity of ACS enzymes and the mechanistic and evolutionary perspective of phosphorylation pattern of Type I ACC synthase enzymes. PMID:23221778

  5. Down regulation of ethylene biosynthesis in apples. Cloning and sequencing of the partial ACC synthase gene in the McIntosh cultivar

    Ethylene is a plant hormone that regulates many aspects of plant growth, development and senescence. 1-aminocyclopropane-1-carboxylate (ACC) synthase has been identified as the key enzyme in the biosynthesis of ethylene. Down regulation of ethylene biosynthesis via transformation with the antisense ACC synthase gene (as has already been proved with tomato) might lengthen the storability of apple. To produce the antisense gene of the apple, RNA was isolated from the McIntosh cultivar and the cDNA synthesized. The cDNA template was amplified by polymerase chain reaction (PCR) using ACC specific primers that have been found to be highly conserved in several plant species. PCR resulted in a 1.1 kb DNA band, which was cloned and sequenced. Sequenced analysis of the McIntosh clones (K1, G1, G2, G3, G4) showed a similarity of 63.5-71.6% with the apple ACC synthase known so far and found comparable homology with the ACC syntheses of other plant species in the databank. 8 refs, 2 tabs

  6. Physical mapping of three fruit ripening genes:Endopolygalacturonase,ACC oxidase and ACC synthase from apple(Malus x domestica) in an apple rootstock A106(Malus sieboldii

    ZHUJIMEI; SEGARDINER; 等

    1995-01-01

    The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.

  7. ACC 260

    ADMIN

    2015-01-01

    ACC 460 Complete Class - NO DQ's To purchase this material click below link http://www.assignmentcloud.com/ACC-460/ACC-460-Complete-Class. For more classes visit www.assignmentcloud.com     ACC 460 Week 1 Individual GASB and FASB  Paper To purchase this material click below link   http://www.assignmentcloud.com/ACC-460/ACC-460-Week-1-Individual-GASB-and-FASB-Paper   Prepare a 350- to 700-word paper comparing and cont...

  8. Molecular cloning, expression, and stress response of the estrogen-related receptor gene (AccERR) from Apis cerana cerana

    Zhang, Weixing; Zhu, Ming; Zhang, Ge; Liu, Feng; Wang, Hongfang; Guo, Xingqi; Xu, Baohua

    2016-04-01

    Estrogen-related receptor (ERR), which belongs to the nuclear receptor superfamily, has been implicated in diverse physiological processes involving the estrogen signaling pathway. However, little information is available on ERR in Apis cerana cerana. In this report, we isolated the ERR gene and investigated its involvement in antioxidant defense. Quantitative real-time polymerase chain reaction (qPCR) revealed that the highest mRNA expression occurred in eggs during different developmental stages. The expression levels of AccERR were highest in the muscle, followed by the rectum. The predicted transcription factor binding sites in the promoter of AccERR suggested that AccERR potentially functions in early development and in environmental stress responses. The expression of AccERR was induced by cold (4 °C), heat (42 °C), ultraviolet light (UV), HgCl2, and various types of pesticides (phoxim, deltamethrin, triadimefon, and cyhalothrin). Western blot was used to measure the expression levels of AccERR protein. These data suggested that AccERR might play a vital role in abiotic stress responses.

  9. Influence of Ginkgo biloba extract on the proliferation, apoptosis of ACC-2 cell and Survivin gene expression in adenoid cystic carcinoma of lacrimal gland

    Li-Xiao Zhou; Yu Zhu

    2012-01-01

    Objective: To explore the influence of extract of Ginkgo biloba (EGB) on the proliferation, apoptosis of ACC-2 cell and Survivin gene expression in adenoid cystic carcinoma (ACC) of lacrimal gland. Methods:ACC-2 cell in human with ACC of lacrimal gland was in vitro cultured. MTT method was used for cell proliferation detection. Annexin V/PI double-staining flow cytometer was used to detect cell apoptosis and cell cycle. Survivin gene expression was analyzed by RT-PCR and Western blotting. Results: EGB had inhibitory effect on the proliferation of ACC-2 cell with significant dose-effect relationship, and there was statistical difference when compared with the control group (P<0.01). The inhibitory concentration 50 % (IC50) is 88 mg/L. The flow cytometer test indicated that EGB can gradually increase ACC-2 cell in G0-G1 stage and decrease it in G2-M and S stage. With the increase of dose, the apoptosis rate of ACC-2 cell was obviously increased (P<0.05 or P<0.01). EGB had certain inhibitory effect on Survivin gene expression of ACC-2 cell, and Survivin gene expression was decreased with the increasing of the EGB concentration (P<0.01). Conclusions:EGB can effectively inhibit Survivin gene expression of ACC-2 cell in human with ACC of lacrimal gland, induce the apoptosis of ACC-2 cell and inhibit tumor cell proliferation.

  10. Elevated expression of fatty acid synthase and nuclear localization of carnitine palmitoyltransferase 1C are common among human gliomas.

    Wakamiya, Tomihiro; Suzuki, Satoshi O; Hamasaki, Hideomi; Honda, Hiroyuki; Mizoguchi, Masahiro; Yoshimoto, Koji; Iwaki, Toru

    2014-10-01

    Fatty acid synthase (FASN) and carnitine palmitoyltransferase 1C (CPT1C), a brain-specific isoform of the CPT1 family, are upregulated in certain types of cancers, including gliomas. Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis, and its phosphorylated form inhibits lipid synthesis. We examined the expression and subcellular localization of these fatty acid metabolism-related molecules in human gliomas. We performed immunostaining of two glioma cell lines (U373MG and U87MG) and 41 surgical specimens of diffuse gliomas with various histological grades (21 with the isocitrate dehydrogenase 1(IDH1) R132H mutation and 20 without the mutation). In the cultured glioma cells, CPT1C and phosphorylated ACC (p-ACC) were mainly localized to the nuclei, whereas FASN localized to the cytoplasm. In the surgical specimens, most glioma tissues showed nuclear staining for CPT1C and p-ACC, and cytoplasmic staining for FASN, regardless of the genetic status of IDH1 and the histological grade. Therefore, elevated cytoplasmic expression of FASN and nuclear localization of CPT1C are common among human diffuse gliomas, which may be regulated by the differential phosphorylation status of ACC in the cellular compartment. PMID:24984811

  11. Atypical expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in subcutaneous adipose tissue of male rats.

    Thumelin, S; Kohl, C; Girard, J; Pégorier, J P

    1999-06-01

    The mRNAs encoding mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mtHMG-CoA synthase), the rate limiting enzyme in ketone body production, are highly expressed in subcutaneous (SC) and, to a lesser extent, in peri-epididymal (PE) rat adipose tissues. This atypical mtHMG-CoA synthase gene expression is dependent on the age (from 9 weeks of age) and sex (higher in male than in female) of the rats. In contrast, the expression of mtHMG-CoA synthase in SC adipose deposit is independent of the nutritional state (fed versus starved) or of the thermic environment (24 degrees C versus 4 degrees C). The expression of mtHMG-CoA synthase is suppressed in SC fat pads of castrated male rats whereas treatment of castrated rats with testosterone restores a normal level of expression. Moreover, testosterone injection induces the expression mtHMG-CoA synthase in SC adipose tissue of age-matched females. The presence of the mtHMG-CoA synthase immunoreactive protein confers to mitochondria isolated from SC adipose deposits, the capacity to produce ketone bodies at a rate similar to that found in liver mitochondria (SC = 13.7 +/- 0.7, liver = 16.4 +/- 1.4 nmol/min/mg prot). mtHMG-CoA synthase is expressed in the stromal vascular fraction (SVF) whatever the adipose deposit considered. While acetyl-CoA carboxylase (ACC) is only expressed in mature adipocytes, the other lipogenic enzymes, fatty acid synthase (FAS) and citrate cleavage enzyme (CCE), are expressed both in SVF cells and mature adipocytes. The expression of lipogenic enzyme genes is markedly reduced in adipocytes but not in SVF cells isolated from 48-h starved male rats. When SVF is subfractionated, mtHMG-CoA synthase mRNAs are mainly recovered in two fractions containing poorly digested structures such as microcapillaries whereas the lowest expression is found in the pre-adipocyte fraction. Interestingly, FAS and CCE mRNAs co-segregate with mtHMG-CoA synthase mRNA. The possible physiological relevance of such

  12. Regulation of Expression of the prb-1b / ACC Deaminase gene by UV-B in Transgenic tomatoes

    Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UWA4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns of the transgene. No ACC deaminase RNA or protein was detected bu RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with alpha-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid , ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots of transformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-B light

  13. Increased expression of fatty acid synthase and acetyl-CoA carboxylase in the prefrontal cortex and cerebellum in the valproic acid model of autism

    Chen, Jianling; Wu, Wei; Fu, Yingmei; Yu, Shunying; Cui, Donghong; Zhao, Min; Du, Yasong; Li, Jijun; Li, Xiaohong

    2016-01-01

    The primary aim of the present study was to investigate alterations in enzymes associated with fatty acid synthesis, namely fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), in the prefrontal cortex and cerebellum of the valproic acid (VPA)-induced animal model of autism. In this model, pregnant rats were given a single intraperitoneal injection of VPA, and prefrontal cortex and cerebellum samples from their pups were analyzed. The results of western blotting and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that the protein and mRNA expression levels of FASN, ACC and phospho-ACC (pACC) were increased in the prefrontal cortex and cerebellum of the VPA model of autism. Furthermore, in the prefrontal cortex and cerebellum of the VPA model of autism, AMPK expression is increased, whereas PI3K and Akt expression are unchanged. This suggests that disorder of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/FASN and/or adenosine 5′-monophosphate-activated protein kinase (AMPK)/ACC pathway may be involved in the pathogenesis of autism. It is hypothesized that fatty acid synthesis participates in autism through PI3K/Akt/FASN and AMPK/ACC pathways. PMID:27602061

  14. Expression of recombinant AccMRJP1 protein from royal jelly of Chinese honeybee in Pichia pastoris and its proliferation activity in an insect cell line

    Main royal jelly protein 1 (MRJP1) is the most abundant member of the main royal jelly protein (MRJP) family among honeybees. Mature MRJP1 cDNA of the Chinese honeybee (Apis cerana cerana MRJP1, or AccMRJP1) was expressed in Pichia pastoris. SDS-PAGE showed that recombinant AccMRJP1 was identical in...

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  18. Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase.

    Martin, Christine J; Timoney, Máire C; Sheridan, Rose M; Kendrew, Steven G; Wilkinson, Barrie; Staunton, James C; Leadlay, Peter F

    2003-12-01

    A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed. PMID:14685317

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  20. Cloning, expression patterns, and preliminary characterization of AccCPR24, a novel RR-1 type cuticle protein gene from Apis cerana cerana.

    Chu, Xiaoqian; Lu, Wenjing; Zhang, Yuanying; Guo, Xingqi; Sun, Rujiang; Xu, Baohua

    2013-11-01

    Cuticular proteins (CPs) are key components of insect cuticle, a structure that plays a pivotal role in insect development and defense. In this study, we cloned the full-length cDNA of a CP gene from Apis cerana cerana (AccCPR24). An amino acid sequence alignment indicated that AccCPR24 contains the conserved Rebers and Riddiford consensus sequence and shares high similarity with the genes from other hymenopteran insects. We then isolated the genomic DNA and found that the first intron, which is present in other CP genes, is absent in AccCPR24. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed that AccCPR24 is highly expressed in the late pupal stage and midgut. Expression was inhibited by an exogenous ecdysteroid in vitro but was enhanced by this hormone in vivo; environmental stressors, such as heavy metals and pesticides, also influenced gene expression. In addition, a disc diffusion assay showed that AccCPR24 enhanced the ability of bacterial cells to resist multiple stresses. We infer from our results that AccCPR24 acts in honeybee development and in protecting these insects from abiotic stresses. PMID:24115354

  1. Enhancement of cellulose production by expression of sucrose synthase in Acetobacter xylinum

    Nakai, Tomonori; Tonouchi, Naoto; Konishi, Teruko; Kojima, Yukiko; Tsuchida, Takayasu; Yoshinaga, Fumihiro; Sakai, Fukumi; Hayashi, Takahisa

    1999-01-01

    Higher plants efficiently conserve energy ATP in cellulose biosynthesis by expression of sucrose synthase, in which the high free energy between glucose and fructose in sucrose can be conserved and used for the synthesis of UDP-glucose. A mixture of sucrose synthase and bacterial cellulose synthase proceeded to form UDP-glucose from sucrose plus UDP and to synthesize 1,4-β-glucan from the sugar nucleotide. The mutant sucrose synthase, which mimics phosphorylated sucrose synthase, enhanced the...

  2. Bacterial phytoene synthase: molecular cloning, expression, and characterization of Erwinia herbicola phytoene synthase.

    Iwata-Reuyl, Dirk; Math, Shivanand K; Desai, Shrivallabh B; Poulter, C Dale

    2003-03-25

    Phytoene synthase (PSase) catalyzes the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to give prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene. These reactions constitute the first pathway specific step in carotenoid biosynthesis. The crtB gene encoding phytoene synthase was isolated from a plasmid containing the carotenoid gene cluster in Erwinia herbicola and cloned into an Escherichia coli expression system. Upon induction, recombinant phytoene synthase constituted 5-10% of total soluble protein. To facilitate purification of the recombinant enzyme, the structural gene for PSase was modified by site-directed mutagenesis to incorporate a C-terminal Glu-Glu-Phe (EEF) tripepetide to allow purification by immunoaffinity chromatography on an immobilized monoclonal anti-alpha-tubulin antibody YL1/2 column. Purified recombinant PSase-EEF gave a band at 34.5 kDa upon SDS-PAGE. Recombinant PSase-EEF was then purified to >90% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The enzyme required Mn(2+) for activity, had a pH optimum of 8.2, and was strongly stimulated by detergent. The concentration of GGPP needed for half-maximal activity was approximately 35 microM, and a significant inhibition of activity was seen at GGPP concentrations above 100 microM. The sole product of the reaction was 15,15'-Z-phytoene. PMID:12641468

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

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

    1989-01-01

    The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar t...... into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein....

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  6. Expression characteristics of CS-ACS1, CS-ACS2 and CS-ACS3, three members of the 1-aminocyclopropane-1-carboxylate synthase gene family in cucumber (Cucumis sativus L.) fruit under carbon dioxide stress.

    Mathooko, F M; Mwaniki, M W; Nakatsuka, A; Shiomi, S; Kubo, Y; Inaba, A; Nakamura, R

    1999-02-01

    We investigated the expression pattern of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes, CS-ACS1, CS-ACS2 and CS-ACS3 in cucumber (Cucumis sativus L.) fruit under CO2 stress. CO2 stress-induced ethylene production paralleled the accumulation of only CS-ACS1 transcripts which disappeared upon withdrawal of CO2. Cycloheximide inhibited the CO2 stress-induced ethylene production but superinduced the accumulation of CS-ACS1 transcript. At higher concentrations, cycloheximide also induced the accumulation of CS-ACS2 and CS-ACS3 transcripts. In the presence of CO2 and cycloheximide, the accumulation of CS-ACS2 transcript occurred within 1 h, disappeared after 3 h and increased greatly upon withdrawal of CO2. Inhibitors of protein kinase and types 1 and 2A protein phosphatases which inhibited and stimulated, respectively, CO2 stress-induced ethylene production had little effect on the expression of these genes. The results presented here identify CS-ACS1 as the main ACC synthase gene responsible for the increased ethylene biosynthesis in cucumber fruit under CO2 stress and suggest that this gene is a primary response gene and its expression is under negative control since it is expressed by treatment with cycloheximide. The results further suggest that the regulation of CO2 stress-induced ethylene biosynthesis by reversible protein phosphorylation does not result from enhanced ACC synthase transcription. PMID:10202812

  7. Crystallization of recombinant 1-amino cyclo propane-1-carboxylate (Acc) oxidase

    Full text. Ethylene is an important harmone in plant biology because it activates gene expression with consequences at all phases of plant growth and development spanning seed germination to fruit ripening and senesense of plant organs. In climacteric fruits, the sharp increase in ethylene production at the onset of ripening is throught to trigger the changes in colour, aroma, texture and flavour. The final step in ethylene biosynthesis is catalyzed by ACC oxidase. Biothechnological methods have been used to inhibit ethylene biosynthesis and ripening in tomato by down-regulating ACC synthase and ACC oxidase gene expression using the antisense RNA strategy. A similar goal has been achieved by overexpressing a bacterial ACC deaminase or a viral-S-adenosylmethionine hydrolase gene, which reduces the availability of the ethylene precursors., ACC and S-adenosylmethionine, respectively. C02 at concentrations commonly found in the intracellular space of plant tissues is required to active ACC oxidase to produce ethylene and can elevate enzyme activity 20-fold in a concentration dependent manner. Consequently, the intracellular ethylene level is modulated from low inactive levels when C02 is not limiting and this may alter gene expression. ACC oxidase undergoes catalytic inactivation as the reaction to make ethylene procedes and this too may involve CO2. It has been suggested that CO2acts as a modulator of ACC oxidase activity and therby helps regulate ethylene levels in the cell and thus may explain many ethylene related phenomena in plant biology. CO2 is know to affect O2 binding in hemoglobin and ribulose bisphosphate carboxylase-oxygenase (Rubisco). Catalytic inactivation is a common phenomena in enzyme turnover, ACC oxidase is a Fe+2/ascorbate requiring enzyme and this makes it a prime candidate for metal ion oxidation-based inactivation. Charentais melon with an antisense ACC oxidase cDNA. A trangenic line exhibits reduction of ethylene production and inhibition of

  8. Crystallization of recombinant 1-amino cyclo propane-1-carboxylate (Acc) oxidase

    Watanabe, L.; Arni, R.K. [UNESP, Sao Jose do Rio Preto, SP (Brazil). Dept. de Fisica; Dilley, D. [Michigan State Univ., East Lansing, MI (United States). Dept. of Biophysics

    1996-12-31

    Full text. Ethylene is an important harmone in plant biology because it activates gene expression with consequences at all phases of plant growth and development spanning seed germination to fruit ripening and senesense of plant organs. In climacteric fruits, the sharp increase in ethylene production at the onset of ripening is throught to trigger the changes in colour, aroma, texture and flavour. The final step in ethylene biosynthesis is catalyzed by ACC oxidase. Biothechnological methods have been used to inhibit ethylene biosynthesis and ripening in tomato by down-regulating ACC synthase and ACC oxidase gene expression using the antisense RNA strategy. A similar goal has been achieved by overexpressing a bacterial ACC deaminase or a viral-S-adenosylmethionine hydrolase gene, which reduces the availability of the ethylene precursors., ACC and S-adenosylmethionine, respectively. C0{sub 2} at concentrations commonly found in the intracellular space of plant tissues is required to active ACC oxidase to produce ethylene and can elevate enzyme activity 20-fold in a concentration dependent manner. Consequently, the intracellular ethylene level is modulated from low inactive levels when C0{sub 2} is not limiting and this may alter gene expression. ACC oxidase undergoes catalytic inactivation as the reaction to make ethylene procedes and this too may involve CO{sub 2}. It has been suggested that CO{sub 2}acts as a modulator of ACC oxidase activity and therby helps regulate ethylene levels in the cell and thus may explain many ethylene related phenomena in plant biology. CO{sub 2} is know to affect O{sub 2} binding in hemoglobin and ribulose bisphosphate carboxylase-oxygenase (Rubisco). Catalytic inactivation is a common phenomena in enzyme turnover, ACC oxidase is a Fe{sup +2}/ascorbate requiring enzyme and this makes it a prime candidate for metal ion oxidation-based inactivation. Charentais melon with an antisense ACC oxidase cDNA. A trangenic line exhibits reduction

  9. Expression of fatty acid synthase in nonalcoholic fatty liver disease.

    Dorn, Christoph; Riener, Marc-Oliver; Kirovski, Georgi; Saugspier, Michael; Steib, Kathrin; Weiss, Thomas S; Gäbele, Erwin; Kristiansen, Glen; Hartmann, Arndt; Hellerbrand, Claus

    2010-01-01

    Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid accumulation which starts with simple hepatic steatosis and may progress toward inflammation (nonalcoholic steatohepatitis [NASH]). Fatty acid synthase (FASN) catalyzes the last step in fatty acid biosynthesis, and thus, it is believed to be a major determinant of the maximal hepatic capacity to generate fatty acids by de novo lipogenesis. The aim of this study was to analyze the correlation between hepatic steatosis and inflammation with FASN expression. In vitro incubation of primary human hepatocytes with fatty acids dose-dependently induced cellular lipid-accumulation and FASN expression, while stimulation with TNF did not affect FASN levels. Further, hepatic FASN expression was significantly increased in vivo in a murine model of hepatic steatosis without significant inflammation but not in a murine NASH model as compared to control mice. Also, FASN expression was not increased in mice subjected to bile duct ligation, an experimental model characterized by severe hepatocellular damage and inflammation. Furthermore, FASN expression was analyzed in 102 human control or NAFLD livers applying tissue micro array technology and immunohistochemistry, and correlated significantly with the degree of hepatic steatosis, but not with inflammation or ballooning of hepatocytes. Quantification of FASN mRNA expression in human liver samples confirmed significantly higher FASN levels in hepatic steatosis but not in NASH, and expression of SREBP1, which is the main transcriptional regulator of FASN, paralleled FASN expression levels in human and experimental NAFLD. In conclusion, the transcriptional induction of FASN expression in hepatic steatosis is impaired in NASH, while hepatic inflammation in the absence of steatosis does not affect FASN expression, suggesting that FASN may serve as a new diagnostic marker or therapeutic target for the progression of NAFLD. PMID:20606731

  10. Novel terpenes generated by heterologous expression of bacterial terpene synthase genes in an engineered Streptomyces host

    YAMADA, YUUKI; Arima, Shiho; Nagamitsu, Tohru; Johmoto, Kohei; Uekusa, Hidehiro; Eguchi, Tadashi; Shin’ya, Kazuo; Cane, David E.; Ikeda, Haruo

    2015-01-01

    Mining of bacterial genome data has revealed numerous presumptive terpene synthases. Heterologous expression of several putative terpene synthase genes in an engineered Streptomyces host has revealed 13 newly discovered terpenes whose GC-MS and NMR data did not match any known compounds in the spectroscopic databases. Each of the genes encoding the corresponding terpene synthases were silent in their parent microorganisms. Heterologous expression and detailed NMR spectroscopic analysis allowe...

  11. Inducible nitric oxide synthase is expressed in synovial fluid granulocytes

    CEDERGREN, J; FORSLUND 2, T; SUNDQVIST 2, T; SKOGH 1, T

    2002-01-01

    The objective of the study was to evaluate the NO-producing potential of synovial fluid (SF) cells. SF from 15 patients with arthritis was compared with blood from the same individuals and with blood from 10 healthy controls. Cellular expression of inducible nitric oxide synthase (iNOS) was analysed by flow cytometry. High-performance liquid chromatography was used to measure l-arginine and l-citrulline. Nitrite and nitrate were measured colourimetrically utilizing the Griess’ reaction. Compared to whole blood granulocytes in patients with chronic arthritis, a prominent iNOS expression was observed in SF granulocytes (P < 0·001). A slight, but statistically significant, increase in iNOS expression was also recorded in lymphocytes and monocytes from SF. l-arginine was elevated in SF compared to serum (257 ± 78 versus 176 ± 65 µmol/l, P = 0·008), whereas a slight increase in l-citrulline (33 ± 11 versus 26 ± 9 µmol/l), did not reach statistical significance. Great variations but no significant differences were observed comparing serum and SF levels of nitrite and nitrate, respectively, although the sum of nitrite and nitrate tended to be elevated in SF (19·2 ± 20·7 versus 8·6 ± 6·5 µmol/l, P = 0·054). Synovial fluid leucocytes, in particular granulocytes, express iNOS and may thus contribute to intra-articular NO production in arthritis. PMID:12296866

  12. Expression of prostaglandin synthases (pgds and pges) during zebrafish gonadal differentiation

    Jørgensen, Anne; Nielsen, John E; Nielsen, Betina Frydenlund;

    2010-01-01

    The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E2 synthase (pges) and especially the lipocalin-type prostaglandin D2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found in...

  13. Expression of prostaglandin synthases (pgds and pges) during zebrafish gonadal differentiation

    Jørgensen, Anne; Nielsen, John E.; Nielsen, Betina F.;

    2010-01-01

    The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E-2 synthase (pges) and especially the lipocalin-type prostaglandin D-2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found ...

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

    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.

  15. ACC 491 Courses/snaptutorial

    charles

    2015-01-01

    For more classes visits www.snaptutorial.com           ACC 491 Week 1 Individual Assignment Generally Accepted Auditing Standards Paper ACC 491 Week 1 DQ 1 ACC 491 Week 1 DQ 2 ACC 491 week 2 Individual Assignments From the Text ACC 491 Week 2 Team Assignment Auditing, Attestation, and Assurance Services Paper ACC 491 Week 2 DQ 1 ACC 491 Week 2 DQ 2 ACC 491 week 3 Individual Assignments From the Text ACC 491 week 3 Te...

  16. ACC 375 Course Tutorial / indigohelp

    roman8034

    2015-01-01

     ACC 375 Entire Course For more classes visit www.indigohelp.com   ACC 375 Week 1 Individual Assignment Understanding Ethics Matrix ACC 375 Week 1 Discussion Question 1 ACC 375 Week 1 Discussion Question 2 ACC 375 Week 2 Team Assignment Sarbanes Oxley Act Training Manual ACC 375 Week 2 Discussion Question 1 ACC 375 Week 2 Discussion Question 2 ACC 375 Week 3 Individual Assignment Ethics Situation 1 Fraudulent Schemes Report ACC 375 Week 3 Team As...

  17. ACC 490 UOP Course Tutorial / acc490dotcom

    geeni

    2015-01-01

    ACC 490 Entire Course For more course tutorials visit www.acc490.com   ACC 490 Week 1 Generally Accepted Auditing Standards Paper ACC 490 Week 1 DQ 1 ACC 490 Week 1 DQ 2 ACC 490 Week 2 Individual Ch. 1 Textbook Exercise ACC 490 Week 2 Learning Team Auditing, Attestation, and Assurance Services Paper ACC 490 Week 2 DQ 1 ACC 490 Week 2 DQ 2 ACC 490 Week 3 Individual Ch. 5, 6, & 7 Textbook Exercises ACC 490 Week 3 Learning Team Ch. 6 &...

  18. ACC 491 UOP Course Tutorial / acc491dotcom

    geeni

    2015-01-01

    ACC 491 Entire Course For more course tutorials visit www.acc491.com   ACC 491 Week 1 Individual Assignment Generally Accepted Auditing Standards Paper ACC 491 Week 1 DQ 1 ACC 491 Week 1 DQ 2 ACC 491 week 2 Individual Assignments From the Text ACC 491 Week 2 Team Assignment Auditing, Attestation, and Assurance Services Paper ACC 491 Week 2 DQ 1 ACC 491 Week 2 DQ 2 ACC 491 week 3 Individual Assignments From the Text ACC 491 week 3 Team Assig...

  19. Expression, crystallization and structure elucidation of γ-terpinene synthase from Thymus vulgaris.

    Rudolph, Kristin; Parthier, Christoph; Egerer-Sieber, Claudia; Geiger, Daniel; Muller, Yves A; Kreis, Wolfgang; Müller-Uri, Frieder

    2016-01-01

    The biosynthesis of γ-terpinene, a precursor of the phenolic isomers thymol and carvacrol found in the essential oil from Thymus sp., is attributed to the activitiy of γ-terpinene synthase (TPS). Purified γ-terpinene synthase from T. vulgaris (TvTPS), the Thymus species that is the most widely spread and of the greatest economical importance, is able to catalyze the enzymatic conversion of geranyl diphosphate (GPP) to γ-terpinene. The crystal structure of recombinantly expressed and purified TvTPS is reported at 1.65 Å resolution, confirming the dimeric structure of the enzyme. The putative active site of TvTPS is deduced from its pronounced structural similarity to enzymes from other species of the Lamiaceae family involved in terpenoid biosynthesis: to (+)-bornyl diphosphate synthase and 1,8-cineole synthase from Salvia sp. and to (4S)-limonene synthase from Mentha spicata. PMID:26750479

  20. ACC 205 NEW TUTORIAL / Uoptutorial

    Richard

    2015-01-01

    For more course tutorials visit www.uoptutorial.com   ACC 205 Week 1 DQ 1 Accounting Equation   ACC 205 Week 1 DQ 2 Accounts   ACC 205 Week 1 Journal Balance Sheet Journal   ACC 205 Week 2 DQ 1 Accounting Cycle   ACC 205 Week 2 DQ 2 Bank Reconciliation   ACC 205 Week 2 Journal Income Statement Journal   ACC 205 Week 3 DQ 1 LIFO vs. FIFO   ACC 205 Week 3 DQ 2 Depreciation   ...

  1. ACC 490 Courses/snaptutorial

    charles

    2015-01-01

    For more classes visits www.snaptutorial.com           ACC 490 Week 1 Generally Accepted Auditing Standards Paper ACC 490 Week 1 – DQ 1   ACC 490 Week 1 – DQ 2   ACC 490 Week 2 Individual Ch. 1 Textbook Exercise ACC 490 Week 2 Learning Team Auditing, Attestation, and Assurance Services Paper ACC 490 Week 2 – DQ 1   ACC 490 Week 2 – DQ 2   ACC 490 Week 3 ...

  2. ACC 375 Courses/snaptutorial

    charles

    2015-01-01

    For more classes visit www.snaptutorial.com   ACC 375 Week 1 Individual Assignment Understanding Ethics Matrix ACC 375 Week 1 Discussion Question 1 ACC 375 Week 1 Discussion Question 2 ACC 375 Week 2 Team Assignment Sarbanes Oxley Act Training Manual ACC 375 Week 2 Discussion Question 1 ACC 375 Week 2 Discussion Question 2 ACC 375 Week 3 Individual Assignment Ethics Situation 1 Fraudulent Schemes Report ACC 375 Week 3 Team Assignment Article Review Ethic...

  3. Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes.

    Jung, W; Yu, O; Lau, S M; O'Keefe, D P; Odell, J; Fader, G; McGonigle, B

    2000-02-01

    Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes. PMID:10657130

  4. The formation of ACC and competition between polyamines and ethylene for SAM

    Ethylene biosynthesis involves the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS). ACC is then converted to ethylene. The genes that encode enzymes in this pathway all belong to a family of genes. Differential transcriptional regulation ...

  5. Dual-level regulation of ACC synthase activity by MPK3/MPK6 cascade and its downstream WRKY transcription factor during ethylene induction in Arabidopsis.

    Guojing Li

    2012-06-01

    Full Text Available Plants under pathogen attack produce high levels of ethylene, which plays important roles in plant immunity. Previously, we reported the involvement of ACS2 and ACS6, two Type I ACS isoforms, in Botrytis cinerea-induced ethylene biosynthesis and their regulation at the protein stability level by MPK3 and MPK6, two Arabidopsis pathogen-responsive mitogen-activated protein kinases (MAPKs. The residual ethylene induction in the acs2/acs6 double mutant suggests the involvement of additional ACS isoforms. It is also known that a subset of ACS genes, including ACS6, is transcriptionally induced in plants under stress or pathogen attack. However, the importance of ACS gene activation and the regulatory mechanism(s are not clear. In this report, we demonstrate using genetic analysis that ACS7 and ACS11, two Type III ACS isoforms, and ACS8, a Type II ACS isoform, also contribute to the B. cinerea-induced ethylene production. In addition to post-translational regulation, transcriptional activation of the ACS genes also plays a critical role in sustaining high levels of ethylene induction. Interestingly, MPK3 and MPK6 not only control the stability of ACS2 and ACS6 proteins via direct protein phosphorylation but also regulate the expression of ACS2 and ACS6 genes. WRKY33, another MPK3/MPK6 substrate, is involved in the MPK3/MPK6-induced ACS2/ACS6 gene expression based on genetic analyses. Furthermore, chromatin-immunoprecipitation assay reveals the direct binding of WRKY33 to the W-boxes in the promoters of ACS2 and ACS6 genes in vivo, suggesting that WRKY33 is directly involved in the activation of ACS2 and ACS6 expression downstream of MPK3/MPK6 cascade in response to pathogen invasion. Regulation of ACS activity by MPK3/MPK6 at both transcriptional and protein stability levels plays a key role in determining the kinetics and magnitude of ethylene induction.

  6. Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

    Sauge-Merle, Sandrine; Cuiné, Stéphan; Carrier, Patrick; Lecomte-Pradines, Catherine; Luu, Doan-Trung; Peltier, Gilles

    2003-01-01

    Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial ...

  7. ACC 491 Courses Tutorial / indigohelp

    sahe

    2015-01-01

      ACC 491 Week 1 Individual Assignment Generally Accepted Auditing Standards Paper ACC 491 Week 1 DQ 1 ACC 491 Week 1 DQ 2 ACC 491 week 2 Individual Assignments From the Text ACC 491 Week 2 Team Assignment Auditing, Attestation, and Assurance Services Paper ACC 491 Week 2 DQ 1 ACC 491 Week 2 DQ 2 ACC 491 week 3 Individual Assignments From the Text ACC 491 week 3 Team Assignments From the Text ACC 491 Week 3 Team Assignment Assessing Materiality and Risk Sim...

  8. ACC 490 Course Tutorial / tutorialrank

    welcome1361

    2015-01-01

    ACC 490 Entire Course (UOP Course) For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 5 Times, Rating: A+   ACC 490 Week 1 Generally Accepted Auditing Standards Paper (UOP Course) ACC 490 Week 1 DQ 1 (UOP Course) ACC 490 Week 1 DQ 2 (UOP Course) ACC 490 Week 2 Individual Ch. 1 Textbook Exercises (UOP Course) ACC 490 Week 2 Learning Team Auditing, Attestation, and Assurance Services Paper (UOP Course) ACC 490 Week 2 DQ 1 (UOP C...

  9. ACC 206 NEW TUTORIAL / Uoptutorial

    Honey Singh

    2015-01-01

    For more course tutorials visit www.uoptutorial.com   ACC 206 Week 1 Assignment Chapter One Problems   ACC 206 Week 1 DQ1 Cash Flows Information   ACC 206 Week 1 DQ2 Apple's Cash Flow   ACC 206 Week 2 Assignment Chapter Two and Three Problems   ACC 206 Week 2 DQ1 Stock Features   ACC 206 Week 2 DQ2 Role of Management Accounting   ACC 206 Week 2 Journal Institute of Management Accounting &nb...

  10. Relationship between inducible nitric oxide synthase expression and angiogenesis in primary gallbladder carcinoma tissue

    Niu, Xin-Jie; wang, Zuo-ren; Wu, Sheng-Li; Geng, Zhi-Min; Zhang, Yun-Feng; Qing, Xing-Lei

    2004-01-01

    AIM: To explore the relationship between angiogenesis and biological behaviors of primary gallbladder carcinoma (PGBC), the relationship between the expression of inducible nitric oxide synthase (iNOS) and biological behaviors of PGBC and its relationship with the expression of iNOS and angiogenesis of PGBC.

  11. Heterologous expression of pentalenene synthase (PSS) from Streptomyces UC5319 in Xanthophyllomyces dendrorhous

    Melillo, Elena; Muntendam, Remco; Quax, Wim J.; Kayser, Oliver

    2012-01-01

    For the first time, the pentalenene synthase (PSS) gene from Streptomyces UC5319 was expressed in Xanthophyllomyces dendrorhous, a native producer of astaxanthin. For the expression of the gene and the concurrent knock out of the native crtE or crtYB genes, two new vectors were engineered and used f

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

    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.

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

    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-[35S]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

  14. ACC 291 NEW Tutorials / acc291dotcom

    modumtejak

    2015-01-01

    For more course tutorials visit www.acc291.com   ACC 291 Final Exam Study Guide Question 207 On January 1, a machine with a useful life of five years and a residual value of $40,000 was purchased for $120,000. What is the depreciation expense for year 2 under the double-declining-balance method of depreciation? IFRS Multiple Choice Question 01 As a recent graduate of State University you're aware that IFRS requires component depreciation for plant assets. ...

  15. ACC 490 Courses Tutorial / indigohelp

    suha

    2015-01-01

    ACC 490 Week 1 Generally Accepted Auditing Standards Paper ACC 490 Week 1 – DQ 1   ACC 490 Week 1 – DQ 2   ACC 490 Week 2 Individual Ch. 1 Textbook Exercise ACC 490 Week 2 Learning Team Auditing, Attestation, and Assurance Services Paper ACC 490 Week 2 – DQ 1   ACC 490 Week 2 – DQ 2   ACC 490 Week 3 Individual Ch. 5, 6, & 7 Textbook Exercises ACC 490 Week 3 Learning TeamCh. 6 & 7 Textbook Exerc...

  16. Effect of aging on expression of nitric oxide synthase I and activity of nitric oxide synthase in rat penis

    Jun-PingSHI; Yong-MeiZHAO; Yu-TongSONG

    2003-01-01

    Aim: To investigate the effect of aging on the expression of nitric oxide synthase I (NOS I) and the activity of NOS in rat penis. Methods: Sixty male rats from 3 age groups (adult, old and senescent) were investigated.The expression of NOS I protein and mRNA in rat penis were detected by Western blot and RT-PCR respectively and the NOS activity, with ultraviolet spectrophotometry. Results: In the old and senescent group, NOS I protein expression was significantly decreased as compared with the adult. NOS I mRNA expression was well correlated with the protein expression. NOS activity was not statistically different between the adult and old groups, but it was significantly reduced in the senescent compared with the adult group (P<0.01). Conclusion: The aging-induced decreases in NOS I expression and NOS activity may be one of the main mechanisms leading to erectile dysfunctionin the senescent rats. ( Asian J Androl 2003 Jun; 5: 117-120)

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

    Bechard Matthew E.

    2003-01-01

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

  18. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy β and originally identified CoA synthase, CoASy α. The transcript specific for CoASy β was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy β. In contrast to CoASy α, which shows ubiquitous expression, CoASy β is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation

  19. ACC 491 Course Tutorial / tutorialrank

    welcome1361

    2015-01-01

    ACC 491 Entire Course (UOP Course) For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 4 Times, Rating: A+   ACC 491 Week 1 Individual Assignment Generally Accepted Auditing Standards Paper (UOP Course) ACC 491 Week 1 DQ 1 (UOP Course) ACC 491 Week 1 DQ 2 (UOP Course) ACC 491 week 2 Individual Assignments From the Text (UOP Course) ACC 491 Week 2 Team Assignment Auditing, Attestation, and Assurance Services Paper (UOP Course) ...

  20. ACC 226 Course Tutorial / tutorialrank

    welcome8888

    2015-01-01

    For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 5 Times, Rating: A+     ACC 226 Week 1 CheckPoint Accounts Receivable and Uncollectible Accounts   ACC 226 Week 1 DQ 1 and DQ 2   ACC 226 Week 2 Assignment Accounting for Depreciation part   ACC 226 Week 2 CheckPoint Ethics and Computing   ACC 226 Week 3 CheckPoint Classifying Liabilities and Preparing Payroll Entries   ACC 226...

  1. ACC 546 Courses Tutorial / indigohelp

    saqe

    2015-01-01

    ACC 546 Week 1 Individual Assignment Auditing Introduction Letter ACC 546 Week 2 Individual Assignment Beginning the Audit Report ACC 546 Week 3 Individual Assignment The Audit Report and Internal Control Evaluation ACC 546 Week 4 Learning Team Assignment Audit Program Design Part I ACC 546 Week 5 Learning Team Assignment Audit Program Design Part II ACC 546 Week 6 Individual Assignment Audit Program Design Part III  

  2. Microsatellite instability in colorectal cancer and association with thymidylate synthase and dihydropyrimidine dehydrogenase expression

    Kruhøffer Mogens; Vainer Ben; Jensen Søren A; Sørensen Jens B

    2009-01-01

    Abstract 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. Met...

  3. Expression of inducible nitric oxide synthase in trigeminal ganglion cells during culture

    Jansen-Olesen, Inger; Zhou, MingFang; Zinck, Tina Jovanovic; Xu, Cang-Bao; Edvinsson, Lars

    2005-01-01

    Nitric oxide (NO) is an important signalling molecule that has been suggested to be a key molecule for induction and maintenance of migraine attacks based on clinical studies, animal experimental studies and the expression of nitric oxide synthase (NOS) immunoreactivity within the trigeminovascul...

  4. Microsatellite instability in colorectal cancer and association with thymidylate synthase and dihydropyrimidine dehydrogenase expression

    Jensen, Søren A; Vainer, Ben; Kruhøffer, Mogens; Sørensen, Jens B

    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...

  5. Molecular Cloning and Bacterial Expression of Germacrene A Synthase cDNA from Crepidiastrum sonchifolium

    2006-01-01

    Germacrene A synthase(GAS) catalyzes the biosynthesis of germacrene A, which is a key precursor for sesquiterpene lactones. Cloning of a novel full-length cDNA encoding GAS from the medicinal plant Crepidiastrum sonchifolium(designated CsGAS) is reported in this study. The cDNA is 1837 bp long and contains a 1680-bp open reading frame encoding a 559 amino-acid protein. The functional expression of the cDNA in Escherichia coli, as an N-terminal thioredoxin fusion protein, with the pET32a vector yielding a recombinant enzyme. Sequence analysis was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco, and the effect of possible involvement of a number of amino acids in sesquiterpene synthase on product specificity was also discussed.

  6. Allotopic Expression of a Gene Encoding FLAG Tagged-subunit 8 of Yeast Mitochondrial ATP Synthase

    I MADE ARTIKA

    2006-03-01

    Full Text Available Subunit 8 of yeast mitochondrial ATP synthase is a polypeptide of 48 amino acids encoded by the mitochondrial ATP8 gene. A nuclear version of subunit 8 gene has been designed to encode FLAG tagged-subunit 8 fused with a mitochondrial signal peptide. The gene has been cloned into a yeast expression vector and then expressed in a yeast strain lacking endogenous subunit 8. Results showed that the gene was successfully expressed and the synthesized FLAG tagged-subunit 8 protein was imported into mitochondria. Following import, the FLAG tagged-subunit 8 protein assembled into functional mitochondrial ATP synthase complex. Furthermore, the subunit 8 protein could be detected using anti-FLAG tag monoclonal antibody.

  7. ACC 250 UOP Tutorial / Uoptutorial

    Ben K. Green

    2015-01-01

    For more course tutorials visit www.uoptutorial.com   ACC 250 Week 1 CheckPoint Choosing Accounting Software ACC 250 Week 1 Assignment Accounting Software Memo ACC 250 Week 2 CheckPoint Bellwether Garden Supply Back Up and Restore Data ACC 250 Week 2 DQ 1 and DQ 2 ACC 250 Week 3 CheckPoint Bellwether Garden Supply Vendor Transactions ACC 250 Week 3 Assignment Bellwether Garden Supply Record a Transaction ACC 250 Week 4 CheckPoint Bellwether Garden Supp...

  8. ACC 250 UOP Courses / uoptutorial

    hani

    2015-01-01

    For more course tutorials visit www.uoptutorial.com     ACC 250 Week 1 CheckPoint Choosing Accounting Software ACC 250 Week 1 Assignment Accounting Software Memo ACC 250 Week 2 CheckPoint Bellwether Garden Supply Back Up and Restore Data ACC 250 Week 2 DQ 1 and DQ 2 ACC 250 Week 3 CheckPoint Bellwether Garden Supply Vendor Transactions ACC 250 Week 3 Assignment Bellwether Garden Supply Record a Transaction ACC 250 Week 4 CheckPoint Bellwether...

  9. ACC 250 UOP TUTORIAL / Uoptutorial

    Cherry

    2015-01-01

    For more course tutorials visit www.uoptutorial.com   ACC 250 Week 1 CheckPoint Choosing Accounting Software ACC 250 Week 1 Assignment Accounting Software Memo ACC 250 Week 2 CheckPoint Bellwether Garden Supply Back Up and Restore Data ACC 250 Week 2 DQ 1 and DQ 2 ACC 250 Week 3 CheckPoint Bellwether Garden Supply Vendor Transactions ACC 250 Week 3 Assignment Bellwether Garden Supply Record a Transaction ACC 250 Week 4 CheckPoint Bellwether Garden Sup...

  10. Perspectives of bacterial ACC deaminase in phytoremediation.

    Arshad, Muhammad; Saleem, Muhammad; Hussain, Sarfraz

    2007-08-01

    Phytoremediation of contaminated soil and water environments is regulated and coordinated by the plant root system, yet root growth is often inhibited by pollutant-induced stress. Prolific root growth could maximize rates of hyperaccumulation of inorganic contaminants or rhizodegradation of organic pollutants, and thus accelerate phytoremediation. Accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as a major limitation in improving phytoremediation efficiency. Recent work shows that bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase regulates ethylene levels in plants by metabolizing its precursor ACC into alpha-ketobutyric acid and ammonia. Plants inoculated with ACC deaminase bacteria or transgenic plants that express bacterial ACC deaminase genes can regulate their ethylene levels and consequently contribute to a more extensive root system. Such proliferation of roots in contaminated soil can lead to enhanced uptake of heavy metals or rhizodegradation of xenobiotics. PMID:17573137

  11. ACC 310 Course Tutorial / tutorialrank

    welcome1351

    2015-01-01

    For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 4 Times, Rating: A+   ASHFORD ACC 310 Week 1 DQ 1 Information for Decision Making and Cost Concepts and Behavior ASHFORD ACC 310 Week 1 DQ 2 Fundamentals of Cost-Volume-Profit Analysis ASHFORD ACC 310 Week 1 Assignment CVP Analysis and Price Changes ASHFORD ACC 310 Week 2 DQ 1 Fundamentals of Cost Accounting for Decision Making ASHFORD ACC 310 Week 2 DQ 2 Fundamentals of Product and S...

  12. Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA

    Yong-Zhong Wang; You-Qing Cao; Jian-Nong Wu; Miao Chen; Xiao-Ying Cha

    2005-01-01

    AIM: To investigate the expression of NOS in gastric carcinoma, and to explore the relationship between the expression of nitric oxide synthases (NOS) and p53, PCNA,pathological features and clinical staging of gastric cancer.METHODS: The activity of NOS protein was investigated in 85 samples of human gastric carcinoma and 25 samples of normal gastric mucosal tissue by biochemical assay. We then examined the expression of NOS, p53, PCNA in 85 samples of human gastric cancer was examined by immunohistochemistry, and NOS mRNA expression in 85 gastric cancer tissue specimens by in situ hybridization.RESULTS: Biochemical assay showed that the activity of NOS was significantly higher in gastric carcinoma than in normal gastric mucosal tissues (t = 0.4161, P<0.01).Immunohistochemistry revealed that endothelial nitric oxide synthase (eNOS) expressed in all samples of normal gastric mucosa, but only 6 cases of 85 gastric cancer specimens showed weak positive immunohistochemical reactions to eNOS (20%). Inducible nitric oxide synthase (iNOS) was expressed strongly in human gastric carcinoma (81.2%). In situ hybridization analysis showed that iNOS mRNA expression was significantly stronger than eNOS mRNA expression in gastric cancer tissue (x2 = 10.23, P<0.01). The expression of iNOS in gastric cancer was associated with differentiation, clinical stages or lymph node metastases (r= 0.3426, P<0.05). However,iNOS expression did not correlate with histological classifications and morphological types. The expression of iNOS was significantly correlated with p53 or PCNA expression (r = 0.3612, P<0.05). The expression of neuronal nitric oxide synthase (nNOS) was not examined by immunohistochemistry and in situ hybridization in gastric cancer specimens and normal gastric mucosa.CONCLUSION: In human gastric cancer, there is an enhanced expression of iNOS, but not of eNOS. NOS promotes the proliferation of tumor cells and plays an important role in gastric cancer spread

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

    Irina Petrache

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

  14. Expression, crystallization and preliminary crystallographic study of octaprenyl pyrophosphate synthase from Helicobacter pylori

    Octaprenyl pyrophosphate synthase from H. pylori has been expressed, purified and crystallized, and a diffraction data set was collected to 2.00 Å resolution. Octaprenyl pyrophosphate synthase (OPPs) is involved in the synthesis of the side chains of ubiquinone and menaquinone and catalyzes consecutive condensation reactions of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate polyprenyl pyrophosphate and pyrophosphate. In order to investigate the roles played by OPPs in the metabolism of ubiquinone and menaquinone and the enzymatic mechanisms of these enzymes, analysis of the structure–function relationship of OPPs from Helicobacter pylori was initiated. The gene for OPPs was cloned, the protein was expressed, purified and crystallized and a diffraction data set was collected to 2.00 Å resolution. The crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 109.33, c = 103.41 Å

  15. Genome-Wide Identification, Characterization and Expression Analysis of the Chalcone Synthase Family in Maize

    Yahui Han; Ting Ding; Bo Su; Haiyang Jiang

    2016-01-01

    Members of the chalcone synthase (CHS) family participate in the synthesis of a series of secondary metabolites in plants, fungi and bacteria. The metabolites play important roles in protecting land plants against various environmental stresses during the evolutionary process. Our research was conducted on comprehensive investigation of CHS genes in maize (Zea mays L.), including their phylogenetic relationships, gene structures, chromosomal locations and expression analysis. Fourteen CHS gen...

  16. ACC 491 Tutorial Course/Uoptutorial

    nina

    2015-01-01

    For more course tutorials visit www.uoptutorial.com     ACC 491 Week 1 Individual Assignment Generally Accepted Auditing Standards Paper ACC 491 Week 1 DQ 1 ACC 491 Week 1 DQ 2 ACC 491 week 2 Individual Assignments From the Text ACC 491 Week 2 Team Assignment Auditing, Attestation, and Assurance Services Paper ACC 491 Week 2 DQ 1 ACC 491 Week 2 DQ 2 ACC 491 week 3 Individual Assignments From the Text ACC 491 week 3 Team Assignments Fr...

  17. Cloning, prokaryotic expression and functional analysis of squalene synthase (SQS) in Magnolia officinalis.

    Zha, Liangping; Liu, Shuang; Su, Ping; Yuan, Yuan; Huang, Luqi

    2016-04-01

    Magnolia officinalis Rehder et Wilson is a traditional Chinese herbal medicine that is used to treat various diseases such as neurosis, anxiety, and stroke. The main secondary metabolites in magnolia bark are phenolic compounds and terpenoids. Squalene synthase plays a significant role in catalyzing two farnesyl diphosphate molecules to form squalene, the first precursor of triterpenoid, phytosterol, and cholesterol biosynthesis. In this study, a full-length cDNA of squalene synthase was cloned from M. officinalis and designated MoSQS (GenBank accession no. KT223496). The gene contains a 1240-bp open reading frame and it encodes a protein with 409 amino acids. Bioinformatic and phylogenetic analysis clearly suggested that MoSQS shared high similarity with squalene synthases among other plants. Prokaryotic expression showed that a transmembrane domain-deleted (385-409 aa) MoSQS mutant (MoSQSΔTM) could be expressed in its soluble form in Escherichia coli Transetta (DE3). GC-MS analysis showed that squalene was detected in an in vitro reaction mixture. These results indicated that MoSQSΔTM was functional, thereby establishing an important foundation for the study of triterpenoid biosynthesis in M. officinalis. PMID:26696600

  18. Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

    Wen-Qin Bai

    Full Text Available Bioactive gibberellins (GAs comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

  19. Molecular cloning and seasonal expression of oyster glycogen phosphorylase and glycogen synthase genes

    Bacca, Helene; Huvet, Arnaud; Fabioux, Caroline; Daniel, Jean-yves; Delaporte, Maryse; Pouvreau, Stephane; van Wormhoudt, A.; Moal, Jeanne

    2005-01-01

    To investigate the control at the mRNA level of glycogen metabolism in the cupped oyster Crassostrea gigas, we report in the present paper the cloning and characterization of glycogen phosphorylase and synthase cDNAs (Cg-GPH and Cg-GYS, respectively, transcripts of main enzymes for glycogen use and storage), and their first expression profiles depending on oyster tissues and seasons. A strong expression of both genes was observed in the labial palps and the gonad in accordance with specific c...

  20. Expression of inducible nitric oxide synthase in carcinomas and sarcomas affecting the oral cavity

    Dominic Augustine; Sekar, B.; Murali, S.; Maya Ramesh; R Nirmal Madhavan; Shankar Gouda Patil; Rao, Roopa S

    2015-01-01

    Context: Inducible nitric oxide synthase (iNOS) is a cytoplasmic enzyme which plays a crucial role in the pathogenesis of oral carcinomas and sarcomas. Aims: The objective of this study was to analyze the immunohistochemical expression of iNOS in carcinomas and sarcomas affecting the oral cavity in order to understand the possible role of iNOS in their biologic behavior and to correlate iNOS expression with lymph node metastasis in carcinomas and sarcomas. Settings and Design: Patients, who a...

  1. ACC 375 Course Tutorial / tutorialrank

    welcome1365

    2015-01-01

                     For more course tutorials visit   www.tutorialrank.com   Tutorial Purchased: 4 Times, Rating: A+       ACC 375 Week 1 Individual Assignment Understanding Ethics Matrix (UOP Course) ACC 375 Week 1 Discussion Question 1 (UOP Course) ACC 375 Week 1 Discussion Question 2 (UOP Course) ACC 375 Week 2 Team Assignment Sarbanes Oxley Act Training Manual (UOP Course) AC...

  2. Differential expression of biphenyl synthase gene family members in fire-blight-infected apple 'Holsteiner Cox'.

    Chizzali, Cornelia; Gaid, Mariam M; Belkheir, Asma K; Hänsch, Robert; Richter, Klaus; Flachowsky, Henryk; Peil, Andreas; Hanke, Magda-Viola; Liu, Benye; Beerhues, Ludger

    2012-02-01

    Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple 'Golden Delicious', nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple 'Holsteiner Cox,' heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple 'Cox Orange,' expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells. PMID:22158676

  3. ACC 205 NEW ASH Tutorial / Uoptutorial

    Albert Camus

    2015-01-01

    For more course tutorials visit www.uoptutorial.com   ACC 205 Week 1 DQ 1 Accounting Equation   ACC 205 Week 1 DQ 2 Accounts   ACC 205 Week 1 Journal Balance Sheet Journal   ACC 205 Week 2 DQ 1 Accounting Cycle   ACC 205 Week 2 DQ 2 Bank Reconciliation   ACC 205 Week 2 Journal Income Statement Journal   ACC 205 Week 3 DQ 1 LIFO vs. FIFO   ACC 205 Week 3 DQ 2 Depreciation   ...

  4. ACC 205new Course Tutorial / tutorialrank

    welcome8888

    2015-01-01

    For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 5 Times, Rating: A+ ACC 205 Week 1 DQ 1 Accounting Equation   ACC 205 Week 1 DQ 2 Accounts   ACC 205 Week 1 Journal Balance Sheet Journal   ACC 205 Week 2 DQ 1 Accounting Cycle   ACC 205 Week 2 DQ 2 Bank Reconciliation   ACC 205 Week 2 Journal Income Statement Journal   ACC 205 Week 3 DQ 1 LIFO vs. FIFO   ACC 205 Week 3...

  5. ACC 205 NEW Tutorial Course/Uoptutorial

    neha

    2015-01-01

    For More Course Tutorials Visit www.uoptutorial.com   ACC 205 Week 1 DQ 1 Accounting Equation   ACC 205 Week 1 DQ 2 Accounts   ACC 205 Week 1 Journal Balance Sheet Journal   ACC 205 Week 2 DQ 1 Accounting Cycle   ACC 205 Week 2 DQ 2 Bank Reconciliation   ACC 205 Week 2 Journal Income Statement Journal   ACC 205 Week 3 DQ 1 LIFO vs. FIFO   ACC 205 Week 3 DQ 2 Depreciation   ...

  6. ACC 205 NEW Course Tutorial / Tutorialrank

    charles

    2015-01-01

    For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 5 Times, Rating: A+ ACC 205 Week 1 DQ 1 Accounting Equation   ACC 205 Week 1 DQ 2 Accounts   ACC 205 Week 1 Journal Balance Sheet Journal   ACC 205 Week 2 DQ 1 Accounting Cycle   ACC 205 Week 2 DQ 2 Bank Reconciliation   ACC 205 Week 2 Journal Income Statement Journal   ACC 205 Week 3 DQ 1 LIFO vs. FIFO   ACC 205 Week 3...

  7. ACC 375 Tutorial Course/Uoptutorial

    hina

    2015-01-01

    For more course tutorials visit www.uoptutorial.com         ACC 375 Week 1 Individual Assignment Understanding Ethics Matrix ACC 375 Week 1 Discussion Question 1 ACC 375 Week 1 Discussion Question 2 ACC 375 Week 2 Team Assignment Sarbanes Oxley Act Training Manual ACC 375 Week 2 Discussion Question 1 ACC 375 Week 2 Discussion Question 2 ACC 375 Week 3 Individual Assignment Ethics Situation 1 Fraudulent Schemes Report ACC 375...

  8. Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer.

    Cathcart, Mary-Clare

    2012-02-01

    BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced\\/absent in human NSCLC protein samples (P < .0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P = .004) and in male patients (P < .05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P < .001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC.

  9. Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens

    Dihydrodipicolinate synthase from the plant pathogen A. tumefaciens has been cloned, expressed, purified and crystallized in its unliganded form, in the presence of its substrate pyruvate and in the presence of pyruvate and the allosteric inhibitor lysine. Diffraction data for the crystals were collected to a maximum resolution of 1.40 Å. Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS (NP-354047.1) from the plant pathogen Agrobacterium tumefaciens (AgT-DHDPS). Enzyme-kinetics studies demonstrate that AgT-DHDPS possesses DHDPS activity in vitro. Crystals of AgT-DHDPS were grown in the unliganded form and in forms with substrate bound and with substrate plus allosteric inhibitor (lysine) bound. X-ray diffraction data sets were subsequently collected to a maximum resolution of 1.40 Å. Determination of the structure with and without substrate and inhibitor will offer insight into the design of novel pesticide agents

  10. Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from the grapevine Vitis vinifera

    Dihydrodipicolinate synthase from the common grapevine V. vinifera has been cloned, expressed, purified and crystallized in the presence of the substrate pyruvate by in-drop hexahistidine-tag cleavage. A diffraction data set has been collected to a resolution of 2.2 Å. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS from the grapevine Vitis vinifera (Vv-DHDPS). Following in-drop cleavage of the hexahistidine tag, cocrystals of Vv-DHDPS with the substrate pyruvate were grown in 0.1 M Bis-Tris propane pH 8.2, 0.2 M sodium bromide, 20%(w/v) PEG 3350. X-ray diffraction data in space group P1 at a resolution of 2.2 Å are presented. Preliminary diffraction data analysis indicated the presence of eight molecules per asymmetric unit (VM = 2.55 Å3 Da−1, 52% solvent content). The pending crystal structure of Vv-DHDPS will provide insight into the molecular evolution in quaternary structure of DHDPS enzymes

  11. Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum

    The cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from T. flavum, a protein which catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, are reported. Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 Å resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3121, with unit-cell parameters a = b = 86.31, c = 118.36 Å. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 Å resolution. The model is under construction

  12. Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from the psychrophile Shewanella benthica

    Dihydrodipicolinate synthase (DHDPS) is an essential oligomeric enzyme of interest to antibiotic discovery research and studies probing the importance of quaternary structure to protein function, stability and dynamics. The cloning, expression, purification and crystallization of DHDPS from the psychrophilic (cold-dwelling) bacterium Shewanella benthica are described. Dihydrodipicolinate synthase (DHDPS) is an oligomeric enzyme that catalyzes the first committed step of the lysine-biosynthesis pathway in plants and bacteria, which yields essential building blocks for cell-wall and protein synthesis. DHDPS is therefore of interest to drug-discovery research as well as to studies that probe the importance of quaternary structure to protein function, stability and dynamics. Accordingly, DHDPS from the psychrophilic (cold-dwelling) organism Shewanella benthica (Sb-DHDPS) was cloned, expressed, purified and crystallized. The best crystals of Sb-DHDPS were grown in 200 mM ammonium sulfate, 100 mM bis-tris pH 5.0–6.0, 23–26%(w/v) PEG 3350, 0.02%(w/v) sodium azide and diffracted to beyond 2.5 Å resolution. Processing of diffraction data to 2.5 Å resolution resulted in a unit cell with space group P212121 and dimensions a = 73.1, b = 84.0, c = 143.7 Å. These studies of the first DHDPS enzyme to be characterized from a bacterial psychrophile will provide insight into the molecular evolution of enzyme structure and dynamics

  13. Zinc Affects Differently Growth, Photosynthesis, Antioxidant Enzyme Activities and Phytochelatin Synthase Expression of Four Marine Diatoms

    Thi Le Nhung Nguyen-Deroche

    2012-01-01

    Full Text Available Zinc-supplementation (20 μM effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase, and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa. Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

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

    Kentaro Wakasa

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

  15. ACC 490 Tutorial Course/Uoptutorial

    nina

    2015-01-01

    For more course tutorials visit www.uoptutorial.com ACC 490 Week 1 Generally Accepted Auditing Standards Paper ACC 490 Week 1 DQ 1 ACC 490 Week 1 DQ 2 ACC 490 Week 2 Individual Ch. 1 Textbook Exercise ACC 490 Week 2 Learning Team Auditing, Attestation, and Assurance Services Paper ACC 490 Week 2 DQ 1 ACC 490 Week 2 DQ 2 ACC 490 Week 3 Individual Ch. 5, 6, & 7 Textbook Exercises ACC 490 Week 3 Learning Team Ch. 6 & 7 Textbook Exercises ACC 490 Week...

  16. Cell-Specific Expression of Homospermidine Synthase, the Entry Enzyme of the Pyrrolizidine Alkaloid Pathway in Senecio vernalis, in Comparison with Its Ancestor, Deoxyhypusine Synthase1

    Moll, Stefanie; Anke, Sven; Kahmann, Uwe; Hänsch, Robert; Hartmann, Thomas; Ober, Dietrich

    2002-01-01

    Pyrrolizidine alkaloids (PAs) are constitutive plant defense compounds with a sporadic taxonomic occurrence. The first committed step in PA biosynthesis is catalyzed by homospermidine synthase (HSS). Recent evidence confirmed that HSS evolved by gene duplication from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of the eukaryotic translation initiation factor 5A. To better understand the evolutionary relationship between these two enzymes, which are involved in completely different biological processes, we studied their tissue-specific expression. RNA-blot analysis, reverse transcriptase-PCR, and immunolocalization techniques demonstrated that DHS is constitutively expressed in shoots and roots of Senecio vernalis (Asteraceae), whereas HSS expression is root specific and restricted to distinct groups of endodermis and neighboring cortex cells located opposite to the phloem. All efforts to detect DHS by immunolocalization failed, but studies with promoter-β-glucuronidase fusions confirmed a general expression pattern, at least in young seedlings of tobacco (Nicotiana tabacum). The expression pattern for HSS differs completely from its ancestor DHS due to the adaptation of HSS to the specific requirements of PA biosynthesis. PMID:12226485

  17. Production of Nitric Oxide and Expression of Inducible Nitric Oxide Synthase in Ovarian Cystic Tumors

    Rosekeila Simões Nomelini

    2008-01-01

    Full Text Available Tumor sections from nonneoplastic (n=15, benign (n=28, and malignant ovarian tumors (n=20 were obtained from 63 women. Immunohistochemistry of the tumor sections demonstrated that inducible nitric oxide synthase (iNOS expression was increased in ovarian cancer samples compared to nonneoplastic or benign tumor samples. Using the Griess method, nitric oxide (NO metabolite levels were also found to be elevated in malignant tumor samples compared to benign tumor samples (P80 μM were more frequent than NO levels <80 μM, and iNOS expression in well-differentiated carcinomas was greater than in moderately/poorly differentiated carcinomas (P<.05. These data suggest an important role for NO in ovarian carcinogenesis.

  18. Effects of niacin on nitric oxide synthase expression in rat lungs exposed to silica

    WANG Shixin; DU Haike; ZHANG Xizhen; CAI Shaoxi; FAN Huaquan; WANG Chang'en

    2004-01-01

    The aim of this study was to evaluate the effects of niacin in diet on the expression of nitric oxide synthase (NOS) in rat lungs of the animal model of silicosis established by direct tracheal instillation of silica particles into rat lungs surgically. The niacin concentration in serum was analyzed by high performance liquid chromatography (HPLC). The expression of inducible nitric oxide synthase (iNOS) protein in paraffin-embedded lung sections was determined by streptavidin/peroxidase (SP) staining. Quantitative analysis by Image-Pro Plus was also performed on the expression of iNOS. The results showed that niacin concentration in serum of the niacin-treated rats was significantly higher than that in the control and silica-treated rats. After 7 days of silica instillation, iNOS integrated optical density (IOD) in rat lungs and total NOS and iNOS activities in bronchoalveolar lavage fluid (BALF) in silica-treated rats rose by 273420.75, 2.61 units/mL and 1.89 units/mL respectively, when compared with those in the control rats. Niacin treatment significantly reduced silica-induced iNOS IOD in rat lung tissues and total NOS and iNOS activities in BALF supernatant by 248292.35, 1.50 units/mL and 0.91 units/mL, respectively, as compared with those in silica-treated rats. Therefore, niacin can effectively attenuate the pathological expression of NOS in rat lung tissues induced by silica particles.

  19. Expression of nitric oxide synthase and guanylate cyclase in the human ciliary body and trabecular meshwork

    WU Ren-yi; MA Ning

    2012-01-01

    Background The role played by the nitric oxide (NO) signaling pathway in the aqueous humor dynamics is still unclear.This study was designed to investigate the expression and distribution of NO synthase (NOS) isoforms and guanylate cyclase (GC) in human ciliary body,trabecular meshwork and the Schlemm's canal.Methods Twelve eyes after corneal transplantation were used.Expression of three NOS isoforms (i.e.neuronal NOS (nNOS),inducible NOS (iNOS) and endothelial NOS (eNOS)) and GC were assessed in 10 eyes by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC.Ciliary bodies were dissected free and the total proteins were extracted.Western blotting was performed to confirm the protein expression of 3 NOS isoforms and GC.Results Expression of 3 NOS isoforms and GC were observed in the ciliary epithelium,ciliary muscle,trabecular meshwork and the endothelium of the Schlemm's canal.Immunoreactivity of nNOS was detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and pigmented epithelial (PE) cells.Protein expressions of 3 NOS isoforms and GC were confirmed in isolated human ciliary body by Western blotting.Conclusions The expression of NOS isoforms and GC in human ciliary body suggest the possible involvement of NO and cyclic guanosine monophosphate (cyclic GMP,cGMP) signaling pathway in the ciliary body,and may play a role in both processes of aqueous humor formation and drainage.

  20. Cloning and Expression of the PHA Synthase Gene From a Locally Isolated Chromobacterium sp. USM2

    Bhubalan, K.

    2010-01-01

    Full Text Available Chromobacterium sp. USM2, a locally isolated bacterium was found to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate, P(3HB-co-3HV copolymer with high 3HV monomer composition. The PHA synthase gene was cloned and expressed in Cupriavidus necator PHB¯4 to investigate the possibilities of incorporating other monomer. The recombinant successfully incorporated 3-hydroxyhexanoate (3HHx monomer when fed with crude palm kernel oil (CPKO as the sole carbon source. Approximately 63 ± 2 wt% of P(3HB-co-3HHx copolymer with 4 mol% of 3HHx was synthesized from 5 g/L of oil after 48 h of cultivation. In addition, P(3HB-co-3HV-co-3HHx terpolymer with 9 mol% 3HV and 4 mol% 3HHx could be synthesized with a mixture of CPKO and sodium valerate. The presence of 3HV and 3HHx monomers in the copolymer and terpolymer was further confirmed with +H-NMR analysis. This locally isolated PHA synthase has demonstrated its ability to synthesize P(3HB-co-3HHx copolymer from a readily available and renewable carbon source; CPKO, without the addition of 3HHx precursors.

  1. The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Delta1-tetrahydrocannabinolic acid synthase from Cannabis sativa L.

    Sirikantaramas, Supaart; Morimoto, Satoshi; Shoyama, Yoshinari; Ishikawa, Yu; Wada, Yoshiko; Shoyama, Yukihiro; Taura, Futoshi

    2004-09-17

    Delta(1)-tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetrahydrocannabinol. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis. PMID:15190053

  2. Expression of thymidylate synthase and glutathione-stransferase π in patients with esophageal squamous cell carcinoma

    Jun-Xing Huang; Feng-Yue Li; Wei Xiao; Zheng-Xiang Song; Rong-Yu Qian; Ping Chen; Eeva Salminen

    2009-01-01

    AIM: To investigate the expression of thymidylate synthase (TS) and glutathione-s-transferase π (GST-π) in esophageal squamous cell carcinoma and their association with the clinicopathologic characteristics. METHODS: Immunohistochemical methods were used to detect the expression of TS and GST-π in surgically resected formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma (ESCC) tissue sections from 102 patients (median age, 58 years) and in 28 normal esophageal mucosa (NEM) samples. The relationship between TS and GST-π expression and clinicopathologic factors was examined. RESULTS: The expression of TS and GST-π was not statistically significantly associated with age of the patients, tumor size, lymph node metastasis, depth of invasion or tumor stage. TS staining was positive in 17.86% of normal esophageal mucosa and in 42.16% of ESCC samples (P < 0.05). The expression level of TS was not only significantly lower in well-differentiated (21.88%) than in poorly-differentiated carcinomas (51.43%, P < 0.05), but was also significantly higher in samples from male patients (46.51%) than from female patients (18.75%, P < 0.05). GST-π was positively stained in 78.57% of normal esophageal mucosa and in 53.92% of ESCC samples (P < 0.05). The expression level of GST-π was also significantly higher in welldifferentiated carcinomas (65.63%) than in poorlydifferentiated carcinomas (35.00%, P < 0.05). CONCLUSION: The expression of TS and of GST-π may be used as molecular markers for the characterization of ESCC. Poorly-differentiated cells showed increased expression of TS and reduced expression of GST-π.

  3. ACC 205 ASH Tutorial/Uoptutorial

    Stella,, Maurizio

    2015-01-01

    For more course tutorials visit www.uoptutorial.com   ACC 205 Week 1 DQ 1 Accounting Equation  ACC 205 Week 1 DQ 2 Accounts  ACC 205 Week 1 Journal Balance Sheet Journal  ACC 205 Week 2 DQ 1 Accounting Cycle  ACC 205 Week 2 DQ 2 Bank Reconciliation  ACC 205 Week 2 Journal Income Statement Journal  ACC 205 Week 3 DQ 1 LIFO vs. FIFO  ACC 205 Week 3 DQ 2 Depreciation  ACC 205 Week 3 Journal Inventory Journal...

  4. ACC 206 NEW Tutorial/Uoptutorial

    Stella,, Maurizio

    2015-01-01

    For more course tutorials visit www.uoptutorial.com   ACC 205 Week 1 DQ 1 Accounting Equation  ACC 205 Week 1 DQ 2 Accounts  ACC 205 Week 1 Journal Balance Sheet Journal  ACC 205 Week 2 DQ 1 Accounting Cycle  ACC 205 Week 2 DQ 2 Bank Reconciliation  ACC 205 Week 2 Journal Income Statement Journal  ACC 205 Week 3 DQ 1 LIFO vs. FIFO  ACC 205 Week 3 DQ 2 Depreciation  ACC 205 Week 3 Journal Inventory Journal...

  5. acc490uopcoursesTutorial /uophelp

    jacobs

    2015-01-01

    For more course tutorials visit www.uophelp.com     ACC 490 Week 1 Generally Accepted Auditing Standards Paper ACC 490 Week 1 – DQ 1  ACC 490 Week 1 – DQ 2 ACC 490 Week 2 Individual Ch. 1 Textbook Exercise ACC 490 Week 2 Learning Team Auditing, Attestation, and Assurance Services Paper ACC 490 Week 2 – DQ 1 ACC 490 Week 2 – DQ 2 ACC 490 Week 3 Individual Ch. 5, 6, & 7 Textbook Exercises ACC 490 Week 3 L...

  6. ACC 250 UOP Tutorial/Uoptutorial

    Giselle Isabel

    2015-01-01

    ACC 250 Week 1 CheckPoint Choosing Accounting Software ACC 250 Week 1 Assignment Accounting Software Memo ACC 250 Week 2 CheckPoint Bellwether Garden Supply Back Up and Restore Data ACC 250 Week 2 DQ 1 and DQ 2 ACC 250 Week 3 CheckPoint Bellwether Garden Supply Vendor Transactions ACC 250 Week 3 Assignment Bellwether Garden Supply Record a Transaction ACC 250 Week 4 CheckPoint Bellwether Garden Supply Payroll ACC 250 Week 4 DQ 1 and DQ 2 ACC 250 Week 5 CheckPoi...

  7. Effect of estrogen on gene expression of fatty acid synthase in periosteum

    ZHENG Rui-min; LIN Shou-qing; LIU Yong; HUANG Man-ting; GONG Wei-yan; WU Zhi-hong

    2009-01-01

    Background Estrogen deficiency contributes to postmenopausal osteoporosis.Periosteum might be a potential target of estrogen,but the underlying mechanism at gene level is far from being elucidated.The objective of this study was to investigate the correlation between estrogen and fatty acid synthase(FAS)expression in periosteum.Methods Human periosteum cells were cultured in vitro.Expressed genes in the substrated cDNA library were verified using semi-quantitative PCR and real-time PCR.The expression of FAS in periosteum of ovarectomized(OVX)SD rats was investigated.Results FAS gene was most significantly expressed in the subtracted cDNA library of periosteal cells screened by semi-quantitative PCR.Low FAS expression was verified by real-time PCR in the estrogen exposed human periosteum rather than in the control.The estradiol levels were(20.81±12.62)pg/ml,(19.64±4.35)pg/ml and(13.47+1.84)pg/ml in the sham group,the control,and the OVX group,respectively.The estradiol levels in the OVX group was significantly lower(P=0.0386).The FAS gene expression in periosteum in the OVX group,sham group,and control group was 3.09±1.97,1.33±0.47 and 1.51±1.32,respectively.The gene expression in the OVX group was significantly higher (P=0.0372).Conclusion Estrogen modulates FAS gene expression in in vitro human perisoteum as well as in in vivo rat periosteum.

  8. Expression of glycogen synthase (GYS) and glycogen synthase kinase 3β (GSK3β) of the Fujian oyster, Crassostrea angulata, in relation to glycogen content in gonad development.

    Zeng, Zhen; Ni, Jianbin; Ke, Caihuan

    2013-01-01

    To investigate the regulation of glycogen metabolism at the mRNA level in Crassostrea angulata, we cloned and characterized glycogen synthase and glycogen synthase kinase 3β cDNAs (Ca-GYS and Ca-GSK3β, respectively), which encode the primary enzymes involved in glycogen storage. We examined their expression profiles in different tissues and during different reproductive stages. The full-length cDNA of GYS was 4771 bp in length with a 2023 bp open reading frame (ORF), predicted to encode a protein of 674 aa. The full-length GSK3β cDNA was 2333 bp long, with an ORF of 1242 bp. High expression levels of both genes were observed in the gonad and the adductor muscle, as compared to the mantle, gill, or visceral mass, which correlates well with the ability to store glucose. The regulation of both genes was correlated with glycogen content via qPCR and in situ hybridization and was dependent upon the stage of the reproductive cycle (initiation stage, maturation stage, ripeness stage). Thus, it appears that the expression of Ca-GYS and Ca-GSK3β is driven by the reproductive cycle of the oyster, reflecting the central role played by glycogen in energy storage and gametogenic development in C. angulata. We suggest that Ca-GYS and Ca-GSK3β can be used as useful molecular markers for identifying the stages of glycogen metabolism and reproduction in C. angulata. PMID:24035883

  9. Heterologous Expression, Purification, and Biochemical Characterization of α-Humulene Synthase from Zingiber zerumbet Smith.

    Alemdar, Semra; Hartwig, Steffen; Frister, Thore; König, Jan Christoph; Scheper, Thomas; Beutel, Sascha

    2016-02-01

    The α-humulene synthase from Zingiber zerumbet Smith was expressed as a polyhistidine-tagged protein in an E. coli BL21(DE3) strain. Induction time and inductor (isopropyl-β-D-thiogalactopyranoside) concentration were optimized. The enzyme was successfully purified directly from cell lysate by NTA affinity column chromatography and careful selection of coordinated metal ion and imidazole elution conditions. Bioactivity assays were conducted with the natural substrate farnesyl diphosphate (FDP) in a two-phase system with in situ extraction of products. The conversion of FDP to α-humulene (~94.5%) and β-caryophyllene (~5.5%) could be monitored by gas chromatography-flame ionization detection (GC-FID). Optimal pH and temperature as well as kinetic parameters K M and k cat were determined using a discontinuous kinetic assay. PMID:26463657

  10. Quinic acids from Aster caucasicus and from transgenic callus expressing a beta-amyrin synthase.

    Pecchia, Paola; Cammareri, Maria; Malafronte, Nicola; Consiglio, M Federica; Gualtieri, Maria Josefina; Conicella, Clara

    2011-11-01

    Several different classes of secondary metabolites, including flavonoids, triterpenoid saponins and quinic acid derivatives, are found in Aster spp. (Fam. Asteraceae). Several Aster compounds revealed biological as well as pharmacological activities. In this work, a phytochemical investigation of A. caucasicus evidenced the presence of quinic acid derivatives, as well as the absence of triterpene saponins. To combine in one species the production of different phytochemicals, including triterpenes, an Agrobacterium-mediated transformation of A. caucasicus was set up to introduce A. sedifolius beta-amyrin synthase (AsOXA1)-encoding gene under the control of the constitutive promoter CaMV35S. The quali-quantitative analysis of transgenic calli with ectopic expression of AsOXA1 showed, in one sample, a negligible amount of triterpene saponins combined with higher amount of quinic acid derivatives as compared with the wild type callus. PMID:22224284

  11. Enhanced expression of constitutive and inducible forms of nitric oxide synthase in autoimmune encephalomyelitis.

    Kim, S; Moon, C; Wie, M B; Kim, H; Tanuma, N; Matsumoto, Y; Shin, T

    2000-06-01

    To elucidate the role of nitric oxide synthase (NOS) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), we analyzed the expression of constitutive neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) in the spinal cords of rats with EAE. We further examined the structural interaction between apoptotic cells and spinal cord cells including neurons and astrocytes, which are potent cell types of nitric oxide (NO) production in the brain. Western blot analysis showed that three forms of NOS significantly increased in the spinal cords of rats at the peak stage of EAE, while small amounts of these enzymes were identified in the spinal cords of rats without EAE. Immunohistochemical study showed that the expression of either nNOS or eNOS increased in the brain cells including neurons and astrocytes during the peak and recovery stages of EAE, while the expression of iNOS was found mainly in the inflammatory macrophages in the perivascular EAE lesions. Double labeling showed that apoptotic cells had intimate contacts with either neurons or astrocytes, which are major cell types to express nNOS and eNOS constitutively. Our results suggest that the three NOS may play an important role in the recovery of EAE. PMID:14612615

  12. Borna disease virus P protein inhibits nitric oxide synthase gene expression in astrocytes

    Borna disease virus (BDV) is one of the potential infectious agents involved in the development of central nervous system (CNS) diseases. Neurons and astrocytes are the main targets of BDV infection, but little is known about the roles of BDV infection in the biological effects of astrocytes. Here we reported that BDV inhibits the activation of inducible nitric oxide synthase (iNOS) in murine astrocytes induced by bacterial LPS and PMA. To determine which protein of BDV is responsible for the regulation of iNOS expression, we co-transfected murine astrocytes with reporter plasmid iNOS-luciferase and plasmid expressing individual BDV proteins. Results from analyses of reporter activities revealed that only the phosphoprotein (P) of BDV had an inhibitory effect on the activation of iNOS. In addition, P protein inhibits nitric oxide production through regulating iNOS expression. We also reported that the nuclear factor kappa B (NF-κB) binding element, AP-1 recognition site, and interferon-stimulated response element (ISRE) on the iNOS promoter were involved in the repression of iNOS gene expression regulated by the P protein. Functional analysis indicated that sequences from amino acids 134 to 174 of the P protein are necessary for the regulation of iNOS. These data suggested that BDV may suppress signal transduction pathways, which resulted in the inhibition of iNOS activation in astrocytes

  13. Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Crock, John E. (Moscow, ID)

    1999-01-01

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) 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 (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene 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 (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  14. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

    Croteau, Rodney Bruce; Crock, John E.

    2005-01-25

    A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) 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 (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene 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 (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

  15. A first insight into the occurrence and expression of functional amoA and accA genes of autotrophic and ammonia-oxidizing bathypelagic Crenarchaeota of Tyrrhenian Sea

    Yakimov, Michail M.; Cono, Violetta La; Denaro, Renata

    2009-05-01

    The autotrophic and ammonia-oxidizing crenarchaeal assemblage at offshore site located in the deep Mediterranean (Tyrrhenian Sea, depth 3000 m) water was studied by PCR amplification of the key functional genes involved in energy (ammonia mono-oxygenase alpha subunit, amoA) and central metabolism (acetyl-CoA carboxylase alpha subunit, accA). Using two recently annotated genomes of marine crenarchaeons, an initial set of primers targeting archaeal accA-like genes was designed. Approximately 300 clones were analyzed, of which 100% of amoA library and almost 70% of accA library were unambiguously related to the corresponding genes from marine Crenarchaeota. Even though the acetyl-CoA carboxylase is phylogenetically not well conserved and the remaining clones were affiliated to various bacterial acetyl-CoA/propionyl-CoA carboxylase genes, the pool of archaeal sequences was applied for development of quantitative PCR analysis of accA-like distribution using TaqMan ® methodolgy. The archaeal accA gene fragments, together with alignable gene fragments from the Sargasso Sea and North Pacific Subtropical Gyre (ALOHA Station) metagenome databases, were analyzed by multiple sequence alignment. Two accA-like sequences, found in ALOHA Station at the depth of 4000 m, formed a deeply branched clade with 64% of all archaeal Tyrrhenian clones. No close relatives for residual 36% of clones, except of those recovered from Eastern Mediterranean, was found, suggesting the existence of a specific lineage of the crenarchaeal accA genes in deep Mediterranean water. Alignment of Mediterranean amoA sequences defined four cosmopolitan phylotypes of Crenarchaeota putative ammonia mono-oxygenase subunit A gene occurring in the water sample from the 3000 m depth. Without exception all phylotypes fell into Deep Marine Group I cluster that contain the vast majority of known sequences recovered from global deep-sea environment. Remarkably, three phylotypes accounted for 91% of all Mediterranean

  16. Regulation of RNA-dependent RNA polymerase 1 and isochorismate synthase gene expression in Arabidopsis.

    Lydia J R Hunter

    Full Text Available BACKGROUND: RNA-dependent RNA polymerases (RDRs function in anti-viral silencing in Arabidopsis thaliana and other plants. Salicylic acid (SA, an important defensive signal, increases RDR1 gene expression, suggesting that RDR1 contributes to SA-induced virus resistance. In Nicotiana attenuata RDR1 also regulates plant-insect interactions and is induced by another important signal, jasmonic acid (JA. Despite its importance in defense RDR1 regulation has not been investigated in detail. METHODOLOGY/PRINCIPAL FINDINGS: In Arabidopsis, SA-induced RDR1 expression was dependent on 'NON-EXPRESSER OF PATHOGENESIS-RELATED GENES 1', indicating regulation involves the same mechanism controlling many other SA- defense-related genes, including pathogenesis-related 1 (PR1. Isochorismate synthase 1 (ICS1 is required for SA biosynthesis. In defensive signal transduction RDR1 lies downstream of ICS1. However, supplying exogenous SA to ics1-mutant plants did not induce RDR1 or PR1 expression to the same extent as seen in wild type plants. Analysing ICS1 gene expression using transgenic plants expressing ICS1 promoter:reporter gene (β-glucuronidase constructs and by measuring steady-state ICS1 transcript levels showed that SA positively regulates ICS1. In contrast, ICS2, which is expressed at lower levels than ICS1, is unaffected by SA. The wound-response hormone JA affects expression of Arabidopsis RDR1 but jasmonate-induced expression is independent of CORONATINE-INSENSITIVE 1, which conditions expression of many other JA-responsive genes. Transiently increased RDR1 expression following tobacco mosaic virus inoculation was due to wounding and was not a direct effect of infection. RDR1 gene expression was induced by ethylene and by abscisic acid (an important regulator of drought resistance. However, rdr1-mutant plants showed normal responses to drought. CONCLUSIONS/SIGNIFICANCE: RDR1 is regulated by a much broader range of phytohormones than previously thought

  17. Hepatic inducible nitric oxide synthase expression increases upon exposure to hypergravity

    Stimulation by a number of conditions, including infection, cytokines, mechanical injury, and hypoxia, can upregulate inducible nitric oxide synthase (iNOS) in hepatocytes. We observed that exposure to hypergravity significantly upregulated the transcription of the hepatic iNOS gene. The aim of this study was to confirm our preliminary data, and to further investigate the distribution of the iNOS protein in the livers of mice exposed to hypergravity. ICR mice were exposed to +3 Gz for 1 h. We investigated the time course of change in the iNOS expression. Hepatic iNOS mRNA expression progressively increased in centrifuged mice from 0 to 12 h, and then decreased rapidly by 18 h. iNOS mRNA levels in the livers of centrifuged mice was significantly higher at 3, 6, and 12 h than in uncentrifuged control mice. The pattern of iNOS protein expression paralleled that of the mRNA expression. At 0 and 1 h, weak cytoplasmic iNOS immunoreactivity was found in some hepatocytes surrounding terminal hepatic venules. It was noted that at 6 h there was an increase in the number of perivenular hepatocytes with moderate to strong cytoplasmic immunoreactivity. The number of iNOS-positive hepatocytes was maximally increased at 12 h. The majority of positively stained cells showed a strong intensity of iNOS expression. The expression levels of iNOS mRNA and protein were significantly increased in the livers of mice exposed to hypergravity. These results suggest that exposure to hypergravity significantly upregulates iNOS at both transcriptional and translational levels

  18. Hepatic inducible nitric oxide synthase expression increases upon exposure to hypergravity

    Kim, H.S. [Sungkyunkwan University School of Medicine, Samsung Medical Center, Department of Pathology and Translational Genomics, Seoul (Korea, Republic of); Republic of Korea Air Force Medical Center, Aerospace Medicine Research Center, Cheongju (Korea, Republic of); Jung, Y.Y. [Sungkyunkwan University School of Medicine, Samsung Medical Center, Department of Pathology and Translational Genomics, Seoul (Korea, Republic of); Do, S.I. [Sungkyunkwan University School of Medicine, Kangbuk Samsung Hospital, Department of Pathology, Seoul (Korea, Republic of)

    2014-08-29

    Stimulation by a number of conditions, including infection, cytokines, mechanical injury, and hypoxia, can upregulate inducible nitric oxide synthase (iNOS) in hepatocytes. We observed that exposure to hypergravity significantly upregulated the transcription of the hepatic iNOS gene. The aim of this study was to confirm our preliminary data, and to further investigate the distribution of the iNOS protein in the livers of mice exposed to hypergravity. ICR mice were exposed to +3 Gz for 1 h. We investigated the time course of change in the iNOS expression. Hepatic iNOS mRNA expression progressively increased in centrifuged mice from 0 to 12 h, and then decreased rapidly by 18 h. iNOS mRNA levels in the livers of centrifuged mice was significantly higher at 3, 6, and 12 h than in uncentrifuged control mice. The pattern of iNOS protein expression paralleled that of the mRNA expression. At 0 and 1 h, weak cytoplasmic iNOS immunoreactivity was found in some hepatocytes surrounding terminal hepatic venules. It was noted that at 6 h there was an increase in the number of perivenular hepatocytes with moderate to strong cytoplasmic immunoreactivity. The number of iNOS-positive hepatocytes was maximally increased at 12 h. The majority of positively stained cells showed a strong intensity of iNOS expression. The expression levels of iNOS mRNA and protein were significantly increased in the livers of mice exposed to hypergravity. These results suggest that exposure to hypergravity significantly upregulates iNOS at both transcriptional and translational levels.

  19. UOP ACC 291 / Assignmentcloud.com

    admin

    2015-01-01

    ACC 291 Complete Class and Final Exams   To purchase this material click below link   http://www.assignmentcloud.com/ACC-291/ACC-291-Complete-Class-Final-Exams   For more classes visit www.assignmentcloud.com

  20. Bacterial Colonization and the Expression of Inducible Nitric Oxide Synthase in Murine Wounds

    Mahoney, Eric; Reichner, Jonathan; Robinson Bostom, Leslie; Mastrofrancesco, Balduino; Henry, William; Albina, Jorge

    2002-01-01

    The expression of inducible nitric oxide synthase (iNOS) in two different murine wound models was investigated. Animals were subjected to either full-thickness linear skin incision with subcutaneous implantation of sterile polyvinyl alcohol sponges, or to 1.5 × 1.5-cm dorsal skin excision. Reverse transcriptase-polymerase chain reaction detected iNOS mRNA in all cell samples retrieved from the sponges. Immunoblotting of lysates of inflammatory cells harvested from the sponges failed to detect iNOS protein, and immunohistochemistry of the incisional wound was mildly positive. Inflammatory cells of excisional wounds stained strongly positive for iNOS. Cutaneous wounds were found to be colonized with Staphylococcus aureus. The detection of iNOS in cells from sponges inoculated in vivo with heat-killed bacteria and the reduction of immunohistochemical signal for iNOS in excisional wounds of animals treated with antibiotics support a role of bacteria in the induction of iNOS in wounds. The expression of iNOS in excisional wounds requires interferon-γ and functional lymphocytes because interferon-γ knockout and SCID-Beige mice exhibited attenuated iNOS staining in excisional wounds. The expression of iNOS in the inflammatory cells of murine wounds is a response to bacterial colonization and not part of the normal repair process elicited by sterile tissue injury. PMID:12466130

  1. Polymorphism of thymidylate synthase gene associated with its protein expression in human colon cancer

    Kai-Huan Yu; Wei-Xing Wang; You-Ming Ding; Hui Li; Ze-Sheng Wang

    2008-01-01

    AIM: To correlate the polymorphisms in the 5'-untranslated region with thymidylate synthase (TS) protein expression in Han Chinese colonic neoplasms.METHODS: Adenocarcinoma samples were from 68 patients who received no treatment before surgery. Tandem repeat length of TS gene was determined by PCR amplification of genomic DNA. Intratumoral TS protein expression was studied immunohistochemically in corresponding sections from paraffin-embedded primary foci. Immunoreactivity was semiquantitatively evaluated by immunoreactivity score (IRS).RESULTS: Double-(2R) and triple-repeated (3R) sequences of the TS gene were found in the cancer tissues. Three genotypes of TS were found: 2R/2R (n = 6), 2R/3R (n = 22) and 3R/3R (n = 40). Patients who were homozygous for triple-repeated (3R/3R) sequences showed significantly higher IRS of TS than patients who were homozygous for double-repeated (2R/2R) sequences or heterozygous patients (2R/3R): 5.73 ± 3.25 vs 2.17 ± 1.47 or 3.77 ± 2.64, P = 0.008 or P = 0.015. But no statistical significance of IRS in cancer tissues was observed between 2R/3R genotype and 2R/2R genotype.CONCLUSION: There is a relationship between TS genotype and TS protein expression in clinical specimens. The data might offer an advantage for selection of Chinese cancer patients to receive fluoropyrimidines treatment.

  2. Molecular identity and gene expression of aldosterone synthase cytochrome P450

    11β-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11β-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolated from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression

  3. Expression Patterns, Activities and Carbohydrate-Metabolizing Regulation of Sucrose Phosphate Synthase, Sucrose Synthase and Neutral Invertase in Pineapple Fruit during Development and Ripening

    Zhang, Xiu-Mei; Wang, Wei; Du, Li-Qing; Xie, Jiang-Hui; Yao, Yan-Li; Sun, Guang-Ming

    2012-01-01

    Differences in carbohydrate contents and metabolizing-enzyme activities were monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv. Comte de paris) during fruit development and ripening. Fructose and glucose of various sections in nearly equal amounts were the predominant sugars in the fruitlets, and had obvious differences until the fruit matured. The large rise of sucrose/hexose was accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a mechanism to increase fruit sink strength by increasing hexose concentrations. Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no. GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid sequences. Homology alignment reveals that the amino acid sequences contain some conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and Ac-ni also indicated distinct patterns related to sugar accumulation and composition of pineapple fruits. It suggests that differential expressions of multiple gene families are necessary for sugar metabolism in various parts and developmental stages of pineapple fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps by generating sugar signals to the cells in a temporally and spatially restricted fashion. PMID:22949808

  4. ACC 491 UOP Course Tutorial / Tutorialrank

    charles

    2015-01-01

    For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 4 Times, Rating: A+   ACC 491 Week 1 Individual Assignment Generally Accepted Auditing Standards Paper (UOP Course) ACC 491 Week 1 DQ 1 (UOP Course) ACC 491 Week 1 DQ 2 (UOP Course) ACC 491 week 2 Individual Assignments From the Text (UOP Course) ACC 491 Week 2 Team Assignment Auditing, Attestation, and Assurance Services Paper (UOP Course) ACC 491 Week 2 DQ 1 (UOP Course) AC...

  5. ACC 375 UOP Course Tutorial / Tutorialrank

    charles

    2015-01-01

    For more course tutorials visit   www.tutorialrank.com   Tutorial Purchased: 4 Times, Rating: A+       ACC 375 Week 1 Individual Assignment Understanding Ethics Matrix (UOP Course) ACC 375 Week 1 Discussion Question 1 (UOP Course) ACC 375 Week 1 Discussion Question 2 (UOP Course) ACC 375 Week 2 Team Assignment Sarbanes Oxley Act Training Manual (UOP Course) ACC 375 Week 2 Discussion Question 1 (UOP Course) ACC 375 We...

  6. Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression.

    Dabrowska, M; Jagielska, E; Cieśla, J; Płucienniczak, A; Kwiatowski, J; Wranicz, M; Boireau, P; Rode, W

    2004-02-01

    The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle. PMID:15030008

  7. Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

    Proudfoot, L; Nikolaev, A. V.; Feng, G.J.; Wei, W Q; Ferguson, M A; Brimacombe, J S; Liew, F. Y.

    1996-01-01

    Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant red...

  8. Glycoprotein Hypersecretion Alters the Cell Wall in Trichoderma reesei Strains Expressing the Saccharomyces cerevisiae Dolichylphosphate Mannose Synthase Gene▿

    Perlińska-Lenart, Urszula; Orłowski, Jacek; Laudy, Agnieszka E.; Zdebska, Ewa; Palamarczyk, Grażyna; Kruszewska, Joanna S.

    2006-01-01

    Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increas...

  9. Co-suppression in transgenic Petunia hybrida expressing chalcone synthase A (chsA)

    LI; Yan; (

    2001-01-01

    [1]Napoli, C., Lemieux, C., Jorgensen, R., Introduction of a chimeric chalone synthase gene into petunia results in reversible cosuppession of homologous genes in trans, The Plant Cell, 1990, 2: 279-289.[2]Van der Krol, A.R., Mur, L.A., Beld, M. M. et al., Flavonnoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression, The Plant Cell, 1990, 2: 291-299.[3]Manika, P.B., Bhadra, U., Birchler, J., Cosuppression in Drosophila: gene silencing of Alcohol dehydrogenase by White-Adh transgene is Polycomb dependent, Cell, 1997, 90: 479-498.[4]de Carvalho Niebel, F., Frendo, P., Van Montagu, M. et al., Post-transcriptional cosuppression of ?-1,3-glucanase transgene expression in homozygous plants, EMBO J., 1992, 11: 2595-2602.[5]Van Blokland, R., Van der Geest, N., Mol, J. N. M. et al., Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover, The Plant Cell, 1994, 6: 861-877.[6]Stam, M., Mol, J. N. M., Kooter, J. M., The silence of genes in transgenic plants, Annals of Bot., 1997, 79: 3-12.[7]Vaucheret, H., Beclin, C., Elmayan, T. et al., Transgene-induced gene silencing in plants, Plant J., 1998, 16(6): 651-659.[8]Shao, L., Li, Y., Yang, M. Z. et al., Transformation of Petunia hybrida with chalcone synthase A (chsA) resulting flower colour alteration and male sterility, Acta Botanica Sinica (in Chinese), 1996, 38(7): 517-524.[9]Koes, R. E., Spelt, C. E., Mol, J. N. M., The chalcone synthase multigene family of Petunia hybrida (V30): differential, light-regulated expression during flower development and UV light induction, Plant Mol. Biol., 1989, 12: 213-225.[10]Drews, G. N., Beals, T. P., Bul, A. Q. et al., Regional and cell-specific expression patterns during petal development, The Plant Cell, 1992, 4: 1383-1404.[11]Martin, C., Gerats, T., Control of pigment biosynthesis genes during petal development, The

  10. Molecular cloning and expression profiling of a chalcone synthase gene from Lamiophlomis rotata

    Qiao Feng; Geng Gui-Gong; Zeng Yang; Xie Hui-Chun; Jin Lan; Shang Jun; Chen Zhi

    2015-06-01

    Lamiophlomis rotata is a renowned Chinese medicinal plant. Chalcone synthase (CHS) is important in flavonoid and isoflavonoid biosynthesis, catalysing the formation of naringenin chalcone in plants. A full-length cDNA encoding the CHS gene was cloned from L. rotata based on the highly conserved CHS gene sequences of Labiatae plants. A blast search showed its homology (named LrCHS) with other CHS genes of Labiate plants. The full-length genomic DNA of LrCHS was 2026 bp with one intron of 651 bp, two exons of 178 bp and 998 bp, flanked by a 73 bp $5'$-UTR and a 126 bp $3'$-UTR. The cDNA sequence of the LrCHS gene had an 1176 bp open reading frame encoding a 391 amino acid protein of 42,798 Da. The CHS protein predicted from L. rotata showed 79–86% identity with CHS of other plant species. We conducted a phylogenetic analysis of nine families containing 48 plants and L. rotata based on the full amino acid sequences of CHS proteins. Consequently, LrCHS was located in the Labiatae branch. Additionally, we examined LrCHS gene expression patterns in different tissues by quantitative real-time PCR with specific primers. The expression analysis showed preferential expression of LrCHS in flowers and leaves during the flowering stage. Total flavonoid content and CHS gene expression exhibited similar patterns during L. rotata organ development. In agreement with its function as an elicitor-responsive gene, LrCHS expression was coordinated by methyl jasmonate and UV light, and induced between 6 and 18 h. These results provide a molecular basis for additional functional studies of LrCHS in L. rotata.

  11. acc226uopcoursesTutorial /uophelp

    keller

    2015-01-01

    For more course tutorials visit www.uophelp.com     ACC 226 Week 1 CheckPoint Accounts Receivable and Uncollectible Accounts ACC 226 Week 1 DQ 1 and DQ 2 ACC 226 Week 2 Assignment Accounting for Depreciation part ACC 226 Week 2 CheckPoint Ethics and Computing ACC 226 Week 3 CheckPoint Classifying Liabilities and Preparing Payroll Entries ACC 226 Week 3 DQ 2 ACC 226 Week 4 Assignment Stocks and Earnings per Share part ACC 226 Week 4 CheckPo...

  12. ACC 226 UOP COURSE TUTORIAL/ UOPHELP

    balumgm

    2015-01-01

    For more course tutorials visit www.uophelp.com     ACC 226 Week 1 CheckPoint Accounts Receivable and Uncollectible Accounts ACC 226 Week 1 DQ 1 and DQ 2 ACC 226 Week 2 Assignment Accounting for Depreciation part ACC 226 Week 2 CheckPoint Ethics and Computing ACC 226 Week 3 CheckPoint Classifying Liabilities and Preparing Payroll Entries ACC 226 Week 3 DQ 2 ACC 226 Week 4 Assignment Stocks and Earnings per Share part ACC 226 Week 4 CheckPo...

  13. ACC 226 Ash Course tutorial/uophelp

    THNSSER

    2015-01-01

    For more course tutorials visit www.uophelp.com   ACC 226 Week 1 CheckPoint Accounts Receivable and Uncollectible Accounts ACC 226 Week 1 DQ 1 and DQ 2 ACC 226 Week 2 Assignment Accounting for Depreciation part ACC 226 Week 2 CheckPoint Ethics and Computing ACC 226 Week 3 CheckPoint Classifying Liabilities and Preparing Payroll Entries ACC 226 Week 3 DQ 2 ACC 226 Week 4 Assignment Stocks and Earnings per Share part ACC 226 Week 4 CheckPoint Stock I...

  14. Relationship between gene expression of nitric oxide synthase and androgens in rat corpus cavernosum

    2000-01-01

    Objective To cladfy the dependence of neural nitric oxide synthase mRNA (nNOSmRNA) and endothelial nitric oxide synthase mRNA (eNOSmRNA) on androgens (testosterone [T] and dihydrotestosterone [DHT]). Methods 160 male Sprague Dawley (SD) rats were divided into Groups A (56 rats, 5 weeks old), B (50 rets,10 weeks old) and C (54 rats, 58 weeks old). Groups A, B and C were all subdivided respectively into five Subgroups. Subgroup 1: intact osntrels; Subgroup 2: castrated; Subgroup 3: castrated with testosterone ubdecanoate 25 mg/kg·mon-1, intramuscular injection, Subgroup 4: castrated with testosterone undecanoate 50 mg/kg·mon-1, intramuscular injection and Subgroup 5: treated with finaeteride 4.5 mg/kg·day-1, orally. Four and ten weeks after treatments described above, one half of the rats were killed. Serum samples were token for measurements of T, free testosterone (FT) and DHT by raclioimmunoassay. Penile samples were treated with liquid nitrogen and then stored at-80℃. nNOSmRNA and eNOSmRNA were detected by semiquantitative reveres-transcription polymerase chain reaction (RT-PCR) and Dot blot. Resulte There was no significant difference between Subgroup 1 and Subgroup 2 or Subgroup 5 in all Groups A, B and C. The expression of penile eNOSmRNA of Group A was significantly increased (4 weeks model) (P<0.05) or increased (10 weeks model) (P>0.05) in Subgroup 2 or 5 compared with those in Subgroup 1.There wes no significant difference between Subgroup 1 and Subgroup 2 or Subgroup 5 of Group B in 4 weeks model (P>0.05). There was an elevation when animals were castrated or treated with finasteride in the 10 weeks model.The expreseion of penile eNOSmRNA of Group C was significantly increased (10 weeks model) (P<0.05) or increased (4 weeks model) in Subgroup 2 compared with those in Subgroup 1.The production of eNOSmRNA in Subgroup 5 was also increased (including 4- and 10-week models). When T was supplied for castration, the penile eNOSmRNA was desreased to

  15. Nitric oxide synthase expression in the opossum superior colliculus: a histochemical, immunohistochemical and biochemical study.

    Giraldi-Guimarães, A; Tenório, F; Brüning, G; Mayer, B; Mendez-Otero, R; Cavalcante, L A

    1999-12-01

    The expression of neuronal nitric oxide synthase (nNOS) in the superior colliculus (SC) of the opossum Didelphis marsupialis was studied by NADPH diaphorase (NADPH-d) histochemistry and nNOS immunohistochemistry. In addition, the activity of nNOS was quantified by measurement of [(3)H]-L-arginine conversion to [(3)H]-L-citrulline in tissue extracts from SC superficial layers in opossums and rats. Our results show that the number of NADPH-d stained cells was small and virtually identical in stratum opticum (SO) and stratum griseum superficiale (SGS) and their staining was very light, particularly in SGS. Neuropil staining was heavier in the stratum zonale (SZ) than in SGS or SO. The intermediate and deep layers contained heavily stained cells and moderate neuropil staining. Surprisingly, nNOS-immunoreactive cells were far more numerous than NADPH-d+ cells in every layer. The production of [(3)H]-L-citrulline from [(3)H]-L-arginine in tissue extracts enriched in superficial layers indicated that nNOS specific activity is as high in the opossum as in the rat. Our results suggest that the location of nNOS-expressing neurons in retino-receptive layers may be related to inter-specific differences in the processing of visual information. PMID:10681601

  16. Expression of the Inducible Nitric Oxide Synthase Isoform in Chorionic Villi in the Early Spontaneous Abortion

    2000-01-01

    To investigate the relationship between inducible nitric oxide synthase (iNOS) and the early spontaneous abortion. , in situ hybridization and immunohistochemistry were used to detect the expression of iNOS in trophoblasts in the early pregnancy with and without spontaneous abortion (group Ⅰ and group Ⅱ ). By light microscopy and computer color magic image analysis system (CMIAS), light density (D) and the positive cell number per statistic square (N/S) in situ hybridization were used to analyze the positive cell index, while total positive cells (N) and the positive unit (Pu) were used in immunohistochemistry. By in situ hybridization, D and N/S in trophoblasts were 0. 35±0. 028, 0. 07±0. 011 respectively in group Ⅰ and 0. 18±0. 016,0. 015±0. 003 in group Ⅱ . In terms of immunohistochemical staining, N and Pu were 0. 058±±0. 007, 11. 94±2. 01 in group Ⅰ and 0. 013±0. 009, 1. 08±0. 35 in group Ⅱ in trophoblasts. Significant differences existed between two groups. It is concluded that the higher nitric oxide produced by the higher expression of iNOS in trophoblasts might play an important role in the early spontaneous abortion.

  17. Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

    Cong, Ming; Zhao, Jianmin; Lü, Jiasen; Ren, Zhiming; Wu, Huifeng

    2016-01-01

    The halophyte Suaeda salsa can grow in heavy metal-polluted areas along intertidal zones having high salinity. Since phytochelatins can eff ectively chelate heavy metals, it was hypothesized that S. salsa possessed a phytochelatin synthase (PCS) gene. In the present study, the cDNA of PCS was obtained from S. salsa (designated as SsPCS) using homologous cloning and the rapid amplification of cDNA ends (RACE). A sequence analysis revealed that SsPCS consisted of 1 916 bp nucleotides, encoding a polypeptide of 492 amino acids with one phytochelatin domain and one phytochelatin C domain. A similarity analysis suggested that SsPCS shared up to a 58.6% identity with other PCS proteins and clustered with PCS proteins from eudicots. There was a new kind of metal ion sensor motif in its C-terminal domain. The SsPCS transcript was more highly expressed in elongated and fibered roots and stems (P mercury exposure significantly enhanced the mRNA expression of SsPCS (P metal sensing capability than the first PCS from Thellungiella halophila. This study provided a new view of halophyte PCS genes in heavy metal tolerance.

  18. Molecular cloning of starch synthase I from maize (W64) endosperm and expression in Escherichia coli.

    Knight, M E; Harn, C; Lilley, C E; Guan, H; Singletary, G W; MuForster, C; Wasserman, B P; Keeling, P L

    1998-06-01

    A full length cDNA clone encoding a starch synthase (zSS) from maize endosperm (inbred line W64) was isolated and characterized. The cDNA clone (Ss1) is 2907 bp in length and contains an open reading frame of 1866 bp corresponding to a polypeptide of 622 amino acid residues including a transit peptide of 39 amino acids. The Ss1 cDNA clone was identified as zSSI by its direct alignment with sequences to: (i) the N-terminus obtained from the granule-associated form of the zSSI polypeptide, (ii) four internal peptide fragments obtained from the granule-associated form of the zSSI protein, and (iii) one internal fragment from the soluble form of the zSSI protein. The deduced amino acid sequence of Ss1 shares 75.7% sequence identity with rice soluble Ss and contains the highly conserved KSGGLGDV putative ADP-Glc binding site. Moreover, Ss1 exhibited significant activity when expressed in E. coli and the expressed protein is recognized by the antibody raised against the granule associated zSSI protein. Ss1 transcripts were detected in endosperm beginning at 15 days after pollination, but were not found in embryo, leaf or root. Maize contains a single copy of the Ss1 gene, which maps close to the Waxy locus of chromosome 9. PMID:9675904

  19. Genome-Wide Identification, Characterization and Expression Analysis of the Chalcone Synthase Family in Maize.

    Han, Yahui; Ding, Ting; Su, Bo; Jiang, Haiyang

    2016-01-01

    Members of the chalcone synthase (CHS) family participate in the synthesis of a series of secondary metabolites in plants, fungi and bacteria. The metabolites play important roles in protecting land plants against various environmental stresses during the evolutionary process. Our research was conducted on comprehensive investigation of CHS genes in maize (Zea mays L.), including their phylogenetic relationships, gene structures, chromosomal locations and expression analysis. Fourteen CHS genes (ZmCHS01-14) were identified in the genome of maize, representing one of the largest numbers of CHS family members identified in one organism to date. The gene family was classified into four major classes (classes I-IV) based on their phylogenetic relationships. Most of them contained two exons and one intron. The 14 genes were unevenly located on six chromosomes. Two segmental duplication events were identified, which might contribute to the expansion of the maize CHS gene family to some extent. In addition, quantitative real-time PCR and microarray data analyses suggested that ZmCHS genes exhibited various expression patterns, indicating functional diversification of the ZmCHS genes. Our results will contribute to future studies of the complexity of the CHS gene family in maize and provide valuable information for the systematic analysis of the functions of the CHS gene family. PMID:26828478

  20. Expression of inducible nitric oxide synthase in lungs of broiler chickens following intravenous cellulose microparticle injection.

    Hamal, K R; Wideman, R; Anthony, N; Erf, G F

    2008-04-01

    Intravenous microparticle (MP) injection is a patented method used to select broilers with a robust pulmonary capacity and improved resistance to pulmonary hypertension syndrome (PHS, ascites). Injected MP become entrapped in the terminal pulmonary arterioles where they elicit an increase in pulmonary arterial pressure attributable to vascular occlusion and focal thrombocyte aggregation. Within 2 to 48 h postinjection perivascular mononuclear cell aggregates begin to form around MP-occluded vessels. Nitric oxide (NO) has been shown to modulate the pulmonary arterial pressure response to MP entrapment, but a role of NO during the more chronic (2 to 48 h) focal inflammatory response has not been evaluated. In this study we determined the time-course of inducible nitric oxide synthase (iNOS) expression in the lungs of MP-injected broilers from PHS-resistant (RES) and PHS-susceptible (SUS) lines. Four-week-old broilers (10 broilers/line per time point) were injected i.v. with a minimally lethal dose of MP, and the right lung was collected at 0 h (no MP) and 2, 24, and 48 h postinjection. Immunohistochemistry revealed that macrophage infiltration increased over time in both lines and was higher in the RES line than the SUS line (P SUS line). For the RES line iNOS mRNA expression increased consistently from 0 to 48 h, but for the SUS line iNOS mRNA expression increased at 2 h, decreased to baseline at 24 h, and increased again by 48 h. The decline in iNOS expression in the SUS line between 2 and 24 h coincides with the interval when most of the MP-induced mortality occurs, which suggests that NO synthesized by iNOS may contribute to lower MP-induced mortality in the RES line when compared with the SUS line. PMID:18339983

  1. Citrus nobiletin suppresses inducible nitric oxide synthase gene expression in interleukin-1β-treated hepatocytes

    Highlights: •Nobiletin is a polymethoxylated flavone that is abundant in citrus peels. •Nobiletin is a major constituent of the Citrus unshiu peel extract. •Nobiletin suppresses induction of NO and reduces iNOS expression in hepatocytes. •Nobiletin reduces the iNOS promoter activity and the DNA-binding activity of NF-κB. -- Abstract: Background: Nobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes. Methods: A C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed. Results: High-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei. Conclusions: The suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases

  2. Citrus nobiletin suppresses inducible nitric oxide synthase gene expression in interleukin-1β-treated hepatocytes

    Yoshigai, Emi [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Machida, Toru [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okuyama, Tetsuya [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Mori, Masatoshi; Murase, Hiromitsu; Yamanishi, Ryota [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okumura, Tadayoshi [Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga (Japan); Department of Surgery, Kansai Medical University, Hirakata, Osaka (Japan); Ikeya, Yukinobu [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga (Japan); Nishino, Hoyoku [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto (Japan); Nishizawa, Mikio, E-mail: nishizaw@sk.ritsumei.ac.jp [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan)

    2013-09-13

    Highlights: •Nobiletin is a polymethoxylated flavone that is abundant in citrus peels. •Nobiletin is a major constituent of the Citrus unshiu peel extract. •Nobiletin suppresses induction of NO and reduces iNOS expression in hepatocytes. •Nobiletin reduces the iNOS promoter activity and the DNA-binding activity of NF-κB. -- Abstract: Background: Nobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes. Methods: A C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed. Results: High-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei. Conclusions: The suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases.

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

    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

  4. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. The GGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest that GGPPS may be one of the candidate genes for prostate cancer.

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

    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.

  6. Changes in the expression of NO synthase isoforms after ozone: the effects of allergen exposure

    Lee June-Hyuk

    2004-06-01

    Full Text Available Abstract Background The functional role of nitric oxide (NO and various nitric oxide synthase (NOS isoforms in asthma remains unclear. Objective This study investigated the effects of ozone and ovalbumin (OVA exposure on NOS isoforms. Methods The expression of inducible NOS (iNOS, neuronal NOS (nNOS, and endothelial NOS (eNOS in lung tissue was measured. Enhanced pause (Penh was measured as a marker of airway obstruction. Nitrate and nitrite in bronchoalveolar lavage (BAL fluid were measured using a modified Griess reaction. Results The nitrate concentration in BAL fluid from the OVA-sensitized/ozone-exposed/OVA-challenged group was greater than that of the OVA-sensitized/saline-challenged group. Methacholine-induced Penh was increased in the OVA-sensitized/ozone-exposed/OVA-challenged group, with a shift in the dose-response curve to the left, compared with the OVA-sensitized/saline-challenged group. The levels of nNOS and eNOS were increased significantly in the OVA-sensitized/ozone-exposed/OVA-challenged group and the iNOS levels were reduced compared with the OVA-sensitized/saline-challenged group. Conclusion In mice, ozone is associated with increases in lung eNOS and nNOS, and decreases in iNOS. None of these enzymes are further affected by allergens, suggesting that the NOS isoforms play different roles in airway inflammation after ozone exposure.

  7. ACC 490 UOP Course Tutorial / Tutorialrank

    charles

    2015-01-01

    For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 5 Times, Rating: A+   ACC 490 Week 1 Generally Accepted Auditing Standards Paper (UOP Course) ACC 490 Week 1 DQ 1 (UOP Course) ACC 490 Week 1 DQ 2 (UOP Course) ACC 490 Week 2 Individual Ch. 1 Textbook Exercises (UOP Course) ACC 490 Week 2 Learning Team Auditing, Attestation, and Assurance Services Paper (UOP Course) ACC 490 Week 2 DQ 1 (UOP Course) ACC 490 Week 2 DQ 2 (UOP Cou...

  8. ACC 310 ASH Tutorial Course/Uoptutorial

    VANI

    2015-01-01

    For more course tutorials visit www.uoptutorial.com   ACC 310 Week 1 DQ 1 Information for Decision Making and Cost Concepts and Behavior ACC 310 Week 1 DQ 2 Fundamentals of Cost-Volume-Profit Analysis ACC 310 Week 1 Assignment CVP Analysis and Price Changes ACC 310 Week 2 DQ 1 Fundamentals of Cost Accounting for Decision Making ACC 310 Week 2 DQ 2 Fundamentals of Product and Service Costing ACC 310 Week 2 Assignment Special Orders ACC 310 Week 3 DQ 1 J...

  9. ACC 250 UOP COURSE TUTORIAL/ UOPHELP

    veeru1

    2015-01-01

    For more course tutorials visit www.uophelp.com   ACC 250 Week 1 CheckPoint Choosing Accounting Software ACC 250 Week 1 Assignment Accounting Software Memo ACC 250 Week 2 CheckPoint Bellwether Garden Supply Back Up and Restore Data ACC 250 Week 2 DQ 1 and DQ 2 ACC 250 Week 3 CheckPoint Bellwether Garden Supply Vendor Transactions ACC 250 Week 3 Assignment Bellwether Garden Supply Record a Transaction ACC 250 Week 4 CheckPoint Bellwether Garden Supply P...

  10. Relationship between the Expression of Thymidylate Synthase,Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase and Survival in Epithelial Ovarian Cancer

    王常玉; 翁艳洁; 王鸿雁; 石英; 马丁

    2010-01-01

    The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression a...

  11. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 106 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

  12. Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

    Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J. (Mount Sinai School of Medicine, New York, NY (USA))

    1988-10-01

    Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 {times} 10{sup 6} recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria.

  13. ACC 310 ASH Course Tutorial / Tutorialrank

    charles

    2015-01-01

    For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 4 Times, Rating: A+ ASHFORD ACC 310 Week 1 DQ 1 Information for Decision Making and Cost Concepts and Behavior ASHFORD ACC 310 Week 1 DQ 2 Fundamentals of Cost-Volume-Profit Analysis ASHFORD ACC 310 Week 1 Assignment CVP Analysis and Price Changes ASHFORD ACC 310 Week 2 DQ 1 Fundamentals of Cost Accounting for Decision Making ASHFORD ACC 310 Week 2 DQ 2 Fundamentals of Product and Service Costing ...

  14. ACC 226 UOP Course Tutorial / Tutorialrank

    charles

    2015-01-01

    For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 5 Times, Rating: A+       ACC 226 Week 1 CheckPoint Accounts Receivable and Uncollectible Accounts   ACC 226 Week 1 DQ 1 and DQ 2   ACC 226 Week 2 Assignment Accounting for Depreciation part   ACC 226 Week 2 CheckPoint Ethics and Computing   ACC 226 Week 3 CheckPoint Classifying Liabilities and Preparing Payroll Entries   ...

  15. An In Planta-Expressed Polyketide Synthase Produces (R)-Mellein in the Wheat Pathogen Parastagonospora nodorum

    Chooi, Yit-Heng; Krill, Christian; Barrow, Russell A.; Chen, Shasha; Trengove, Robert; Oliver, Richard P.; Solomon, Peter S

    2014-01-01

    Parastagonospora nodorum is a pathogen of wheat that affects yields globally. Previous transcriptional analysis identified a partially reducing polyketide synthase (PR-PKS) gene, SNOG_00477 (SN477), in P. nodorum that is highly upregulated during infection of wheat leaves. Disruption of the corresponding SN477 gene resulted in the loss of production of two compounds, which we identified as (R)-mellein and (R)-O-methylmellein. Using a Saccharomyces cerevisiae yeast heterologous expression syst...

  16. Effects of pneumonectomy on nitric oxide synthase expression and perivascular edema in the remaining lung of rats

    M.N. Samano; R. Pazetti; Prado, C.M.; I.C. Tibério; Saldiva, P H N; L.F.P. Moreira; P.M. Pêgo-Fernandes; F.B. Jatene; J.C. Das-Neves-Pereira

    2009-01-01

    Pneumonectomy is associated with high mortality and high rates of complications. Postpneumonectomy pulmonary edema is one of the leading causes of mortality. Little is known about its etiologic factors and its association with the inflammatory process. The purpose of the present study was to evaluate the role of pneumonectomy as a cause of pulmonary edema and its association with gas exchange, inflammation, nitric oxide synthase (NOS) expression and vasoconstriction. Forty-two non-specific pa...

  17. Elevated expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in primary sclerosing cholangitis : Implications for cholangiocarcinogenesis

    Ishii, Yasutaka

    2014-01-01

    Cholangiocarcinoma (CCA) occurs frequently in primary sclerosing cholangitis (PSC). Cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) induced by inflammation are believed to mediate prostaglandin E2 (PGE2) production thereby promoting carcinogenesis. Their expression in PSC-associated CCA tissues and non-neoplastic bile duct epithelial cells (BDECs) in PSC was investigated. COX-2 and mPGES-1 levels in 15 PSC patients (7 with CCA) were scored using immunohistochemica...

  18. Mechanisms of suppression of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells by andrographolide

    Chiou, Wen-Fei; Chen, Chieh-Fu; Lin, Jin-Jung

    2000-01-01

    Andrographolide, an active component found in leaves of Andrographis paniculata, has been reported to exhibit nitric oxide (NO) inhibitory property in endotoxin-stimulated macrophages, however, the detailed mechanisms remain unclear. In the present study we investigated the effect of andrographolide on the expression of inducible NO synthase (iNOS) mRNA, protein, and enzyme activity in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) plus interferon-γ (IFN-γ).RAW 264.7 cells sti...

  19. Nitric oxide production and the expression of two nitric oxide synthases in the avian retina.

    Tekmen-Clark, Merve; Gleason, Evanna

    2013-05-01

    Nitric oxide (NO) is known to exert multiple effects on the function of many retinal neurons and their synapses. Therefore, it is equally important to understand the potential sources of NO within the retina. To explore this, we employ a combination of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) based NO detection and immunohistochemistry for the NO synthetic enzymes, neuronal and endothelial nitric oxide synthase (nNOS and eNOS). We find DAF signals in photoreceptors, horizontal cells, amacrine cells, efferent synapses, Müller cells, and cells in the ganglion cell layer (GCL). nNOS immunoreactivity was consistent with the DAF signal with the exception that horizontal cells and Müller cells were not clearly labeled. eNOS-like immunoreactivity (eNOS-LI) was more widespread with photoreceptors, horizontal cells, occasional bipolar cells, amacrine cells, Müller cells, and cells in the GCL all showing labeling. Double labeling with antibodies raised against calretinin, syntaxin, and glutamine synthetase confirmed that horizontal cells, amacrine cells, and Müller cells (respectively) were expressing eNOS-LI. Although little or no nNOS labeling is observed in horizontal cells or Müller cells, the expression of eNOS-LI is consistent with the ability of these cells to produce NO. Together these results suggest that the capability to produce NO is widespread in the chicken retina. We propose that multiple forms of regulation for nNOS and eNOS play a role in the patterning of NO production in the chicken retina. PMID:23721886

  20. Expression of an(E)-β-farnesene synthase gene from Asian peppermint in tobacco affected aphid infestation

    Xiudao; Yu; Yongjun; Zhang; Youzhi; Ma; Zhaoshi; Xu; Genping; Wang; Lanqin; Xia

    2013-01-01

    Aphids are major agricultural pests that cause significant yield losses in crop plants each year.(E)-β-farnesene(EβF) is the main or only component of an alarm pheromone involved in chemical communication within aphid species and particularly in the avoidance of predation. EβF also occurs in the essential oil of some plant species, and is catalyzed by EβF synthase. By using oligonucleotide primers designed from the known sequence of an EβF synthase gene from black peppermint(Mentha × piperita), two cDNA sequences, MaβFS1 and MaβFS2, were isolated from Asian peppermint(Mentha asiatica). Expression pattern analysis showed that the MaβFS1 gene exhibited higher expression in flowers than in roots, stems and leaves at the transcriptional level. Overexpression of MaβFS1 in tobacco plants resulted in emission of pure EβF ranging from 2.62 to 4.85 ng d-1g-1of fresh tissue. Tritrophic interactions involving peach aphids(Myzus persicae), and predatory lacewing(Chrysopa septempunctata) larvae demonstrated that transgenic tobacco expressing MaβFS1 had lower aphid infestation. This result suggested that the EβF synthase gene from Asian peppermint could be a good candidate for genetic engineering of agriculturally important crop plants.

  1. Expression of an (E-β-farnesene synthase gene from Asian peppermint in tobacco affected aphid infestation

    Xiudao Yu

    2013-10-01

    Full Text Available Aphids are major agricultural pests that cause significant yield losses in crop plants each year. (E-β-farnesene (EβF is the main or only component of an alarm pheromone involved in chemical communication within aphid species and particularly in the avoidance of predation. EβF also occurs in the essential oil of some plant species, and is catalyzed by EβF synthase. By using oligonucleotide primers designed from the known sequence of an EβF synthase gene from black peppermint (Mentha × piperita, two cDNA sequences, MaβFS1 and MaβFS2, were isolated from Asian peppermint (Mentha asiatica. Expression pattern analysis showed that the MaβFS1 gene exhibited higher expression in flowers than in roots, stems and leaves at the transcriptional level. Overexpression of MaβFS1 in tobacco plants resulted in emission of pure EβF ranging from 2.62 to 4.85 ng d− 1 g− 1 of fresh tissue. Tritrophic interactions involving peach aphids (Myzus persicae, and predatory lacewing (Chrysopa septempunctata larvae demonstrated that transgenic tobacco expressing MaβFS1 had lower aphid infestation. This result suggested that the EβF synthase gene from Asian peppermint could be a good candidate for genetic engineering of agriculturally important crop plants.

  2. Analysis of the Expression and Activity of Nitric Oxide Synthase from Marine Photosynthetic Microorganisms.

    Foresi, Noelia; Correa-Aragunde, Natalia; Santolini, Jerome; Lamattina, Lorenzo

    2016-01-01

    Nitric oxide (NO) functions as a signaling molecule in many biological processes in species belonging to all kingdoms of life. In animal cells, NO is synthesized primarily by NO synthase (NOS), an enzyme that catalyze the NADPH-dependent oxidation of L-arginine to NO and L-citrulline. Three NOS isoforms have been identified, the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) and one inducible (iNOS). Plant NO synthesis is complex and is a matter of ongoing investigation and debate. Despite evidence of an Arg-dependent pathway for NO synthesis in plants, no plant NOS homologs to animal forms have been identified to date. In plants, there is also evidence for a nitrate-dependent mechanism of NO synthesis, catalyzed by cytosolic nitrate reductase. The existence of a NOS enzyme in the plant kingdom, from the tiny single-celled green alga Ostreococcus tauri was reported in 2010. O. tauri shares a common ancestor with higher plants and is considered to be part of an early diverging class within the green plant lineage.In this chapter we describe detailed protocols to study the expression and characterization of the enzymatic activity of NOS from O. tauri. The most used methods for the characterization of a canonical NOS are the analysis of spectral properties of the oxyferrous complex in the heme domain, the oxyhemoglobin (oxyHb) and citrulline assays and the NADPH oxidation for in vitro analysis of its activity or the use of fluorescent probes and Griess assay for in vivo NO determination. We further discuss the advantages and drawbacks of each method. Finally, we remark factors associated to the measurement of NOS activity in photosynthetic organisms that can generate misunderstandings in the interpretation of results. PMID:27094418

  3. Cyclooxygenase 2 and neuronal nitric oxide synthase expression in the renal cortex are not interdependent in states of salt deficiency

    Castrop, H; Kammerl, M; Mann, Birgitte;

    2000-01-01

    Neuronal nitric oxide synthase (nNOS) and cyclooxygenase-2 (COX-2) expression in the kidney are localized to the cortical thick ascending limb of the loop of Henle (cTALH), including the macula region, and increase after salt restriction. Because of the similar localization and regulation of n...... prostanoid excretion. These findings suggest that under these conditions the control of nNOS and COX-2 gene expression in the macula densa regions of the kidney cortex are not dependent on each other....

  4. acc310ashcoursesTutorial /uophelp

    hakkins

    2015-01-01

    For more course tutorials visit www.uophelp.com   ACC 310 Week 1 DQ 1 Information for Decision Making and Cost Concepts and Behavior(1-A+ ACC 310 Week 1 DQ 2 Fundamentals of Cost-Volume-Profit Analysis    (no ratg ) ACC 310 Week 1 Assignment CVP Analysis and Price Changes   (2A) ACC 310 Week 2 DQ 1 Fundamentals of Cost Accounting for Decision Making (no ratg ) ACC 310 Week 2 DQ 2 Fundamentals of Product and Service Costing  (2B+) ACC 31...

  5. Cloning, expression, and characterization of recombinant nitric oxide synthase-like protein from Bacillus anthracis

    Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with L-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of L-arginine, N ω-hydroxy-L-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein

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

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

    1991-01-01

    Prostaglandin H (PGH) synthase (EC 1.14.99.1) 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 (0.1-1 micrograms/ml) inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-i...

  7. An in planta-expressed polyketide synthase produces (R)-mellein in the wheat pathogen Parastagonospora nodorum.

    Chooi, Yit-Heng; Krill, Christian; Barrow, Russell A; Chen, Shasha; Trengove, Robert; Oliver, Richard P; Solomon, Peter S

    2015-01-01

    Parastagonospora nodorum is a pathogen of wheat that affects yields globally. Previous transcriptional analysis identified a partially reducing polyketide synthase (PR-PKS) gene, SNOG_00477 (SN477), in P. nodorum that is highly upregulated during infection of wheat leaves. Disruption of the corresponding SN477 gene resulted in the loss of production of two compounds, which we identified as (R)-mellein and (R)-O-methylmellein. Using a Saccharomyces cerevisiae yeast heterologous expression system, we successfully demonstrated that SN477 is the only enzyme required for the production of (R)-mellein. This is the first identification of a fungal PKS that is responsible for the synthesis of (R)-mellein. The P. nodorum ΔSN477 mutant did not show any significant difference from the wild-type strain in its virulence against wheat. However, (R)-mellein at 200 μg/ml inhibited the germination of wheat (Triticum aestivum) and barrel medic (Medicago truncatula) seeds. Comparative sequence analysis identified the presence of mellein synthase (MLNS) homologues in several Dothideomycetes and two sodariomycete genera. Phylogenetic analysis suggests that the MLNSs in fungi and bacteria evolved convergently from fungal and bacterial 6-methylsalicylic acid synthases. PMID:25326302

  8. Cloning and Expression of Poly(hydroxyalkanoate) Synthase Genes from Photosynthetic bacterium Allochromatium vinosum ATCC 35206

    Poly(hydroxyalkanoate) (PHA) synthases catalyze the polymerization of beta-hydroxy fatty acids to form PHA biopolyesters. These enzymes are grouped into four classes (classes I to IV) based on their subunit composition and substrate specificity. Since PHA biopolymers are naturally synthesized by b...

  9. Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90.

    Averna, Monica; Stifanese, Roberto; De Tullio, Roberta; Salamino, Franca; Bertuccio, Mara; Pontremoli, Sandro; Melloni, Edon

    2007-12-01

    Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced. PMID:17970747

  10. Identification, expression and serological evaluation of the recombinant ATP synthase beta subunit of Mycoplasma pneumoniae

    Nuyttens Hélène

    2010-08-01

    Full Text Available Abstract Background Mycoplasma pneumoniae is responsible for acute respiratory tract infections (RTIs common in children and young adults. As M. pneumoniae is innately resistant to β-lactams antibiotics usually given as the first-line treatment for RTIs, specific and early diagnosis is important in order to select the right treatment. Serology is the most used diagnostic method for M. pneumoniae infections. Results In this study, we identified the M. pneumoniae ATP synthase beta subunit (AtpD by serologic proteome analysis and evaluated its usefulness in the development of a serological assay. We successfully expressed and purified recombinant AtpD (rAtpD protein, which was recognised by serum samples from M. pneumoniae-infected patient in immunoblots. The performance of the recombinant protein rAtpD was studied using a panel of serum samples from 103 infected patients and 86 healthy blood donors in an in-house IgM, IgA and IgG enzyme-linked immunosorbent assay (ELISA. The results of this assay were then compared with those of an in-house ELISA with a recombinant C-terminal fragment of the P1 adhesin (rP1-C and of the commercial Ani Labsystems ELISA kit using an adhesin P1-enriched whole-cell extract. Performances of the rAtpD and rP1-C antigen combination were further assessed by binary logistic regression analysis. We showed that combination of rAtpD and rP1-C discriminated maximally between the patients infected with M. pneumoniae (children and adults and the healthy subjects for the IgM class, performing better than the single recombinant antigens or the commercial whole-cell extract. Conclusion These results suggest that AtpD can be used as an antigen for the immunodiagnosis of early and acute M. pneumoniae infection in association with adhesin P1, providing an excellent starting point for the development of point-of-care diagnostic assays.

  11. A Riboswitch Regulates Expression of the Coenzyme B12-Independent Methionine Synthase in Mycobacterium tuberculosis: Implications for Differential Methionine Synthase Function in Strains H37Rv and CDC1551▿ †

    Warner, Digby F.; Savvi, Suzana; Mizrahi, Valerie; Dawes, Stephanie S.

    2007-01-01

    We observed vitamin B12-mediated growth inhibition of Mycobacterium tuberculosis strain CDC1551. The B12 sensitivity was mapped to a polymorphism in metH, encoding a coenzyme B12-dependent methionine synthase. Vitamin B12-resistant suppressor mutants of CDC1551 containing mutations in a B12 riboswitch upstream of the metE gene, which encodes a B12-independent methionine synthase, were isolated. Expression analysis confirmed that the B12 riboswitch is a transcriptional regulator of metE in M. ...

  12. Monoterpene synthases from common sage (Salvia officinalis)

    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.

  13. Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

    Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD2 and its non-enzymatic dehydration products, PGJ2 series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD2 and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD2 from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE2 remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize Δ12-PGJ2 increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor γ (PPARγ) agonists including troglitazone and Δ12-PGJ2. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPARγ signaling pathway without the contribution of endogenous pro-adipogenic prostanoids. -- Highlights: ► Cultured preadipocytes were transfected with sense lipocalin-type PGD

  14. Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

    Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Nishimura, Kohji [Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Jisaka, Mitsuo; Nagaya, Tsutomu [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Shono, Fumiaki [Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima 770-8514 (Japan); Yokota, Kazushige, E-mail: yokotaka@life.shimane-u.ac.jp [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan)

    2012-02-15

    Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD{sub 2} and its non-enzymatic dehydration products, PGJ{sub 2} series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD{sub 2} and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD{sub 2} from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE{sub 2} remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize {Delta}{sup 12}-PGJ{sub 2} increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists including troglitazone and {Delta}{sup 12}-PGJ{sub 2}. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPAR{gamma} signaling pathway without the contribution of endogenous pro-adipogenic prostanoids

  15. Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity.

    Lanone, Sophie; Manivet, Philippe; Callebert, Jacques; Launay, Jean-Marie; Payen, Didier; Aubier, Michel; Boczkowski, Jorge; Mebazaa, Alexandre

    2002-01-01

    Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity. PMID:12097137

  16. Aloe vera toxic effects: expression of inducible nitric oxide synthase (iNOS) in testis of Wistar rat

    Samira Asgharzade; Mahmoud Rafieian-kopaei; Amin Mirzaeian; Somaye Reiisi; Loghman Salimzadeh

    2015-01-01

    Objective(s): Nitric oxide (NO), a product of inducible nitric oxide synthase (iNOS), contributes in germ cell apoptosis. This study was aimed to evaluate the effects of Aloe vera gel (AVG) on male Wistar rat reproductive organ, serum NO level, and expression of iNOS gene in leydig cells. Materials and Methods: Adult male Wistar rats (n=36) were used for experiments in three groups. The experimental groups were orally administered with the AVG extract solution once-daily as follow: 150 mg.kg-...

  17. Alteration of syncytiotrophoblast mitochondria function and endothelial nitric oxide synthase expression in the placenta of rural residents.

    Rivero Osimani, Valeria L; Valdez, Susana R; Guiñazú, Natalia; Magnarelli, Gladis

    2016-06-01

    The impact of environmental organophosphate (OP) pesticide exposure on respiratory complexes, enzymatic antioxidant defense activities, and oxidative damage markers in the syncytiotrophoblast and cytotrophoblast mitochondria was evaluated. Placental progesterone (PG) levels and endothelial nitric oxide synthase (eNOS) expression were studied. Samples from women non-exposed (control group-CG) and women living in a rural area (rural group-RG) were collected during pesticide spraying season (RG-SS) and non-spraying season (RG-NSS). In RG-SS, the exposure biomarker placental carboxylesterase decreased and syncytiotrophoblast cytochrome c oxidase activity increased, while 4-hydroxynonenal levels decreased. PG levels decreased in RG-SS and in the RG. Nitric oxide synthase expression decreased in RG, RG-SS and RG-NSS. No significant changes in mitochondrial antioxidant enzyme activities were found. These results suggest that the alteration of syncytiotrophoblast mitochondrial complex IV activity and steroidogenic function may be associated to pesticide exposure. Reduction in placental PG and eNOS expression may account for low newborn weight in RG. PMID:26939719

  18. Further studies of auxin and ACC induced feminization in the cucumber plant using ethylene inhibitors

    Takahashi, H.; Jaffe, M. J.

    1984-01-01

    The present study was designed to establish the role of an essential hormone controlling sex expression in cucumber. A potent anti-ethylene agent, AgNO3, completely inhibited pistillate flower formation caused by IAA, ACC or ethephon. Inhibitors of ethylene biosynthesis, AVG and CoCl2 also suppressed feminization due to exogenous IAA or ACC. Though AVG also suppressed ethephon-induced feminization, this may be due to the second effect of AVG rather than the effect on ACC biosynthesis. These results confirm that ethylene is a major factor regulating feminization and that exogenous auxin induces pistillate flower formation through its stimulation of ethylene production, rather than ACC production.

  19. Effect of vardenafil on nitric oxide synthase expression in the paraventricular nucleus of rats without sexual stimulation.

    Shin, M-S; Ko, I-G; Kim, S-E; Kim, B-K; Kim, C-J; Kim, D-H; Yoon, S-J; Kim, K-H

    2012-05-01

    Vardenafil hydrochloride (HCl) is a potent and selective phosphodiesterase type-5 (PDE-5) inhibitor that enhances nitric oxide (NO)-mediated relaxation of human corpus cavernosum and NO-induced rabbit penile erection, and enhances erectile function in patients. In the present study, the effect of vardenafil on nitric oxide synthase (NOS) and neuronal NOS expressions in the paraventricular nucleus (PVN) of rats without sexual stimulation was investigated using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal NOS (nNOS) immunohistochemistry and western blot analysis. The present results showed that NOS and nNOS expression in the PVN was increased by vardenafil treatment as the dose- and duration-dependently without sexual stimulation. The phosphodiesterase type-5 inhibitor, vardenafil, augmented NOS expression in the brain without sexual stimulation. The present study suggests that sexual behaviour can be directly modulated by neurotransmitters such as nitric oxide. PMID:21950284

  20. Expression of nitric oxide synthase in the spinal cord after selective brachial plexus injury

    Na Liu; Feng Li; Longju Chen; Wutian Wu

    2006-01-01

    BACKGROUND: Some researches showed that motoneurons in spinal cord anterior horn wound die following brachial plexus injury, but the concrete mechanism of motoneurons death remains unclear.OBJECTIVE: To observe the expression of nitric oxide synthase (NOS) and survival of C7 motoneurons in spinal cord of rats after selective brachial plexus injury.DESIGN: A randomized controlled animal experiment.SETTING: Department of Anatomy, Sun Yet-sen Medical College, Sun Yet-sen University.MATERIALS: Totally 35 adult healthy male Sprague-Dawley rats with the body mass of 200-300 g were provided by Experimental Animal Center, Sun Yet-sen Medical College, Sun Yat-sen University. The rats were divided into control group (n =5) and experimental group (n=30) by random number table method, and the experimental group was divided into three injury subgroups: anterior root avulsion group, dorsal root transection group and spinal cord hemisection group, 10 rats in each group. There were horse anti-neuronal NOS (Nnos) polycolonal antibody (Sigma company) and nicotina mideadeninedinucleotide phosphate (NADPH-d) (SigmaCompany).METHODS: The experiment was performed at Department of Anatomy, Sun Yet-sen Medical College, Sun Yet-sen University between September 2004 and April 2005. ①After anesthetizing the rats, the spinous process of second thoracic vertebra as a marker, the vertebra was exposed from C5 to T1 and the lamina of vertebra was unclenched, and spinal dura mater was carved to expose the spinal nerve dorsal roots of C5-T1.The right ventral root of C7 was avulsed, and the residual root was removed in anterior root avulsion group. The right ventral root of C7 was avulsed and the right dorsal roots of brachial plexus (C5-T1) were cut off in dorsal root transection group. In spinal cord hemisection group, the hemisection between the C5 and C6 spinal segment on right side and avulsion of right ventral root of C7 were made. In the control group, the vertebra from C5 to T1 was

  1. ACC 490 V4 Course Tutorial / tutorialrank

    welcome1362

    2015-01-01

    ACC 490 Version 4 Entire Course UOP Course For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 5 Times, Rating: A+   ACC 490 Version 4 Week 1 Generally Accepted Auditing Standards Paper (UOP Course) ACC 490 Version 4 Week 1 Individual Assignment Ch 1 and Ch 4 Questions (UOP Course) ACC 490 Version 4 Week 1 Team Assignment Ch 1 and Ch 4 Questions (UOP Course) ACC 490 Version 4 Week 2 Individual Assignment Ch 6, Ch 7 and Ch 9 Exercise (U...

  2. ACC 490 V4 Course Tutorial / Tutorialrank

    charles

    2015-01-01

    For more course tutorials visit www.tutorialrank.com Tutorial Purchased: 5 Times, Rating: A+   ACC 490 Version 4 Week 1 Generally Accepted Auditing Standards Paper (UOP Course) ACC 490 Version 4 Week 1 Individual Assignment Ch 1 and Ch 4 Questions (UOP Course) ACC 490 Version 4 Week 1 Team Assignment Ch 1 and Ch 4 Questions (UOP Course) ACC 490 Version 4 Week 2 Individual Assignment Ch 6, Ch 7 and Ch 9 Exercise (UOP Course) ACC 490 Version 4 Week 2 Team As...

  3. Astrocytes and microglia express inducible nitric oxide synthase in mice with experimental allergic encephalomyelitis

    Tran, E H; Hardin-Pouzet, H; Verge, G;

    1997-01-01

    Nitric oxide (NO), produced by inducible NO synthase (iNOS), may play a role in inflammatory demyelinating diseases of the central nervous system (CNS). We show upregulation of iNOS mRNA in CNS of SJL/J mice with experimental allergic encephalomyelitis (EAE). Using antibodies against mouse i...... microglia rather than astrocytes are implicated in demyelinating pathology, we propose that microglial NO may be cytopathic whereas astrocyte-derived NO may be protective in EAE....

  4. Cis-regulatory Evolution of Chalcone-Synthase Expression in the Genus Arabidopsis

    de Meaux, J. (Juliette); Pop, A.(National Institute for Physics and Nuclear Engineering, Bucharest, Romania); Mitchell-Olds, T.

    2006-01-01

    The contribution of cis-regulation to adaptive evolutionary change is believed to be essential, yet little is known about the evolutionary rules that govern regulatory sequences. Here, we characterize the short-term evolutionary dynamics of a cis-regulatory region within and among two closely related species, A. lyrata and A. halleri, and compare our findings to A. thaliana. We focused on the cis-regulatory region of chalcone synthase (CHS), a key enzyme involved in the synthesis of plant sec...

  5. ASH ACC 205 NEW Tutorials /ashacc205dotcom

    yamini63

    2015-01-01

    ACC 205 Entire Course(New)   For more course tutorials visit www.ashacc205.com   ACC 205 Week 1 DQ 1 Accounting Equation ACC 205 Week 1 DQ 2 Accounts ACC 205 Week 1 Journal Balance Sheet Journal ACC 205 Week 2 DQ 1 Accounting Cycle ACC 205 Week 2 DQ 2 Bank Reconciliation ACC 205 Week 2 Journal Income Statement Journal ACC 205 Week 3 DQ 1 LIFO vs. FIFO ACC 205 Week 3 DQ 2 Depreciation ACC 205 Week 3 Journal Inventory Journal ACC...

  6. ACC 205 NEW Ash Course tutorial/uophelp

    RGNSDD

    2015-01-01

    ACC 205 Week 1 DQ 1 Accounting Equation ACC 205 Week 1 DQ 2 Accounts ACC 205 Week 1 Exercise Assignment Basic Accounting Equations ACC 205 Week 1 Journal Balance Sheet Journal ACC 205 Week 2 DQ 1 Accounting Cycle ACC 205 Week 2 DQ 2 Bank Reconciliation ACC 205 Week 2 Exercise Assignment Revenue and Expenses ACC 205 Week 2 Journal Income Statement Journal ACC 205 Week 3 DQ 1 LIFO vs ACC 205 Week 3 DQ 2 Depreciation ACC 205 Week 3 Exercise Assignment Inventory A...

  7. Geranyl diphosphate synthase from mint

    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.

  8. Geranyl diphosphate synthase from mint

    Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

    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.

  9. ACC 375 uop course tutorial/uop help

    jeeven

    2015-01-01

    For more course tutorials visit www.uophelp.com   ACC 375 Week 1 Individual Assignment Understanding Ethics Matrix ACC 375 Week 1 Discussion Question 1 ACC 375 Week 1 Discussion Question 2 ACC 375 Week 2 Team Assignment Sarbanes Oxley Act Training Manual ACC 375 Week 2 Discussion Question 1 ACC 375 Week 2 Discussion Question 2 ACC 375 Week 3 Individual Assignment Ethics Situation 1 Fraudulent Schemes Report ACC 375 Week 3 Team Assignment Article Review Et...

  10. Isolation of developing secondary xylem specific cellulose synthase genes and their expression profiles during hormone signalling in Eucalyptus tereticornis

    Balachandran Karpaga Raja Sundari; Modhumita Ghosh Dasgupta

    2014-08-01

    Cellulose synthases (CesA) represent a group of -1, 4 glycosyl transferases involved in cellulose biosynthesis. Recent reports in higher plants have revealed that two groups of CesA gene families exist, which are associated with either primary or secondary cell wall deposition. The present study aimed at identifying developing secondary xylem specific cellulose synthase genes from Eucalyptus tereticornis, a species predominantly used in paper and pulp industries in the tropics. The differential expression analysis of the three EtCesA genes using qRT-PCR revealed 49 to 87 fold relative expression in developing secondary xylem tissues. Three full length gene sequences of EtCesA1, EtCesA2 and EtCesA3 were isolated with the size of 2940, 3114 and 3123 bp, respectively. Phytohormone regulation of all three EtCesA genes were studied by exogenous application of gibberellic acid, naphthalene acetic acid, indole acetic acid and 2, 4-epibrassinolide in internode tissues derived from three-month-old rooted cuttings. All three EtCesA transcripts were upregulated by indole acetic acid and gibberellic acid. This study demonstrates that the increased cellulose deposition in the secondary wood induced by hormones can be attributed to the upregulation of xylem specific CesAs.

  11. Expression in Arabidopsis of a Strawberry Linalool Synthase Gene Under the Control of the Inducible Potato P12 Promoter

    YANG Li-mei; Per Mercke; Joop J A van Loon; FANG Zhi-yuan; Marcel Dicke; Maarten A Jongsma

    2008-01-01

    To investigate the role of inducible linalool in Arabidopsis-insect interactions, the FANESl linalool synthase (LIS) cDNA from strawberry with plastid targeting and a synthetic intron (LIS') was placed under the control of the wound inducible proteinase inhibitor 2 (PI2) promoter from potato. The construct pBin-PP12-LIS' was transformed to Arabidopsis thaliana ecotype Columbia O. Kanamycin resistant T0 seedlings were confirmed for the presence and transcription of the LIS' gene by PCR analysis on genomic DNA and by RT-PCR analysis on RNA. Genomic and RT-PCR products were sequenced to confirm correct splicing of the synthetic intron. The expression of active linalool synthase by the PP12-LIS' gene construct in the transgenic lines was assessed by measuring linalool emission using solid phase micro-extraction (SPME) GC-MS measurements after induction with methyl jasmonate. Among 30 tested independent T2 transgenic lines, 10 exhibited linalool production.Linalool expression could be induced by methyl jasmonate treatment, but not by diamondback moth larvae.

  12. Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in association with neovascularization in human primary astrocytoma

    PAN Jian-wei; ZHAN Ren-ya; TONG Ying; ZHOU Yong-qing; ZHANG Ming

    2005-01-01

    Objective: To investigate the relationship between the expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and angiogenesis in primary astrocytoma. Methods: Thirty-seven primary astrocytomas and 4 astrocytic hyperplasia samples were collected and divided into three groups according to histological grade. The expression of eNOS, VEGF and factor Ⅷ related antigen (FVIIIRAg) were assayed by immunohistochemistry. Microvascular density was assessed by FVIIIRAg immunoreactivity. The intensity of immunoreactivity was graded according to the percentage of positive tumor cells. Results: No eNOS and VEGF were expressed in the astrocytes and vascular endothelium in astrocytic hyperplasia.The expression of eNOS or VEGF was light in low-grade astrocytoma and strong in glioblastoma. eNOS expression in astrocytoma was very positively correlated with VEGF. eNOS and VEGF expression in anaplastic astrocytoma was median in contrast to the low grade astrocytoma and glioblastoma. Lower microvascular density was found in low grade astrocytoma than that in higher grade malignant ones. The expressions of eNOS and VEGF were correlated with microvascular density and tumor malignancy.Conclusion: This finding suggests that eNOS and VEGF may have cooperative effect in tumor angiogenesis and play an important role in the pathogenesis of primary astrocytoma.

  13. Airborne Cloud Computing Environment (ACCE)

    Hardman, Sean; Freeborn, Dana; Crichton, Dan; Law, Emily; Kay-Im, Liz

    2011-01-01

    Airborne Cloud Computing Environment (ACCE) is JPL's internal investment to improve the return on airborne missions. Improve development performance of the data system. Improve return on the captured science data. The investment is to develop a common science data system capability for airborne instruments that encompasses the end-to-end lifecycle covering planning, provisioning of data system capabilities, and support for scientific analysis in order to improve the quality, cost effectiveness, and capabilities to enable new scientific discovery and research in earth observation.

  14. Minocycline attenuates experimental colitis in mice by blocking expression of inducible nitric oxide synthase and matrix metalloproteinases

    In addition to its antimicrobial activity, minocycline exerts anti-inflammatory effects in several disease models. However, whether minocycline affects the pathogenesis of inflammatory bowel disease has not been determined. We investigated the effects of minocycline on experimental colitis and its underlying mechanisms. Acute and chronic colitis were induced in mice by treatment with dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS), and the effect of minocycline on colonic injury was assessed clinically and histologically. Prophylactic and therapeutic treatment of mice with minocycline significantly diminished mortality rate and attenuated the severity of DSS-induced acute colitis. Mechanistically, minocycline administration suppressed inducible nitric oxide synthase (iNOS) expression and nitrotyrosine production, inhibited proinflammatory cytokine expression, repressed the elevated mRNA expression of matrix metalloproteinases (MMPs) 2, 3, 9, and 13, diminished the apoptotic index in colonic tissues, and inhibited nitric oxide production in the serum of mice with DSS-induced acute colitis. In DSS-induced chronic colitis, minocycline treatment also reduced body weight loss, improved colonic histology, and blocked expression of iNOS, proinflammatory cytokines, and MMPs from colonic tissues. Similarly, minocycline could ameliorate the severity of TNBS-induced acute colitis in mice by decreasing mortality rate and inhibiting proinflammatory cytokine expression in colonic tissues. These results demonstrate that minocycline protects mice against DSS- and TNBS-induced colitis, probably via inhibition of iNOS and MMP expression in intestinal tissues. Therefore, minocycline is a potential remedy for human inflammatory bowel diseases.

  15. Morphological alterations and NO-synthase expression in the heart after continuous light exposure of rats

    Paulis, L.; Važan, R.; Šimko, F.; Pecháňová, Olga; Styk, J.; Babál, P.; Janega, P.

    2007-01-01

    Roč. 56, Suppl.2 (2007), S71-S76. ISSN 0862-8408 Grant ostatní: VEGA(SK) 1/3429/06; VEGA(SK) 2/6148/26; VEGA(SK) 2/5110/25; -(SK) 29/2007; -(SK) 30/2007; -(SK) SP51/0280900/0280901; -(SK) APVT-51-027404; -(SK) APVT51-018004 Institutional research plan: CEZ:AV0Z50110509 Keywords : myocardium * collagen I/III * nitric oxide synthase Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 1.505, year: 2007

  16. Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18

    Pičmanová, M. (Martina); Reňák, D. (David); Feciková, J. (Jana); P. Růžička; Mikšátková, P.; Lapčík, O.; Honys, D. (David)

    2013-01-01

    Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of phenolic secondary metabolites, isoflavonoids. These compounds, which are well-known for their benefits to human health and plant defence, are produced mostly in legumes. However, more than 200 of them have been described in 59 other plant families without any knowledge of their respective IFS orthologue genes (with the sole exception of sugar beet). In this study, we selected IFS from Pisum sativum L. (CYP93C18) for fu...

  17. Evaluating the Effect of Expressing a Peanut Resveratrol Synthase Gene in Rice

    Zheng, Shigang; Zhao, Shanchang; Li, Zhen; Wang, Qingguo; Yao, Fangyin; Yang, Lianqun; Pan, Jiaowen; Liu, Wei

    2015-01-01

    Resveratrol (Res) is a type of natural plant stilbenes and phytoalexins that only exists in a few plant species. Studies have shown that the Res could be biosynthesized and accumulated within plants, once the complete metabolic pathway and related enzymes, such as the key enzyme resveratrol synthase (RS), existed. In this study, a RS gene named PNRS1 was cloned from the peanut, and the activity was confirmed in E. coli. Using transgenic approach, the PNRS1 transgenic rice was obtained. In T3 ...

  18. Heterologous expression and product identification of Colletotrichum lagenarium polyketide synthase encoded by the PKS1 gene involved in melanin biosynthesis.

    Fujii, I; Mori, Y; Watanabe, A; Kubo, Y; Tsuji, G; Ebizuka, Y

    1999-08-01

    The Colletotrichum lagenarium PKS1 gene was expressed in the heterologous fungal host, Aspergillus oryzae, under the starch-inducible alpha-amylase promoter to identify the direct product of polyketide synthase (PKS) encoded by the PKS1 gene. The main compound produced by an A. oryzae transformant was isolated and characterized to be 1,3,6,8-tetrahydroxynaphthalene (T4HN) as its tetraacetate. Since the PKS1 gene was cloned from C. lagenarium to complement the nonmelanizing albino mutant, T4HN was assumed to be an initial biosynthetic intermediate, and thus the product of the PKS reaction, but had not been isolated from the fungus. The production of T4HN by the PKS1 transformant unambiguously identified the gene to encode a PKS of pentaketide T4HN. In addition, tetraketide orsellinic acid and pentaketide isocoumarin were isolated, the latter being derived from a pentaketide monocyclic carboxylic acid, as by-products of the PKS1 PKS reaction. Production of the pentaketide carboxylic acid provided insights into the mechanism for the PKS1 polyketide synthase reaction to form T4HN. PMID:10501004

  19. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of molybdopterin synthase from Thermus thermophilus HB8

    The molybdopterin synthase from T. thermophilus HB8 was cloned, expressed, purified and crystallized. The crystals belong to space group P21 and diffracted to a resolution of 1.64 Å. Thermus thermophilus is a Gram-negative aerobic thermophilic eubacterium which can grow at temperatures ranging from 323 to 355 K. In addition to their importance in thermostability or adaptation strategies for survival at high temperatures, the thermostable enzymes in thermophilic organisms contribute to a wide range of biotechnological applications. The molybdenum cofactor in all three kingdoms consists of a tricyclic pyranopterin termed molybdopterin that bears the cis-dithiolene group responsible for molybdenum ligation. The crystals of molybdopterin synthase from T. thermophilus HB8 belong to the primitive monoclinic space group P21, with unit-cell parameters a = 33.94, b = 103.32, c = 59.59 Å, β = 101.3°. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit

  20. The organ-specific expression of terpene synthase genes contributes to the terpene hydrocarbon composition of chamomile essential oils

    Irmisch Sandra

    2012-06-01

    Full Text Available Abstract Background The essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in Europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. It is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. Chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases (TPS, the key enzymes in constructing terpene carbon skeletons. Results Here, we present the identification and characterization of five TPS enzymes contributing to terpene biosynthesis in chamomile (Matricaria recutita. Four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons (−-(E-β-caryophyllene (MrTPS1, (+-germacrene A (MrTPS3, (E-β-ocimene (MrTPS4 and (−-germacrene D (MrTPS5. A fifth TPS, the multiproduct enzyme MrTPS2, was mainly expressed in roots and formed several Asteraceae-specific tricyclic sesquiterpenes with (−-α-isocomene being the major product. The TPS transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding TPS products isolated from these organs suggesting that the spatial regulation of TPS gene expression qualitatively contribute to terpene composition. Conclusions The terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. While the products of MrTPS1, MrTPS2, MrTPS4 and MrTPS5 accumulate in the oils without further chemical alterations, (+-germacrene A produced by MrTPS3 accumulates only in trace amounts, indicating that it is converted into another compound like matricine. Thus, MrTPS3, but also the other TPS genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils.

  1. Hydrocellular foam dressing promotes wound healing along with increases in hyaluronan synthase 3 and PPARα gene expression in epidermis.

    Takumi Yamane

    Full Text Available BACKGROUND: Hydrocellular foam dressing, modern wound dressing, induces moist wound environment and promotes wound healing: however, the regulatory mechanisms responsible for these effects are poorly understood. This study was aimed to reveal the effect of hydrocellular foam dressing on hyaluronan, which has been shown to have positive effects on wound healing, and examined its regulatory mechanisms in rat skin. METHODOLOGY/PRINCIPAL FINDINGS: We created two full-thickness wounds on the dorsolateral skin of rats. Each wound was covered with either a hydrocellular foam dressing or a film dressing and hyaluronan levels in the periwound skin was measured. We also investigated the mechanism by which the hydrocellular foam dressing regulates hyaluronan production by measuring the gene expression of hyaluronan synthase 3 (Has3, peroxisome proliferator-activated receptor α (PPARα, and CD44. Hydrocellular foam dressing promoted wound healing and upregulated hyaluronan synthesis, along with an increase in the mRNA levels of Has3, which plays a primary role in hyaluronan synthesis in epidermis. In addition, hydrocellular foam dressing enhanced the mRNA levels of PPARα, which upregulates Has3 gene expression, and the major hyaluronan receptor CD44. CONCLUSIONS/SIGNIFICANCE: These findings suggests that hydrocellular foam dressing may be beneficial for wound healing along with increases in hyaluronan synthase 3 and PPARα gene expression in epidermis. We believe that the present study would contribute to the elucidation of the mechanisms underlying the effects of hydrocellular foam dressing-induced moist environment on wound healing and practice evidence-based wound care.

  2. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na6[TeW6O24] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P212121 and P1211 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3121. The crystals of latent cgAUS1 belonged to space group P1211 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na6[TeW6O24] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  3. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette, E-mail: annette.rompel@univie.ac.at [Universität Wien, Althanstrasse 14, 1090 Wien (Austria)

    2015-05-22

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2{sub 1}2{sub 1}2{sub 1} and P12{sub 1}1 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3{sub 1}21. The crystals of latent cgAUS1 belonged to space group P12{sub 1}1 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  4. Effects of pneumonectomy on nitric oxide synthase expression and perivascular edema in the remaining lung of rats

    M.N. Samano

    2009-11-01

    Full Text Available Pneumonectomy is associated with high mortality and high rates of complications. Postpneumonectomy pulmonary edema is one of the leading causes of mortality. Little is known about its etiologic factors and its association with the inflammatory process. The purpose of the present study was to evaluate the role of pneumonectomy as a cause of pulmonary edema and its association with gas exchange, inflammation, nitric oxide synthase (NOS expression and vasoconstriction. Forty-two non-specific pathogen-free Wistar rats were included in the study. Eleven animals died during or after the procedure, 21 were submitted to left pneumonectomy and 10 to sham operation. These animals were sacrificed after 48 or 72 h. Perivascular pulmonary edema was more intense in pneumonectomized rats at 72 h (P = 0.0131. Neutrophil density was lower after pneumonectomy in both groups (P = 0.0168. There was higher immunohistochemical expression of eNOS in the pneumonectomy group (P = 0.0208, but no statistically significant difference in the expression of iNOS. The lumen-wall ratio and pO2/FiO2 ratio did not differ between the operated and sham groups after pneumonectomy. Left pneumonectomy caused perivascular pulmonary edema with no elevation of immunohistochemical expression of iNOS or neutrophil density, suggesting the absence of correlation with the inflammatory process or oxidative stress. The increased expression of eNOS may suggest an intrinsic production of NO without signs of vascular reactivity.

  5. Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth, early flowering and increased seed yield.

    Tata, Sandeep Kumar; Jung, Jihye; Kim, Yoon-Ha; Choi, Jun Young; Jung, Ji-Yul; Lee, In-Jung; Shin, Jeong Sheop; Ryu, Stephen Beungtae

    2016-01-01

    Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy. PMID:25644367

  6. Caloric restriction increases internal iliac artery and penil nitric oxide synthase expression in rat: Comparison of aged and adult rats

    Emin Ozbek

    2013-09-01

    Full Text Available Because of the positive corelation between healthy cardiovascular system and sexual life we aimed to evaluate the effect of caloric restriction (CR on endothelial and neuronal nitric oxide synthase (eNOS, nNOS expression in cavernousal tissues and eNOS expression in the internal iliac artery in young and aged rats. Young (3 mo, n = 7 and aged (24 mo, n = 7 male Sprague-Dawley rats were subjected to 40% CR and were allowed free access to water for 3 months. Control rats (n = 14 fed ad libitum had free access to food and water at all times. On day 90, rats were sacrified and internal iliac arteries and penis were removed and parafinized, eNOS and nNOS expression evaluated with immunohistochemistry. Results were evaluated semiquantitatively. eNOS and nNOS expression in cavernousal tis- sue in CR rats were more strong than in control group in both young and old rats. eNOS expression was also higher in the internal iliac arteries of CR rats than in control in young and old rats. As a result of our study we can say that there is a positive link between CR and neurotransmitter of erection in cavernousal tissues and internal iliac arteries. CR has beneficial effect to prevent sexual dysfunction in young and old animals and possible humans.

  7. Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases

    Saito Koji

    2005-08-01

    Full Text Available Abstract Background In Arabidopsis, ETO1 (ETHYLENE-OVERPRODUCER1 is a negative regulator of ethylene evolution by interacting with AtACS5, an isoform of the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthases (ACC synthase or ACS, in ethylene biosynthetic pathway. ETO1 directly inhibits the enzymatic activity of AtACS5. In addition, a specific interaction between ETO1 and AtCUL3, a constituent of a new type of E3 ubiquitin ligase complex, suggests the molecular mechanism in promoting AtACS5 degradation by the proteasome-dependent pathway. Because orthologous sequences to ETO1 are found in many plant species including tomato, we transformed tomato with Arabidopsis ETO1 to evaluate its ability to suppress ethylene production in tomato fruits. Results Transgenic tomato lines that overexpress Arabidopsis ETO1 (ETO1-OE did not show a significant delay of fruit ripening. So, we performed yeast two-hybrid assays to investigate potential heterologous interaction between ETO1 and three isozymes of ACC synthases from tomato. In the yeast two-hybrid system, ETO1 interacts with LE-ACS3 as well as AtACS5 but not with LE-ACS2 or LE-ACS4, two major isozymes whose gene expression is induced markedly in ripening fruits. According to the classification of ACC synthases, which is based on the C-terminal amino acid sequences, both LE-ACS3 and AtACS5 are categorized as type 2 isozymes and possess a consensus C-terminal sequence. In contrast, LE-ACS2 and LE-ACS4 are type 1 and type 3 isozymes, respectively, both of which do not possess this specific C-terminal sequence. Yeast two-hybrid analysis using chimeric constructs between LE-ACS2 and LE-ACS3 revealed that the type-2-ACS-specific C-terminal tail is required for interaction with ETO1. When treated with auxin to induce LE-ACS3, seedlings of ETO1-OE produced less ethylene than the wild type, despite comparable expression of the LE-ACS3 gene in the wild type. Conclusion These results suggest that ETO1

  8. UOP ACC 422 / Assignmentcloud.com

    admn

    2015-01-01

    ACC 422 Week 1 DQ 1 To purchase this material click http://www.assignmentcloud.com/ACC-422/ACC-422-Week-1-DQ-1 Consider how an organization must manage cash, receivables, and inventory. Which of the three variables is the most important to manage? Is one more susceptible to fraud and errors than the others? Explain your answer. How would a misstatement in each affect the organization? For more classes visit www.assignmentcloud.com

  9. Effect of Dexamethasone on Nitric Oxide Synthase and Caspase-3 Gene Expressions in Endotoxemia in Neonate Rat Brain

    HUA WANG; YU-BIN WU; XIU-HUA DU

    2005-01-01

    Objective To investigate the gene and protein expressions of three isoforms of nitric oxide synthase (NOS) and gene expression of Caspase-3, and effect of dexamethasone on them in neonatal rats with lipopolysaccharide (LPS)-induced endotoxemic brain damage. Methods Expressions of the three isoforms of NOS and caspase-3 mRNA in the brain were investigated by RT-PCR in postnatal 7-day wistar rats with acute endotoxemia by intraperitoneal administration of LPS. Regional distributions of NOSs were examined by immunohistochemical technique. Results nNOS and Caspase-3 mRNA were obviously detected. eNOS mRNA was faintly expressed, but iNOS mRNA was undetectable in the control rat brain. The expressions of NOS mRNA of three isoforms were weak 2 h after LPS (5 mg/mg) delivery, peaked at 6 h, and thereafter, reduced gradually up to 24 h. The expression intensity was in the order of nNOS> iNOS> eNOS. Widespread nNOS, scattered eNOS distribution and negative iNOS were identified in the control rat brain and all isoforms of NOS could be induced by LPS which reached the apex at 24 h in the order of nNOS> iNOS> eNOS as detected by immunostaining. Although Caspase-3 mRNA could be found in all groups, DNA fragmentation was only seen at 6 h and 24 h. The expressions of NOS and Caspase-3 mRNA were inhibited in the rat brain when dexamethasone was administrated. Conclusion LPS-induced NO production induces apoptosis of neurons through mechanism involving the Caspase-3 activation, which may play an important role in the pathogenesis of brain damage during endotoxemia, and neuro-protective effects of dexamethasone may be partially realized by inhibiting the expression of NOS mRNA.

  10. Evaluating the Effect of Expressing a Peanut Resveratrol Synthase Gene in Rice

    Li, Zhen; Wang, Qingguo; Yao, Fangyin; Yang, Lianqun; Pan, Jiaowen; Liu, Wei

    2015-01-01

    Resveratrol (Res) is a type of natural plant stilbenes and phytoalexins that only exists in a few plant species. Studies have shown that the Res could be biosynthesized and accumulated within plants, once the complete metabolic pathway and related enzymes, such as the key enzyme resveratrol synthase (RS), existed. In this study, a RS gene named PNRS1 was cloned from the peanut, and the activity was confirmed in E. coli. Using transgenic approach, the PNRS1 transgenic rice was obtained. In T3 generation, the Res production and accumulation were further detected by HPLC. Our data revealed that compared to the wild type rice which trans-resveratrol was undetectable, in transgenic rice, the trans-resveratrol could be synthesized and achieved up to 0.697 μg/g FW in seedlings and 3.053 μg/g DW in seeds. Furthermore, the concentration of trans-resveratrol in transgenic rice seedlings could be induced up to eight or four-fold higher by ultraviolet (UV-C) or dark, respectively. Simultaneously, the endogenous increased of Res also showed the advantages in protecting the host plant from UV-C caused damage or dark-induced senescence. Our data indicated that Res was involved in host-defense responses against environmental stresses in transgenic rice. Here the results describes the processes of a peanut resveratrol synthase gene transformed into rice, and the detection of trans-resveratrol in transgenic rice, and the role of trans-resveratrol as a phytoalexin in transgenic rice when treated by UV-C and dark. These findings present new outcomes of transgenic approaches for functional genes and their corresponding physiological functions, and shed some light on broadening available resources of Res, nutritional improvement of crops, and new variety cultivation by genetic engineering. PMID:26302213

  11. Evaluating the Effect of Expressing a Peanut Resveratrol Synthase Gene in Rice.

    Shigang Zheng

    Full Text Available Resveratrol (Res is a type of natural plant stilbenes and phytoalexins that only exists in a few plant species. Studies have shown that the Res could be biosynthesized and accumulated within plants, once the complete metabolic pathway and related enzymes, such as the key enzyme resveratrol synthase (RS, existed. In this study, a RS gene named PNRS1 was cloned from the peanut, and the activity was confirmed in E. coli. Using transgenic approach, the PNRS1 transgenic rice was obtained. In T3 generation, the Res production and accumulation were further detected by HPLC. Our data revealed that compared to the wild type rice which trans-resveratrol was undetectable, in transgenic rice, the trans-resveratrol could be synthesized and achieved up to 0.697 μg/g FW in seedlings and 3.053 μg/g DW in seeds. Furthermore, the concentration of trans-resveratrol in transgenic rice seedlings could be induced up to eight or four-fold higher by ultraviolet (UV-C or dark, respectively. Simultaneously, the endogenous increased of Res also showed the advantages in protecting the host plant from UV-C caused damage or dark-induced senescence. Our data indicated that Res was involved in host-defense responses against environmental stresses in transgenic rice. Here the results describes the processes of a peanut resveratrol synthase gene transformed into rice, and the detection of trans-resveratrol in transgenic rice, and the role of trans-resveratrol as a phytoalexin in transgenic rice when treated by UV-C and dark. These findings present new outcomes of transgenic approaches for functional genes and their corresponding physiological functions, and shed some light on broadening available resources of Res, nutritional improvement of crops, and new variety cultivation by genetic engineering.

  12. [Full-length cDNA cloning of flavonol synthase genes of Carthamus tinctorius and construction plant expression vector].

    Yang, Wen-ting; Liu, Xiu-ming; Wan, Qiu; Yao, Na; Wang, Nan; Zhang, Xue-meng; Jiao, Zhong-da; Li, Hai-yan; Li, Xiao-kun

    2015-02-01

    Flavonol synthase (FLS) is one of the key enzymes in flavonoids metabolic pathways. In this study, middle sequence was obtained from Carthamus tinctorius transcriptome sequencing results. Full-length cDNAs of FLS was cloned from petals of C. tinctorius to FLS by using RT-PCR and RACE technology. Its full-length cDNA was 1,201 bp, with an open reading frame of 1,101 bp and 336 encoded amino acids. The phylogenetic analysis showed that, FLS gene encoded amino acids in C. tinctorius were highly homologous with amino acids in congeneric Compositae species, especially Rudbeckia laciniata. The pBASTA-FLS plant expression vector was successfully built by the molecular biology method, which lays a foundation for further studying biology functions of the gene and biosynthesis mechanism of flavonoids. PMID:26137682

  13. Gene structure, phylogeny and expression profile of the sucrose synthase gene family in cacao (Theobroma cacao L.)

    Fupeng Li; Chaoyun Hao; Lin Yan; Baoduo Wu; Xiaowei Qin; Jianxiong Lai; Yinghui Song

    2015-09-01

    In higher plants, sucrose synthase (Sus, EC 2.4.1.13) is widely considered as a key enzyme involved in sucrose metabolism. Although, several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, to date detailed information about the Sus genes is lacking for cacao. This study reports the identification of six novel Sus genes from economically important cacao tree. Analyses of the gene structure and phylogeny of the Sus genes demonstrated evolutionary conservation in the Sus family across cacao and other plant species. The expression of cacao Sus genes was investigated via real-time PCR in various tissues, different developmental phases of leaf, flower bud and pod. The Sus genes exhibited distinct but partially redundant expression profiles in cacao, with TcSus1, TcSus5 and TcSus6, being the predominant genes in the bark with phloem, TcSus2 predominantly expressing in the seed during the stereotype stage. TcSus3 and TcSus4 were significantly detected more in the pod husk and seed coat along the pod development, and showed development dependent expression profiles in the cacao pod. These results provide new insights into the evolution, and basic information that will assist in elucidating the functions of cacao Sus gene family.

  14. Elevated expression of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in primary sclerosing cholangitis: ιmplications for cholangiocarcinogenesis.

    Ishii, Yasutaka; Sasaki, Tamito; Serikawa, Masahiro; Minami, Tomoyuki; Okazaki, Akihito; Yukutake, Masanobu; Ishigaki, Takashi; Kosaka, Keiichi; Mouri, Teruo; Yoshimi, Satoshi; Shimizu, Akinori; Tsuboi, Tomofumi; Chayama, Kazuaki

    2013-10-01

    Cholangiocarcinoma (CCA) occurs frequently in primary sclerosing cholangitis (PSC). Cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) induced by inflammation are believed to mediate prostaglandin E2 (PGE2) production thereby promoting carcinogenesis. Their expression in PSC-associated CCA tissues and non-neoplastic bile duct epithelial cells (BDECs) in PSC was investigated. COX-2 and mPGES-1 levels in 15 PSC patients (7 with CCA) were scored using immunohistochemical staining. The results were compared with those obtained in CCA tissues and non-neoplastic BDECs (controls) of 15 sporadic CCA patients. Non-neoplastic BDECs from large and small bile ducts were investigated separately. The mRNA expression levels of COX-2 and mPGES-1 in CCA tissues were analyzed by quantitative polymerase chain reaction. Ki-67 immunostaining was performed to evaluate cell proliferation. COX-2 was strongly expressed in PSC-associated CCA tissues and non-neoplastic BDECs in PSC. This expression was significantly upregulated in both compared with sporadic CCA tissues and non-neoplastic BDECs in sporadic CCA (both Pcholangiocarcinogenesis. PMID:23900502

  15. Gene structure, phylogeny and expression profile of the sucrose synthase gene family in cacao (Theobroma cacao L.).

    Li, Fupeng; Hao, Chaoyun; Yan, Lin; Wu, Baoduo; Qin, Xiaowei; Lai, Jianxiong; Song, Yinghui

    2015-09-01

    In higher plants, sucrose synthase (Sus, EC 2.4.1.13) is widely considered as a key enzyme involved in sucrose metabolism. Although, several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, to date detailed information about the Sus genes is lacking for cacao. This study reports the identification of six novel Sus genes from economically important cacao tree. Analyses of the gene structure and phylogeny of the Sus genes demonstrated evolutionary conservation in the Sus family across cacao and other plant species. The expression of cacao Sus genes was investigated via real-time PCR in various tissues, different developmental phases of leaf, flower bud and pod. The Sus genes exhibited distinct but partially redundant expression profiles in cacao, with TcSus1, TcSus5 and TcSus6, being the predominant genes in the bark with phloem, TcSus2 predominantly expressing in the seed during the stereotype stage. TcSus3 and TcSus4 were significantly detected more in the pod husk and seed coat along the pod development, and showed development dependent expression profiles in the cacao pod. These results provide new insights into the evolution, and basic information that will assist in elucidating the functions of cacao Sus gene family. PMID:26440085

  16. Citrate-release-mediated aluminum resistance is coupled to the inducible expression of mitochondrial citrate synthase gene in Paraserianthes falcataria.

    Osawa, Hiroki; Kojima, Katsumi

    2006-05-01

    Aluminum (Al) resistance in some leguminous plants is achieved by enhanced citrate release from roots. Enhancement requires several hours for complete activation and is postulated to involve Al-responsive genes or components. We examined the mechanism of Al-induced citrate release by studying the relationship between citrate release and expression of the mitochondrial citrate synthase (mCS) gene in three leguminous trees. Root elongation in Leucaena leucocephala (Lam.) de Wit was arrested within 24 h by 30 microM Al, whereas root elongation in Paraserianthes falcataria (L.) Neilson and Acacia mangium Willd. was inhibited mangium maintained enhanced release and accumulation of citrate for at least 28 days in response to Al treatment. Aluminum increased the accumulation of mCS transcripts in P. falcataria roots, but not in L. leucocephala roots, and thus up-regulation decreased following removal of Al. Lanthanum did not alter the expression level of mCS. Aluminum increased mCS activity concomitantly with enhanced mCS gene expression in P. falcataria, whereas it did not affect mCS activity in L. leucocephala. Aluminum content in root apices of P. falcataria was increased by cycloheximide, supporting the idea that de novo synthesis of proteins is a prerequisite for Al resistance. Our findings suggest that Al-inducible expression of mCS coupled with enhanced citrate release mediates Al resistance in P. falcataria. PMID:16452070

  17. Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

    Proudfoot, L; Nikolaev, A V; Feng, G J; Wei, W Q; Ferguson, M A; Brimacombe, J S; Liew, F Y

    1996-10-01

    Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania parasites through their ability to regulate the expression of iNOS by macrophages. PMID:8855295

  18. Modulation of Inducible Nitric Oxide Synthase Expression by the Attaching and Effacing Bacterial Pathogen Citrobacter rodentium in Infected Mice

    Vallance, Bruce A.; Deng, Wanyin; De Grado, Myriam; Chan, Crystal; Jacobson, Kevan; Finlay, B. Brett

    2002-01-01

    Citrobacter rodentium belongs to the attaching and effacing family of enteric bacterial pathogens that includes both enteropathogenic and enterohemorrhagic Escherichia coli. These bacteria infect their hosts by colonizing the intestinal mucosal surface and intimately attaching to underlying epithelial cells. The abilities of these pathogens to exploit the cytoskeleton and signaling pathways of host cells are well documented, but their interactions with the host's antimicrobial defenses, such as inducible nitric oxide synthase (iNOS), are poorly understood. To address this issue, we infected mice with C. rodentium and found that iNOS mRNA expression in the colon significantly increased during infection. Immunostaining identified epithelial cells as the major source for immunoreactive iNOS. Finding that nitric oxide (NO) donors were bacteriostatic for C. rodentium in vitro, we examined whether iNOS expression contributed to host defense by infecting iNOS-deficient mice. Loss of iNOS expression caused a small but significant delay in bacterial clearance without affecting tissue pathology. Finally, immunofluorescence staining was used to determine if iNOS expression was localized to infected cells by staining for the C. rodentium virulence factor, translocated intimin receptor (Tir), as well as iNOS. Interestingly, while more than 85% of uninfected epithelial cells expressed iNOS, fewer than 15% of infected (Tir-positive) cells expressed detectable iNOS. These results demonstrate that both iNOS and intestinal epithelial cells play an active role in host defense during C. rodentium infection. However, the selective expression of iNOS by uninfected but not infected cells suggests that this pathogen has developed mechanisms to locally limit its exposure to host-derived NO. PMID:12379723

  19. Responses of ethylene and ACC in rice grains to soil moisture and their relations to grain filling

    2008-01-01

    The objectives of this study were to-investigate ethylene and 1-aminocylopropane-1-carboxylic acid (ACC) in rice grains and root bleeding sap during the grain filling period and their relationship to the grain filling rate.Two high lodging-resistant rice (Oryza sativa L.) cultivars were grown in pots or tanks.Three treatments,including well watered (WW),moderate soil-drying (MD) and severe soil-drying (SD),were conducted from 9 days of post-anthesis until maturity.The effects of chemical regulators on the concentrations of ethylene and ACC in the grains were also studied.The results show that MD significantly increased the grainfilling rate and grain weight,whereas SD significantly reduced the grain-filling rate and grain weight.Concentrations of ethylene and ACC in the grains were very high at the early grain filling stage and then sharply decreased during the linear period of grain growth.MD reduced the ACC concentrations and ethylene evolution rate,whereas SD remarkably increased the ACC concentrations and ethylene evolution rate.Both the ethylene evolution rate in rice grains and the ACC concentrations in the root-bleeding sap were significantly and positively correlated with the ACC concentrations in rice grains.The ethylene evolution rate was significantly and negatively correlated with the grain-filling rate.The application of amino-ethoxyvinylglycine (AVG),an inhibitor of ethylene synthesis,at 9-13 days of postanthesis significantly reduced the ACC concentrations and ethylene evolution rate of grains,but significantly enhanced the activities of sucrose synthase,ADP glucose pyrophosphorylase and soluble starch synthase.The results were reversed when ethephon,an ethylenereleasing agent,was applied.The results suggest that moderate soil drying during the grain-filling period in rice could inhibit the production of ethylene and ACC and therefore accelerate grain filling and increase grain weight.

  20. Lentiviral-mediated over-expression of hyaluronan synthase-1 (HAS-1) decreases the cellular inflammatory response and results in regenerative wound repair

    Caskey, Robert C.; Allukian, Myron; Lind, Robert C.; Herdrich, Benjamin J.; Xu, Junwang; Radu, Antoneta; Mitchell, Marc E.; Liechty, Kenneth W.

    2013-01-01

    Fetal wounds have been found to have increased levels of high-molecular-weight hyaluronan (HMW-HA) compared with those of adults. The primary enzyme responsible for producing HMW-HA is hyaluronic acid synthase-1 (HAS-1). We hypothesized that over-expression of HAS-1 in adult dermal wounds would decr

  1. Developmental evolution of flowering plant pollen tube cell walls: callose synthase (CalS gene expression patterns

    Abercrombie Jason M

    2011-07-01

    Full Text Available Abstract Background A number of innovations underlie the origin of rapid reproductive cycles in angiosperms. A critical early step involved the modification of an ancestrally short and slow-growing pollen tube for faster and longer distance transport of sperm to egg. Associated with this shift are the predominantly callose (1,3-β-glucan walls and septae (callose plugs of angiosperm pollen tubes. Callose synthesis is mediated by callose synthase (CalS. Of 12 CalS gene family members in Arabidopsis, only one (CalS5 has been directly linked to pollen tube callose. CalS5 orthologues are present in several monocot and eudicot genomes, but little is known about the evolutionary origin of CalS5 or what its ancestral function may have been. Results We investigated expression of CalS in pollen and pollen tubes of selected non-flowering seed plants (gymnosperms and angiosperms within lineages that diverged below the monocot/eudicot node. First, we determined the nearly full length coding sequence of a CalS5 orthologue from Cabomba caroliniana (CcCalS5 (Nymphaeales. Semi-quantitative RT-PCR demonstrated low CcCalS5 expression within several vegetative tissues, but strong expression in mature pollen. CalS transcripts were detected in pollen tubes of several species within Nymphaeales and Austrobaileyales, and comparative analyses with a phylogenetically diverse group of sequenced genomes indicated homology to CalS5. We also report in silico evidence of a putative CalS5 orthologue from Amborella. Among gymnosperms, CalS5 transcripts were recovered from germinating pollen of Gnetum and Ginkgo, but a novel CalS paralog was instead amplified from germinating pollen of Pinus taeda. Conclusion The finding that CalS5 is the predominant callose synthase in pollen tubes of both early-diverging and model system angiosperms is an indicator of the homology of their novel callosic pollen tube walls and callose plugs. The data suggest that CalS5 had transient expression

  2. ACC 205(NEW) UOP COURSE TUTORIAL/UOPHELP

    MADHU

    2015-01-01

    For more course tutorials visit www.uophelp.com   ACC 205 Week 1 DQ 1 Accounting Equation ACC 205 Week 1 DQ 2 Accounts ACC 205 Week 1 Exercise Assignment Basic Accounting Equations ACC 205 Week 1 Journal Balance Sheet Journal ACC 205 Week 2 DQ 1 Accounting Cycle ACC 205 Week 2 DQ 2 Bank Reconciliation ACC 205 Week 2 Exercise Assignment Revenue and Expenses ACC 205 Week 2 Journal Income Statement Journal ACC 205 Week 3 DQ 1 LIFO vs. FIFO (New) ...

  3. ACC 491 uop course tutorial/uop help

    petor nex

    2015-01-01

    For more course tutorials visit www.uophelp.com     ACC 491 Week 1 Individual Assignment Generally Accepted Auditing Standards Paper ACC 491 Week 1 DQ 1 ACC 491 Week 1 DQ 2 ACC 491 week 2 Individual Assignments From the Text ACC 491 Week 2 Team Assignment Auditing, Attestation, and Assurance Services Paper ACC 491 Week 2 DQ 1 ACC 491 Week 2 DQ 2 ACC 491 week 3 Individual Assignments From the Text ACC 491 week 3 Team Assignments From the Text ...

  4. ACC 490 uop course tutorial/uop help

    antony

    2015-01-01

    For more course tutorials visit www.uophelp.com     ACC 490 Week 1 Generally Accepted Auditing Standards Paper ACC 490 Week 1 – DQ 1  ACC 490 Week 1 – DQ 2 ACC 490 Week 2 Individual Ch. 1 Textbook Exercise ACC 490 Week 2 Learning Team Auditing, Attestation, and Assurance Services Paper ACC 490 Week 2 – DQ 1 ACC 490 Week 2 – DQ 2 ACC 490 Week 3 Individual Ch. 5, 6, & 7 Textbook Exercises ACC 490 Week 3 L...

  5. Inhaled nitric oxide decreases pulmonary endothelial nitric oxide synthase expression and activity in normal newborn rat lungs

    Thông Hua-Huy

    2016-02-01

    Full Text Available Inhaled nitric oxide (iNO is commonly used in the treatment of very ill pre-term newborns. Previous studies showed that exogenous NO could affect endothelial NO synthase (eNOS activity and expression in vascular endothelial cell cultures or adult rat models, but this has never been fully described in newborn rat lungs. We therefore aimed to assess the effects of iNO on eNOS expression and activity in newborn rats. Rat pups, post-natal day (P 0 to P7, and their dams were placed in a chamber containing NO at 5 ppm (iNO-5 ppm group or 20 ppm (iNO-20 ppm group, or in room air (control group. Rat pups were sacrificed at P7 and P14 for evaluation of lung eNOS expression and activity. At P7, eNOS protein expression in total lung lysates, in bronchial and arterial sections, was significantly decreased in the iNO-20 ppm versus control group. At P14, eNOS expression was comparable among all three groups. The amounts of eNOS mRNA significantly differed at P7 between the iNO-20 ppm and control groups. NOS activity decreased in the iNO-20 ppm group at P7 and returned to normal levels at P14. There was an imbalance between superoxide dismutase and NOS activities in the iNO-20 ppm group at P7. Inhalation of NO at 20 ppm early after birth decreases eNOS gene transcription, protein expression and enzyme activity. This decrease might account for the rebound phenomenon observed in patients treated with iNO.

  6. Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-β-farnesene

    Crock, John; Wildung, Mark; Croteau, Rodney

    1997-01-01

    (E)-β-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-β-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a...

  7. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    Severson, Eric A.; Kwon, Mike; Hilgarth, Roland S.; Parkos, Charles A. [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States); Nusrat, Asma, E-mail: anusrat@emory.edu [Epithelial Pathobiology Research Unit, Dept. of Pathology, Emory University, Atlanta, GA 30322 (United States)

    2010-07-02

    The Apical Junctional Complex (AJC) encompassing the tight junction (TJ) and adherens junction (AJ) plays a pivotal role in regulating epithelial barrier function and epithelial cell proliferative processes through signaling events that remain poorly characterized. A potential regulator of AJC protein expression is Glycogen Synthase Kinase-3 (GSK-3). GSK-3 is a constitutively active kinase that is repressed during epithelial-mesenchymal transition (EMT). In the present study, we report that GSK-3 activity regulates the structure and function of the AJC in polarized model intestinal (SK-CO15) and kidney (Madin-Darby Canine Kidney (MDCK)) epithelial cells. Reduction of GSK-3 activity, either by small molecule inhibitors or siRNA targeting GSK-3 alpha and beta mRNA, resulted in increased permeability to both ions and bulk solutes. Immunofluorescence labeling and immunoblot analyses revealed that the barrier defects correlated with decreased protein expression of AJC transmembrane proteins Occludin, Claudin-1 and E-cadherin without influencing other TJ proteins, Zonula Occludens-1 (ZO-1) and Junctional Adhesion Molecule A (JAM-A). The decrease in Occludin and E-cadherin protein expression correlated with downregulation of the corresponding mRNA levels for these respective proteins following GSK-3 inhibition. These observations implicate an important role of GSK-3 in the regulation of the structure and function of the AJC that is mediated by differential modulation of mRNA transcription of key AJC proteins, Occludin, Claudin-1 and E-cadherin.

  8. Glycogen Synthase Kinase 3 (GSK-3) influences epithelial barrier function by regulating Occludin, Claudin-1 and E-cadherin expression

    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.

  9. Functional Expression of Electron Transport Chain and FoF1-ATP Synthase in Optic Nerve Myelin Sheath.

    Bartolucci, Martina; Ravera, Silvia; Garbarino, Greta; Ramoino, Paola; Ferrando, Sara; Calzia, Daniela; Candiani, Simona; Morelli, Alessandro; Panfoli, Isabella

    2015-11-01

    Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and β subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration. PMID:26334391

  10. Molecular characterization and expression analyses of an anthocyanin synthase gene from Magnolia sprengeri Pamp.

    Shi, Shou-Guo; Li, Shan-Ju; Kang, Yong-Xiang; Liu, Jian-Jun

    2015-01-01

    Anthocyanin synthase (ANS), which catalyzes the conversion of colorless leucoanthocyanins into colored anthocyanins, is a key enzyme in the anthocyanin biosynthetic pathway. It plays important roles in plant development and defense. An ANS gene designated as MsANS was cloned from Magnolia sprengeri using rapid amplification of complementary DNA (cDNA) ends technology. The full-length MsANS is 1171-bp long and contains a 1080-bp open reading frame encoding a 360 amino acid polypeptide. In a sequence alignment analysis, the deduced MsANS protein showed high identity to ANS proteins from other plants: Prunus salicina var. cordata (74 % identity), Ampelopsis grossedentata (74 % identity), Pyrus communis (73 % identity), and Prunus avium (73 % identity). A structural analysis showed that MsANS belongs to 2-oxoglutarate (2OG)- and ferrous iron-dependent oxygenase family because it contains three binding sites for 2OG. Real-time quantitative polymerase chain reaction analyses showed that the transcript level of MsANS was 26-fold higher in red petals than in white petals. The accumulation of anthocyanins in petals of white, pink, and red M. sprengeri flowers was analyzed by HPLC. The main anthocyanin was cyanidin-3-o-glucoside chloride, and the red petals contained the highest concentration of this pigment. PMID:25315387

  11. Chalcone synthase genes from milk thistle (Silybum marianum): isolation and expression analysis.

    Sanjari, Sepideh; Shobbar, Zahra Sadat; Ebrahimi, Mohsen; Hasanloo, Tahereh; Sadat-Noori, Seyed-Ahmad; Tirnaz, Soodeh

    2015-12-01

    Silymarin is a flavonoid compound derived from milk thistle (Silybum marianum) seeds which has several pharmacological applications. Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids; thereby, the identification of CHS encoding genes in milk thistle plant can be of great importance. In the current research, fragments of CHS genes were amplified using degenerate primers based on the conserved parts of Asteraceae CHS genes, and then cloned and sequenced. Analysis of the resultant nucleotide and deduced amino acid sequences led to the identification of two different members of CHS gene family,SmCHS1 and SmCHS2. Third member, full-length cDNA (SmCHS3) was isolated by rapid amplification of cDNA ends (RACE), whose open reading frame contained 1239 bp including exon 1 (190 bp) and exon 2 (1049 bp), encoding 63 and 349 amino acids, respectively. In silico analysis of SmCHS3 sequence contains all the conserved CHS sites and shares high homology with CHS proteins from other plants.Real-time PCR analysis indicated that SmCHS1 and SmCHS3 had the highest transcript level in petals in the early flowering stage and in the stem of five upper leaves, followed by five upper leaves in the mid-flowering stage which are most probably involved in anthocyanin and silymarin biosynthesis. PMID:26690515

  12. Chalcone synthase genes from milk thistle (Silybum marianum): isolation and expression analysis

    Sepideh Sanjari; Zahra Sadat Shobbar; Mohsen Ebrahimi; Tahereh Hasanloo; Seyed-Ahmad Sadat-Noor; Soodeh Tirnaz

    2015-12-01

    Silymarin is a flavonoid compound derived from milk thistle (Silybum marianum) seeds which has several pharmacological applications. Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids; thereby, the identification of encoding genes in milk thistle plant can be of great importance. In the current research, fragments of genes were amplified using degenerate primers based on the conserved parts of Asteraceae genes, and then cloned and sequenced. Analysis of the resultant nucleotide and deduced amino acid sequences led to the identification of two different members of gene family, 1 and 2. Third member, full-length cDNA (3) was isolated by rapid amplification of cDNA ends (RACE), whose open reading frame contained 1239 bp including exon 1 (190 bp) and exon 2 (1049 bp), encoding 63 and 349 amino acids, respectively. In silico analysis of SmCHS3 sequence contains all the conserved CHS sites and shares high homology with CHS proteins from other plants. Real-time PCR analysis indicated that 1 and 3 had the highest transcript level in petals in the early flowering stage and in the stem of five upper leaves, followed by five upper leaves in the mid-flowering stage which are most probably involved in anthocyanin and silymarin biosynthesis.

  13. Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4

    Ji-Hee Kim; Ga-Young Park; Soo Young Bang; Sun Young Park; Soo-Kyung Bae; YoungHee Kim

    2014-01-01

    Crocin is a water-soluble carotenoid pigment that is primarily used in various cuisines as a seasoning and coloring agent, as well as in traditional medicines for the treatment of edema, fever, and hepatic disorder. In this study, we demonstrated that crocin markedly induces the expression of heme oxygenase-1 (HO-1) which leads to an anti-inflammatory response. Crocin inhibited inducible nitric oxide synthase (iNOS) expression and nitric oxide production via downregulation of nuclear factor k...

  14. Expression of inducible nitric oxide synthase is increased in patients with heart failure due to ischemic disease

    Ferreiro C.R.

    2004-01-01

    Full Text Available The objective of the present study was to determine the relationship between nitric oxide synthases (NOS and heart failure in cardiac tissue from patients with and without cardiac decompensation. Right atrial tissue was excised from patients with coronary artery disease (CAD and left ventricular ejection fraction (LVEF 60% (N = 10 during cardiac surgery. NOS activity was measured by the conversion of L-[H³]-arginine to L-[H³]-citrulline. Gene expression was quantified by the competitive reverse transcription-polymerase chain reaction. Both endothelial NOS (eNOS activity and expression were significantly reduced in failing hearts compared to non-failing hearts: 0.36 ± 0.18 vs 1.51 ± 0.31 pmol mg-1 min-1 (P < 0.0001 and 0.37 ± 0.08 vs 0.78 ± 0.09 relative cDNA absorbance at 320 nm (P < 0.0001, respectively. In contrast, inducible NOS (iNOS activity and expression were significantly higher in failing hearts than in non-failing hearts: 4.00 ± 0.90 vs 1.54 ± 0.65 pmol mg-1 min-1 (P < 0.0001 and 2.19 ± 0.27 vs 1.43 ± 0.13 cDNA absorbance at 320 nm (P < 0.0001, respectively. We conclude that heart failure down-regulates both eNOS activity and expression in cardiac tissue from patients with LVEF <35%. In contrast, iNOS activity and expression are increased in failing hearts and may represent an alternative mechanism for nitric oxide production in heart failure due to ischemic disease.

  15. Localisation and endocrine control of hyaluronan synthase (HAS) 2, HAS3 and CD44 expression in sheep granulosa cells.

    Chavoshinejad, R; Marei, W F A; Hartshorne, G M; Fouladi-Nashta, A A

    2016-04-01

    The aim of the present study was to investigate the hormonal regulation of hyaluronan (HA) components in sheep granulosa cells. HA components are present in the reproductive tract and have a range of physical and signalling properties related to reproductive function in several species. First, abattoir-derived ovaries of sheep were used to determine the localisation of HA synthase (HAS) 1-3 and CD44 proteins in antral follicles. Staining for HAS1-3 and CD44 proteins was most intense in the granulosa layer. Accordingly, the expression of HAS2, HAS3 and CD44 mRNA was measured in cultured granulosa cells exposed to 0-50ngmL(-1) of 17β-oestradiol and different combinations of oestradiol, gonadotropins, insulin-like growth factor (IGF)-1 and insulin for 48-96h (1ngmL(-1) FSH, 10ngmL(-1) insulin, 10ngmL(-1) IGF-1, 40ngmL(-1) E2 and 25ngmL(-1) LH.). mRNA expression was quantified by real-time polymerase chain reaction using a fold induction method. The results revealed that the hormones tested generally stimulated mRNA expression of the genes of interest in cultured granulosa cells. Specifically, oestradiol, when combined with IGF-1, insulin and FSH, stimulated HAS2 mRNA expression. Oestradiol and LH had synergistic effects in increasing HAS3 mRNA expression. In conclusion, we suggest that the hormones studied differentially regulate HAS2, HAS3 and CD44 in ovine granulosa cells in vitro. Further work is needed to address the signalling pathways involved. PMID:25427133

  16. Alfalfa Cellulose synthase gene expression under abiotic stress: a Hitchhiker's guide to RT-qPCR normalization.

    Gea Guerriero

    Full Text Available Abiotic stress represents a serious threat affecting both plant fitness and productivity. One of the promptest responses that plants trigger following abiotic stress is the differential expression of key genes, which enable to face the adverse conditions. It is accepted and shown that the cell wall senses and broadcasts the stress signal to the interior of the cell, by triggering a cascade of reactions leading to resistance. Therefore the study of wall-related genes is particularly relevant to understand the metabolic remodeling triggered by plants in response to exogenous stresses. Despite the agricultural and economical relevance of alfalfa (Medicago sativa L., no study, to our knowledge, has addressed specifically the wall-related gene expression changes in response to exogenous stresses in this important crop, by monitoring the dynamics of wall biosynthetic gene expression. We here identify and analyze the expression profiles of nine cellulose synthases, together with other wall-related genes, in stems of alfalfa plants subjected to different abiotic stresses (cold, heat, salt stress at various time points (e.g. 0, 24, 72 and 96 h. We identify 2 main responses for specific groups of genes, i.e. a salt/heat-induced and a cold/heat-repressed group of genes. Prior to this analysis we identified appropriate reference genes for expression analyses in alfalfa, by evaluating the stability of 10 candidates across different tissues (namely leaves, stems, roots, under the different abiotic stresses and time points chosen. The results obtained confirm an active role played by the cell wall in response to exogenous stimuli and constitute a step forward in delineating the complex pathways regulating the response of plants to abiotic stresses.

  17. GABAB Receptors Expressed in Human Aortic Endothelial Cells Mediate Intracellular Calcium Concentration Regulation and Endothelial Nitric Oxide Synthase Translocation

    Xu-Ping Wang

    2014-01-01

    Full Text Available GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i in a number of cells (e.g., retina, airway epithelium and smooth muscle, but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA, in cultured human aortic endothelial cells (HAECs, and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

  18. Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

    Ouyang Danming; Hu Yongxuan; Li Mulan; Zeng Xiaojun; He Zhixiong; Yuan Caijia

    2008-01-01

    Objective: To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosoma japonicum,(SjAslp) and transfer it into mammalian cells to express the objective protein. Methods: By polymerase chain reaction (PCR) technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK+/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1(+). After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Resnlts: The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion: SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.

  19. An Arabidopsis callose synthase

    Ostergaard, Lars; Petersen, Morten; Mattsson, Ole;

    2002-01-01

    unclear whether callose synthases can also produce cellulose and whether plant cellulose synthases may also produce beta-1,3-glucans. We describe here an Arabidopsis gene, AtGsl5, encoding a plasma membrane-localized protein homologous to yeast beta-1,3-glucan synthase whose expression partially......Beta-1,3-glucan polymers are major structural components of fungal cell walls, while cellulosic beta-1,4-glucan is the predominant polysaccharide in plant cell walls. Plant beta-1,3-glucan, called callose, is produced in pollen and in response to pathogen attack and wounding, but it has been...

  20. Cloning, expression and characterization of histidine-tagged biotin synthase of Mycobacterium tuberculosis.

    Magwamba, Clement Chedza; Rukseree, Kamolchanok; Palittapongarnpim, Prasit

    2016-05-01

    The emergence of Mycobacterium tuberculosis strains that are resistant to the current anti-tuberculosis (TB) drugs necessitates a need to develop a new class of drugs whose targets are different from the current ones. M. tuberculosis biotin synthase (MtbBS) is one such target that is essential for the survival of the bacteria. In this study, MtbBS was cloned, overexpressed and purified to homogeneity for biochemical characterization. It is likely to be a dimer in its native form. Its pH and temperature optima are 8.0 and 37 °C, respectively. Km for DTB and SAM was 2.81 ± 0.35 and 9.95 ± 0.98 μM, respectively. The enzyme had a maximum velocity of 0.575 ± 0.015 μM min(-1), and a turn-over of 0.0935 min(-1). 5'-deoxyadenosine (dAH), S-(5'-Adenosyl)-l-cysteine (AdoCy) and S-(5'-Adenosyl)-l-homocysteine (AdoHcy) were competitive inhibitors of MtbBS with the following inactivation parameters: Ki = 24.2 μM, IC50 = 267.4 μM; Ki = 0.84 μM, IC50 = 9.28 μM; and Ki = 0.592 μM, IC50 = 6.54 μM for dAH, AdoCy and AdoHcy respectively. dAH could inhibit the growth of M. tuberculosis H37Ra with an MIC of 392.6 μg/ml. This information should be useful for the discovery of inhibitors of MtbBS. PMID:27156617

  1. Expression of the Grifola frondosa Trehalose Synthase Gene and Improvement of Drought-Tolerance in Sugarcane (Saccharum officinarum L.)

    2006-01-01

    Trehalose is a nonreducing disaccharide of glucose that functions as a protectant in the stabilization of biological structures and enhances stress tolerance to abiotic stresses in organisms. We report here the expression of a Grifola frondosa trehalose synthase (TSase) gene for improving drought tolerance in sugarcane (Saccharum officinarum L.). The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and transferred into sugarcane by Agrobacterium tumefaciens EHA105. The transgenic plants accumulated high levels of trehalose, up to 8.805-12.863 mg/g fresh weight, whereas it was present at undetectable level in nontransgenic plants. It has been reported that transgenic plants transformed with Escherichia coli TPS (trehalose-6-phosphatesynthase) and/or TPP (trehalose-6-phosphate phosphatase) are severely stunted and have root morphologic alterations. Interestingly, our transgenic sugarcane plants had no obvious morphological changes and no growth inhibition in the field. Trehalose accumulation in 35S-35S: TSase plants resulted in increased drought tolerance, as shown by the drought and the drought physiological indexes, such as the rate of bound water/free water, plasma membrane permeability, malondialdehyde content, chlorophyll a and b contents,and activity of SOD and POD of the excised leaves. These results suggest that transgenic plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought.

  2. Identification of the trehalose-6-phosphate synthase gene family in winter wheat and expression analysis under conditions of freezing stress

    D. W. Xie; X. N. Wang; L. S. Fu; J. Sun; W. Zheng; Z. F. Li

    2015-03-01

    Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in plants. Trehalose contents are potentially modulated by trehalose-6-phosphate synthase (TPS), which is a key enzyme in the trehalose biosynthetic pathway. Using available wheat expressed sequence tag sequence information from NCBI and two wheat genome databases, we identified 12 wheat TPS genes and performed a comprehensive study on their structural, evolutionary and functional properties. The estimated divergence time of wheat TPS gene pairs and wheat–rice orthologues suggested that wheat and rice have a common ancestor. The number of TPS genes in the wheat genome was estimated to be at least 12, which is close to the number found in rice, Arabidopsis and soybean. Moreover, it has been reported earlier in other plants that TPS genes respond to abiotic stress, however, our study mainly analysed the TPS gene family under freezing conditions in winter wheat, and determined that most of the TPS gene expression in winter wheat was induced by freezing conditions, which further suggested that wheat TPS genes were involved in winter wheat freeze-resistance signal transduction pathways. Taken together, the current study represents the first comprehensive study of TPS genes in winter wheat and provides a foundation for future functional studies of this important gene family in Triticeae.

  3. Triptolide Inhibits Cyclooxygenase-2 and Inducible Nitric Oxide Synthase Expression in Human Colon Cancer and Leukemia Cells

    Xiangmin TONG; Shui ZHENG; Jie JIN; Lifen ZHU; Yinjun LOU; Hangping YAO

    2007-01-01

    Triptolide (TP), a traditional Chinese medicine, has been reported to be effective in the treatment of autoimmune diseases and exerting antineoplastic activity in several human tumor cell lines. This study investigates the antitumor effect of TP in human colon cancer cells (SW114) and myelocytic leukemia (K562), and elucidates the possible molecular mechanism involved. SW114 and K562 cells were treated with different doses of TP (0, 5, 10, 20, or 50 ng/ml). The cell viability was assessed by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide (MTT). Results demonstrated that TP inhibited the proliferation of both tumor cell lines in a dose-dependent manner. To further investigate its mechanisms, the products prostaglandin E2 (PGE2) and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay (ELISA). Our data showed that TP strongly inhibited the production of NO and PGE2. Consistent with these results, the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) was up-regulated both at the mRNA level and the protein expression level, as shown by real-time RT-PCR and Western blotting. These results indicated that the inhibition of the inflammatory factor COX-2 and iNOS activity could be involved in the antitumor mechanisms of TP.

  4. Expression and purification of cysteine mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear magnetic resonance study

    Jiafu Liu; Kejiang Lin; Chenyun Guo; Hongchang Gao; Yihe Yao; Donghai Lin

    2008-01-01

    Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the only member of the lipocalin superfamily that displays enzymatic activity. It binds lipophilic ligands with high affinity and also can catalyze PGH2 to produce PGD2. Three cysteine residues, Cys 65 , Cys 89 , and Cys 186 in L-PGDS, are conserved among all species, of which Cys 89 and Cys 186 residues form a disulfide bridge. In this study, we clarified the effects of thiol groups on the structure of the protein and investigated the structural significance of Cys residues of rat L-PGDS by site-directed mutagenesis. Four mutants were constructed by substituting Cys residues with alanine to identify the correct formation of disulfide bonds among these three residues. The effects of thiol groups on the structure of rat L-PGDS were also identified by these mutants. Analysis of HSQC experiments indicated that these enzymes were all properly folded with well defined tertiary structures. As the first step towards the 3-D nuclear magnetic resonance solution structure, we optimized expression of recombinant rat L-PGDS in Escherichia coli and established an efficient and economic purification protocol yielding large amounts of pure isotopically labeled rat L-PGDS. The results of assignments indicated that the wild-type rat L-PGDS obtained using this expression system was suitable for determination of 3-D nuclear magnetic resonance solution structure.

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

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

    2011-01-01

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

  6. Expression of nitric oxide synthase and transforming growth factor-beta in crush-injured tendon and synovium

    Adam Curtis

    1992-01-01

    Full Text Available THIS study examined the expression of inducible nitric oxide synthase (iNOS and transforming growth factor-beta (TGF-β in macrophage infiltrates within crush-injured digital flexor tendon and synovium of control rats and rats treated with N(G-nitro-l-arginine methyl ester (L-NAME (5 mg/kg. Release of TGF-β from organ cultures of tendon, muscle, and synovium, and the effects of L-NAME treatment (in vitro and in vivo, on adhesion of peritoneal macrophages to epitenon monolayers were also investigated. The results showed that during normal tendon healing the levels of TGF-β are high at first and gradually decrease after 3 weeks of injury to slightly above control uninjured levels. However, when L-NAME was administered at the time of injury, the macrophage infiltrates were expressing high levels of TGF-β even at 5 weeks after the injury, with no evidence of reduction. In the standard injury, iNOS activity was greatest at the acute phase of the inflammatory response and then gradually returned to normal. Treatment with L-NAME, however, resulted in inhibition of iNOS activity at 3 days and a reduction in the activity at the later time points examined after injury. We also found greatly increased levels of adhesion of peritoneal macrophages from L-NAME-treated rats to epitenon monolayers in vitro, which reflect a chronic imbalance in expression of TGF-β, which is overexpressed, and nitric oxide, which is underexpressed. The results of the current study show that formation of nitric oxide is an important event in the course of tendon healing since its inhibition results in chronic inflammation and fibrosis due to an imbalance in TGF-β expression in vivo.

  7. Expression of nitric oxide synthase and transforming growth factor-beta in crush-injured tendon and synovium.

    Darmani, Horma; Crossan, James; McLellan, Sarah D; Meek, Dominic; Adam, Curtis

    2004-12-01

    This study examined the expression of inducible nitric oxide synthase (iNOS) and transforming growth factor-beta (TGF-beta) in macrophage infiltrates within crush-injured digital flexor tendon and synovium of control rats and rats treated with N(G)-nitro-1-arginine methyl ester (L-NAME) (5 mg/kg). Release of TGF-beta from organ cultures of tendon, muscle, and synovium, and the effects of L-NAME treatment (in vitro and in vivo), on adhesion of peritoneal macrophages to epitenon monolayers were also investigated. The results showed that during normal tendon healing the levels of TGF-beta are high at first and gradually decrease after 3 weeks of injury to slightly above control uninjured levels. However, when L-NAME was administered at the time of injury, the macrophage infiltrates were expressing high levels of TGF-beta even at 5 weeks after the injury, with no evidence of reduction. In the standard injury, iNOS activity was greatest at the acute phase of the inflammatory response and then gradually returned to normal. Treatment with L-NAME, however, resulted in inhibition of iNOS activity at 3 days and a reduction in the activity at the later time points examined after injury. We also found greatly increased levels of adhesion of peritoneal macrophages from L-NAME-treated rats to epitenon monolayers in vitro, which reflect a chronic imbalance in expression of TGF-beta, which is overexpressed, and nitric oxide, which is underexpressed. The results of the current study show that formation of nitric oxide is an important event in the course of tendon healing since its inhibition results in chronic inflammation and fibrosis due to an imbalance in TGF-beta expression in vivo. PMID:15770044

  8. Jinggangmycin increases fecundity of the brown planthopper, Nilaparvata lugens (Stål) via fatty acid synthase gene expression.

    Li, Lei; Jiang, Yiping; Liu, Zongyu; You, Linlin; Wu, You; Xu, Bing; Ge, Linquan; Stanley, David; Song, Qisheng; Wu, Jincai

    2016-01-01

    The antibiotic jinggangmycin (JGM) is mainly used in controlling the rice sheath blight, Rhizoctonia solani, in China. JGM also enhances reproduction of the brown planthopper (BPH), Nilaparvata lugens (Stål). To date, however, molecular mechanisms of the enhancement are unclear. Our related report documented the influence of foliar JGM sprays on ovarian protein content. Here, we used isobaric tags for relative and absolute quantitation (iTRAQ) protocols to analyze ovarian proteins of BPH females following JGM spray (JGM-S) and topical application (JGM-T). We recorded changes in expression of 284 proteins (142↑ and 142↓) in JGM-S compared to the JGM-S control group (S-control); 267 proteins were differentially expressed (130↑ and 137↓) in JGM-T compared to the JGM-T control group (T-control), of which, 22 proteins were up-regulated in both groups. Comparing the JGM-S to the JGM-T group, 114 proteins were differentially expressed (62↑ and 52↓). Based on the biological significance of fatty acids, pathway annotation and enrichment analysis, we designed a dsRNA construct to silence a gene encoding fatty acid synthase (FAS). FAS was more highly expressed in JGM-S vs S-control and JGM-S vs JGM-T groups. The dsFAS treatment reduced fecundity by about 46% and reduced ovarian and fat body fatty acid concentrations in JGM-S-treated females relative to controls. We infer FAS provides critically needed fatty acids to support JGM-enhanced fecundity in BPH. PMID:26388431

  9. The role of the erythroid-specific delta-aminolevulinate synthase gene expression in erythroid heme synthesis.

    Meguro, K; Igarashi, K; Yamamoto, M; Fujita, H; Sassa, S

    1995-08-01

    Using antisense technology, the effects of suppressed gene expression of the erythroid-specific delta-aminolevulinate (ALA) synthase (ALAS-E) on heme synthesis, expression of mRNAs encoding an erythroid-specific transcription factor NF-E2, other heme pathway enzymes, and beta-globin were examined in murine erythroleukemia (MEL) cells. In MEL cells in which an antisense ALAS-E RNA was expressed (AS clone), sense ALAS-E mRNA levels in both untreated and dimethylsulfoxide (DMSO)-treated cells were decreased compared with their respective controls. Heme synthesis in AS clones was decreased in proportion to the suppressed levels of ALAS-E mRNA. In addition, mRNAs for ALA dehydratase, porphobilinogen deaminase, ferrochelatase (FeC), and beta-globin were also decreased in AS clones. There was a strong correlation between the level of ALAS-E mRNA and most of the mRNAs of the heme pathway enzymes and beta-globin. There was a decrease in the mRNA level of p45, but not of mafK, which are the large and the small subunits of NF-E2, respectively, in AS clones. Treatment of AS cells with hemin and ALA in the presence of DMSO partially restored the suppressed mRNA levels for beta-globin and FeC and heme content, respectively. These findings thus indicate that heme formation, which is determined by the level of ALAS-E, plays an essential role on gene expression of many proteins necessary for erythroid development. PMID:7620186

  10. Expression of Nitric Oxide Synthase Isoenzyme in Lung Tissue of Smokers with and without Chronic Obstructive Pulmonary Disease

    Wen-Ting Jiang

    2015-01-01

    Full Text Available Background: It has been demonstrated that only 10%-20% cigarette smokers finally suffer chronic obstructive pulmonary disease (COPD. The underlying mechanism of development remains uncertain so far. Nitric oxide (NO has been found to be closely associated with the pathogenesis of COPD, the alteration of NO synthase (NOS expression need to be revealed. The study aimed to investigate the alterations of NOS isoforms expressions between smokers with and without COPD, which might be helpful for identifying the susceptibility of smokers developing into COPD. Methods: Peripheral lung tissues were obtained from 10 nonsmoker control subjects, 15 non-COPD smokers, and 15 smokers with COPD. Neuronal NOS (nNOS, inducible NOS (iNOS, and endothelial NOS (eNOS mRNA and protein levels were measured in each sample by using real-time polymerase chain reaction and Western blotting. Results: INOS mRNA was significantly increased in patients with COPD compared with nonsmokers and smokers with normal lung function (P < 0.001, P = 0.001, respectively. iNOS protein was also higher in COPD patients than nonsmokers and smokers with normal lung function (P < 0.01 and P = 0.01, respectively. However, expressions of nNOS and eNOS did not differ among nonsmokers, smokers with and without COPD. Furthermore, there was a negative correlation between iNOS protein level and lung function parameters forced expiratory volume in 1 s (FEV 1 (% predicted (r = −0.549, P = 0.001 and FEV 1 /forced vital capacity (%, r = −0.535, P = 0.001. Conclusions: The expression of iNOS significantly increased in smokers with COPD compared with that in nonsmokers or smokers without COPD. The results suggest that iNOS might be involved in the pathogenesis of COPD, and may be a potential marker to identify the smokers who have more liability to suffer COPD.